AU2022396272A1 - Antibodies against ctla-4 and methods of use thereof - Google Patents

Antibodies against ctla-4 and methods of use thereof Download PDF

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AU2022396272A1
AU2022396272A1 AU2022396272A AU2022396272A AU2022396272A1 AU 2022396272 A1 AU2022396272 A1 AU 2022396272A1 AU 2022396272 A AU2022396272 A AU 2022396272A AU 2022396272 A AU2022396272 A AU 2022396272A AU 2022396272 A1 AU2022396272 A1 AU 2022396272A1
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Matthew Chang
Wayne A. Marasco
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Dana Farber Cancer Institute Inc
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Abstract

The present invention is directed to antibodies against human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) and methods of use thereof.

Description

ANTIBODIES AGAINST CTLA-4 AND METHODS OF USE THEREOF
[0001] This application claims priority to U.S. Provisional Application No. 63/282,887, filed on November 24, 2021, the entire contents of which are incorporated herein by reference.
[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.
SEQUENCE LISTING
[0004] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on [ ], is named [ ] and is [ ] bytes in size.
FIELD OF THE INVENTION
[0005] This invention is directed to antibodies against human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) and methods of use thereof.
BACKGROUND
[0006] CTLA-4 is an inhibitory receptor acting as a major negative regulator of T-cell responses. The affinity of CTLA4 for its natural B7 family ligands, CD80 and CD86, is stronger that the affinity of their cognate stimulatory coreceptor CD28.
SUMMARY OF THE INVENTION [0007] The invention provides for human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) and methods of use thereof.
[0008] Embodiments are drawn to an isolated antibody or fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 26, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 61, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 63; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 30, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69; or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; or (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 39, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; or (I) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78; or (g) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 79, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 81; or (h) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 82, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 84; or (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 85, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or j) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 90; or (k) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 91, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 92, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93; or (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 58, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 60, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 94, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 95, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 96; or (m) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 413, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 414, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 415, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 455, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 456, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 457; or (n) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 416, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 417, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 418, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 458, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 459, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 460; or (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 419, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 420, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 421, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 461, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 462, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 463; or (p) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 422, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 423, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 424, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 464, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 465, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 466; or (q) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 425, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 426, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 427, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 467, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 468, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 469; or (r) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 428, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or (s) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 432, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 433, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 473, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 474, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 475; or (t) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 434, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 435, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 436, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 476, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 477, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 478; or (u) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 437, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 438, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 439, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 479, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 480, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 481; or (v) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 440, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 441, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 442, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 482, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 483, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 484; or (w) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 443, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 444, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 445, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 485, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 486, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 487; or (x) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 446, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 447, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 448, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 488, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 489, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 490; or (y) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 449, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 450, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 451, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 491, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 492, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 493; or (z) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 452, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 453, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 454, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 494, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 495, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 496; or (aa) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 857, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 858, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 859, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 892, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 893, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 894; or (bb) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 860, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 861, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 862, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 895, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 896, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 897; or (cc) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 863, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 864, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 865, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 898, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 899, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 900; or (dd) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 866, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 867, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 868, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 901, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 902, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 903; or (ee) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 869, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 870, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 871, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 904, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 905, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 906; or (ff) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 872, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 873, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 874, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 907, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 908, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 909; or (gg) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 875, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 876, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 877, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 910, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 911, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 912; or (hh) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 878, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 1000, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 879, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 913, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 914, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 915; or (ii) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 880, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 881, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 882, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 916, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 917, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 918; or (jj) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 883, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 884, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 885, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 919, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 920, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 921; or (kk) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 886, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 887, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 888, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 922, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 923, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 924; or (11) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927.
[0009] Aspects of the invention are further drawn towards an isolated scFv antibody, that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising: (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 26, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 61, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 63; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 30, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69; or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; or (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 39, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; or (I) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78; or (g) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 79, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 81; or (h) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 82, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 84; or (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 85, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or (j) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 90; or (k) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 91, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 92, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93; or (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 58, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 60, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 94, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 95, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 96; or (m) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 413, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 414, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 415, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 455, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 456, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 457; or (n) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 416, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 417, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 418, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 458, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 459, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 460; or (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 419, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 420, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 421, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 461, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 462, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 463; or (p) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 422, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 423, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 424, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 464, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 465, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 466; or (q) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 425, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 426, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 427, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 467, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 468, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 469; or (r) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 428, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or (s) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 432, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 433, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 473, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 474, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 475; or (t) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 434, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 435, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 436, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 476, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 477, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 478; or (u) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 437, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 438, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 439, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 479, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 480, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 481 ; or (v) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 440, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 441, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 442, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 482, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 483, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 484; or (w) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 443, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 444, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 445, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 485, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 486, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 487; or (x) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 446, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 447, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 448, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 488, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 489, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 490; or (y) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 449, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 450, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 451, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 491, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 492, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 493; or (z) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 452, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 453, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 454, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 494, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 495, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 496; or (aa) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 857, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 858, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 859, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 892, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 893, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 894; or (bb) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 860, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 861, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 862, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 895, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 896, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 897; or (cc) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 863, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 864, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 865, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 898, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 899, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 900; or (dd) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 866, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 867, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 868, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 901, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 902, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 903; or (ee) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 869, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 870, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 871, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 904, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 905, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 906; or (ff) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 872, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 873, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 874, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 907, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 908, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 909; or (gg) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 875, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 876, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 877, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 910, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 911, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 912; or (hh) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 878, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 1000, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 879, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 913, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 914, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 915; or (ii) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 880, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 881, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 882, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 916, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 917, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 918; or (jj) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 883, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 884, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 885, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 919, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 920, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 921; or (kk) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 886, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 887, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 888, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 922, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 923, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 924; or (11) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927.
[0010] Still further, aspects of the invention are drawn to an isolated antibody or fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, and 1023 and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 368, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, and 1024.
[0011] Also, aspects of the invention are drawn to an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, and 1023 and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 368, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, and 1024.
[0012] For example, an embodiment is drawn to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 2.
[0013] Another embodiment is drawn to an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 2.
[0014] A further embodiment is drawn to an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 3, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 4. [0015] An embodiment is also drawn to an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 3, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 4.
[0016] An embodiment is drawn to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA- 4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 5, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 6.
[0017] Also, embodiments are drawn to an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 5, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 6.
[0018] Still further, embodiments are drawn to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 7, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 8.
[0019] Also, embodiments are drawn to an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 7, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 8.
[0020] Further embodiments are drawn to an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 9, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 10. [0021] Also, embodiments are drawn to an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 9, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 10.
[0022] Embodiments are drawn to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA- 4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 11, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 12.
[0023] Embodiments are also drawn to an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 11, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 12.
[0024] Further, embodiments are drawn towards an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 13, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 14.
[0025] Still further, embodiments are drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 13, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 14.
[0026] Also, embodiments are drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 15, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 16. [0027] Still further, embodiments are drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 15, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 16.
[0028] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 17, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 18.
[0029] Further, embodiments are drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 17, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 18.
[0030] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 19, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 20.
[0031] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 19, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 20.
[0032] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 21, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 22. [0033] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 21, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 22.
[0034] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 23, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 24.
[0035] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 23, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 24.
[0036] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 385, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 386.
[0037] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 385, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 386.
[0038] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 387, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 388. [0039] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 387, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 388.
[0040] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 389, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 390.
[0041] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 389, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 390.
[0042] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 391, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 392.
[0043] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 391, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 392.
[0044] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 393, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 394. [0045] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 393, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 394.
[0046] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 395, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 396.
[0047] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 395, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 396.
[0048] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 397, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 398.
[0049] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 397, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 398.
[0050] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 399, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 400. [0051] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 399, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 400.
[0052] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 401, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 402.
[0053] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 401, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 402.
[0054] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 403, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 404.
[0055] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 403, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 404.
[0056] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 405, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 406. [0057] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 405, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 406.
[0058] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 407, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 408.
[0059] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 407, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 408.
[0060] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 409, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 410.
[0061] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 409, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 410.
[0062] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 411, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 412. [0063] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 411, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 412.
[0064] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1001, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1002.
[0065] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1001, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1002.
[0066] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1003, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1004.
[0067] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1003, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1004.
[0068] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1005, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1006.
[0069] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1005, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1006.
[0070] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1007, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1008.
[0071] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1007, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1008.
[0072] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1009, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1010.
[0073] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1009, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1010. [0074] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1011, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1012.
[0075] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1011, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1012.
[0076] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1013, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1014.
[0077] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1013, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1014.
[0078] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1015, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1016.
[0079] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1015, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1016.
[0080] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1017, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1018.
[0081] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1017, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1018.
[0082] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1019, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1020.
[0083] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1019, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1020.
[0084] Embodiments are also drawn towards n isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1021, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1022. [0085] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1021, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1022.
[0086] Embodiments are also drawn towards an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1023, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1024.
[0087] Embodiments are also drawn towards an isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1023, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1024.
[0088] Aspects of the invention are also drawn towards an isolated monoclonal antibody or antigen-binding fragment of claims 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, or 79, wherein the isolated monoclonal antibody or antigen-binding fragment comprises a wildtype Fc or a modified Fc.
[0089] Further, aspects of the invention are drawn to an isolated bispecific antibody comprising an antibody or fragment as described herein, and a second antigenbinding fragment having specificity to a molecule on an immune cell. For example, the molecule is selected from the group consisting of CCR4, B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, PD-1, TIM3, 4-1BB, TIGIT, VISTA, HEVM, BTLA, CD47, PDL1, MICA, MICB, and KIR.
[0090] In embodiments, the fragment and the second fragment each is independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. [0091] Embodiments can further comprise a Fc fragment. For example, the Fc fragment comprises a wildtype Fc fragment or a modified Fc fragment.
[0092] Aspects of the invention are drawn towards a nucleic acid encoding an antibody as described herein.
[0093] Still further, aspects of the invention are drawn towards a nucleic acid encoding a bispecific antibody as described herein.
[0094] Also, aspects of the invention are drawn towards a pharmaceutical composition comprising the antibody or fragment thereof as described herein, and a pharmaceutically acceptable carrier or excipient.
[0095] Embodiments can further comprise at least one additional therapeutic agent. For example, the therapeutic agent can be a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine.
[0096] Aspects of the invention are drawn to a pharmaceutical composition comprising the bispecific antibody as described herein, and a pharmaceutically acceptable carrier or excipient.
[0097] In embodiments, the pharmaceutical composition can further comprise at least one additional therapeutic agent. For example, the therapeutic agent can be a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine.
[0098] Further, aspects of the invention are drawn to an isolated cell comprising one or more polynucleotide(s) encoding an antibody or fragment as described herein.
[0099] Also, aspects of the invention are drawn towards an isolated cell comprising one or more polynucleotide(s) encoding a bispecific antibody or fragment thereof as described herein.
[00100] Further, aspects of the invention are drawn towards a vector comprising a nucleic acid as described herein.
[00101] Also, aspects of the invention are drawn to a cell comprising a vector as described herein. [00102] Aspects of the invention are drawn to methods of treating a cancer in a subject. For example, the method comprises administering to a subject a therapeutically effective amount of the CTLA-4 antibody or fragment thereof as described herein. For example, the method comprises administering to a subject a therapeutically effective amount of a CTLA-4 bispecific antibody as described herein.
[00103] Aspects of the invention are also drawn towards a chimeric antigen receptor (CAR). In embodiments, the CAR comprises an intracellular signaling domain, a transmembrane domain and an extracellular domain, wherein the extracellular domain is an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), wherein the monoclonal antibody or fragment thereof comprises a heavy chain, light chain, or combination thereof, wherein the heavy chain comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 26, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 61, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 63; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 30, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69; or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; or (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 39, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; or (I) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78; or (g) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 79, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 81; or (h) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 82, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 84; or (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 85, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or j) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 90; or (k) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 91, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 92, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93; or (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 58, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 60, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 94, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 95, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 96; or (m) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 413, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 414, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 415, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 455, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 456, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 457; or (n) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 416, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 417, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 418, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 458, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 459, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 460; or (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 419, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 420, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 421, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 461, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 462, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 463; or (p) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 422, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 423, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 424, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 464, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 465, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 466; or (q) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 425, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 426, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 427, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 467, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 468, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 469; or (r) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 428, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or (s) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 432, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 433, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 473, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 474, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 475; or (y) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 434, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 435, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 436, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 476, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 477, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 478; or (u) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 437, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 438, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 439, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 479, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 480, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 481 ; or (v) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 440, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 441, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 442, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 482, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 483, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 484; or (w) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 443, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 444, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 445, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 485, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 486, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 487; or (x) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 446, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 447, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 448, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 488, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 489, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 490; or (y) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 449, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 450, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 451, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 491, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 492, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 493; or (z) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 452, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 453, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 454, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 494, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 495, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 496; or (aa) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 857, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 858, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 859, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 892, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 893, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 894; or (bb) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 860, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 861, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 862, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 895, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 896, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 897; or (cc) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 863, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 864, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 865, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 898, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 899, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 900; or (dd) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 866, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 867, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 868, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 901, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 902, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 903; or (ee) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 869, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 870, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 871, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 904, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 905, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 906; or (ff) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 872, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 873, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 874, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 907, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 908, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 909; or (gg) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 875, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 876, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 877, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 910, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 911, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 912; or (hh) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 878, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 1000, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 879, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 913, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 914, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 915; or (ii) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 880, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 881, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 882, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 916, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 917, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 918; or (jj) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 883, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 884, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 885, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 919, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 920, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 921; or (kk) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 886, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 887, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 888, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 922, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 923, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 924; or (11) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927. For example, the transmembrane domain comprises CD28.
[00104] In embodiments, the transmembrane domain further comprises a stalk region positioned between the extracellular domain and the transmembrane domain.
[00105] Embodiments can further comprise one or more additional costimulatory molecules positioned between the transmembrane domain and the intracellular signaling domain. For example, the costimulatory molecules is CD28, 4-1BB, ICOS, or 0X40. For example, the intracellular signaling domain comprises a CD3 zeta chain.
[00106] In embodiments, the antibody is a Fab or a scFV. [00107] Aspects of the invention are also drawn to a nucleic acid encoding a CAR as described herein. In embodiments, the nucleic acid can further comprise a nucleic acid encoding a polypeptide positioned after the intracellular signaling domain. For example, the polypeptide is an antibody or a cytokine, such as an scFV.
[00108] Aspescts of the invention are drawn towards a nucleic acid encoding a CAR, wherein the CAR comprises an intracellular signaling domain, a transmembrane domain and an extracellular domain, further comprising a nucleic acid encoding a polypeptide positioned after the intracellular signaling domain, wherein the polypeptide comprises an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T- Lymphocyte Associated Protein 4 (CTLA-4), wherein the monoclonal antibody or fragment thereof comprises a heavy chain, light chain, or combination thereof, wherein the heavy chain comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 26, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 61, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 63; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 30, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69; or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; or (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 39, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; or (I) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78; or (g) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 79, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 81; or (h) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 82, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 84; or (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 85, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or (j) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 90; or (k) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 91, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 92, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93; or (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 58, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 60, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 94, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 95, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 96; or (m) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 413, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 414, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 415, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 455, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 456, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 457; or (n) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 416, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 417, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 418, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 458, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 459, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 460; or (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 419, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 420, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 421, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 461, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 462, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 463; or (p) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 422, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 423, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 424, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 464, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 465, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 466; or (q) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 425, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 426, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 427, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 467, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 468, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 469; or (r) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 428, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or (s) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 432, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 433, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 473, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 474, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 475; or (t) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 434, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 435, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 436, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 476, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 477, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 478; or (u) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 437, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 438, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 439, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 479, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 480, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 481 ; or (v) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 440, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 441, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 442, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 482, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 483, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 484; or (w) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 443, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 444, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 445, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 485, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 486, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 487; or (x) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 446, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 447, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 448, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 488, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 489, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 490; or (y) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 449, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 450, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 451, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 491, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 492, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 493; or (z) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 452, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 453, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 454, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 494, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 495, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 496; or (aa) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 857, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 858, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 859, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 892, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 893, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 894; or (bb) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 860, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 861, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 862, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 895, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 896, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 897; or (cc) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 863, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 864, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 865, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 898, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 899, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 900; or (dd) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 866, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 867, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 868, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 901, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 902, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 903; or (ee) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 869, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 870, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 871, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 904, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 905, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 906; or (ff) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 872, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 873, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 874, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 907, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 908, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 909; or (gg) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 875, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 876, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 877, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 910, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 911, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 912; or (hh) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 878, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 1000, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 879, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 913, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 914, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 915; or (ii) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 880, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 881, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 882, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 916, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 917, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 918; or (jj) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 883, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 884, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 885, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 919, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 920, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 921; or (kk) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 886, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 887, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 888, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 922, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 923, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 924; or (11) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927..
[00109] Aspects of the invention are drawn towards a vector comprising the nucleic acid as describd herein.
[00110] Aspects of the invention are also drawn to a cell comprising a vector as described herein.
[00111] Aspects of the invention are drawn towards a genetically engineered cell which expresses and bears on the cell surface membrane a chimeric antigen receptor as described herein. For example, the cell is a T-cell or an NK cell. For example, the T cell is CD4+ or CD8+. [00112] Embodiments can further comprise a mixed population of CD4+ and CD8 cells+.
[00113] Aspects of the invention are also drawn towards a genetically engineered cell which express and bear on the cell surface membrane a chimeric antigen receptor, and which is further engineered to express and secrete a polypeptide, wherein polypeptide is an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T- Lymphocyte Associated Protein 4 (CTLA-4), wherein the monoclonal antibody or fragment thereof comprises a heavy chain, light chain, or combination thereof, wherein the heavy chain comprises (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 26, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 61, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 63; or (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 30, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66; or (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69; or (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; or (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 39, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; or (I) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78; or (g) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 79, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 81; or (h) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 82, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 84; or (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 85, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or (j) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 90; or (k) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 91, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 92, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93; or (1) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 58, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 60, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 94, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 95, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 96; or (m) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 413, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 414, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 415, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 455, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 456, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 457; or (n) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 416, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 417, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 418, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 458, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 459, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 460; or (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 419, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 420, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 421, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 461, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 462, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 463; or (p) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 422, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 423, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 424, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 464, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 465, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 466; or (q) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 425, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 426, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 427, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 467, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 468, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 469; or (r) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 428, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or (s) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 432, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 433, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 473, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 474, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 475; or (t) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 434, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 435, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 436, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 476, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 477, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 478; or (u) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 437, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 438, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 439, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 479, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 480, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 481 ; or (v) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 440, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 441, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 442, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 482, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 483, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 484; or (w) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 443, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 444, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 445, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 485, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 486, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 487; or (x) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 446, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 447, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 448, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 488, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 489, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 490; or (y) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 449, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 450, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 451, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 491, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 492, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 493; or (z) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 452, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 453, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 454, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 494, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 495, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 496; or (aa) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 857, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 858, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 859, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 892, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 893, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 894; or (bb) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 860, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 861, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 862, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 895, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 896, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 897; or (cc) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 863, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 864, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 865, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 898, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 899, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 900; or (dd) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 866, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 867, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 868, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 901, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 902, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 903; or (ee) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 869, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 870, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 871, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 904, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 905, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 906; or (ff) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 872, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 873, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 874, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 907, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 908, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 909; or (gg) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 875, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 876, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 877, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 910, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 911, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 912; or (hh) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 878, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 1000, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 879, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 913, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 914, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 915; or (ii) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 880, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 881, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 882, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 916, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 917, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 918; or (jj) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 883, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 884, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 885, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 919, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 920, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 921; or (kk) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 886, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 887, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 888, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 922, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 923, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 924; or (11) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927.
