Antibodies to CTLA-4 and methods of use thereof

Abstract

The present invention is directed to antibodies against human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and methods of use thereof. TIFF2024541476000124.tif84160

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 282,887, filed 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, and the disclosures of these publications are incorporated by reference into this application in order to more fully describe the state of the art known to those skilled in the art 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 all and any copyright rights whatsoever.

[0004] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. This ASCII copy, created above in [], is named [] and is [] bytes in size.

[0005] FIELD OF THEINVENTION The present invention is directed to antibodies against human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and methods of use thereof. [Background technology]

[0006] background CTLA-4 is an inhibitory receptor that acts as a major negative regulator of T cell responses. The affinity of CTLA4 for the natural B7 family ligands CD80 and CD86 is stronger than that of their cognate stimulatory coreceptor CD28. Summary of the Invention

[0007] The present invention provides human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and methods of use thereof.

[0008] Embodiments include 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, 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 CDR2, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:95. CDR2, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 461. 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 CDR2, 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 CDR2 comprising the amino acid sequence of SEQ ID NO: 479 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 CDR2, 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 CDR3 comprising the amino acid sequence of SEQ ID NO: 892 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 CDR2, 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, 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, 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 (ll) 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 present invention relates to an isolated antibody or fragment thereof comprising the CDR3.

[0009] A further embodiment of the invention relates to 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:33. 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 CDR2, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:95. CDR2, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 461. 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 CDR2, 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 CDR2 comprising the amino acid sequence of SEQ ID NO: 479 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 CDR2, 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 CDR3 comprising the amino acid sequence of SEQ ID NO: 892 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 CDR2, 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, and 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, 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 (ll) 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 present invention relates to an isolated scFv antibody comprising a CDR3.

[0010] Yet another aspect of the present invention is an isolated antibody or fragment thereof that binds to human cytotoxic T-lymphocyte associated protein 4 (CTLA-4), the antibody or fragment thereof being 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. The present invention relates to an isolated antibody or fragment thereof comprising a heavy 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 an aspect of the invention is an isolated scFv antibody that binds to human cytotoxic T-lymphocyte associated protein 4 (CTLA-4), comprising an antibody 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, one embodiment relates 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 that is about 95% identical to SEQ ID NO:1, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:2.

[0013] Another embodiment relates 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 that is about 95% identical to SEQ ID NO:1, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:2.

[0014] A further embodiment relates 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 that is about 95% identical to SEQ ID NO:3, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:4.

[0015] One embodiment also relates 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 that is about 95% identical to SEQ ID NO:3, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:4.

[0016] One embodiment relates 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 that is about 95% identical to SEQ ID NO:5, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:6.

[0017] An embodiment also relates 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 that is about 95% identical to SEQ ID NO:5, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:6.

[0018] Furthermore, embodiments relate 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 that is about 95% identical to SEQ ID NO:7, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:8.

[0019] An embodiment also relates 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 that is about 95% identical to SEQ ID NO:7, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:8.

[0020] A further embodiment relates 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 that is about 95% identical to SEQ ID NO:9, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:10.

[0021] An embodiment also relates 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 that is about 95% identical to SEQ ID NO:9, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:10.

[0022] An embodiment relates 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 that is about 95% identical to SEQ ID NO:11, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:12.

[0023] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:11, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:12.

[0024] Further embodiments relate 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 that is about 95% identical to SEQ ID NO:13, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:14.

[0025] Furthermore, an embodiment relates 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 that is about 95% identical to SEQ ID NO:13, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:14.

[0026] Also, embodiments relate 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 that is about 95% identical to SEQ ID NO:15, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:16.

[0027] Furthermore, an embodiment relates 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 that is about 95% identical to SEQ ID NO:15, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:16.

[0028] The embodiments also relate 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 that is about 95% identical to SEQ ID NO:17, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:18.

[0029] Further embodiments relate 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 that is about 95% identical to SEQ ID NO:17, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:18.

[0030] Embodiments also relate 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 that is about 95% identical to SEQ ID NO:19, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:20.

[0031] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:19, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:20.