[00114] Other objects and advantages of this invention are readily apparent from the ensuing description.
BRIEF DESCRIPTION OF THE FIGURES
[00115] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
[00116] FIG. 1 shows binding ELISA with PEG purified phage at varying dilutions.
[00117] FIG. 2 shows FACS confirms CTLA-4 phages can bind to CTLA-4-expressing
1G5 Jurkat cells. [00118] FIG. 3 provides anti-CTLA-4 antibody amino acid (AA) and nucleotide (Nuc) sequences. “HC” is heavy chain, and “LC” is light chain.
[00119] FIG. 4 - FIG. 8 provides anti-CTLA-4 antibody amino acid (AA) and nucleotide (Nuc) sequences.
[00120] FIG. 9 - FIG. 11 provides anti-CTLA-4 antibody amino acid (AA) and nucleotide (Nuc) sequences.
[00121] FIG. 12 shows percent competition with CD80 hFc for binding to CTLA4 on 1G5 Jurkat. 1G5 Jurkat cells were transduced to overexpress CTLA4. Cells were incubated with soluble antibody at the indicated concentration, washed, then incubated with labeled CD80- Fc. Samples were read on a BD FACSCanto II. Decrease in fluorescence was used to indicate blockade of CD80 binding to the CTLA4 on the surface of the cells.
[00122] FIG. 13 shows a ribbon diagram adapted from Metzler, William J., et al. “Solution Structure of Human CTLA-4 and Delineation of a CD80/CD86 Binding Site Conserved in CD28.”
[00123] FIG. 14 shows ribbon diagrams adapted from Stamper, Carin C., et al. “Crystal Structure of the B7-1/CTLA-4 Complex That Inhibits Human Immune Responses.” Nature.
[00124] FIG. 15 shows schematics of mechanisms to suppress immune response.
[00125] FIG. 16 shows ribbon diagrams of existing therapies: Ipilimumab- IgGl and Tremelimumab- IgG2. Panels A and B show ribbon diagrams adapted from Ramagopal, Udupi A., et al. “Structural Basis for Cancer Immunotherapy by the First-in-Class Checkpoint Inhibitor Ipilimumab.” Panel C shows a ribbon diagram adapted from He, Mengnan, et al. “Remarkably Similar CTLA-4 Binding Properties of Therapeutic Ipilimumab and Tremelimumab Antibodies.”
[00126] FIG. 17 shows a chart of exemplary panning results for anti-CTLA4 antibodies. Rounds: 1, lb, 2, 2b, 3, 3b. Mouse CTLA4 were introduced in rounds 3 and 3b. 10 unique antibodies were the result.
[00127] FIG. 18 shows a chart of exemplary kinetic analysis of aCTLA4 antibodies.
[00128] FIG. 19 shows a chart of exemplary competition analysis of aCTLA4 antibodies. [00129] FIG. 20 shows a graph of FACS of aCTLA4 antibodies binding to T cells. E1-D7 binds significantly more CTLA4 on T cells compared to T2-C10. An anti-CTLA4 ab from biolegend was used at a single point (recommended staining cone) for the control. Ipi IgG is an antibody cloned in the lab to represent ipilimumab, it is not a commercial prep or biosimilar.
[00130] FIG. 21 shows exemplary FACS of aCTLA4 Abs binding to T Cells.
[00131] FIG. 22 shows exemplary graphs of ELISA results of mouse/human CTLA4 cross reactivity. E1-D7 cross reacts with mouse CTLA4.
[00132] FIG. 23 shows a schematic of exemplary mechanisms of T cells adapted from P. Ott et al. 2014.
[00133] FIG. 24 shows a schematic of an MLR assay. Schematic courtesy of Explicyte. Protocol: Co-culture of CD4+ T cells with monocyte-derived DCs each from different donors. This indicated allorecognition of the dendritic cells leads to activation of T cells. DCs expressing PDL1 can suppress T cell activation. Addition of immune checkpoint inhibitors can recover T cell from suppression. We investigated whether anti-PDl enhances T cell activation. Measured IFNy and IL2 cytokine production, indicators of T cell activation.
[00134] FIG. 25 shows data from immunophenotyping of monocytes, imMo-DCs, and mMo-DCs adapted from Miltenyi. In this protocol Isolate CD14+ monocytes using Miltenyi CD 14+ microbeads were cultured in Miltenyi Mo-DC media (pre-prepared media with GM- CSF + IL4) and cultured for 5 days. Then, TNF-a (1000 U/ml), IL-ip (5 ng/ml), IL-6 (10 ng/ml) and prostaglandin E2 (PGE2) (1 pM) was added and cultured for 2 days to mature DC. mMo-DCs express various DC markers that are involved in the formation of immunological synapses between DCs and naive T cells, including CD80, CD86, and MHC II (HLA-DR). Mature Mo-DCs also expressed DC activation markers CD83, CD40, and CCR7.
[00135] FIG. 26 shows data and analysis of DC donor AD staining.
[00136] FIG. 27 shows graphs of exemplary MLR1 data: measuring IFNy production. We investigated if E1-D7 suppressed T cell activation. A decrease in IFNy is seen in wells treated with E1-D7 compared to T cells + DC only, anti-PDl treatment, and irrelevant Ab control.
An absorbance value of 4 was used to represent saturation.
[00137] FIG. 28 shows graphs of exemplary data of the combination of aCTLA4/aPD-l MLR: IFNy production.
[00138] FIG. 29 shows an exemplary schematic of the second panning using CTLA4- PMPLs.
[00139] FIG. 30 shows exemplary graphs of data from binding ELISA with PEG purified phage at varying dilutions.
[00140] FIG. 31 shows exemplary FACS confirming our CTLA4 phages can bind to CTLA-4 expressing 1G5 Jurkat cells.
[00141] FIG. 32 shows a chart of exemplary kinetic screening data of aCTLA4 scFv-Fc sup.
[00142] FIG. 33 shows an exemplary binding curve for hCTLA4.
[00143] FIG. 34 shows an exemplary binding curve for mCTLA4.
[00144] FIG. 35 shows a BSA negative control binding curve.
[00145] FIG. 36 shows a chart of data and observations. The data is arranged in decreasing order of Association response at 25nM concentration of antibodies. All candidate antibodies have higher dissociation rates than Ipilimumab and BiolegendaCTLA4 (commercial controls). Most candidates seem to have a higher association response than the commercial controls. 4 candidates (highlighted in the table) have lower dissociation constants than E1-D7 (Taylor’s Antibody - Lab Control). E2-D10 and E1-A12 have identical heavy chains but have different families of light chains. The difference in their response rates is mediated by light chain binding. E2-H10 also belongs to the same Vh family as the two above.
[00146] FIG. 37 shows a chart of exemplary data from a competition assay. CD80 blockade: All candidate antibodies block CD80 binding to some extent, but none more than Ipilimumab, so I selected for candidates that have higher blockade (lower values of second association) than E1-D7 (lab control, Taylor’s candidate). CD86 blockade: All candidates have negative values of second association, signaling they all block CD86 completely (normalized values = 0). I selected for candidates that have negative values closer to zero when compared to Ipilimumab because higher negative values could indicate antibody dissociation. E1-A8 and E2-A4 belong to the same germline family. E1-D7 blockade: Highlighted candidates block E1-D7 binding better than Ipilimumab. Ipilimumab blockade: Highlighted candidates block Ipilimumab binding better than E1-D7. All candidates that block CD80 and CD86 performed relatively poorly in Kinetics.
[00147] FIG. 38 shows an exemplary graph of CTLA4 binding. Jurkat cells were transduced with hCTLA4 and used to generate a binding curve.
[00148] FIG. 39 shows a schematic of a PROMEGA lucierfase reporter assay adapted from www. moleculardevices . com/ en/ assets/ app-note/br/ characterize-biologics-for-immune- checkpoint-blockade-with-reporter-bioassays-using-spectramax-microplate-readers#gref.
[00149] FIG. 40 shows graphs of exemplary Promega CTLA4 bioassay data. Anti-CTLA4 scFv-Fcs were tested against an Ipi biosimilar IgG. Plates were read using a BMG polarstar omega with an interval of either 1 second (left column) or 10 seconds (right column). El- B10, E1-A8, E2-A4, and E1-D7 show similar activity at about 40% of Ipi. On plate 2, E2-G9 shows comparable activity to ipi however E2-H10, E1-A4, and E2-H12 show minimal improvement.
[00150] FIG. 41 shows an illustration of a bispecific design. BsAbs constructed with the IgGl hinge show a mix of monomer and dimer when expressed so to increase dimerization frequency we switched to an IgG3 hinge. We initially built two constructs, one with a (G4S)5 linker preceding the IgG3 hinge and one without the leading linker. The second linker was maintained at 5 repeats to provide ample space for the 2nd scFv to reach binding sites without interference from the hinge.
[00151] FIG. 42 shows sequences of IgG3 hinge linker sequences.
[00152] FIG. 43 shows a picture of an exemplary labeled SDS Page Gel for Non-reduced and Reduced ElD7-IgG3 hinge-P4B3M3 and P4B3M3-IgG3 hinge-E!D7 bsAbs.
[00153] FIG. 44 shows a chart of exemplary data. SA sensors were loaded with biotinylated CTLA4 or PD1 and the indicated abs were allowed to associate/disassociate. Due to sensor drift, the off rates of the PD1 loaded sensors are unreliable. [00154] FIG. 45 shows data from exemplary dual binding assays. CTLA4+, PD1+, or CTLA4+PD1+ Jurkat cells were first coated with the indicated amount of mono or bispecific protein. Soluble protein was next added and allowed to bind to free arms. As shown here, monospecific abs are not able to bind significant amounts of soluble antigen, however when bound to PD1 expressing cells, the bsAbs are able to capture soluble CTLA4 from solution.
[00155] FIG. 46 shows non-limiting, exemplary data from a competition assay for Ab (including combination mAbs) vs CD80 hFc for CTLA4+/PD1+ 1G5 Jurkat. BsAb demonstrates remarkably improved competition of CD80 binding to PD1+CTLA4+ jurkat cells when compared to a monospecific antibody and a cocktail.
[00156] FIG. 47 shows non-limiting, exemplary data from a % competition assay with CD80 hFc for binding to CTLA4 on 1G5 Jurkat. CD80 competition assay using CTLA4 transduced Jurkat cells: Four bispecific constructs were tested for CD80 competition. Two had the full 5,5 linker configuration and two had the abbreviated 0,5 linker. We also switched the order of the aPDl/aCTLA4 abs in the final construct. As shown here, E1-D7 and El- D7+P4B3m3 combination therapy leads to moderate competition, however the creation of the bsAb shows a pronounced increase in CD80 competition, likely due to the additional binding potential generated by the anti-PDl domain.
[00157] FIG. 48 shows photos and illustrations of clear cell renal cell carcinoma panel (A) characteristics (adapted from Li et al, 2019), panel (B) model for cellular differentiation (adapted from Tun et.al, 2010), and panel (C) epidemiology (adapted from Hsieh et al, 2019).
[00158] FIG. 49 shows a graph of OS of ccRCC patients adapted from Feng et al., 2019.
[00159] FIG. 50 shows graphs of the representation of immune populations of ccRCC adapted from Chevrier et al., 2017.
[00160] FIG. 51 shows illustrations and graphs of the CTLA4 signaling pathway. Panel A shows an illustration of the CTLA4 signalling pathway. Panel B shows a graph CTLA4 overexpression in ccRCC (adapted from Lie et al, 2020). Panel C shows an illustration of Ipilimumab and CTLA4 blockade.
[00161] FIG. 52 shows pictures and illustrations of the PD1 signalling pathway adapted from Kim et al., 2021. Panel A shows the PD1-PDL1 signalling pathway. Panel B shows histology images of PD1 overexpression in ccRCC. Panel C shows an illustration of PD1 blockage and non-limiting effects.
[00162] FIG. 53 shows illustrations of dual checkpoint blockade by bispecific antibodies. Panel A shows a schematic dual CTLA4/PDL checkpoint blockade. Panel B shows nonlimiting, exemplary bispecific antibody formats.
[00163] FIG. 54 shows a table of exemplary anti-CTLA4/antiPDl bispecifics used in the clinic.
[00164] FIG. 55 shows a schematic of a non-limiting, exemplary experimental design to evaluate the binding affinity of anti-CTLA4 candidates to human and mouse CTLA4 at various concentrations using ELISA.
[00165] FIG. 56 shows non-limiting data and results of ELISA binding curve. Panel A shows ELISA binding curve to indicate binding affinity of anti-CTLA4 candidates to hCTLA4. Panel B shows ELISA binding curve to indicate binding affinity of anti-CTLA4 candidates to mCTLA4. Panel C shows ELISA binding curve to indicate binding affinity of anti-CTLA4 candidates to BSA (negative control).
[00166] FIG. 57 shows a non-limiting, exemplary experimental design to characterize the kinetic properties of the antibody candidates using BLI-Octet.
[00167] FIG. 58 shows non-limiting, data indicating the kinetic properties of anti-CTLA4 candidates. All tested candidates have higher dissociation rates when compared to Ipilimumab, even when they have a higher association response. Among the candidates, C, B, and G appear to have the lowest dissociation rates. KD - Dissociation constant, Kon - Rate of association, Kon - Rate of association.
[00168] FIG. 59 shows a schematic of a non-limiting, exemplary experimental design to determine the ability of anti-CTLA4 antibodies to block CD80/CD86/Ipilimumab binding and determine the similarity in the localization of antibodies and ligands on the surface of CTLA4.
[00169] FIG. 60 shows a chart of the percentage blockade of CD80/CD86/Ipilimumab binding to CTLA4 by anti-CTLA4 candidates. CD80: Ipilimumab blocks CD80 to the highest extent. The candidates that show the highest CD80 blockade: I, E, J. CD86: All candidates show 100% blockade of CD86 binding. This could be due to documented lower affinity of CD86 binding to CTLA4. Ipilimumab: All candidates block the binding of Ipilimumab to CTLA4 to some extent, even those that cross-react with mCTLA4.
[00170] FIG. 61 shows ribbon diagrams Ipilimumab complexes adapted from He M, Chai Y, Qi J, Zhang CWH, Tong Z, Shi Y, Yan J, Tan S, Gao GF. Remarkably similar CTLA-4 binding properties of therapeutic ipilimumab and tremelimumab antibodies. Oncotarget. 2017 May 19;8(40):67129-67139. doi: 10.18632/oncotarget.18004. PMID: 28978021; PMCID: PMC5620161. Panel A shows Ipilimumab complexed with hCTLA4 - Contact residues highlighted. Panel B shows Ipilimumab complexed with mCTLA4 - Aligned epitope highlighted.
[00171] FIG. 62 shows ribbon diagrams of CD80 complexes adapted from Stamper CC, Zhang Y, Tobin JF, Erbe DV, Ikemizu S, Davis SJ, Stahl ML, Seehra J, Somers WS, Mosyak L. Crystal structure of the B7-1/CTLA-4 complex that inhibits human immune responses. Nature. 2001 Mar 29;410(6828):608-l l. doi: 10.1038/35069118. Erratum in: Nature 2001 May 31 ;411(6837):617. PMID: 11279502. Panel A shows CD80 complexed with hCTLA4 - Contact residues highlighted. Panel B shows CD 80 complexed with mCTLA4 - Aligned epitope highlighted.
[00172] FIG. 63 shows a non-limiting, exemplary schematic of an experimental design to determine the binding affinity of the candidate antibodies to Jurkat cells that express CTLA4.
[00173] FIG. 64 shows a non-limiting, exemplary binding curve of the binding affinity of anti-CTLA4 candidates to CTLA4+ Jurkat cells.
[00174] FIG. 65 shows a non-limiting, exemplary schematic of an experimental design to assess the biological significance of antibody binding in facilitating CD28 signalling.
[00175] FIG. 66 shows non-limiting, exemplary graphs of fold induction of Luciferase vs. concentration of anti-CTLA4 candidates. Across both plates, Ipilimumab shows the highest induction of Luciferase expression. Five of the 8 tested candidates: E, H, J, F, and I, show a similar response.
[00176] FIG. 67 shows anti-CTLA-4 antibody germline alignment.
[00177] FIG. 68 shows anti-CTLA-4 antibody amino acid sequences. [00178] FIG. 69 shows anti-CTLA-4 antibody nucleic acid sequences.
DETAILED DESCRIPTION
[00179] Abbreviations and Definitions
[00180] Detailed descriptions of one or more embodiments are provided herein. It is to be understood, however, that the present invention may be embodied in various forms.
Therefore, specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the claims and as a representative basis for teaching one skilled in the art to employ the present invention in any appropriate manner.
[00181] The singular forms “a”, “an” and “the” include plural reference unless the context clearly dictates otherwise. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
[00182] Wherever any of the phrases “for example,” “such as,” “including” and the like are used herein, the phrase “and without limitation” is understood to follow unless explicitly stated otherwise. Similarly, “an example,” “exemplary” and the like are understood to be nonlimiting.
[00183] The term “substantially” allows for deviations from the descriptor that do not negatively impact the intended purpose. Descriptive terms are understood to be modified by the term “substantially” even if the word “substantially” is not explicitly recited.
[00184] The terms “comprising” and “including” and “having” and “involving” (and similarly “comprises”, “includes,” “has,” and “involves”) and the like are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the common United States patent law definition of “comprising” and is therefore interpreted to be an open term meaning “at least the following,” and is also interpreted not to exclude additional features, limitations, aspects, etc. Thus, for example, “a process involving steps a, b, and c” means that the process includes at least steps a, b and c. Wherever the terms “a” or “an” are used, “one or more” is understood, unless such interpretation is nonsensical in context.
[00185] The term “about” is used herein to mean approximately, roughly, around, or in the region of. When the term “about” is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term “about” is used herein to modify a numerical value above and below the stated value by a variance of 20 percent up or down (higher or lower).
[00186] CTLA-4
[00187] Human antibody-phage screens were performed against human CTLA-4 (rounds 1- 3) or muring CTLA-4 (Round 3). The first set of antibodies was discovered via panning against the soluble protein, and the second set was discovered by PMPL panning.