[0032] Embodiments also relate 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 that is about 95% identical to SEQ ID NO:21, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:22.

[0033] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:21, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:22.

[0034] The embodiments also relate 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 that is about 95% identical to SEQ ID NO:23, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:24.

[0035] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:23, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:24.

[0036] Embodiments also relate 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:385, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:386.

[0037] The embodiment also relates 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:385, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:386.

[0038] Embodiments also relate 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:387, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:388.

[0039] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:387, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:388.

[0040] Embodiments also relate 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:389, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:390.

[0041] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:389, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:390.

[0042] Embodiments also relate 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 that is about 95% identical to SEQ ID NO:391, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:392.

[0043] The embodiment also relates 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:391, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:392.

[0044] Embodiments also relate 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:393, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:394.

[0045] The embodiment also relates 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:393, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:394.

[0046] Embodiments also relate 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:395, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:396.

[0047] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:395, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:396.

[0048] Embodiments also relate 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:397, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:398.

[0049] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:397, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:398.

[0050] Embodiments also relate 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 that is about 95% identical to SEQ ID NO:399, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:400.

[0051] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:399, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:400.

[0052] Embodiments also relate 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:401, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:402.

[0053] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:401, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:402.

[0054] Embodiments also relate 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:403, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:404.

[0055] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:403, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:404.

[0056] Embodiments also relate 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:405, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:406.

[0057] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:405, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:406.

[0058] Embodiments also relate 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:407, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:408.

[0059] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:407, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:408.

[0060] Embodiments also relate 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:409, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:410.

[0061] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:409, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:410.

[0062] Embodiments also relate 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:411, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO:412.

[0063] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:411, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:412.

[0064] The embodiments also relate 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 that is about 95% identical to SEQ ID NO:1001, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1002.

[0065] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:1001, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1002.

[0066] Embodiments also relate 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 that is about 95% identical to SEQ ID NO:1003, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1004.

[0067] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:1003, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1004.

[0068] The embodiments also relate 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 that is about 95% identical to SEQ ID NO:1005, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1006.

[0069] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:1005, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1006.

[0070] Embodiments also relate 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 that is about 95% identical to SEQ ID NO:1007, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1008.

[0071] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:1007, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1008.

[0072] Embodiments also relate 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 that is about 95% identical to SEQ ID NO:1009, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1010.

[0073] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:1009, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1010.

[0074] The embodiments also relate 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 that is about 95% identical to SEQ ID NO:1011, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1012.

[0075] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:1011, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1012.

[0076] Embodiments also relate 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 that is about 95% identical to SEQ ID NO:1013, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1014.

[0077] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:1013, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1014.

[0078] The embodiments also relate 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 that is about 95% identical to SEQ ID NO:1015, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1016.

[0079] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:1015, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1016.

[0080] Embodiments also relate 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 that is about 95% identical to SEQ ID NO:1017, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1018.

[0081] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:1017, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1018.

[0082] Embodiments also relate 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 that is about 95% identical to SEQ ID NO:1019, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1020.

[0083] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:1019, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1020.

[0084] Embodiments also relate 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 that is about 95% identical to SEQ ID NO:1021, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1022.

[0085] The embodiment also relates 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 that is about 95% identical to SEQ ID NO:1021, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO:1022.

[0086] Embodiments also relate 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: 1023, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 1024.

[0087] The embodiment also relates 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 that is about 95% identical to SEQ ID NO: 1023, and the light chain comprises an amino acid sequence that is about 95% identical to SEQ ID NO: 1024.

[0088] An embodiment of the invention also relates to an isolated monoclonal antibody or antigen-binding fragment of claim 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, which comprises a wild-type or modified Fc.

[0089] A further aspect of the invention relates to an isolated bispecific antibody comprising an antibody or fragment described herein and a second antigen-binding fragment having specificity for 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, each of the fragment and the second fragment is independently selected from a Fab fragment, a single chain variable fragment (scFv), or a single domain antibody.

[0091] The embodiment may further comprise an Fc fragment, for example, the Fc fragment comprises a wild-type Fc fragment or a modified Fc fragment.