[00188] The binding activity have been characterized for a panel of human monoclonal antibodies against human CTLA-4. Anti-CTLA-4 immunotherapy is being evaluated as part of the burgeoning field of ImmunoOncology. The anti-CTLA-4 mAbs described herein can have therapeutic potential. CTLA-4 antibodies have been identified with varying affinity towards CTLA-4.
[00189] Aspects of the invention provide isolated antibodies specific against CTLA-4. The term “isolated” as used herein with respect to cells, nucleic acids, such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term “isolated” can also refer to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. For example, an “isolated nucleic acid” can include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. “Isolated” can also refer to cells or polypeptides which are isolated from other cellular proteins or tissues. Isolated polypeptides can include both purified and recombinant polypeptides. The isolated antibodies were identified through the use of a 27 billion member human single-chain antibody (scFv) phage library panned against soluble or paramagnetic proteoliposomes displayed CTLA4 as the selection target. These antibodies represent a new class of monoclonal antibodies against CTLA-4. The monoclonal CTLA-4 antibodies discussed herein can also be used in the construction of multi-specific antibodies or as the payload for a genetically engineered cell, such as a CAR-T cell, a CAR-NK cell, or a genetically engineered B cell.
[00190] Unique recombinant monoclonal CTLA-4 antibodies are described herein. “Recombinant” as it pertains to polypeptides (such as antibodies) or polynucleotides refers to a form of the polypeptide or polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides that would not normally occur together.
[00191] The amino acid sequence of the monoclonal CTLA-4 antibodies are provided herein (see Tables 1-26); the amino acid sequences of the heavy and light chain complementary determining regions CDRs of the CTLA-4 antibodies are underlined (CDR1). underlined and bolded (CDR2). or underlined, italiciz.ed, and bolded (CDR3) below:
[00192] The nucleic acid sequence of the monoclonal CTLA-4 antibodies are provided herein (see Tables 27-52): [00193] The amino acid sequences of the heavy and light chain complementary determining regions of the CTLA-4 antibodies are shown in Table 53A-B below:
Table 53A. Heavy chain (VH) complementary determining regions (CDRs) of the CTLA-4 antibodies
Table 53B. Light chain (VL) complementary determining regions (CDRs) of the CTLA-4 antibodies
[00194] The nucleic acid sequences of the heavy and light chain complementary determining regions of the CTLA-4 antibodies are shown in Table 54A-B below:
Table 54A. Heavy chain (VH) complementary determining regions (CDRs) of the CTLA-4 antibodies
Table 54B. Light chain (VL) complementary determining regions (CDRs) of the CTLA-4 antibodies
[00195] The amino acid sequences of the heavy and light chain framework regions of the
CTLA4 antibodies are shown in Table 55A-B below:
Table 55A. Heavy chain (VH) framework regions (FRs) of the CTLA-4 antibodies
Table 55B. Light chain (VL) framework regions (FRs) of the CTLA-4 antibodies
[00196] The nucleic acid sequences of the heavy and light chain framework regions of the
CTLA-4 antibodies are shown in Table 56A-B below:
Table 56A. Heavy chain (VH) framework regions (FRs) of the CTLA-4 antibodies
Table 56B. Light chain (VL) framework regions (FRs) of the CTLA-4 antibodies
[00197] The CTLA-4 antibodies described herein bind to CTLA-4. In one embodiment, the CTLA-4 antibodies have high affinity and high specificity for CTLA-4. Some embodiments also feature antibodies that have a specified percentage identity or similarity to the amino acid or nucleotide sequences of the anti- CTLA-4 antibodies described herein. For example, “homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence, which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, the antibodies can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher amino acid sequence identity when compared to a specified region or the full length of any one of the anti-CTLA-4 antibodies described herein. For example, the antibodies can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher nucleic acid identity when compared to a specified region or the full length of any one of the anti-CTLA-4 antibodies described herein. Sequence identity or similarity to the nucleic acids and proteins of the present invention can be determined by sequence comparison and/or alignment by methods known in the art, for example, using software programs known in the art, such as those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. For example, sequence comparison algorithms (i.e. BLAST or BLAST 2.0), manual alignment or visual inspection can be utilized to determine percent sequence identity or similarity for the nucleic acids and proteins of the present invention.
[00198] “Polypeptide” as used herein can encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, can refer to “polypeptide” herein, and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. “Polypeptide” can also refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. As to amino acid sequences, one of skill in the art will readily recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds, deletes, or substitutes a single amino acid or a small percentage of amino acids in the encoded sequence is collectively referred to herein as a "conservatively modified variant". In some embodiments the alteration results in the substitution of an amino acid with a chemically similar amino acid.
Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants of the anti-CTLA-4 antibodies disclosed herein can exhibit increased cross-reactivity to CTLA-4 in comparison to an unmodified CTLA-4 antibody.
[00199] For example, a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in an immunoglobulin polypeptide is replaced with another amino acid residue from the same side chain family. In another embodiment, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members.
[00200] Antibodies
[00201] As used herein, an “antibody” or “antigen-binding polypeptide” can refer to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a whole antibody and any antigen binding fragment or a single chain thereof. For example, “antibody” can include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule having biological activity of binding to the antigen. Non-limiting examples a complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework (FR) region, or any portion thereof, or at least one portion of a binding protein. As used herein, the term "antibody" can refer to an immunoglobulin molecule and immunologically active portions of an immunoglobulin (Ig) molecule, i.e., a molecule that contains an antigen binding site that specifically binds (immunoreacts with) an antigen. By "specifically binds" or "immunoreacts with" is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides. [00202] The terms “antibody fragment” or “antigen-binding fragment”, as used herein, is a portion of an antibody such as F(ab’)2, F(ab)2, Fab', Fab, Fv, scFv and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. The term “antibody fragment” can include aptamers (such as spiegelmers), minibodies, and diabodies. The term “antibody fragment” can also include any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. Antibodies, antigen-binding polypeptides, variants, or derivatives described herein include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, epitope-binding fragments, e.g., Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, dAb (domain antibody), minibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies.
[00203] A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light chains (VL) of immunoglobulins. A single chain Fv ("scFv") polypeptide molecule is a covalently linked VH:VL heterodimer, which can be expressed from a gene fusion including VH- and VL-encoding genes linked by a peptide- encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). In some aspects, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker. A number of methods have been described to discern chemical structures for converting the naturally aggregated, but chemically separated, light and heavy polypeptide chains from an antibody V region into an scFv molecule, which will fold into a three dimensional structure substantially similar to the structure of an antigen-binding site. See, e.g., U.S. Patent No. 5,091,5 13; No. 5,892,019; No. 5,132,405; and No. 4,946,778, each of which are incorporated by reference in their entireties.
[00204] Very large naive human scFv libraries have been and can be created to offer a large source of rearranged antibody genes against a plethora of target molecules. Smaller libraries can be constructed from individuals with infectious diseases in order to isolate diseasespecific antibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43 (1992); Zebedee et al, Proc. Natl. Acad. Sci. USA 89:3 175-79 (1992)). [00205] Antibodies of the invention can also be modified to produce mosaic antibodies. A mosaic antibody is one in which the external amino acid residues of an antibody of one species are rationally replaced or "mosaicked" by the external amino acid residues of an antibody of a second species such that the antibody of the first species is not immunogenic in the second species, thereby reducing the immunogenicity of the antibody. Since the antigenicity of a protein depends primarily on its surface properties, the immunogenicity of an antibody can be reduced by substituting exposed residues that differ from those typically found in antibodies of another mammalian species. Reasonable substitution of this external residue should have little or no effect on the internal domain or on inter-domain contacts. Thus, since the changes are limited to variable region framework residues, ligand binding properties should not be affected. This process is called "mosaicism" since only the outer surface or skin of the antibody is altered and the supporting residues remain undisturbed.
[00206] The "mosaicing" process utilizes sequence data for human antibody variable domains compiled by available Kabat et al (1987) Sequences of Proteins of Immunological interest, 4th ed., Bethesda, Md., National Institutes of Health, updates to this database, and other accessible U.S. and foreign databases (nucleic acids and Proteins). Non-limiting examples of methods for generating mosaic antibodies include EP 519596; U.S. Pat. No. 6,797,492; and is described in Padlan et al, 1991.
[00207] Antibody molecules obtained from humans fall into five classes of immunoglobulins: IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (y, p, a, 6, s) with some subclasses among them (e.g., yl-y4). Certain classes have subclasses as well, such as IgGi, IgG2, IgGi and IgGi and others. The immunoglobulin subclasses (isotypes) e.g., IgGi, IgG2, IgGi, IgGi, IgGs, etc. are well characterized and are known to confer functional specialization. With regard to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of molecular weight approximately 23,000 Daltons, and two identical heavy chain polypeptides of molecular weight 53,000-70,000. The four chains are typically joined by disulfide bonds in a “Y” configuration wherein the light chains bracket the heavy chains starting at the mouth of the “Y” and continuing through the variable region. Immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGi, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of an immunoglobulin molecule. [00208] Light chains are classified as either kappa or lambda (K, Z). Each heavy chain class can be bound with either a kappa or lambda light chain. In general, the light and heavy chains are covalently bonded to each other, and the “tail” portions of the two heavy chains are bonded to each other by covalent disulfide linkages or non-covalent linkages when the immunoglobulins are generated either by hybridomas, B cells, or genetically engineered host cells. In the heavy chain, the amino acid sequences run from an N-terminus at the forked ends of the Y configuration to the C-terminus at the bottom of each chain.
[00209] Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. The variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. The term "antigen-binding site," or "binding portion" can refer to the part of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by amino acid residues of the N-terminal variable ("V") regions of the heavy ("H") and light ("L") chains. Three highly divergent stretches within the V regions of the heavy and light chains, referred to as "hypervariable regions," are interposed between more conserved flanking stretches known as "framework regions," or "FRs". Thus, the term "FR" can refer to amino acid sequences which are naturally found between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, the three hypervariable regions of a light chain and the three hypervariable regions of a heavy chain are disposed relative to each other in three dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen, and the three hypervariable regions of each of the heavy and light chains are referred to as "complementarity -determining regions," or "CDRs." VH and VL regions, which contain the CDRs, as well as frameworks (FRs) of the CTLA-4 antibodies are shown in Table lA-Table 12B.
[00210] The six CDRs present in each antigen-binding domain are short, non-contiguous sequences of amino acids that are specifically positioned to form the antigen-binding domain as the antibody assumes its three dimensional configuration in an aqueous environment. The remainder of the amino acids in the antigen-binding domains, the FR regions, show less inter- molecular variability. The framework regions largely adopt a [3-sheet conformation and the CDRs form loops which connect, and in some cases form part of, the [3-sheet structure. The framework regions act to form a scaffold that provides for positioning the CDRs in correct orientation by inter-chain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs provides a surface complementary to the epitope on the immunoreactive antigen, which promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the framework regions, respectively, can be readily identified for a heavy or light chain variable region by one of ordinary skill in the art, since they have been previously defined (See, “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., U.S. Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).
[00211] Where there are two or more definitions of a term which is used and/or accepted within the art, the definition of the term as used herein is intended to include all such meanings unless explicitly stated to the contrary. A specific example is the use of the term “complementarity determining region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. This particular region has been described by Kabat et al., U.S. Dept, of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either definition to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth in the table below as a comparison. The exact residue numbers which encompass a particular CDR will vary depending on the sequence and size of the CDR. Those skilled in the art can routinely determine which residues comprise a particular CDR given the variable region amino acid sequence of the antibody. [00212] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. The skilled artisan can unambiguously assign this system of “Kabat numbering” to any variable domain sequence, without reliance on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system set forth by Kabat et al., U.S. Dept, of Health and Human Services, “Sequence of Proteins of Immunological Interest” (1983).
[00213] In addition to table above, the Kabat number system describes the CDR regions as follows: CDR-H1 begins at approximately amino acid 31 (i.e., approximately 9 residues after the first cysteine residue), includes approximately 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins at approximately the thirty third amino acid residue after the end of CDR- H2; includes 3-25 amino acids; and ends at the sequence W-G-X-G, where X is any amino acid. CDR-L1 begins at approximately residue 24 (i.e., following a cysteine residue); includes approximately 10-17 residues; and ends at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues. CDR-L3 begins at approximately the thirty third residue after the end of CDR-L2 (i.e., following a cysteine residue); includes approximately 7-11 residues and ends at the sequence F or W-G-X-G, where X is any amino acid.
[00214] As used herein, the term "epitope" can include any protein determinant that can specifically bind to an immunoglobulin, a scFv, or a T-cell receptor. The variable region allows the antibody to selectively recognize and specifically bind epitopes on antigens. For example, the VL domain and VH domain, or subset of the complementarity determining regions (CDRs), of an antibody combine to form the variable region that defines a three dimensional antigen-binding site. This quaternary antibody structure forms the antigenbinding site present at the end of each arm of the Y. Epitopic determinants can consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics. For example, antibodies can be raised against N- terminal or C- terminal peptides of a polypeptide. More specifically, the antigen-binding site is defined by three CDRs on each of the VH and VL chains (i.e. CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In one embodiment, the antibodies can be directed to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) having Ann UniProt Reference No: Pl 6410 (CTLA-4_HUMAN) (223 amino acid residues in length), comprising the amino acid sequence of SEQ ID NO: [ ]:
[00215] As used herein, the terms "immunological binding," and "immunological binding properties" can refer to the non-covalent interactions of the type which occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity of immunological binding interactions can be expressed in terms of the dissociation constant (Kd) of the interaction, wherein a smaller K 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 antigenbinding site/antigen complex formation and dissociation, wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the "on rate constant" (Kon) and the "off rate constant" (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. (See Nature 361 : 186-87 (1993)). The ratio of Koff /Kon enables the cancellation of all parameters not related to affinity, and is equal to the equilibrium binding constant, KD. (See, generally, Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the invention can specifically bind to a CTLA4 epitope when the equilibrium binding constant (KD) is <1 pM, <10 pM, < 10 nM, < 10 pM, or < 100 pM to about 1 pM, as measured by kinetic assays such as radioligand binding assays or similar assays known to those skilled in the art, such as BIAcore or Octet (BLI). For example, in some embodiments, the KD is between about IE- 12 M and a KD about IE-11 M. In some embodiments, the Kois between about IE- 11 M and a KD about IE- 10 M. In some embodiments, the KD is between about IE- 10 M and a KD about IE-9 M. In some embodiments, the KD is between about IE-9 M and a KD about IE-8 M. In some embodiments, the KD is between about IE-8 M and a KD about IE-7 M. In some embodiments, the KD is between about IE- 7 M and a KD about IE-6 M. For example, in some embodiments, the Kois about IE-12 M while in other embodiments the Kois about 1E- 11 M. In some embodiments, the KD is about IE- 10 M while in other embodiments the KD is about IE-9 M. In some embodiments, the KD is about IE-8 M while in other embodiments the KD is about IE-7 M. In some embodiments, the KD is about IE-6 M while in other embodiments the KD is about IE- 5 M. In some embodiments, for example, the KD is about 3 E-ll M, while in other embodiments the Kois about 3E-12 M. In some embodiments, the KD is about 6E-11 M. “Specifically binds” or “has specificity to,” can refer to an antibody that binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to “specifically bind” to an epitope when it binds to that epitope, via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. [00216] For example, the CTLA4 antibody can be monovalent or bivalent, and can comprise a single or double chain. Functionally, the binding affinity of the CTLA4 antibody is within the range of 1 () M to 10 12 M. For example, the binding affinity of the CTLA4 antibody is from 10 6 M to 10 12 M, from 10 7 M to 10 12 M, from 10 8 M to 10 12 M, from 10 9 M to 10 12 M, from 10 5 M to 10 11 M, from 10 6 M to 10 11 M, from 10 7 M to 10 " M. from 10 8 M to 10 1 1 M. from 10 9 M to 10 1 1 M. from 10 10 M to 10 1 1 M. from IO 5 M to 10 l0M. from 10 6 M to 10 10 M. from 10 7 M to 10 10 M. from 10 8 M to 10 l0M. from 10 9 M to 10 10 M, from 10 5 M to 10 9 M, from 10 6 M to 10 9M, from 10 7 M to 10 9 M, from 10 8 M to 10 9 M, from I0 5 M to 10 8 M, from 10 6 M to 10 8 M, from 10 7 M to 10 8 M, from 10 5 M to 10 7 M, from 10 6 M to 10 7 M, or from 10 5 M to 10 6 M.
[00217] A CTLA-4 protein, or a derivative, fragment, analog, homolog or ortholog thereof, can be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components. A CTLA-4 protein or a derivative, fragment, analog, homolog, or ortholog thereof, coupled to a proteoliposome can be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
[00218] Those skilled in the art will recognize that it is possible to determine, without undue experimentation, if a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by ascertaining whether the former prevents the latter from binding to CTLA-4. For example, if the human monoclonal antibody being tested competes with the human monoclonal antibody of the invention, as shown by a decrease in binding by the human monoclonal antibody of the invention, then it is likely that the two monoclonal antibodies bind to the same, or to a closely related, epitope.
[00219] Another way to determine whether a human monoclonal antibody has the specificity of a human monoclonal antibody of the invention is to pre-incubate the human monoclonal antibody of the invention with the CTLA-4 protein, with which it is normally reactive, and then add the human monoclonal antibody being tested to determine if the human monoclonal antibody being tested is inhibited in its ability to bind CTLA-4. If the human monoclonal antibody being tested is inhibited then, in all likelihood, it has the same, or functionally equivalent, epitopic specificity as the monoclonal antibody of the invention. Screening of human monoclonal antibodies of the invention can be also carried out by utilizing CTLA-4 and determining whether the test monoclonal antibody is able to neutralize CTLA-4.
[00220] Various procedures known within the art can be used for the production of polyclonal or monoclonal antibodies directed against a protein of the invention, or against derivatives, fragments, analogs homologs or orthologs thereof. (See, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).
[00221] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of immune serum. Subsequently, or alternatively, the specific antigen which is the target of the immunoglobulin sought, or an epitope thereof, can be immobilized on a column to purify the immune specific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).
[00222] The term “monoclonal antibody” or “mAb” or “Mab” or “monoclonal antibody composition”, as used herein, can refer to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
[00223] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.
[00224] The immunizing agent can include the protein antigen, a fragment thereof or a fusion protein thereof. For example, peripheral blood lymphocytes can be used if cells of human origin are desired, or spleen cells or lymph node cells can be used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59- 103). Immortalized cell lines can be transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. For example, rat or mouse myeloma cell lines are employed. The hybridoma cells can be cultured in a suitable culture medium that contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
[00225] Immortalized cell lines that are useful are those that fuse efficiently, support stable high-level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. For example, immortalized cell lines can be murine myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center (San Diego, California) and the American Type Culture Collection (Manassas, Virginia). Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies. (See Kozbor, J. Immunol, 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).
[00226] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against the antigen. For example, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem, 107:220 (1980). Moreover, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies having a high degree of specificity and a high binding affinity for the target antigen.