[0092] Aspects of the invention pertain to nucleic acids encoding the antibodies described herein.

[0093] A further aspect of the invention pertains to nucleic acids encoding the bispecific antibodies described herein.

[0094] Aspects of the invention also relate to pharmaceutical compositions comprising an antibody or fragment thereof described herein and a pharma- ceutically acceptable carrier or excipient.

[0095] The embodiment may further comprise at least one additional therapeutic agent. For example, the therapeutic agent may be a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine.

[0096] An aspect of the invention pertains to a pharmaceutical composition comprising a bispecific antibody as described herein and a pharma- ceutically 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, an siRNA, a small molecule, or a cytokine.

[0098] Further aspects of the invention pertain to isolated cells comprising one or more polynucleotides encoding the antibodies or fragments described herein.

[0099] Aspects of the invention also relate to isolated cells comprising one or more polynucleotides encoding the bispecific antibodies or fragments thereof described herein.

[0100] Further aspects of the invention pertain to vectors comprising the nucleic acids described herein.

[0101] Aspects of the invention also relate to cells comprising the vectors described herein.

[0102] Aspects of the invention relate to methods of treating cancer in a subject. For example, the method includes administering to the subject a therapeutically effective amount of a CTLA-4 antibody or fragment thereof described herein. For example, the method includes administering to the subject a therapeutically effective amount of a CTLA-4 bispecific antibody described herein.

[0103] Aspects of the present invention also relate to chimeric antigen receptors (CARs). 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), and the monoclonal antibody or fragment thereof comprises a heavy chain, a light chain, or a 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 74. CDR2, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 92. CDR2, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:459. 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 CDR2 comprising the amino acid sequence of SEQ ID NO: 427 CDR3, 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 CDR3 comprising the amino acid sequence of SEQ ID NO: 476 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 CDR2, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 494. 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 CDR2, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 907. CDR1, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 908, and sequence; 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, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 882. 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 (ll) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, CDR1, 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.

[0104] In embodiments, the transmembrane domain further comprises a stalk region disposed between the extracellular domain and the transmembrane domain.

[0105] Embodiments can further include one or more additional costimulatory molecules disposed between the transmembrane domain and the intracellular signaling domain. For example, the costimulatory molecule is CD28, 4-1BB, ICOS, or OX40. For example, the intracellular signaling domain includes the CD3 zeta chain.

[0106] In embodiments, the antibody is a Fab or scFv.

[0107] Aspects of the present invention also relate to nucleic acids encoding the CAR described herein. In embodiments, the nucleic acid can further comprise a nucleic acid encoding a polypeptide disposed after the intracellular signaling domain. For example, the polypeptide is an antibody or a cytokine, such as an scFV.

[0108] Aspects of the invention relate to nucleic acids encoding a CAR, the CAR comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain, and further comprising a nucleic acid encoding a polypeptide located after the intracellular signaling domain, the polypeptide comprising an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the monoclonal antibody or fragment thereof comprising a heavy chain, a light chain, or a combination thereof, the heavy chain 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, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 73. CDR1, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 92. CDR2, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:459. 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 CDR2 comprising the amino acid sequence of SEQ ID NO: 427 CDR3, 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 CDR3 comprising the amino acid sequence of SEQ ID NO: 476 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 CDR2, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 494. 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 CDR2, 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 sequence; 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. 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 (ll) 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;

[0109] Aspects of the invention pertain to vectors comprising the nucleic acids described herein.

[0110] Aspects of the invention also relate to cells comprising the vectors described herein.

[0111] An aspect of the invention relates to a genetically engineered cell that expresses and carries on its cell surface membrane a chimeric antigen receptor as described herein. For example, the cell is a T cell or a NK cell. For example, the T cell is CD4+ or CD8+.

[0112] Embodiments may further include a mixed population of CD4+ and CD8+ cells.

[0113] Aspects of the invention also relate to genetically engineered cells that express and retain a chimeric antigen receptor on a cell surface membrane and are further engineered to express and secrete a polypeptide, wherein the 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, a light chain, or a 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 CDR3 comprising the amino acid sequence of SEQ ID NO:65, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 73. CDR1, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 92. CDR2, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:459. 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 CDR2 comprising the amino acid sequence of SEQ ID NO: 427 CDR3, 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 CDR3 comprising the amino acid sequence of SEQ ID NO: 476 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 CDR2, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 494. 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 CDR2, 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 907. CDR1, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 908, and SEQ ID NO:; 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, and a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 882. 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 (ll) a VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, the VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, the VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and the VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927.

[0114] Other objects and advantages of the present invention will become readily apparent from the following description. [Brief description of the drawings]

[0115] 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.

[0116] [Figure 1-1] Binding ELISA using various dilutions of PEG-purified phage is shown. [Figure 1-2] A continuation of Figure 1-1 is shown.

[0117] [Figure 2-1] FACS confirming that CTLA-4 phage can bind to CTLA-4 expressing 1G5 Jurkat cells is shown. [Figure 2-2] A continuation of Figure 2-1 is shown.

[0118] [Figure 3-1] The amino acid (AA) and nucleotide (Nuc) sequences of anti-CTLA-4 antibodies are provided. "HC" is the heavy chain and "LC" is the light chain. [Figure 3-2] A continuation of Figure 3-1 is shown. [Figure 3-3] A continuation of Figure 3-2 is shown. [Diagram 3-4] A continuation of Figure 3-3 is shown.

[0119] [Figure 4-1] The amino acid (AA) and nucleotide (Nuc) sequences of the anti-CTLA-4 antibodies are provided. [Figure 4-2] A continuation of Figure 4-1 is shown. [Figure 4-3] This is a continuation of Figure 4-2. [Figure 4-4] This is a continuation of Figure 4-3. [Figure 4-5] This is a continuation of Figure 4-4. [Figure 4-6]A continuation of Figure 4-5 is shown. [Figure 5-1] The amino acid (AA) and nucleotide (Nuc) sequences of the anti-CTLA-4 antibodies are provided. [Figure 5-2] A continuation of Figure 5-1 is shown. [Figure 5-3] This is a continuation of Figure 5-2. [Figure 5-4] This is a continuation of Figure 5-3. [Figure 5-5] This is a continuation of Figure 5-4. [Figure 5-6] A continuation of Figure 5-5 is shown. [Figure 5-7] A continuation of Figure 5-6 is shown. [Figure 5-8] A continuation of Figure 5-7 is shown. [Figure 5-9] This is a continuation of Figure 5-8. [Figure 5-10] This is a continuation of Figure 5-9. [Figure 5-11] A continuation of Figure 5-10 is shown. [Figure 5-12] This is a continuation of Figure 5-11. [Figure 5-13] A continuation of Figure 5-12 is shown. [Figure 5-14] This is a continuation of Figure 5-13. [Figure 6-1] The amino acid (AA) and nucleotide (Nuc) sequences of the anti-CTLA-4 antibodies are provided. [Figure 6-2] A continuation of Figure 6-1 is shown. [Figure 6-3] A continuation of Figure 6-2 is shown. [Figure 6-4] This is a continuation of Figure 6-3. [Figure 6-5] This is a continuation of Figure 6-4. [Figure 6-6] A continuation of Figure 6-5 is shown. [Figure 7-1] The amino acid (AA) and nucleotide (Nuc) sequences of the anti-CTLA-4 antibodies are provided. [Figure 7-2] A continuation of Figure 7-1 is shown. [Figure 7-3] A continuation of Figure 7-2 is shown. [Figure 7-4] This is a continuation of Figure 7-3. [Figure 7-5] This is a continuation of Figure 7-4. [Figure 7-6] This is a continuation of Figure 7-5. [Figure 7-7] This is a continuation of Figure 7-6. [Figure 7-8] A continuation of Figure 7-7 is shown. [Figure 7-9] This is a continuation of Figure 7-8. [Figure 7-10] This is a continuation of Figure 7-9. [Figure 7-11] A continuation of Figure 7-10 is shown. [Figure 7-12] A continuation of Figure 7-11 is shown. [Figure 7-13] This is a continuation of Figure 7-12. [Figure 7-14] This is a continuation of Figure 7-13. [Figure 8-1] The amino acid (AA) and nucleotide (Nuc) sequences of the anti-CTLA-4 antibodies are provided. [Figure 8-2] A continuation of Figure 8-1 is shown. [Figure 8-3] This is a continuation of Figure 8-2. [Figure 8-4] This is a continuation of Figure 8-3. [Figure 8-5] This is a continuation of Figure 8-4. [Figure 8-6] This is a continuation of Figure 8-5. [Figure 8-7] This is a continuation of Figure 8-6. [Figure 8-8] This is a continuation of Figure 8-7.