[00227] After the desired hybridoma cells are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640 medium. Alternatively, the hybridoma cells can be grown in vivo as ascites in a mammal.
[00228] The monoclonal antibodies secreted by the subclones can be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
[00229] Monoclonal antibodies can also be made by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567 (incorporated herein by reference in its entirety). DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a source of such DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. The DNA also can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (See U.S. Patent No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
[00230] Fully human antibodies, for example, are antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CDRs, arise from human genes. Such antibodies are termed "human antibodies" or "fully human antibodies". Human monoclonal antibodies, such as fully human and humanized antibodies, can be prepared by using trioma technique; the human B-cell hybridoma technique (see Kozbor, et al, 1983 Immunol Today 4: 72); and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al, 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be utilized and can be produced by using human hybridomas (see Cote, et al, 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
[00231] “Humanized antibodies” can be antibodies from a non-human species (such as mouse), whose amino acid sequences (for example, in the CDR regions) have been modified to increase their similarity to antibody variants produced in humans. Antibodies can be humanized by methods known in the art, such as CDR-grafting. See also, Safdari et al., (2013) Biotechnol Genet Eng Rev., ' 29:175-86. In addition, humanized antibodies can be produced in transgenic plants, as an an inexpensive production alternative to existing mammalian systems. For example, the transgenic plant can be a tobacco plant, i.e., Nicotiania benthamiana, and Nicotiana tabaccum. The antibodies are purified from the plant leaves. Stable transformation of the plants can be achieved through the use of Agrobacterium tumefaciens or particle bombardment. For example, nucleic acid expression vectors containing at least the heavy and light chain sequences are expressed in bacterial cultures, i. e. , A. tumefaciens strain BLA4404, via transformation. Infiltration of the plants can be accomplished via injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and by centrifugation. Isolation and purification of the antibodies can be readily be performed by many of the methods known to the skilled artisan in the art. Other methods for antibody production in plants are described in, for example, Fischer et al., Vaccine, 2003, 21:820-5; and Ko et al, Current Topics in Microbiology and Immunology, Vol. 332, 2009, pp. 55-78. As such, the invention further provides any cell or plant comprising a vector that encodes the antibody of the invention, or produces the antibody of the invention.
[00232] Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska. et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332, which is incorporated by reference in its entirety). “Humanization” (also called Reshaping or CDR-grafting) is a well-established technique understood by the skilled artisan for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (commonly rodent) and for improving their activation of the human immune system (See, for example, Hou S, Li B, Wang L, Qian W, Zhang D, Hong X, Wang H, Guo Y (July 2008). "Humanization of an anti-CD34 monoclonal antibody by complementarity-determining region grafting based on computer-assisted molecular modeling". J Biochem. 144 (1): 115-20).
[00233] In addition, antibodies (such as human antibodies) can also be produced using other techniques, including phage display libraries. (See Hoogenboom and Winter, J. Mol. Biol, 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13: 65-93 (1995).
[00234] Human antibodies can additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen. (See, PCT publication no. W094/02602 and U.S. Patent No. 6,673,986). The endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome. The human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments. An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications. A non-limiting example of such a nonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed in PCT publication nos. WO96/33735 and WO96/34096. This animal produces B cells which secrete fully human immunoglobulins. The antibodies can be obtained directly from the animal after immunization with an immunogen of interest, as, for example, a preparation of a polyclonal antibody, or alternatively from immortalized B cells derived from the animal, such as hybridomas producing monoclonal antibodies. Additionally, the genes encoding the immunoglobulins with human variable regions can be recovered and expressed to obtain the antibodies directly, or can be further modified to obtain analogs of antibodies such as, for example, single chain Fv (scFv) molecules.
[00235] Thus, using such a technique, therapeutically useful IgG, IgA, IgM and IgE antibodies can be produced. For an overview of this technology for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publication nos. WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Creative BioLabs (Shirley, NY) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described herein.
[00236] An example of a method of producing a nonhuman host, exemplified as a mouse, lacking expression of an endogenous immunoglobulin heavy chain is disclosed in U.S. Patent No. 5,939,598. It can be obtained by a method, which includes deleting the J segment genes from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent formation of a transcript of a rearranged immunoglobulin heavy chain locus, the deletion being effected by a targeting vector containing a gene encoding a selectable marker; and producing from the embryonic stem cell a transgenic mouse whose somatic and germ cells contain the gene encoding the selectable marker.
[00237] One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. This method includes introducing an expression vector that contains a nucleotide sequence encoding a heavy chain into one mammalian host cell in culture, introducing an expression vector containing a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody containing the heavy chain and the light chain.
[00238] In a further improvement on this procedure, a method for identifying a clinically relevant epitope on an immunogen and a correlative method for selecting an antibody that binds immunospecifically to the relevant epitope with high affinity, is disclosed in PCT publication No. WO99/53049.
[00239] The antibody of interest can also be expressed by a vector containing a DNA segment encoding the single chain antibody described herein. Vectors include, but are not limited to, chemical conjugates such as described in WO 93/64701, which has targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine), viral vectors (e.g. a DNA or RNA viral vector), fusion proteins such as described in PCT/US 95/02140 (WO 95/22618), which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage, viral vectors, etc. The vectors can be chromosomal, non- chromosomal or synthetic. Retroviral vectors can also be used, and include moloney murine leukemia viruses. DNA viral vectors can also be used, and include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (See Geller, A. I. et al, J. Neurochem, 64:487 (1995); Lim, F., et al, in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al, Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., et al, Proc Natl. Acad. Sci USA 87: 1149 (1990), Adenovirus Vectors (see LeGal LaSalle et al, Science, 259:988 (1993); Davidson, et al, Nat. Genet 3 :219 (1993); Yang, et al, J. Virol. 69:2004 (1995) and Adeno-associated Virus Vectors (see Kaplitt, M. G.. et al, Nat. Genet. 8: 148 (1994).
[00240] Pox viral vectors introduce the gene into the cell’s cytoplasm. Avipox virus vectors result in only a short term expression of the nucleic acid. Adenovirus vectors, adeno- associated virus vectors, and herpes simplex virus (HSV) vectors can be used for introducing the nucleic acid into neural cells. The adenovirus vector results in a shorter term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The particular vector chosen will depend upon the target cell and the condition being treated. The introduction can be by standard techniques, e.g. infection, transfection, transduction or transformation. Examples of modes of gene transfer include e.g., naked DNA, CaPCh precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.
[00241] The vector can be employed to target essentially any desired target cell. For example, stereotaxic injection can be used to direct the vectors (e.g. adenovirus, HSV) to a desired location. Additionally, the particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system, such as a SynchroMed Infusion System. A method based on bulk flow, termed convection, has also proven effective at delivering large molecules to extended areas of the brain and can be useful in delivering the vector to the target cell. (See Bobo et al, Proc. Natl. Acad. Sci. USA 91 :2076-2080 (1994); Morrison et al, Am. J. Physiol. 266:292-305 (1994)). Other methods that can be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection, and oral or other known routes of administration.
[00242] These vectors can be used to express large quantities of antibodies that can be used in a variety of ways. For example, to detect the presence of CTLA4 in a sample. The antibody can also be used to try to bind to and disrupt a CTLA4 activity.
[00243] In embodiments, the antibodies herein can be delivered to a subject using a genebased approach. For example, the nucleotide sequence of an anti-CTLA4 antibody as described herein can be delivered into a subject using a vector, DNA or RNA. See, for example, Deal, Cailin E., Andrea Carfi, and Obadiah J. Plante. "Advancements in mRNA Encoded Antibodies for Passive Immunotherapy." Vaccines 9.2 (2021): 108. Such methods can allow for the design and generation of more complex antibody molecules with improved and/or added functions.
[00244] Viral vectors such as adenovirus (Ad) and adeno-associated virus (AAV) have been engineered for in vivo mAb expression by replacing part of their genome with nucleic acid sequences encoding the antibody of interest. The term “viral vector” can refer to a nucleic acid vector construct that includes at least one element of viral origin and has the capacity to be packaged into a viral vector particle. The viral vector can contain the nucleic acid encoding an antibody, antigen-binding portion thereof, or CAR as described herein in place of non-essential viral genes. The vector and/or particle may be utilized for the purpose of transferring any nucleic acids into cells either in vitro or in vivo.
[00245] Retroviruses are a common tool for gene delivery. In embodiments, a retrovirus is used to deliver a polynucleotide encoding the antibody to a cell. The term “retrovirus” can refer to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into a host genome. Once the virus is integrated into the host genome, it is referred to as a “provirus.” The provirus serves as a template for RNA polymerase II and directs the expression of RNA molecules which encode the structural proteins and enzymes needed to produce new viral particles.
[00246] Illustrative retroviruses suitable for use in particular embodiments, include, but are not limited to: Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), Spumavirus, Friend murine leukemia virus, Murine Stem Cell Virus (MSCV) and Rous Sarcoma Virus (RSV)) and lentivirus. [00247] The term “lentivirus” can refer to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1, and HIV type 2); visna-maedi virus (VMV) virus; the caprine arthritisencephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV based vector backbones (i.e., HIV cis-acting sequence elements) are preferred. In particular embodiments, a lentivirus is used to deliver a polynucleotide comprising a CAR to a cell.
[00248] Gene transfer of the antibody nucleotide sequences themselves, as DNA or RNA, is an alternative option for transient in vivo antibody production. For example, plasmid DNA approaches can utilie an electroporation component to enhance transfection efficiency following IM delivery. Also, mRNA delivery can be used for in vivo expression of antibodies. In embodiments, the mRNA encoding the antibody of interest can be modified, such as optimization of the mRNA codon usage, and lipid nanoparticles can be used for mRNA delivery.
[00249] In an embodiment, the antibodies described herein can be full-length antibodies, containing an Fc region similar to wild-type Fc regions that bind to Fc receptors.
[00250] Techniques can be adapted for the production of single-chain antibodies specific to an antigenic protein of the invention (See e.g., U.S. Patent No. 4,946,778). In addition, methods can be adapted for the construction of Fab expression libraries (See e.g., Huse, et al, 1989 Science 246: 1275-1281) to allow rapid and effective identification of monoclonal Fab fragments with the desired specificity for a protein or derivatives, fragments, analogs or homologs thereof. Antibody fragments that contain the idiotypes to a protein antigen can be produced by techniques known in the art including, but not limited to: (i) an F(ab')2 fragment produced by pepsin digestion of an antibody molecule; (ii) an Fab fragment generated by reducing the disulfide bridges of an F(ab')2 fragment; (iii) an Fab fragment generated by the treatment of the antibody molecule with papain and a reducing agent and (iv) Fv fragments. [00251] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies can, for example, target immune system cells to unwanted cells (see U.S. Patent No. 4,676,980), and for treatment of HIV infection (See PCT Publication Nos. W091/00360; WO92/20373). The antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4- mercaptobutyrimidate and those disclosed, for example, in U.S. Patent No. 4,676,980. [00252] The antibody of the invention can be modified with respect to effector function, so as to enhance, e.g., the effectiveness of the antibody in treating cancer. For example, cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated can have improved internalization capability and/or increased complement-mediated cell killing and antibodydependent cellular cytotoxicity (ADCC). (See Caron et al, J. Exp Med., 176: 1 191-1 195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities. (See Stevenson et al, Anti-Cancer Drug Design, 3 : 219-230 (1989)). In one embodiment, the antibody of the invention has modifications of the Fc region, such that the Fc region does not bind to the Fc receptors. For example, the Fc receptor is Fey receptor. Antibodies with modification of the Fc region such that the Fc region does not bind to Fey, but still binds to neonatal Fc receptor are useful as described herein.
[00253] In certain embodiments, an antibody of the invention can comprise an Fc variant comprising an amino acid substitution which alters the antigen-independent effector functions of the antibody, in particular the circulating half-life of the antibody. Such antibodies exhibit either increased or decreased binding to FcRn when compared to antibodies lacking these substitutions, therefore, have an increased or decreased half-life in serum, respectively. Fc variants with improved affinity for FcRn are anticipated to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where long half-life of the administered antibody is desired, e.g., to treat a chronic disease or disorder. In contrast, Fc variants with decreased FcRn binding affinity are expected to have shorter haltlives, and such molecules are also useful, for example, for administration to a mammal where a shortened circulation time can be advantageous, e.g., for in vivo diagnostic imaging or in situations where the starting antibody has toxic side effects when present in the circulation for prolonged periods. Fc variants with decreased FcRn binding affinity are also less likely to cross the placenta and, thus, are also useful in the treatment of diseases or disorders in pregnant women. In addition, other applications in which reduced FcRn binding affinity can be desired include those applications in which localization to the brain, kidney, and/or liver is desired. In one embodiment, the Fc variant-containing antibodies can exhibit reduced transport across the epithelium of kidney glomeruli from the vasculature. In another embodiment, the Fc variant-containing antibodies can exhibit reduced transport across the blood brain barrier (BBB) from the brain, into the vascular space. In one embodiment, an antibody with altered FcRn binding comprises an Fc domain having one or more amino acid substitutions within the "FcRn binding loop" of an Fc domain. The FcRn binding loop is comprised of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions with altered FcRn binding activity are disclosed in PCT Publication No. WO05/047327 which is incorporated by reference herein. In certain exemplary embodiments, the antibodies, or fragments thereof, of the invention comprise an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E and S304D (EU numbering).
[00254] In some embodiments, mutations are introduced to the constant regions of the mAh such that the antibody dependent cell-mediated cytotoxicity (ADCC) activity of the mAh is altered. For example, the mutation is an LALA mutation in the CH2 domain. In one embodiment, the antibody (e.g., a human mAh, or a bispecific Ab) contains mutations on one scFv unit of the heterodimeric mAh, which reduces the ADCC activity. In another embodiment, the mAh contains mutations on both chains of the heterodimeric mAh, which completely ablates the ADCC activity. For example, the mutations introduced into one or both scFv units of the mAh are LALA mutations in the CH2 domain. These mAbs with variable ADCC activity can be optimized such that the mAbs exhibits maximal selective killing towards cells that express one antigen that is recognized by the mAh, however exhibits minimal killing towards the second antigen that is recognized by the mAh.
[00255] In other embodiments, antibodies of the invention for use in the diagnostic and treatment methods described herein have a constant region, e.g., an IgGi , IgG2, or IgG4 heavy chain constant region, which can be altered to reduce or eliminate glycosylation. For example, an antibody of the invention can also comprise an Fc variant comprising an amino acid substitution which alters the glycosylation of the antibody. For example, the Fc variant can have reduced glycosylation (e.g., N- or O-linked glycosylation). In some embodiments, the Fc variant comprises reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within a glycosylation motif, for example, an N-linked glycosylation motif that contains the amino acid sequence NXT or NXS. In a particular embodiment, the antibody comprises an Fc variant with an amino acid substitution at amino acid position 228 or 299 (EU numbering). In more particular embodiments, the antibody comprises an IgGl or IgG4 constant region comprising an S228P and a T299A mutation (EU numbering). [00256] Exemplary amino acid substitutions which confer reduced or altered glycosylation are described in PCT Publication No, W005/018572, which is incorporated by reference herein in its entirety. In some embodiments, the antibodies of the invention, or fragments thereof, are modified to eliminate glycosylation. Such antibodies, or fragments thereof, can be referred to as "agly" antibodies, or fragments thereof, (e.g. "agly" antibodies). While not wishing to be bound by theory "agly" antibodies, or fragments thereof, can have an improved safety and stability profile in vivo. Exemplary agly antibodies, or fragments thereof, comprise an aglycosylated Fc region of an IgGi antibody which is devoid of Fc-effector function thereby eliminating the potential for Fc mediated toxicity to the normal vital tissues and cells that express CTLA4. In yet other embodiments, antibodies of the invention, or fragments thereof, comprise an altered glycan. For example, the antibody can have a reduced number of fucose residues on an N-glycan at Asn297 of the Fc region, i.e., is afucosylated. In another embodiment, the antibody can have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.
[00257] The invention also is directed to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).
[00258] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Non-limiting examples include 212Bi, 133I, 131In, 90Y, and 186Re.
[00259] Conjugates of the antibody and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro- 2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al, Science 238: 1098 (1987). Carbon- 14-labeled l-isothiocyanatobenzyl-3- methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. (See PCT Publication No. WO94/11026, and U.S. Patent No. 5,736,137).
[00260] Those of ordinary skill in the art understand that a large variety of possible moieties can be coupled to the resultant antibodies or to other molecules of the invention. (See, for example, "Conjugate Vaccines", Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).
[00261] Coupling can be accomplished by any chemical reaction that will bind the two molecules so long as the antibody and the other moiety retain their respective activities. This linkage can include many chemical mechanisms, for instance covalent binding, affinity binding, intercalation, coordinate binding, and complexation. In one embodiment, binding is, covalent binding. Covalent binding can be achieved either by direct condensation of existing side chains or by the incorporation of external bridging molecules. Many bivalent or polyvalent linking agents are useful in coupling protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative coupling agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes and hexamethylene diamines. This listing is not intended to be exhaustive of the various classes of coupling agents known in the art but, rather, is exemplary of the more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133 : 1335-2549 (1984); Jansen et al., Immunological Reviews 62: 185-216 (1982); and Vitetta et al, Science 238: 1098 (1987)). Non-limiting examples of linkers are described in the literature. (See, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Patent No. 5,030,719, describing use of halogenated acetyl hydrazide derivative coupled to an antibody by way of an oligopeptide linker. Non-limiting examples of useful linkers that can be used with the antibodies of the invention include: (i) EDC (l-ethyl-3- (3 -dimethyl aminopropyl) carbodiimide hydrochloride; (ii) SMPT (4- succinimidyloxycarbonyl-alpha-methyl- alpha-(2-pridyl-dithio)-toluene (Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6 [3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat #21651G); (iv) Sulfo- LC-SPDP (sulfosuccinimidyl 6 [3-(2- pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat. #2165-G); and (v) sulfo- NHS ( -hydroxysulfo-succinimide: Pierce Chem. Co., Cat. #24510) conjugated to EDC. [00262] The linkers described herein contain components that have different attributes, thus leading to conjugates with differing physio-chemical properties. For example, sulfo- NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester containing linkers are less soluble than sulfo-NHS esters. Further, the linker SMPT contains a sterically hindered disulfide bond, and can form conjugates with increased stability. Disulfide linkages, are in general, less stable than other linkages because the disulfide linkage is cleaved in vitro, resulting in less conjugate available. Sulfo-NHS, in particular, can enhance the stability of carbodimide couplings. Carbodimide couplings (such as EDC) when used in conjunction with sulfo-NHS, forms esters that are more resistant to hydrolysis than the carbodimide coupling reaction alone.
[00263] The antibodies disclosed herein can also be formulated as immunoliposomes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al, Proc. Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al, Proc. Natl Acad. Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.
[00264] Non-limiting examples of useful liposomes can be generated by the reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al, J. Biol. Chem, 257: 286-288 (1982) via a disulfide-interchange reaction.