[0120] [Figure 9-1] The amino acid (AA) and nucleotide (Nuc) sequences of the anti-CTLA-4 antibodies are provided. [Figure 9-2] A continuation of Figure 9-1 is shown. [Figure 9-3] A continuation of Figure 9-2 is shown. [Figure 9-4] This is a continuation of Figure 9-3. [Figure 10-1] The amino acid (AA) and nucleotide (Nuc) sequences of the anti-CTLA-4 antibodies are provided. [Figure 10-2] A continuation of Figure 10-1 is shown. [Figure 10-3] A continuation of Figure 10-2 is shown. [Figure 10-4] A continuation of Figure 10-3 is shown. [Figure 10-5] A continuation of Figure 10-4 is shown. [Figure 10-6] A continuation of Figure 10-5 is shown. [Figure 10-7] A continuation of Figure 10-6 is shown. [Figure 10-8] A continuation of Figure 10-7 is shown. [Figure 10-9] A continuation of Figure 10-8 is shown. [Figure 10-10] A continuation of Figure 10-9 is shown. [Figure 10-11] Continuation of Figure 10-10 is shown. [Figure 10-12] Continuation of Figure 10-11 is shown. [Figure 11-1] The amino acid (AA) and nucleotide (Nuc) sequences of the anti-CTLA-4 antibodies are provided. [Figure 11-2] A continuation of Figure 11-1 is shown. [Figure 11-3] A continuation of Figure 11-2 is shown. [Figure 11-4] A continuation of Figure 11-3 is shown. [Figure 11-5] A continuation of Figure 11-4 is shown. [Figure 11-6] A continuation of Figure 11-5 is shown. [Figure 11-7] This is a continuation of Figure 11-6. [Figure 11-8] This is a continuation of Figure 11-7. [Figure 11-9] This is a continuation of Figure 11-8. [Figure 11-10] This is a continuation of Figure 11-9. [Figure 11-11] A continuation of Figure 11-10 is shown. [Figure 11-12] Continuation of Figure 11-11 is shown. [Figure 11-13] Continuation of Figure 11-12. [Figure 11-14] Continuation of Figure 11-13. [Figure 11-15]Continuation of Figure 11-14 is shown. [Figure 11-16] Continuation of Figure 11-15. [Figure 11-17] Continuation of Figure 11-16. [Figure 11-18] Continuation of Figure 11-17. [Figure 11-19] Continuation of Figure 11-18. [Figure 11-20] Continuation of Figure 11-19. [Figure 11-21] A continuation of Figure 11-20 is shown. [Figure 11-22] A continuation of Figure 11-21 is shown. [Figure 11-23] A continuation of Figure 11-22 is shown. [Figure 11-24] This is a continuation of Figure 11-23. [Figure 11-25] Continuation of Figure 11-24. [Figure 11-26] Continuation of Figure 11-25. [Figure 11-27] Continuation of Figure 11-26. [Figure 11-28] Continuation of Figure 11-27.

[0121] [Figure 12-1] Percent competition with CD80 hFc for binding to CTLA4 on 1G5 Jurkat is shown. 1G5 Jurkat cells were transduced to overexpress CTLA4. Cells were incubated with the indicated concentrations of soluble antibodies, washed, and then incubated with labeled CD80-Fc. Samples were read on a BD FACSCanto II. A decrease in fluorescence was used to indicate blocking of CD80 binding to CTLA4 on the cell surface. [Figure 12-2] A continuation of Figure 12-1 is shown. [Figure 12-3] A continuation of Figure 12-2 is shown. [Figure 12-4] A continuation of Figure 12-3 is shown. [Figure 12-5] A continuation of Figure 12-4 is shown. [Figure 12-6] A continuation of Figure 12-5 is shown.

[0122] [Figure 13] Ribbon diagram edited from Metzler, William J., et al. "Solution Structure of Human CTLA-4 and Delineation of a CD80 / CD86 Binding Site Conserved in CD28."

[0123] [Figure 14-1] Stamper, Carin C., et al. "Crystal Structure of the B7-1 / CTLA-4 Complex That Inhibits Human Immune Responses". Ribbon diagram adapted from Nature. [Figure 14-2] A continuation of Figure 14-1 is shown.

[0124] [Figure 15-1] 1 shows a schematic diagram of the mechanism of suppressing immune responses. [Figure 15-2] A continuation of Figure 15-1 is shown.

[0125] [Figure 16A] Figure 16 shows ribbon diagrams of existing therapies Ipilimumab-IgG1 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." [Figure 16B] See legend to Figure 16A. [Figure 16C] Panel C shows a ribbon diagram adapted from He, Mengnan, et al. "Remarkably Similar CTLA-4 Binding Properties of Therapeutic Ipilimumab and Tremelimumab Antibodies."

[0126] [Figure 17] A chart of exemplary panning results for anti-CTLA4 antibodies is shown. Rounds: 1, 1b, 2, 2b, 3, 3b. Mouse CTLA4 was introduced in rounds 3 and 3b. 10 unique antibodies were the outcome.

[0127] [Figure 18] 1 shows a chart of an exemplary kinetic analysis of an aCTLA4 antibody.

[0128] [Figure 19] 1 shows a chart of an exemplary competition analysis of aCTLA4 antibody.

[0129] [Figure 20] Shown is a FACS graph of aCTLA4 antibody binding to T cells. E1-D7 binds significantly more CTLA4 on T cells compared to T2-C10. Anti-CTLA4 ab obtained from Biolegend was used as a single point (recommended staining concentration) for control. Ipi IgG is an antibody cloned in the laboratory to represent ipilimumab and is not a commercial preparation or biosimilar.

[0130] [Figure 21-1] 1 shows an exemplary FACS of aCTLA4 Ab binding to T cells. [Figure 21-2] A continuation of Figure 21-1 is shown.

[0131] [Figure 22] 1 shows an exemplary graph of mouse / human CTLA4 cross-reactivity ELISA results. E1-D7 cross-reacts with mouse CTLA4.

[0132] [Diagram 23] FIG. 1 shows a schematic diagram of an exemplary mechanism for T cells, adapted from P. Ott et al. 2014.

[0133] [Figure 24]Schematic diagram of the MLR assay. Schematic diagram courtesy of Explicyte. Protocol: Co-culture of CD4+ T cells and monocyte-derived DCs, each obtained from a different donor. This showed that dendritic cell allorecognition leads to T cell activation. DCs expressing PDL1 can suppress T cell activation. Addition of immune checkpoint inhibitors can rescue T cells from suppression. We investigated whether anti-PD1 enhances T cell activation. Production of IFNγ and IL2 cytokines, indicators of T cell activation, was measured.

[0134] [Diagram 25] Data of immunophenotyping of monocytes, imMo-DCs, and mMo-DCs are shown, compiled from Miltenyi. In this protocol, CD14+ monocytes isolated using Miltenyi CD14+ microbeads were cultured in Miltenyi Mo-DC medium (pre-prepared medium containing GM-CSF+IL4) and cultured for 5 days. DCs were then matured by adding TNF-α (1000U / ml), IL-1β (5ng / ml), IL-6 (10ng / ml), and prostaglandin E2 (PGE2) (1μM) and culturing for 2 days. mMo-DCs express various DC markers involved in the formation of immune 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.

[0135] [Figure 26] Data and analysis of DC donor AD staining are shown.