[00265] Multispecific Antibodies (Bispecific and Trispecific)
[00266] Multispecific antibodies are antibodies that can recognize two or more different antigens. For example, a bi-specific antibody (bsAb) is an antibody comprising two variable domains or scFv units such that the resulting antibody recognizes two different antigens. For example, a trispecific antibody (tsAb) is an antibody comprising two variable domains or scFv units such that the resulting antibody recognizes three different antigens. This invention provides for multispecific antibodies, such as bi-specific and trispecific antibodies, that recognize CTLA-4 and a second antigen and/or a third antigen. In one embodiment, multispecific antibodies (e.g., bi-specific antibodies and trispecific antibodies) can comprise CTLA-4 specific fusion proteins encompassing the antibodies described herein. Exemplary second and.or third antigens include tumor associated antigens (e.g., LINGO1), cytokines (e.g., IL-12 (IL-12A (p35 subunit) protein sequence having NCBI Reference No. NP_000873.2; IL-12B (p40 subunit) protein sequence having NCBI Reference No. NP_002178.2); IL-18 (protein sequence having NCBI Reference no. NP_001553.1); IL-15 (protein sequence having NCBI Reference No. NP_000576.1); IL-7 (protein sequence having NCBI Reference No. NP_000871.1); IL-2 (protein sequence having NCBI Reference No. NP_000577.2); and IL-21 (protein sequence having NCBI Reference No. NP_068575.1)), cytokine cognate receptors (eg., IL-12R), and cell surface receptors. Non-limiting examples of second and/or third antigens include CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX- 40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM (or BTLA), CD47, PDL1, MICA, MICB, and CD73. In one embodiment, the bispecific and trispecific antibodies comprise CTLA-4 fusion proteins. For example, the fusion protein comprises an antibody comprising a variable domain or scFv unit and a ligand or antigen and/or a third ligand or antigen as described herein such that the resulting antibody recognizes an antigen and binds to the ligand-specific receptor. Exemplary antibody compositions (e.g., VH and/or VL sequences or fragments thereof) that are useful for the design of CTLA-4 fusion proteins as described herein include, but are not limited to, anti-CAIX antibodies described in PCT/US2006/046350 and PCT/US2015/067178; anti- CXCR4 antibodies described in PCT/US20006/005691; anti-CCR4 antibodies described in PCT/US2008/088435, PCT/US2013/039744, and PCT/US2015/054202; anti-PD-Ll antibodies described in PCT/US2008/088435 and PCT/US2020/062815; anti-PD-1 antibodies described in PCT/US2020/037791 and PCT/US2020/037781; anti-GITR antibodies described in PCT/US2017/043504; anti-claudin-4 antibodies described in PCT/US2019/022272; and anti-MUCl antibodies described in PCT/US2020/037783 (each of the applications which are incorporated by reference in their entireties). In one embodiment, the fusion protein further comprises a constant region, and/or a linker as described herein. Different formats of multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies such as a fusion protein comprises an antibody that recognizes CTLA-4 and a ligand) are described herein. Ligands can be tumor associated antigens (e.g., LINGO1, ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), MUC1, MSLN, CD 19, CD20, CD30, CD40, CD22, RAGE-1, MN-CA IX, RET1, RET2 (AS), prostate specific antigen (PSA), TAG-72, PAP, p53, Ras, prostein, PSMA, survivin, 9D7, prostate-carcinoma tumor antigen-1 (PCTA-1), GAGE, MAGE, mesothelin, 0-catenin, TGF-0RII, BRCA1/2, SAP-1, HPV-E6, HPV-E7 (see also, PCT/US2015/067225 and PCT/US2019/022272 for additional tumor-associated surface antigens, which are incorporated by reference in their entireties)); cytokines (e.g., IL- 12 (IL- 12A (p35 subunit) protein sequence having NCBI Reference No. NP_000873.2; IL-12B (p40 subunit) protein sequence having NCBI Reference No. NP_002178.2); IL-18 (protein sequence having NCBI Reference no. NP_001553.1); IL-15 (protein sequence having NCBI Reference No. NP_000576.1); IL-7 (protein sequence having NCBI Reference No. NP_000871.1); IL-2 (protein sequence having NCBI Reference No. NP_000577.2); and IL- 21 (protein sequence having NCBI Reference No. NP_068575.1)); CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3, OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27, VISTA, B7H3, B7H4, HEVM (or BTLA), CD47, PDL1, MICA, MICB, and CD73. Different formats of bispecific or trispecific antibodies are also provided herein. In some embodiments, each of the anti-CTLA-4 fragment and the second antigenspecific fragment and/or the third antigen-specific fragment is each independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. In some embodiments, the bispecific or trispecific antibody further includes a Fc fragment (e.g., as described in PCT/US2015/021529 and PCT/US2019/023382, each of which are incorporated by reference in their entireties). A bispecific or trispecific antibody of the invention can comprise a heavy chain and a light chain combination or scFv of the CTLA-4 antibodies described herein.
[00267] Multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies) of the invention (for example, an anti-CTLA-4-scFv fusion protein) can be constructed using methods known art. In some embodiments, the bi-specific antibody is a single polypeptide wherein the two scFv fragments are joined by a long linker polypeptide, of sufficient length to allow intramolecular association between the two scFv units to form an antibody. In other embodiments, the bi-specific antibody is more than one polypeptide linked by covalent or non-covalent bonds. In some embodiments, the amino acid linker depicted herein (GGGGSGGGGS; “(G4S)2” (SEQ ID NO: [ ])) can be generated with a longer G4S linker to improve flexibility. For example, the linker can also be “(G4S)3” (e.g., GGGGSGGGGSGGGGS (SEQ ID NO: [ ])); “(G4S)4” (e.g., GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: [ ]); “(G4S)5” (e.g., GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: [ ])); “(G4S)6” (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: [ ])); “(G4S)7” (e g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: [ ])); and the like. For example, use of the (G4S)5 linker can provide more flexibility to a ligand described herein and can improve expression. In some embodiments, the linker can also be (GS)n, (GGS)n, (GGGS)n, (GGSG)n, (GGSGG)n, or (GGGGS)n, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Non-limiting examples of linkers known to those skilled in the art that can be used to construct the fusions described herein can be found in U.S. Patent No. 9,708,412; U.S. Patent Application Publication Nos. US 20180134789 and US 20200148771; and PCT Publication No. WO2019051122 (each of which are incorporated by reference in their entireties).
[00268] In another embodiment, the multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies such as anti-CTLA-4-scFv fusions) can be constructed using the "knob into hole" method (Ridgway et al, Protein Eng 7:617-621 (1996)). In this method, the Ig heavy chains of the two different variable domains are reduced to selectively break the heavy chain pairing while retaining the heavy-light chain pairing. The two heavy -light chain heterodimers that recognize two different antigens/ligands or three different antigens/ligands are mixed to promote heteroligation pairing, which is mediated through the engineered "knob into holes" of the CH3 domains.
[00269] In another embodiment the multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies such as anti-CTLA-4-scFv fusions) can be constructed through exchange of heavy-light chain dimers from two or more different antibodies to generate a hybrid antibody where the first heavy -light chain dimer recognizes CTLA-4 and the second heavy -light chain dimer recognizes a second antigen and/or third antigen. The mechanism for heavy -light chain dimer is similar to the formation of human IgG4, which also functions as a bispecific molecule. Dimerization of IgG heavy chains is driven by intramolecular force, such as the pairing the CH3 domain of each heavy chain and disulfide bridges. Presence of a specific amino acid in the CH3 domain (R409) has been shown to promote dimer exchange and construction of the IgG4 molecules. Heavy chain pairing is also stabilized further by interheavy chain disulfide bridges in the hinge region of the antibody. Specifically, in IgG4, the hinge region contains the amino acid sequence Cys-Pro-Ser-Cys (in comparison to the stable IgGl hinge region which contains the sequence Cys-Pro-Pro-Cys) at amino acids 226- 230. This sequence difference of Serine at position 229 has been linked to the tendency of IgG4 to form intrachain disulfides in the hinge region (Van der Neut Kolfschoten, M. et al, 2007, Science 317: 1554-1557 and Labrijn, A.F. et al, 2011, Journal of Immunol 187:3238- 3246).
[00270] The multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies such as anti-CTLA-4-scFv fusions) of the invention can be created through introduction of the R409 residue in the CH3 domain and the Cys-Pro-Ser-Cys sequence in the hinge region of antibodies that recognize CTLA-4 or a second and/or third antigen, so that the heavy-light chain dimers exchange to produce an antibody molecule with one heavy-light chain dimer recognizing CTLA-4 and the second heavy-light chain dimer recognizing a second and/or third antigen, wherein the second and/or third antigen (or ligand) is any antigen (or ligand) disclosed herein. Known IgG4 molecules can also be altered such that the heavy and light chains recognize CTLA-4 or a second and/or third antigen, as disclosed herein. Use of this method for constructing the multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies such as anti-CTLA-4-scFv fusions) of the invention can be beneficial due to the intrinsic characteristic of IgG4 molecules wherein the Fc region differs from other IgG subtypes in that it interacts poorly with effector systems of the immune response, such as complement and Fc receptors expressed by certain white blood cells. This specific property makes these IgG4-based multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies such as anti-CTLA-4-scFv fusions) attractive for therapeutic applications, in which the antibody is required to bind the target(s) and functionally alter the signaling pathways associated with the target(s), however not trigger effector activities.
[00271] The multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies such as anti-CTLA-4-scFv fusions) described herein can be engineered with a non-depleting heavy chain isotype, such as IgGl-LALA or stabilized IgG4 or one of the other non-depleting variants. In some embodiments, mutations are introduced to the constant regions of the bsAb such that the antibody dependent cell-mediated cytotoxicity (ADCC) activity of the bsAb is altered. For example, the mutation is a LALA mutation in the CH2 domain. In one aspect, the multispecific antibody (e.g., bispecific antibodies and trispecific antibodies such as anti- CTLA-4-scFv fusions) contains mutations on one scFv unit of the heterodimeric multispecific antibody, which reduces the ADCC activity. In another aspect, the multispecific antibody (e.g., bispecific antibodies and trispecific antibodies such as anti-CTLA-4-scFv fusions) contains mutations on both chains of the heterodimeric multispecific antibody, which completely ablates the ADCC activity. For example, the mutations introduced in one or both scFv units of the multispecific antibody (e.g., bispecific antibodies and trispecific antibodies such as anti-CTLA-4-scFv fusions) are LALA mutations in the CH2 domain. These multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies such as anti- CTLA-4-scFv fusions) with variable ADCC activity can be optimized such that the multispecific antibodies exhibit maximal selective killing towards cells that express one antigen that is recognized by the multispecific antibody, however, exhibits minimal killing towards the second antigen that is recognized by the multispecific antibody.
[00272] The multispecific antibodies (e.g., bispecific antibodies) described herein can be engineered as modular tetrameric bispecific antibodies (tBsAb). See, for example, WO 2018/071913, which is incorporated by reference herein in its entirety. For example, the tetravalent antibody can be a dimer of a bispecific scFv fragment including a first binding site for a first antigen, and a second binding site for a second antigen. In embodiments, the anti- CTLA-4 antibody can be the first binding site for a first antigen. In embodiments, the anti- CTLA-4 antibody can be the second binding site for a second antigen. The two binding sites can be joined together via a linker domain. In embodiments, the scFv fragment is a tandem scFv, the linker domain includes an immunoglobulin hinge region (e.g., an IgGl, an IgG2, an IgG3, or an IgG4 hinge region) amino acid sequence. In embodiments, the immunoglobulin hinge region amino acid sequence can be flanked by a flexible linker amino acid sequence, e.g., having the linker amino acid sequence (GGGS)xi-e, (GGGGS)xi-e, or GSAGSAAGSGEF. In embodiments, the linker domain includes at least a portion of an immunoglobulin Fc domain, e.g., an IgGl, an IgG2, an IgG3, or an IgG4 Fc domain. In embodiments, the at least a portion of the immunoglobulin Fc domain does not include a CH2 domain. In embodiments, the at least a portion of the immunoglobulin Fc domain can be a CH2 domain. An exemplary CH2 domain amino acid sequence includes APELLGGPDVFLF (SEQ ID NO: [ ]). The Fc domain can be linked to the C-terminus of an immunoglobulin hinge region (e.g., an IgGl, an IgG2, an IgG3, or an IgG4 hinge region) amino acid sequence. The linker domain can include a flexible linker amino acid sequence (e.g., (GGGS)xi-e, (GGGGS)xi-e, or GSAGSAAGSGEF (SEQ ID NO: [ ])) at one terminus or at both termini. [00273] In embodiments, the multispecific antibodies (e.g., bispecific antibodies) described herein can be engineered with a wild type Fc region, or a modified Fc region. For example, embodiments can comprise an IgGs CH2 domain. In other embodiments, the multispecific antibodies (e.g., bispecific antibodies) are engineered without an Fc domain and/or Fc fragment.
[00274] In embodiments, the tBsAb can be specific for CTLA-4, and also a target selected from the group consisting of B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CCR4, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, PD-L1, PD-1, TIM3, 4-1BB, TIGIT, VISTA, HEVM, BTLA, MICA, MICB, and KIR.
[00275] The multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies such as anti-CTLA-4-scFv fusions) disclosed herein can be useful in treatment of chronic infections, diseases, or medical conditions, for example, cancer.
[00276] Use of Antibodies Against CTLA-4 [00277] Antibodies of the invention specifically binding a CTLA-4 protein, or a fragment thereof, can be administered for the treatment of a CTLA-4 associated disease or disorder. A "CTLA-4-associated disease or disorder" includes disease states and/or symptoms associated with a disease state, where increased levels of CTLA-4 and/or activation of cellular signaling pathways involving CTLA-4 are found. In some embodiments, the cancer can be lung cancer, kidney cancer, ovarian cancer, prostate cancer, colon cancer, breast cancer, cervical cancer, uterine cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, glioblastoma multiforme, cutaneous squamous cell carcinoma, melanoma, renal cancer, such as clear cell renal cell carcinoma, or stomach cancer.
[00278] Antibodies of the invention, including bi-specific, polyclonal, monoclonal, humanized and fully human antibodies, can be used as therapeutic agents. Such agents will generally be employed to treat cancer in a subject, increase vaccine efficiency or augment a natural immune response. An antibody preparation, for example, one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target. Administration of the antibody can abrogate or inhibit or interfere with an activity of the CTLA-4 protein.
[00279] Pharmaceutical Compositions
[00280] Antibodies of the invention specifically binding a CTLA-4 protein or fragment thereof can be administered for the treatment of a cancer in the form of pharmaceutical compositions. Principles and considerations involved in preparing therapeutic pharmaceutical compositions comprising the antibody, as well as guidance in the choice of components are provided, for example, in: Remington: The Science And Practice Of Pharmacy 20th ed. (Alfonso R._Gennaro, et al, editors) Mack Pub. Co., Easton, Pa., 2000; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.
[00281] A specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the particular antibodies, variant or derivative thereof used, the patient's age, body weight, general health, sex, and diet, and the time of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.
[00282] A therapeutically effective amount of an antibody of the invention can be the amount needed to achieve a therapeutic objective. As noted herein, this can be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target. The amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. The dosage administered to a subject (e.g., a patient) of the antigen-binding polypeptides described herein is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight, between 0.1 mg/kg and 20 mg/kg of the patient's body weight, or 1 mg/kg to 10 mg/kg of the patient's body weight. Human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the disclosure may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention can be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Common dosing frequencies can range, for example, from twice daily to once a week.
[00283] Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, based upon the variable-region sequences of an antibody, peptide molecules can be designed that retain the ability to bind the target protein sequence. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology. (See, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). The formulation can also contain more than one active compound as necessary for the particular indication being treated, for example, those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can comprise an agent that enhances its function, such as, for example, a cytotoxic agent, cytokine (e.g. IL-15), chemotherapeutic agent, or growth- inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. [00284] The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
[00285] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
[00286] Sustained-release preparations can be prepared. Suitable examples of sustained- release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. , films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers ofL-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. While polymers such as ethylenevinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
[00287] The antibodies or agents of the invention (also referred to herein as "active compounds"), and derivatives, fragments, analogs and homologs thereof, can be incorporated into pharmaceutical compositions suitable for administration. Such pharmaceutical compositions can comprise the antibody or agent and a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Non-limiting examples of such carriers or diluents include water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. [00288] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
[00289] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In embodiments, the composition is sterile and is fluid to the extent that easy syringeability exists. It can be stable under the conditions of manufacture and storage and can be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents can be included, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
[00290] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. For example, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. [00291] Oral compositions can include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
[00292] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
[00293] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
[00294] The compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
[00295] In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polygly colic acid, collagen, poly orthoesters, and polylactic acid. Methods for preparation of such formulations are apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.
[00296] Oral or parenteral compositions can be formulated in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
[00297] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
[00298] Diagnostics
[00299] An antibody according to the invention can be used as an agent for detecting the presence of CTLA-4 (or a protein fragment thereof) in a sample. For example, the antibody can contain a detectable label. Antibodies can be polyclonal or monoclonal. An intact antibody, or a fragment thereof (e.g., Fab, scFv, or F(ab)2) can be used. The term "labeled", with regard to the probe or antibody, can encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" can include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term "biological sample", therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an analyte mRNA includes Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations.
[00300] Procedures for conducting immunoassays are described, for example in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, NJ, 1995; "Immunoassay", E. Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, CA, 1996; and "Practice and Theory of Enzyme Immunoassays", P. Tijssen, Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-analyte protein antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
[00301] Antibodies directed against a CTLA-4 protein (or a fragment thereof) can be used in methods known within the art relating to the localization and/or quantitation of a CTLA-4 protein (e.g., for use in measuring levels of the CTLA-4 protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like). In a given embodiment, antibodies specific to a CTLA-4 protein, or derivative, fragment, analog or homolog thereof, that contain the antibody derived antigen binding domain, are utilized as pharmacologically active compounds (referred to herein as "therapeutics").
[00302] An antibody of the invention specific for a CTLA-4 protein can be used to isolate a CTLA-4 polypeptide by standard techniques, such as immunoaffinity, chromatography or immunoprecipitation. Antibodies directed against a CTLA-4 protein (or a fragment thereol) can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen.
[00303] Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, [3-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125I, 131I, 35S, 32P or 3H.
[00304] Cellular therapies
[00305] Cellular therapies are also provided here. For example, cellular therapies can include cells, such as T cells NK cells, or B cells, genetically engineered to express an anti- CTLA4 antibody as described herein. A “genetically engineered cell” can refer to any cell of any organism that is modified, transformed, or manipulated by addition or modification of a gene, a DNA or RNA sequence, or protein or polypeptide. Isolated cells, host cells, and genetically engineered cells can include isolated immune cells, such as NK cells, T cells, and B cells, that contain the DNA or RNA sequences encoding an antibody as described herein, and/or a chimeric receptor or chimeric receptor complex that is expressed on the cell surface. [00306] A “T cell" is a cell of the immune system that matures in the thymus and produces T cell receptors (TCR). T cells can be naive cells (not in contact with the antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127 and CD45RA and reduced expression of CD45RO compared to T CM ), memory T cells (T M ) (contacted with antigen and long-living) and effector cells (in contact with the antigen, cytotoxic). T M can be further subdivided into subpopulations of central memory T cells (T CM , increased expression of CD62L, CCR7, CD28, CD127, CD45RO and CD95 and decreased expression of CD54RA compared to naive T cells) and effector memory T cells (T EM , reduced expression of CD62L, CCR7, CD28, CD45RA and increased expression of CD 127 compared to naive T cells or T CM ). Effector T cells (T E ) can refer to CD8 + antigen-contacted cytotoxic T lymphocytes that have reduced expression of CD62L, CCR7, CD28 and are positive for granzyme and perforin compared to T CM .
[00307] T-cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue and tumours. In certain embodiments, T-cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled person, such as FICOLL™ separation. In one embodiment, cells from the circulating blood of a subject are obtained by apheresis. The apheresis product typically contains lymphocytes, including T-cells, monocytes, granulocytes, B-cells, other nucleated white blood cells, red blood cells and platelets. In one embodiment, the cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or media for subsequent processing. In one embodiment of the invention, the cells are washed with PBS. In an alternative embodiment, the washed solution lacks calcium and/or magnesium or can lack many if not all divalent cations. As would be appreciated by those of ordinary skill in the art, a washing step can be accomplished by methods known to those in the art, such as by using a semi-automated flow- through centrifuge, for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the like. After washing, the cells can be resuspended in a variety of biocompatible buffers or other saline solution with or without buffer. In certain embodiments, the undesirable components of the apheresis sample can be removed and the cell directly resuspended in culture media.
[00308] In embodiments, T-cells are isolated from PBMCs. PBMCs may be isolated from buffy coats obtained by density gradient centrifugation of whole blood, for instance centrifugation through a LYMPHOPREP™ gradient, a PERCOLL™ gradient or a FICOLL™ gradient. T-cells may be isolated from PBMCs by depletion of the monocytes, for instance by using CD14 DYNABEADS®. In some embodiments, red blood cells may be lysed prior to the density gradient centrifugation.
[00309] An "NK cell" is a large granular lymphocyte, being a cytotoxic lymphocyte derived from the common lymphoid progenitor which does not naturally comprise an antigen-specific receptor (e.g. a T-cell receptor or a B-cell receptor). NK cells can be characterised by their CD3-, CD56+ phenotype. In embodiments, NK cell can refer to any known NK cell or any NK-like cell or any cell having the characteristics of an NK cell. Thus primary NK cells can be used or, in another embodiment, a NK cell known in the art that has previously been isolated and cultured can be used. Thus an NK cell-line can be used. A number of different NK cells are known and reported in the literature and any of these could be used, or a cellline may be prepared from a primary NK cell, for example by viral transformation (Vogel et al. 2014, Leukemia 28:192-195). Suitable NK cells include (but are by no means limited to), in addition to NK-92, the NK-YS, NK-YT, MOTN-1 , NKL, KHYG-1 , HANK-1 , or NKG cell lines. Variants of other cell lines may also be used.
[00310] A “B-cell” is a B lymphocyte that, subsequent to activation, can produce antibody molecules. Activation of a B cell can be, without limitation, by recognition of an antigen by an antigen specific immunoglobulin receptor on the B cell surface or by a non-specific stimulus, e.g., a B cell mitogen such as lipopolysaccharide or pokeweed mitogen. Thus, a B cell can be a “virgin” B lymphocyte that has never previously been activated or a “memory” B lymphocyte that has previously been activated or the progeny of such a B lymphocyte. [00311] Cellular therapies, such as chimeric antigen receptor (CAR) T-cell therapies or chimeric antigen receptor (CAR) NK-cell therapies, are also provided herein. CAR T-cell therapies redirect a patient’s T-cells and NK cells to kill tumor cells by the exogenous expression of a CAR on a T-cell or NK cell, for example. For example, when expressed in a T or NK cell, CARs have the ability to redirect T or NK cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies. The non-MHC-restricted antigen recognition gives T or NK cells expressing CARs the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape.
[00312] A CAR can be a membrane spanning fusion protein that links the antigen recognition domain of an antibody to the intracellular signaling domains of the T-cell receptor and co-receptor. A suitable cell can be used, for example, that can secrete an anti- CTLA-4 antibody of the invention (or alternatively engineered to express an anti-CTLA-4 antibody as described herein to be secreted). The anti- CTLA-4 “payloads” to be secreted, can be, for example, minibodies, scFvs, IgG molecules, bispecific fusion molecules, and other antibody fragments as described herein. Upon contact or engineering, the cell described herein can then be introduced to a patient in need of a treatment by infusion therapies known to one of skill in the art. The patient can have a CTLA-4-associated disease or disorder as described herein, such as chronic infections or cancer. The cell (e.g., a T cell or NK cell) can be, for instance, T lymphocyte, a CD4+ T cell, a CD8+ T cell, or the combination thereof, without limitation. Exemplary CARs and CAR factories useful in aspects of the invention include those disclosed in, for example, PCT/US2015/067225 and PCT/US2019/022272, each of which are hereby incorporated by reference in their entireties. In one embodiment, the CTLA-4 antibodies discussed herein can be used in the construction of multi-specific antibodies or as the payload for a genetically engineered cell, such as a CAR-T cell, a CARNK cell, or a genetically engineered B cell. For example, in one embodiment, the anti-CTLA-4 antibodies discussed herein can be used for the targeting of the CARS (i.e., as the targeting moiety). In another embodiment, the anti- CTLA-4 antibodies discussed herein can be used as the targeting moiety, and a different CTLA-4 antibody that targets a different epitope can be used as the payload. In another embodiment, the payload can be an immunomodulatory antibody payload. [00313] Methods of Treatment
[00314] As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e. , not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can refer to prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
[00315] The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a cancer (for example, if an early detection cancer biomarker is identified in such a subject), or other cell proliferation-related diseases or disorders. Such diseases or disorders include but are not limited to, e.g., those diseases or disorders associated with aberrant expression of CTLA-4. For example, the methods are used to treat, prevent or alleviate a symptom of cancer. In an embodiment, the methods are used to treat, prevent or alleviate a symptom of a solid tumor. Non-limiting examples of other tumors that can be treated by compositions described herein comprise lung cancer (e.g., nonsmall cell lung cancer or lung adenocarcinoma), gastric cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, esophageal squamous cell carcinoma, nasopharyngeal carcinoma, glioblastoma multiforme, squamous cell carcinoma, melanoma, renal cell carcinoma, urothelial carcinoma, hepatocellular carcinoma, malignant pleural mesothelioma, and liquid tumors or stomach cancer. Additionally, the methods of the invention can be used to treat hematologic cancers such as leukemia and lymphoma. Alternatively, the methods can be used to treat, prevent or alleviate a symptom of a cancer that has metastasized. For example, cancers that can be treated or prevented or for which symptons can be alleviated include B-cell chronic lymphocytic leukemia (CLL), non-smallcell lung cancer, melanoma, ovarian cancer, lymphoma, or renal-cell cancer. Cancers that can also be treated or prevented or for which symptons can be alleviated include those solid tumors with a high mutation burden and WBC in filtrate. [00316] Accordingly, in one aspect, the invention provides methods for preventing, treating or alleviating a symptom cancer or a cell proliferative disease or disorder in a subject by administering to the subject a monoclonal antibody, scFv antibody or bi- specific antibody of the invention. For example, an anti- CTLA-4 antibody can be administered in therapeutically effective amounts.
[00317] Subjects at risk for cancer or cell proliferation-related diseases or disorders can include patients who have a family history of cancer or a subject exposed to a known or suspected cancer-causing agent. Administration of a prophylactic agent can occur prior to the manifestation of cancer such that the disease is prevented or, alternatively, delayed in its progression.
[00318] In another aspect, tumor cell growth is inhibited by contacting a cell with an anti- CTLA4 antibody of the invention. The cell can be any cell that expresses CTLA-4.
[00319] Also included in the invention are methods of increasing or enhancing an immune response to an antigen. An immune response is increased or enhanced by administering to the subject a monoclonal antibody, scFv antibody, or bi-specific antibody of the invention. The immune response is augmented for example by augmenting antigen specific T effector function. The antigen is a viral (e.g. HIV), bacterial, parasitic or tumor antigen. The immune response is a natural immune response. By natural immune response is meant an immune response that is a result of an infection. The infection is a chronic infection. Increasing or enhancing an immune response to an antigen can be measured by a number of methods known in the art. For example, an immune response can be measured by measuring any one of the following: T cell activity, T cell proliferation, T cell activation, production of effector cytokines, and T cell transcriptional profile. Alternatively, the immune response is a response induced due to a vaccination.
[00320] Accordingly, in another aspect the invention provides a method of increasing vaccine efficiency by administering to the subject a monoclonal antibody or scFv antibody of the invention and a vaccine. The antibody and the vaccine are administered sequentially or concurrently. The vaccine is a tumor vaccine a bacterial vaccine or a viral vaccine.
[00321] Combinatory Methods
[00322] Compositions of the invention as described herein can be administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with the compositions of the disclosure include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alpha-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogen mustard) and thiotepa); steroids and combinations (e.g., bethamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).
[00323] In additional embodiments, the compositions of the invention as described herein can be administered in combination with cytokines. Cytokines that may be administered with the compositions include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL- 10, IL- 12, IL-13, IL-15, anti-CD40, CD40L, and TNF-a.
[00324] In additional embodiments, the compositions described herein can be administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
[00325] In some embodiments, the compositions described herein can be administered in combination with other immunotherapeutic agents. Non-limiting examples of immunotherapeutic agents include simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab, detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab, ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab, girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab, indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, namatumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab, pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab, rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab, siltuximab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49, and 3F8.
[00326] The invention provides for methods of treating cancer in a patient by administering two antibodies that bind to the same epitope of the CTLA-4 protein or, alternatively, two different epitopes of the CTLA-4 protein. Alternatively, the cancer can be treated by administering a first antibody that binds to CTLA-4 and a second antibody that binds to a protein other than CTLA-4. In other embodiments, the cancer can be treated by administering a bispecific antibody that binds to CTLA-4 and that binds to a protein other than CTLA-4. For example, the other protein other than CTLA-4 can include, but is not limited to, IL-12, IL-12R, IL-2, IL-2R, IL-15, IL-15R, IL-7, IL-7R, IL-21, or IL-21R. For example, the other protein other than CTLA-4 is a tumor- associated antigen; the other protein other than CTLA-4 can also be a cytokine.
[00327] In some embodiments, the invention provides for the administration of an anti-PD- 1 antibody alone or in combination with an additional antibody that recognizes another protein other than CTLA-4, with cells that are capable of effecting or augmenting an immune response. For example, these cells can be peripheral blood mononuclear cells (PBMC), or any cell type that is found in PBMC, e.g., cytotoxic T cells, macrophages, and natural killer (NK) cells.
[00328] Additionally, the invention provides administration of an antibody that binds to the CTLA4 protein and an anti-neoplastic agent, such as a small molecule, a growth factor, a cytokine or other therapeutics including biomolecules such as peptides, peptidomimetics, peptoids, polynucleotides, lipid-derived mediators, small biogenic amines, hormones, neuropeptides, and proteases. Small molecules include, but are not limited to, inorganic molecules and small organic molecules. Suitable growth factors or cytokines include an IL-2, GM-CSF, IL-12, and TNF-alpha. Small molecule libraries are known in the art. (See, Lam, Anticancer Drug Des., 12: 145, 1997.)
[00329] Chimeric antisen receptor (CAR) T-cell therapies
[00330] Cellular therapies, such as chimeric antigen receptor (CAR) T-cell therapies, are also provided herein. CAR T-cell therapies redirect a patient’s T-cells to kill tumor cells by the exogenous expression of a CAR on a T-cell, for example. A CAR can be a membrane spanning fusion protein that links the antigen recognition domain of an antibody to the intracellular signaling domains of the T-cell receptor and co-receptor. A suitable cell can be used, for example, that can secrete an anti-CTLA-4 antibody of the present invention (or alternatively engineered to express an anti-CTLA-4 antibody as described herein to be secreted). The anti-CTLA-4 “payloads” to be secreted, can be, for example, minibodies, ScFvs, IgG molecules, bispecific fusion molecules, and other antibody fragments as described herein.
[00331] Solid tumors offer unique challenges for CAR-T therapies. Some barriers to CAR- T effectiveness in solid tumors include heterogeneous antigen expression, insufficient tissue homing, activation, persistence, and the immunosuppressive tumor microenvironment. Unlike blood cancers, tumor-associated target proteins are overexpressed between the tumor and healthy tissue resulting in on-target/off-tumor T-cell killing of healthy tissues. Furthermore, immune repression in the tumor microenvironment (TME) limits the activation of CAR-T cells towards killing the tumor. Upon such contact or engineering, the cell can then be introduced to a cancer patient in need of a treatment by infusion therapies known to one of skill in the art. The cancer patient may have a cancer of any of the types as disclosed herein. The cell (e.g., a T cell) can be, for instance, a tumor-infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or the combination thereof, without limitation.
[00332] Exemplary CARs and CAR factories useful in aspects of the invention include those disclosed in, for example, PCT/US2015/067225 and PCT/US2019/022272, each of which are hereby incorporated by reference in their entireties. For example, CAR-T cells can be generated according to methods known in the art using lentivirus systems (via transduction), retrovirus systems (via transfection (electroporation)), and transposon systems (via PiggyBac). Useful for promoters for payloads that can be used in the generating of CAR-Ts or CAR NKs include, for example, constitutive promoters (where the promoter is the same as for CAR-T or CAR NK, such as EFla then IRES or 2A); inducible promoters (where the promoter is different from the promoter for CAR-T, such as NF AT, IL-2 prom); and genetically engineered promoters (such as a CTLA-4 locus “knock in” of cytokine and/or a promoter that is under the control of an endogenous promoter). In one embodiment, the CTLA-4 antibodies or the CTLA-4 fusion proteins discussed herein can be used in the construction of multi-specific antibodies or as the payload for a genetically engineered cell, such as a CAR-T cell, CAR NK cell, or genetically engineered B cell. For example, in one embodiment, the anti-CTLA-4 antibodies or the CTLA-4 fusion proteins discussed herein can be used for the targeting of the CARS (i.e., as the targeting moiety). In one embodiment, the anti-CTLA-4 antibodies or the CTLA-4 fusion proteins discussed herein can be used as a payload to be secreted by a genetically engineered cell, such as a CAR-T cell, a CAR NK cell, or a genetically engineered B cell. In another embodiment, the anti-CTLA-4 antibodies or the CTLA-4 fusion proteins discussed herein can be used as the targeting moiety, and a different CTLA-4 antibody that targets a different epitope can be used as the payload. In another embodiment, the payload can be an immunomodulatory antibody payload. In some embodiments, the CTLA-4 antibodies or the CTLA-4 fusion proteins as described herein for use in CAR-T compositions are not high-affinity CTLA-4 antibodies (for example, so that the antibody does not bind strongly to its CTLA-4 target). For example, the CTLA-4 antibodies or the CTLA-4 fusion proteins described herein can be used as a payload secreted by the genetically engineered cell, such as a CAR-T cell, a CAR-NK cell, or a genetically engineered B cell, with the two targeting moieties (for example, tumor-associated surface antigens) selected for a specific cancer (i.e. MSLN and MUC1 for ovarian cancer). Nonlimiting examples of a tumor-associated surface antigen include ErbB2 (HER2/neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), MUC1, MSLN, CD 19, CD20, CD30, CD40, CD22, RAGE-1, MN-CA IX, RET1, RET2 (AS), prostate specific antigen (PSA), TAG-72, PAP, p53, Ras, prostein, PSMA, survivin, 9D7, prostate-carcinoma tumor antigen-1 (PCTA-1), GAGE, MAGE, mesothelin, 0-catenin, TGF-0RII, BRCA1/2, SAP-1, HPV-E6, HPV-E7 (see also, PCT/US2015/067225 and PCT/US2019/022272 for additional tumor-associated surface antigens, which are incorporated by reference in their entireties). Exemplary armored CAR-T cells are listed in the table below.
CART Payload Format Promoter Publication
PSMA DN-TGFb Molecular Therapy 26: 1855 (2018)
CD19 PD-1 nature biotechnology 36:847 (2018)
Mucl6
CD19 IL-18 Cell Reports 23:2130 (2018)
Mucl6
CD19 IL-12 fusion IRES Scientific REPOrTS 7: 10541 (2017)
[00333] In one embodiment, bispecific (or dual -targeted) CAR-Ts are provided. In another embodiment, the CAR-T is an engineered cell comprising a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular ligand binding domain that is specific for a first antigen and a second antigen on the surface of a cancer cell, wherein the first antigen comprises CXCR4 and the second antigen comprises CLDN4, or the first antigen comprises CAIX and the second antigen comprises CD70, or the first antigen comprises MUC1 and the second antigen comprises Msln. For example, the anti-CTLA-4 antibodies or the CTLA-4 fusion proteins described herein can be used as a payload for a genetically engineered cell, such as a CAR-T, CAR-NK cell or genetically engineered B cell as described herein. In one embodiment, a CXCR4/CLDN4 dual targeting CAR-T with an anti-CTLA-4 fusion payload can be used for breast cancer. In one embodiment, a CAIX/CD70 dual targeting CAR-T with an anti-CTLA-4 fusion payload can be used for clear cell renal cell carcinoma (ccRCC). In one embodiment, a MUCl/Msln dual targeting CAR-T with an anti-CTLA-4 fusion payload can be used for ovarian cancer.
[00334] Diagnostic Assays
[00335] The anti-CTLA-4 antibodies can be used diagnostically to, for example, monitor the development or progression of cancer as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment and/or prevention regimen.
[00336] In some embodiments, for diagnostic purposes, the anti-CTLA-4 antibody of the invention is linked to a detectable moiety, for example, so as to provide a method for detecting a cancer cell in a subject at risk of or suffering from a cancer.
[00337] The detectable moieties can be conjugated directly to the antibodies or fragments, or indirectly by using, for example, a fluorescent secondary antibody. Direct conjugation can be accomplished by standard chemical coupling of, for example, a fluorophore to the antibody or antibody fragment, or through genetic engineering. Chimeras, or fusion proteins can be constructed which contain an antibody or antibody fragment coupled to a fluorescent or bioluminescent protein. For example, Casadei, et al, (Proc Natl Acad Sci U S A. 1990 Mar; 87(6): 2047 -51) describe a method of making a vector construct capable of expressing a fusion protein of aequorin and an antibody gene in mammalian cells.