[0136] [Figure 27] Graph of exemplary MLR1 data: Measurement of IFNγ production. We investigated whether E1-D7 suppresses T cell activation. A decrease in IFNγ is seen in wells treated with E1-D7 compared to T cells+DC only, anti-PD1 treatment, and irrelevant Ab control. An absorbance value of 4 was used to represent saturation.

[0137] [Figure 28] 1 shows exemplary data for the aCTLA4 / aPD-1 MLR combination: IFNγ production graph.

[0138] [Figure 29] FIG. 1 shows an exemplary schematic of the second panning using CTLA4-PMPL.

[0139] [Diagram 30] 1 shows an exemplary graph of data from a binding ELISA using various dilutions of PEG-purified phage.

[0140] [Figure 31-1] 1 shows an exemplary FACS assay confirming that our CTLA4 phage can bind to CTLA-4 expressing 1G5 Jurkat cells. [Figure 31-2] A continuation of Figure 31-1 is shown.

[0141] [Diagram 32] 1 shows a chart of exemplary kinetic screening data for aCTLA4 scFv-Fc sup.

[0142] [Diagram 33] 1 shows an exemplary binding curve for hCTLA4.

[0143] [Diagram 34] 1 shows an exemplary binding curve for mCTLA4.

[0144] [Diagram 35] The binding curve for the BSA negative control is shown.

[0145] [Diagram 36]A chart of the data and observations is shown. The data is arranged in descending order of binding response at 25 nM concentration of antibody. All candidate antibodies have higher dissociation rates than ipilimumab and Biolegend aCTLA4 (commercial control). Most candidates appear to have higher binding responses than the commercial control. Four candidates (highlighted in the table) have lower dissociation constants than E1-D7 (Taylor's antibody-laboratory control). E2-D10 and E1-A12 have identical heavy chains but light chains from different families. The difference in their response rates is mediated by light chain binding. E2-H10 also belongs to the same Vh family as the above two.

[0146] [Figure 37] A chart showing exemplary data from the competitive assay is shown. CD80 blocking: All candidate antibodies block CD80 binding to some extent, but less than ipilimumab, therefore we selected candidates with higher blocking (lower secondary binding values) than E1-D7 (laboratory control, Taylor's candidate). CD86 blocking: All candidates have negative secondary binding values, indicating that all completely block CD86 (normalized value=0). Since higher negative values ​​may indicate antibody dissociation, we selected candidates with negative values ​​closer to 0 when compared to ipilimumab. E1-A8 and E2-A4 belong to the same germline family. E1-D7 blocking: The highlighted candidates block E1-D7 binding better than ipilimumab. Ipilimumab blocking: The highlighted candidates block ipilimumab binding better than E1-D7. All CD80 and CD86 blocking candidates performed relatively poorly in kinetics.

[0147] [Figure 38] 1 shows an exemplary graph of CTLA4 binding. Jurkat cells were transduced with hCTLA4 and used to generate binding curves.

[0148] [Figure 39]A schematic of the PROMEGA luciferase 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.

[0149] [Diagram 40] Graphs of exemplary Promega CTLA4 bioassay data are shown. Anti-CTLA4 scFv-Fc was tested against Ipi biosimilar IgG. Plates were read using a BMG polarstar omega at either 1 second (left column) or 10 second (right column) intervals. E1-B10, E1-A8, E2-A4, and E1-D7 show similar activity at about 40% of Ipi. In plate 2, E2-G9 shows comparable activity to Ipi, while E2-H10, E1-A4, and E2-H12 show minimal improvement.

[0150] [Diagram 41] A diagram of the bispecific design is shown. BsAbs constructed with an IgG1 hinge show a mixture of monomers and dimers when expressed we switched to an IgG3 hinge to increase the frequency of dimer formation. We first built two constructs, one with a (G4S)5 linker preceding the IgG3 hinge and one without a preceding linker. The second linker was kept at 5 repeats to provide enough space for the second scFv to reach the binding site without interference from the hinge.

[0151] [Figure 42-1] The sequence of the IgG3 hinge linker sequence is shown. [Figure 42-2] A continuation of Figure 42-1 is shown.