[00338] As used herein, the term "labeled", with regard to the probe or antibody, can encompass direct labeling of the probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently -labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" is intended to include tissues, cells and biological fluids isolated from a subject (such as a biopsy), as well as tissues, cells and fluids present within a subject. That is, the detection method of the invention can be used to detect cells that express CTLA-4 in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of CTLA-4 include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. Furthermore, in vivo techniques for detection of CTLA-4 include introducing into a subject a labeled anti-CTLA-4 antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
[00339] In the case of "targeted" conjugates, that is, conjugates which contain a targeting moiety — a molecule or feature designed to localize the conjugate within a subject or animal at a particular site or sites, localization can refer to a state when an equilibrium between bound, "localized", and unbound, "free" entities within a subject has been essentially achieved. The rate at which such equilibrium is achieved depends upon the route of administration. For example, a conjugate administered by intravenous injection can achieve localization within minutes of injection. On the other hand, a conjugate administered orally can take hours to achieve localization. Alternatively, localization can simply refer to the location of the entity within the subject or animal at selected time periods after the entity is administered. By way of another example, localization is achieved when an moiety becomes distributed following administration.
[00340] It is understood that a reasonable estimate of the time to achieve localization can be made by one skilled in the art. Furthermore, the state of localization as a function of time can be followed by imaging the detectable moiety (e.g., a light-emitting conjugate) according to the methods of the invention, such as with a photodetector device. The "photodetector device" used should have a high enough sensitivity to enable the imaging of faint light from within a mammal in a reasonable amount of time, and to use the signal from such a device to construct an image.
[00341] In cases where it is possible to use light-generating moieties which are extremely bright, and/or to detect light-generating fusion proteins localized near the surface of the subject or animal being imaged, a pair of "night- vision" goggles or a standard high- sensitivity video camera, such as a Silicon Intensified Tube (SIT) camera (e.g., from Hammamatsu Photonic Systems, Bridgewater, N.J.), can be used. More typically, however, a more sensitive method of light detection is required.
[00342] In extremely low light levels the photon flux per unit area becomes so low that the scene being imaged no longer appears continuous. Instead, it is represented by individual photons which are both temporally and spatially distinct form one another. Viewed on a monitor, such an image appears as scintillating points of light, each representing a single detected photon. By accumulating these detected photons in a digital image processor over time, an image can be acquired and constructed. In contrast to conventional cameras where the signal at each image point is assigned an intensity value, in photon counting imaging the amplitude of the signal carries no significance. The objective is to simply detect the presence of a signal (photon) and to count the occurrence of the signal with respect to its position over time.
[00343] At least two types of photodetector devices, described below, can detect individual photons and generate a signal which can be analyzed by an image processor. Reduced-Noise Photodetection devices achieve sensitivity by reducing the background noise in the photon detector, as opposed to amplifying the photon signal. Noise is reduced primarily by cooling the detector array. The devices include charge coupled device (CCD) cameras referred to as "backthinned", cooled CCD cameras. In the more sensitive instruments, the cooling is achieved using, for example, liquid nitrogen, which brings the temperature of the CCD array to approximately -120°C. "Backthinned" refers to an ultra- thin backplate that reduces the path length that a photon follows to be detected, thereby increasing the quantum efficiency. A particularly sensitive backthinned cryogenic CCD camera is the "TECH 512", a series 200 camera available from Photometries, Ltd. (Tucson, Ariz.).
[00344] "Photon amplification devices" amplify photons before they hit the detection screen. This class includes CCD cameras with intensifiers, such as microchannel intensifiers. A microchannel intensifier typically contains a metal array of channels perpendicular to and co-extensive with the detection screen of the camera. The microchannel array is placed between the sample, subject, or animal to be imaged, and the camera. Most of the photons entering the channels of the array contact a side of a channel before exiting. A voltage applied across the array results in the release of many electrons from each photon collision. The electrons from such a collision exit their channel of origin in a "shotgun" pattern, and are detected by the camera.
[00345] Even greater sensitivity can be achieved by placing intensifying microchannel arrays in series, so that electrons generated in the first stage in turn result in an amplified signal of electrons at the second stage. Increases in sensitivity, however, are achieved at the expense of spatial resolution, which decreases with each additional stage of amplification. An exemplary microchannel intensifier-based single-photon detection device is the C2400 series, available from Hamamatsu.
[00346] Image processors process signals generated by photodetector devices which count photons in order to construct an image which can be, for example, displayed on a monitor or printed on a video printer. Such image processors are typically sold as part of systems which include the sensitive photon-counting cameras described above, and accordingly, are available from the same sources. The image processors are usually connected to a personal computer, such as an IBM-compatible PC or an Apple Macintosh (Apple Computer, Cupertino, Calif), which may or may not be included as part of a purchased imaging system. Once the images are in the form of digital files, they can be manipulated by a variety of image processing programs (such as "ADOBE PHOTOSHOP", Adobe Systems, Adobe Systems, Mt. View, Calif) and printed.
[00347] In an embodiment, the biological sample contains protein molecules from the test subject. One exemplary biological sample is a peripheral blood leukocyte sample isolated by conventional means from a subject.
[00348] The invention also encompasses kits for detecting the presence of CTLA-4 or a CTLA-4-expressing cell in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting a cancer or tumor cell (e.g., an anti-CTLA-4 scFv or monoclonal antibody) in a biological sample; means for determining the amount of CTLA-4 in the sample; and means for comparing the amount of CTLA-4 in the sample with a standard. The standard is, in some embodiments, a non-cancer cell or cell extract thereof. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect cancer in a sample.
[00349] Other Embodiments [00350] While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
[00351] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
[00352] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.
EXAMPLE 1
[00353] Described herein are anti-CTLA-4 antibodies identified via phage display that block CTLA-4/CD80 binding. Further, a yeast display library was also generated from this panning campaign to further interrogate the results of the 2nd and 3rd rounds of panning. After sorting clones that bound higher than the bulk, clones were sequenced and additional clones were identified that demonstrate an improved binding curve compared to the original ones found in the ELISA screens of the third round.
[00354] Embodiments herein can have commercial applications as a stand-alone antibody therapy or in combination with other therapies.
EXAMPLE 2 - aCTLA-4 abs from PMPL panning (first group)
[00355] As shown in FIG. 1, anti-CTLA-4 phage binding assay was performed. Assay performed was medium-scale phagemid rescue using PEG + NaCl to precipitate out the phages and purify them.
[00356] With the purified phage, a binding curve from ELISA data was created. We diluted each of the 9 phage PMPL candidates + E1-D7 starting at 2E13 phagemid particles/mL and decreasing 5 fold. We added these phages in triplicates for CTLA-4 Fc- coated plate and CAIX Fc coated plate.
[00357] As shown in FIG. 2, FACS confirm that CTLA-4 phages can bind to CTLA-4 expressing 1G5 Jurkat cells. The negative control was cells + secondary antibody (red box), and the positive comparison/control was current E1-D7 candidate (green box).
[00358] For the purposes of filtering out candidates, we will consider any CTLA-4 phage candidate that has over 25% positive cells as positive. Out of our 9 possible phage candidates, there was only one comparable candidate B10 (circled in blue) as it is over 75% positive. There were also some partial positives, these are circled in orange and are B6 and
C12, which are over 25% positive.
EXAMPLE 3
[00359] Soluble proteins utilized herein include:
[00360] Human CTLA-4 Human CTLA-4, His Tag (CT4-H5229) is expressed from human 293 cells (HEK293). It contains AA Ala 37 - Phe 162 (Accession # NP_005205.2)
1 maclgfqrhk aqlnlatrtw pctll ffll f ipvfckamhv aqpavvlas s rgias fvcey 61 aspgkatevr vtvlrqadsq vtevcaatym mgneltfldd s ictgts sgn qvnltiqglr 121 amdtglyick velmypppyy Igigngtqiy vidpepcpds dfllwilaav s sgl ffys fl 181 Itavs ls kml kkrsplttgv yvkmpptepe cekqfqpyfi pin
[00361] Mouse CTLA-4, His Tag (CT4-M52H5) is expressed from human 293 cells (HEK293). It contains AA Glu 36 - Phe 162 (Accession # NP_033973).
1 maclglrryk aqlqlps rtw pfvalltll f ipvfseaiqv tqpsvvlas s hgvas fpcey 61 spshntdevr vtvlrqtndq mtevcattft e kntvgfldy pfcsgtfnes rvnltiqglr 121 avdtglylck velmypppyf vgmgngtqiy vidpepcpds dfllwilvav s lgl ffys fl 181 vtavs ls kml kkrsplttgv yvkmpptepe cekqfqpyfi pin
[00362]
[00363] Full length CTLA4 was cloned using cDNA from Transomic (BC074893)
EXAMPLE 4 - aCTLA-4
[00364] CTLA4 Overview:
[00365] -Protein receptor that functions as an immune checkpoint
[00366] -Overall structure: Extracellular V domain, Transmembrane domain, cytoplasmic tail
[00367] -Homodimer when membrane-bound: Monomer in soluble form
[00368] CTLA4 Binding:
[00369] -CD28 homolog
[00370] -binds to CD-80/86 (B7-1/2)
[00371] -highly conserved MYPPY motif involved in binding
[00372] Mechanisms to Suppress Immune Response (Fig. 15)
[00373] -Still much debate: Ligand competition, negative signaling, ligand removal, effects on T cell mobility
[00374] Background (Fig. 23):
[00375] - T cells are important mediators to anti-tumor immunity [00376] - They express inhibitory immune checkpoints: these immune checkpoints serve to limit T cell activation, preventing collateral damage to the body by unrestrained T cell activity
[00377] - PD1 protein is expressed on the surface of T cells: Upon ligation to its ligands PDL-1 and PDL-2, a negative pathway is promoted to inhibit T cell function [00378] - Upon ligation to its ligands PDL-1 and PDL-2, a negative pathway is promoted to inhibit T cell function: Tumor cells also express PDL1 which can facilitate tumor escape.
[00379] MLR Assay (Fig. 24):
[00380] - Co-culture of CD4+ T cells with monocyte-derived DCs each from different donors: Allorecognition of the dendritic cells leads to activation of T cells
[00381] - DCs expressing PDL1 can suppress T cell activation: Addition of immune checkpoint inhibitors can recover T cell from suppression
[00382] - Investigate whether anti-PDl enhances T cell activation
[00383] - Measure IFNy and IL2 cytokine production, indicators of T cell activation
[00384] Protocol:
[00385] -Isolate CD14+ monocytes using Miltenyi CD14+ microbeads
[00386] - Culture in Miltenyi Mo-DC media (pre-prepared media with GM-CSF +
IL4)
[00387] - Culture for 5 days, then add: TNF-a (1000 U/ml), IL-ip (5 ng/ml), IL-6 (10 ng/ml) and prostaglandin E2 (PGE2) (1 pM) and culture for 2 days to mature DC
[00388] - mMo-DCs express various DC markers that are involved in the formation of immunological synapses between DCs and naive T cells, including CD80, CD86, and MHC II (HLA-DR). Mature Mo-DCs also expressed DC activation markers CD83, CD40, and CCR7
[00389] Protocol Continued:
[00390] - Isolate T cells day of MLR experiment ( CD4+ negative selection kit
StemCell)
[00391] - 100, 000 T cells and 10, 000 MoDC cells per well for MLR
[00392] - Antibodies are added at desired concentration and culture is incubated for 5 days
[00393] - Supernatant is saved for ELISA screening (IL2 and IFNy)
[00394] aCTLA4 Antibodies MLR 1 & 2:
[00395] MLR1: [00396] - 2 T cell Donors, 2 DC Donors
[00397] - DC cultured for 7 days- Secondary cytokines added on day 5
[00398] - Cells combined and cultured for 4 days
[00399] - Positive control: aPD-1 Antibody
[00400] - Negative control: aSARS2 Antibody
[00401] MLR 2:
[00402] - Testing 2 aCTLA4 antibodies (D7, CIO) in combination with aPD-1 antibody.
[00403] - 4 plates: 2 T cell donors, 2 concentrations of aPDl
[00404] - Used aPD-1 media to make antibody dilutions (starting at lOOnM, 5 4-fold dilutions): 5nM aPD-1 media and lOOnM aPD-1 media
[00405] - 100000 T cells + 10000 MoDC per well
[00406] - 50ul of antibody combined with 50ul T cells first
[00407] - Ab/T cells then added to DC
[00408] - Controls: aPD-1 + cells, aPD-1 high + cells, DC + T cell
[00409] - Incubate for 5 days
[00410] FACS to confirm that our CTLA4 phages can bind to CTLA-4 expressing 1G5 Jurkat cells (Fig. 31)
[00411] - To ensure we were able to find a suitable candidate, we went back and screened a 2nd plate of CTLA4 PMPL phage colonies and combined them with data from plate El. All unique colonies were cloned into scFv-Fc expression vectors and transfected into Expi293F cells for high throughput screening via Octet.
[00412] - SA sensors were loaded with biotinyated CTLA4. scFv-Fc cone in expi293 supernatant was measured via IgG quantification ELISA (Bethyl) and used to normalize samples. Binding of scFv-Fc in sup was measured using an OctetRed96.
[00413] Non-Limiting Examples of Categories Considered analyzing data here:
[00414] Very Important
[00415] 1. CD80 blockade
[00416] 2. CD86 blockade
[00417] 3. Dissociation constant (Kd)
[00418] Moderately Important
[00419] 1. Mouse Cross Reactivity
[00420] Of low Importance
[00421] 1. Response [00422] Exemplary Selected Candidate:
[00423] E1-A8
[00424] Non-Limiting Reasons for Candidate Selection:
[00425] Shows highest CD80 blockade amongst the shortlisted candidates
[00426] CD86 blockade is comparable to Ipilimumab
[00427] Shows Significant Cross-Reactivity with Mouse CTLA4
[00428] Has higher Response than both Ipilimumab and E1-D7
[00429] Shortcomings:
[00430] Shows a relatively higher dissociation rate when compared to most other antibodies tested
[00431] Notes:
[00432] E1-A8 has an identical heavy chain to E2-A4 which shows amongst the highest response rates and a low dissociation constant.
[00433] E1-A8 is derived from the same V gene (IGHV3) but a different allele as
Ipilimumab
EXAMPLE 5 - Engineering and Characterization of an antiPDl/anti-CTLA4 bispecific antibody
[00434] Dual Checkpoint Blockade by Bispecific Antibodies (Fig. 53):
[00435] Without wishing to be bound by theory, advantages can comprise: lower therapeutic dosage, cooperative binding, targeted therapy, and reduced manufacturing cost.
[00436] Non-limiting Overview of goals described herein:
[00437] 1. Discovery and characterization of new anti-CTLA4 antibody:
[00438] 1A: Kinetic Screening and Characterization
[00439] IB: Epitope binning
[00440] 1C: Cell binding assay
[00441] ID: Biological assay
[00442] 2. Engineering and Characterization of antiPDl/anti-CTLA4 bispecific antibody
[00443] 3. Characterization of antiPDl/anti-CTLA4 bispecific antibody as a CAR T payload
[00444] hCTLA2 ELISA binding curve data and results (Fig.56 panel A):
[00445] -ELISA binding curve to demonstrate binding affinity of anti-CTLA4 candidates to hCTLA4. [00446] - All candidates have comparable binding affinity to hCTLA4 at the highest concentration.
[00447] - Most candidates show comparable binding affinity to ipilimumab
[00448] -Top 3: I, J, H
[00449] -A, E, I have the best saturating concentrations
[00450] mCTLA4 ELISA binding curve data and results (Fig. 56 Panel B)
[00451] - Candidates that show high cross-reactivity: E, F, A
[00452] - Candidates that show moderate cross-reactivity: J, H
[00453] - Ipilimumab and Biolegend anti-CTLA4 do not show cross-reactivity true to the literature
[00454] BSA Negative Curve (Fig. 56 Panel C)
[00455] -indicates binding affinity of anti-CTLA4 candidates to BSA (negative control)
[00456] Binding affinity of anti-CTLA4 candidates to CTLA4+ Jurkat Cells (Fig. 64)
[00457] - Apparent inconsistency in the binding affinities of the antibodies to cells vs. to soluble protein.
[00458] - J and H have a much higher binding affinity and a lower saturating concentration to Ipilimumab
[00459] - E and F have comparable binding characteristics to ipilimumab
[00460] Non-Limiting, Exemplary Conclusions from Data Described Herein:
[00461] - 5 anti-CTLA4 antibodies screened from a library of different candidates show:
[00462] a. significant binding affinity to CTLA4
[00463] b. ability to block CTLA4 interaction with CD80/86
[00464] c. biological functional relevance by facilitating CD80/CD28 signaling in effector cells.
[00465] Without wishing to be bound by theory, the following experiments can supplement the data described herein:
[00466] - Further experiments with the bispecific format to determine the most suited anti-CTLA4 antibody for the antiPDl/anti-CTLA4 bispecific
[00467] - In-vitro assays to characterize the Bispecific antibody
[00468] - Characterization and efficacy studies of the Bispecific as a payload in CAR
T cell therapy. EQUIVALENTS
[00469] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims (1)

  1. What is claimed:
    1. An isolated antibody or fragment thereof that binds to human Cytotoxic T- Lymphocyte Associated Protein 4 (CTLA-4) comprising:
    (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 26, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 61, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 63; or
    (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 30, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66; or
    (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69; or
    (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; or
    (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 39, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; or
    (f) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78; or
    (g) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 79, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 81; or
    (h) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 82, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 84; or
    (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 85, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or
    (j) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and a VL CDR3 comprising the amino acid sequence of
    SEQ ID NO: 90; or
    (k) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 91, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 92, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93; or
    (l) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 58, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 60, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 94, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 95, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 96; or
    (m) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 413, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 414, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 415, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 455, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 456, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 457; or
    (n) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 416, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 417, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 418, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 458, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 459, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 460; or
    (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 419, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 420, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 421, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 461, a VL CDR2 comprising the amino acid
    - 171 - sequence of SEQ ID NO: 462, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 463; or
    (p) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 422, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 423, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 424, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 464, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 465, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 466; or
    (q) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 425, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 426, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 427, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 467, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 468, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 469; or
    (r) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 428, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or
    (s) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 432, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 433, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 473, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 474, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 475; or
    (t) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 434, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 435, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 436, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 476, a VL CDR2 comprising the amino acid
    - 172 - sequence of SEQ ID NO: 477, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 478; or
    (u) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 437, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 438, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 439, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 479, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 480, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 481; or
    (v) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 440, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 441, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 442, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 482, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 483, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 484; or
    (w) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 443, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 444, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 445, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 485, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 486, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 487; or
    (x) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 446, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 447, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 448, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 488, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 489, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 490; or
    (y) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 449, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 450, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 451, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 491, a VL CDR2 comprising the amino acid
    - 173 - sequence of SEQ ID NO: 492, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 493; or
    (z) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 452, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 453, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 454, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 494, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 495, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 496; or
    (aa) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 857, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 858, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 859, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 892, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 893, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 894; or
    (bb) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 860, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 861, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 862, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 895, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 896, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 897; or
    (cc) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 863, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 864, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 865, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 898, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 899, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 900; or
    (dd) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 866, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 867, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 868, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 901, a VL CDR2 comprising the amino acid
    - 174 - sequence of SEQ ID NO: 902, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 903; or
    (ee) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 869, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 870, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 871, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 904, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 905, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 906; or
    (ff) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 872, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 873, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 874, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 907, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 908, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 909; or
    (gg) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 875, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 876, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 877, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 910, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 911, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 912; or
    (hh) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 878, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 1000, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 879, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 913, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 914, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 915; or
    (ii) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 880, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 881, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 882, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 916, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 917, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 918; or
    (jj) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 883, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 884, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 885, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 919, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 920, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 921; or
    (kk) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 886, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 887, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 888, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 922, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 923, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 924; or
    (11) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927. An isolated scFv antibody, that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising:
    (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 26, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 61, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 63; or
    (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 30, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66; or
    (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69; or
    (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; or
    (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 39, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; or
    (I) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78; or
    (g) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, a VL CDR1 comprising the
    - 177 - amino acid sequence of SEQ ID NO: 79, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 81; or
    (h) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 82, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 84; or
    (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 85, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or
    (j) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 90; or
    (k) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 91, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 92, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93; or
    (l) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 58, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 60, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 94, a VL CDR2 comprising the amino acid
    - 178 - sequence of SEQ ID NO: 95, and a VL CDR3 comprising the amino acid sequence of
    SEQ ID NO: 96; or
    (m) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 413, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 414, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 415, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 455, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 456, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 457; or
    (n) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 416, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 417, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 418, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 458, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 459, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 460; or
    (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 419, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 420, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 421, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 461, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 462, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 463; or
    (p) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 422, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 423, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 424, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 464, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 465, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 466; or
    (q) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 425, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 426, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 427, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 467, a VL CDR2 comprising the amino acid
    - 179 - sequence of SEQ ID NO: 468, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 469; or
    (r) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 428, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or
    (s) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 432, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 433, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 473, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 474, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 475; or
    (t) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 434, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 435, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 436, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 476, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 477, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 478; or
    (u) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 437, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 438, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 439, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 479, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 480, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 481; or
    (v) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 440, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 441, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 442, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 482, a VL CDR2 comprising the amino acid
    - 180 - sequence of SEQ ID NO: 483, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 484; or
    (w) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 443, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 444, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 445, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 485, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 486, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 487; or
    (x) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 446, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 447, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 448, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 488, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 489, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 490; or
    (y) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 449, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 450, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 451, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 491, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 492, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 493; or
    (z) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 452, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 453, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 454, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 494, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 495, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 496; or
    (aa) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 857, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 858, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 859, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 892, a VL CDR2 comprising the amino acid
    - 181 - sequence of SEQ ID NO: 893, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 894; or
    (bb) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 860, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 861, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 862, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 895, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 896, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 897; or
    (cc) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 863, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 864, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 865, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 898, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 899, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 900; or
    (dd) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 866, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 867, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 868, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 901, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 902, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 903; or
    (ee) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 869, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 870, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 871, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 904, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 905, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 906; or
    (ff) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 872, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 873, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 874, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 907, a VL CDR2 comprising the amino acid
    - 182 - sequence of SEQ ID NO: 908, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 909; or
    (gg) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 875, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 876, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 877, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 910, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 911, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 912; or
    (hh) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 878, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 1000, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 879, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 913, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 914, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 915; or
    (ii) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 880, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 881, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 882, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 916, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 917, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 918; or
    (jj) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 883, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 884, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 885, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 919, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 920, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 921; or
    (kk) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 886, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 887, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 888, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 922, a VL CDR2 comprising the amino acid
    - 183 - sequence of SEQ ID NO: 923, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 924; or
    (11) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927. An isolated antibody or fragment thereof that binds to human Cytotoxic T- Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, and 1023 and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 368, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, and 1024. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, and 1023 and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 368, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, and 1024. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an
    - 184 - amino acid sequence about 95% identical to SEQ ID NO: 1, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 2. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 2. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 3, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 4. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 3, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 4. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 5, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 6. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 5, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 6. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an
    - 185 - amino acid sequence about 95% identical to SEQ ID NO: 7, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 8. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 7, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 8. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 9, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 10. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 9, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 10. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 11, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 12. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 11, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 12. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 13, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 14. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 13, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 14. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 15, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 16. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 15, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 16. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 17, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 18. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 17, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 18. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 19, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 20. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 19, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 20. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 21, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 22. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 21, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 22. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 23, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 24. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 23, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 24. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an
    - 188 - amino acid sequence about 95% identical to SEQ ID NO: 385, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 386. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 385, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 386. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 387, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 388. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 387, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 388. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 389, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 390. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 389, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 390. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an
    - 189 - amino acid sequence about 95% identical to SEQ ID NO: 391, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 392. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 391, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 392. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 393, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 394. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 393, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 394. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 395, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 396. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 395, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 396. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an
    - 190 - amino acid sequence about 95% identical to SEQ ID NO: 397, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 398. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 397, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 398. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 399, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 400. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 399, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 400. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 401, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 402. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 401, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 402. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an
    - 191 - amino acid sequence about 95% identical to SEQ ID NO: 403, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 404. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 403, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 404. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 405, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 406. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 405, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 406. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 407, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 408. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 407, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 408. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an
    - 192 - amino acid sequence about 95% identical to SEQ ID NO: 409, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 410. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 409, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 410. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 411, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 412. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 411, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 412. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1001, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1002. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1001, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1002. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an
    - 193 - amino acid sequence about 95% identical to SEQ ID NO: 1003, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1004. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1003, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1004. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1005, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1006. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1005, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1006. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1007, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1008. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1007, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1008. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an
    - 194 - amino acid sequence about 95% identical to SEQ ID NO: 1009, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1010. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1009, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1010. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1011, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1012. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1011, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1012. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1013, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1014. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1013, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1014. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an
    - 195 - amino acid sequence about 95% identical to SEQ ID NO: 1015, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1016. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1015, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1016. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1017, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1018. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1017, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1018. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1019, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1020. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1019, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1020. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an
    - 196 - amino acid sequence about 95% identical to SEQ ID NO: 1021, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1022. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1021, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1022. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1023, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1024. An isolated scFv antibody that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4) comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1023, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1024. An isolated monoclonal antibody or antigen-binding fragment of claims 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, or 79, wherein the isolated monoclonal antibody or antigen-binding fragment comprises a wildtype Fc or a modified Fc. An isolated bispecific antibody comprising an antibody or fragment of any one of claims 1-81, and a second antigen-binding fragment having specificity to a molecule on an immune cell. The bispecific antibody of claim 82, wherein the molecule is selected from the group consisting of CCR4, B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, PD-1, TIM3, 4- IBB, TIGIT, VISTA, HEVM, BTLA, CD47, PDL1, MICA, MICB, and KIR.
    - 197 - The bispecific antibody of claim 82, wherein the fragment and the second fragment each is independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. The bispecific antibody of claim 82, further comprising a Fc fragment. The bispecific antibody of claim 85, wherein the Fc fragment comprises a wildtype Fc fragment or a modified Fc fragment. A nucleic acid encoding the antibody according to any one of claims 1-81. A nucleic acid encoding the bispecific antibody according to any one of claims 82-86. A pharmaceutical composition comprising the antibody or fragment thereof according to any one of claims 1-81, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition of claim 89, further comprising at least one additional therapeutic agent. The pharmaceutical composition of claim 90, wherein the therapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 82-86, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition of claim 92, further comprising at least one additional therapeutic agent. The pharmaceutical composition of claim 93, wherein the therapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine. An isolated cell comprising one or more polynucleotide(s) encoding the antibody or fragment thereof of any one of claims 1-81. An isolated cell comprising one or more polynucleotide(s) encoding the bispecific antibody or fragment thereof of any one of claims 82-86. The isolated cell of claim 95 or claim 96, wherein the isolate cell is an immune cell.
    - 198 - The isolated cell of claim 97, wherein the immune cell is a T cell, an NK cell, or a B cell. A vector comprising the nucleic acid of claims 87 or 88. A cell comprising the vector of claim 99. The cell of claim 100, wherein the cell is an immune cell. The cell of claim 101, wherein the immune cell is a T cell, an NK cell, or a B cell. A method of treating a cancer in a subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of the CTLA-4 antibody or fragment thereof of any one of claims 1-81. A method of treating a cancer in a subject in need thereof, the method comprising administering to a subject a therapeutically effective amount of the CTLA-4 bispecific antibody of any one of claims 82-86. A chimeric antigen receptor (CAR) comprising an intracellular signaling domain, a transmembrane domain and an extracellular domain, wherein the extracellular domain is an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), wherein the monoclonal antibody or fragment thereof comprises a heavy chain, light chain, or combination thereof, wherein the heavy chain comprises
    (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 26, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 61, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 63; or
    (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 30, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a VL CDR2 comprising the amino acid
    - 199 - sequence of SEQ ID NO: 65, and a VL CDR3 comprising the amino acid sequence of
    SEQ ID NO: 66; or
    (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69; or
    (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; or
    (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 39, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; or
    (I) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78; or
    (g) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 79, a VL CDR2 comprising the amino acid
    - 200 - sequence of SEQ ID NO: 80, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 81; or
    (h) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 82, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 84; or
    (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 85, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or
    (j) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 90; or
    (k) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 91, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 92, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93; or
    (l) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 58, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 60, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 94, a VL CDR2 comprising the amino acid
    - 201 - sequence of SEQ ID NO: 95, and a VL CDR3 comprising the amino acid sequence of
    SEQ ID NO: 96; or
    (m) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 413, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 414, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 415, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 455, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 456, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 457; or
    (n) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 416, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 417, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 418, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 458, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 459, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 460; or
    (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 419, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 420, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 421, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 461, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 462, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 463; or
    (p) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 422, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 423, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 424, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 464, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 465, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 466; or
    (q) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 425, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 426, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 427, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 467, a VL CDR2 comprising the amino acid
    - 202 - sequence of SEQ ID NO: 468, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 469; or
    (r) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 428, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or
    (s) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 432, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 433, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 473, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 474, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 475; or
    (t) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 434, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 435, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 436, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 476, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 477, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 478; or
    (u) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 437, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 438, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 439, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 479, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 480, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 481; or
    (v) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 440, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 441, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 442, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 482, a VL CDR2 comprising the amino acid
    - 203 - sequence of SEQ ID NO: 483, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 484; or
    (w) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 443, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 444, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 445, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 485, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 486, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 487; or
    (x) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 446, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 447, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 448, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 488, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 489, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 490; or
    (y) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 449, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 450, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 451, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 491, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 492, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 493; or
    (z) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 452, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 453, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 454, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 494, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 495, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 496; or
    (aa) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 857, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 858, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 859, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 892, a VL CDR2 comprising the amino acid
    - 204 - sequence of SEQ ID NO: 893, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 894; or
    (bb) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 860, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 861, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 862, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 895, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 896, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 897; or
    (cc) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 863, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 864, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 865, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 898, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 899, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 900; or
    (dd) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 866, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 867, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 868, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 901, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 902, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 903; or
    (ee) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 869, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 870, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 871, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 904, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 905, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 906; or
    (ff) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 872, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 873, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 874, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 907, a VL CDR2 comprising the amino acid
    - 205 - sequence of SEQ ID NO: 908, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 909; or
    (gg) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 875, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 876, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 877, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 910, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 911, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 912; or
    (hh) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 878, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 1000, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 879, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 913, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 914, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 915; or
    (ii) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 880, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 881, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 882, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 916, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 917, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 918; or
    (jj) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 883, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 884, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 885, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 919, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 920, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 921; or
    (kk) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 886, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 887, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 888, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 922, a VL CDR2 comprising the amino acid
    - 206 - sequence of SEQ ID NO: 923, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 924; or
    (11) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927. The CAR of claim 105, wherein the transmembrane domain further comprises a stalk region positioned between the extracellular domain and the transmembrane domain. The CAR or claim 105, wherein the transmembrane domain comprises CD28. The CAR of claim 105, further comprising one or more additional costimulatory molecules positioned between the transmembrane domain and the intracellular signaling domain The CAR of claim 105, wherein the costimulatory molecules is CD28, 4-1BB, ICOS, or 0X40. The CAR of claim 105, wherein the intracellular signaling domain comprises a CD3 zeta chain. The CAR of claim 105, wherein the antibody is a Fab or a scFV. A nucleic acid encoding the CAR of any one of claims 105-111. The nucleic acid of claim 112, further comprising a nucleic acid encoding a polypeptide positioned after the intracellular signaling domain. The nucleic acid of claim 113, wherein the polypeptide is an antibody or a cytokine. The nucleic acid of claim 114, wherein the antibody is a scFV. A nucleic acid encoding a CAR, wherein the CAR comprises an intracellular signaling domain, a transmembrane domain and an extracellular domain, further comprising a nucleic acid encoding a polypeptide positioned after the intracellular signaling domain,
    - 207 - wherein the polypeptide comprises an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), wherein the monoclonal antibody or fragment thereof comprises a heavy chain, light chain, or combination thereof, wherein the heavy chain comprises
    (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 26, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 61, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 63; or
    (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 30, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66; or
    (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69; or
    (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; or
    - 208 - (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 39, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; or
    (I) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78; or
    (g) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 79, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 81; or
    (h) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 82, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 84; or
    (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 85, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or
    - 209 - (j) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 90; or
    (k) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 91, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 92, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93; or
    (l) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 58, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 60, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 94, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 95, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 96; or
    (m) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 413, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 414, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 415, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 455, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 456, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 457; or
    (n) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 416, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 417, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 418, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 458, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 459, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 460; or
    - 210 - (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 419, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 420, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 421, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 461, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 462, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 463; or
    (p) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 422, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 423, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 424, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 464, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 465, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 466; or
    (q) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 425, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 426, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 427, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 467, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 468, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 469; or
    (r) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 428, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or
    (s) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 432, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 433, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 473, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 474, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 475; or
    - 211 - (t) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 434, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 435, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 436, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 476, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 477, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 478; or
    (u) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 437, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 438, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 439, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 479, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 480, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 481; or
    (v) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 440, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 441, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 442, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 482, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 483, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 484; or
    (w) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 443, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 444, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 445, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 485, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 486, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 487; or
    (x) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 446, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 447, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 448, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 488, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 489, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 490; or
    - 212 - (y) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 449, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 450, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 451, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 491, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 492, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 493; or
    (z) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 452, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 453, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 454, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 494, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 495, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 496; or
    (aa) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 857, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 858, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 859, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 892, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 893, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 894; or
    (bb) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 860, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 861, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 862, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 895, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 896, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 897; or
    (cc) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 863, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 864, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 865, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 898, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 899, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 900; or
    - 213 - (dd) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 866, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 867, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 868, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 901, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 902, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 903; or
    (ee) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 869, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 870, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 871, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 904, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 905, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 906; or
    (ff) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 872, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 873, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 874, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 907, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 908, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 909; or
    (gg) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 875, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 876, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 877, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 910, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 911, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 912; or
    (hh) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 878, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 1000, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 879, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 913, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 914, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 915; or
    - 214 - (ii) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 880, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 881, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 882, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 916, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 917, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 918; or
    (jj) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 883, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 884, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 885, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 919, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 920, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 921; or
    (kk) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 886, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 887, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 888, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 922, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 923, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 924; or
    (11) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927. A vector comprising the nucleic acid of any one of claims 112-116. A cell comprising the vector of claim 117. The cell of claim 118, wherein the cell is an immune cell. A genetically engineered cell which expresses and bears on the cell surface membrane the chimeric antigen receptor of any one of claims 105-111.
    - 215 - The cell of claim 120, wherein the immune cell is a T-cell, an NK cell or a B cell. The cell of claim 121, wherein the T cell is CD4+ or CD8+. The genetically engineered cell of claim 122, which comprises a mixed population of CD4+ and CD8 cells+. A genetically engineered cell which express and bear on the cell surface membrane a chimeric antigen receptor, and which is further engineered to express and secrete a polypeptide, wherein polypeptide is an isolated monoclonal antibody or antigenbinding fragment thereof that binds to human Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA-4), wherein the monoclonal antibody or fragment thereof comprises a heavy chain, light chain, or combination thereof, wherein the heavy chain comprises
    (a) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 26, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 61, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 63; or
    (b) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 30, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66; or
    (c) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 31, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 32, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 33, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 67, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 68, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 69; or
    - 216 - (d) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 34, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 35, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 36, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 70, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 71, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 72; or
    (e) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 38, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 39, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 73, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 74, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 75; or
    (I) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 40, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 41, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 42, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 76, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 77, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 78; or
    (g) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 43, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 44, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 45, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 79, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 81; or
    (h) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 46, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 47, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 48, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 82, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 83, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 84; or
    - 217 - (i) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 85, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 86, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 87; or
    (j) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 52, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 53, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 54, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 88, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 89, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 90; or
    (k) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 91, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 92, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 93; or
    (l) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 58, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 59, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 60, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 94, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 95, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 96; or
    (m) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 413, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 414, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 415, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 455, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 456, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 457; or
    - 218 - (n) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 416, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 417, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 418, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 458, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 459, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 460; or
    (o) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 419, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 420, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 421, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 461, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 462, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 463; or
    (p) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 422, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 423, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 424, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 464, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 465, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 466; or
    (q) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 425, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 426, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 427, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 467, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 468, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 469; or
    (r) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 428, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or
    - 219 - (s) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 432, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 433, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 473, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 474, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 475; or
    (t) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 434, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 435, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 436, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 476, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 477, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 478; or
    (u) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 437, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 438, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 439, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 479, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 480, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 481; or
    (v) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 440, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 441, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 442, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 482, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 483, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 484; or
    (w) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 443, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 444, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 445, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 485, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 486, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 487; or
    - 220 - (x) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 446, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 447, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 448, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 488, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 489, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 490; or
    (y) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 449, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 450, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 451, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 491, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 492, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 493; or
    (z) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 452, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 453, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 454, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 494, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 495, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 496; or
    (aa) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 857, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 858, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 859, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 892, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 893, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 894; or
    (bb) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 860, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 861, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 862, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 895, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 896, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 897; or
    - 221 - (cc) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 863, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 864, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 865, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 898, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 899, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 900; or
    (dd) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 866, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 867, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 868, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 901, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 902, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 903; or
    (ee) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 869, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 870, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 871, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 904, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 905, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 906; or
    (ff) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 872, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 873, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 874, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 907, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 908, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 909; or
    (gg) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 875, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 876, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 877, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 910, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 911, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 912; or
    - 222 - (hh) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 878, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 1000, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 879, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 913, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 914, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 915; or
    (ii) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 880, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 881, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 882, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 916, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 917, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 918; or
    (jj) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 883, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 884, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 885, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 919, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 920, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 921; or
    (kk) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 886, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 887, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 888, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 922, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 923, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 924; or
    (11) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927.
    - 223 -
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