[0152] [Diagram 43]Photographs of exemplary labeled SDS Page Gels of non-reduced and reduced E1D7-IgG3-hinge-P4B3M3 and P4B3M3-IgG3-hinge-E1D7 bsAb are shown.

[0153] [Diagram 44] A chart of exemplary data is shown. SA sensors were loaded with biotinylated CTLA4 or PD1 and allowed to bind / dissociate the indicated antibodies. Due to sensor drift, the off-rates of sensors loaded with PD1 are unreliable.

[0154] [Diagram 45] Exemplary dual binding assay data are shown. CTLA4+, PD1+, or CTLA4+PD1+ Jurkat cells were first coated with the indicated amounts of monospecific or bispecific proteins. Soluble protein was then added and allowed to bind to the free arm. As shown here, monospecific antibodies cannot bind large amounts of soluble antigen, but BsAbs can capture soluble CTLA4 from solution when bound to PD1 expressing cells.

[0155] [Figure 46] Non-limiting exemplary data from an Ab (including mAb combinations) vs. CD80 hFc competition assay for CTLA4+ / PD1+1G5 Jurkat. BsAbs demonstrate significantly increased competition for CD80 binding to PD1+CTLA4+Jurkat cells when compared to monospecific antibodies and cocktails.

[0156] [Figure 47]Non-limiting exemplary data of % competition assay with CD80 hFc for binding to CTLA4 on 1G5 Jurkat are shown. CD80 competition assay using CTLA4 transduced Jurkat cells: Four bispecific constructs were tested for CD80 competition. Two had the complete 5,5 linker arrangement and two had the omitted 0,5 linker. We also swapped the order of aPD1 / aCTLA4 abs in the final construct. As shown here, E1-D7 and E1-D7+P4B3m3 combination therapy results in moderate competition, while the BsAb construct shows a significant increase in CD80 competition, likely due to the additional binding potential generated by the anti-PD1 domain.

[0157] [Figure 48-1] Photographs and illustrations of panel (A) characteristics (adapted from Li et al., 2019), panel (B) cell differentiation model (adapted from Tun et al., 2010), and panel (C) epidemiology (adapted from Hsieh et al., 2019) of clear cell renal cell carcinoma are shown. [Figure 48-2] A continuation of Figure 48-1 is shown.

[0158] [Figure 49] A graph of OS for ccRCC patients edited from Feng et al., 2019 is shown.

[0159] [Figure 50] A graph depicting immune populations in ccRCC adapted from Chevrier et al., 2017 is shown.

[0160] [Figure 51A] 51 shows diagrams and graphs of the CTLA4 signaling pathway. Panel A shows a diagram of the CTLA4 signaling pathway. [Figure 51B] Panel B shows a graph of CTLA4 overexpression in ccRCC (adapted from Lie et al., 2020). [Figure 51C] Panel C shows a diagram of ipilimumab and CTLA4 blockade.

[0161] [Figure 52A] Figure 52 shows photographs and diagrams of the PD1 signaling pathway compiled from Kim et al., 2021. Panel A shows the PD1-PDL1 signaling pathway. [Figure 52B] Panel B shows histological images of PD1 overexpression in ccRCC. [Figure 52C] Panel C shows an illustration of PD1 blockade and non-limiting effects.

[0162] [Figure 53A] Figure 53 shows an illustration of dual checkpoint blockade with bispecific antibodies. Panel A shows a schematic of dual CTLA4 / PDL checkpoint blockade. [Figure 53B] Panel B shows non-limiting exemplary bispecific antibody formats.

[0163] [Figure 54] FIG. 1 shows a table of exemplary anti-CTLA4 / anti-PD1 bispecific antibodies in clinical use.

[0164] [Figure 55] FIG. 1 shows a schematic diagram of a non-limiting exemplary study design for evaluating the binding affinity of anti-CTLA4 candidates to various concentrations of human and mouse CTLA4 using ELISA.

[0165] [Figure 56-1] Non-limiting data and results of ELISA binding curves are shown. Panel A shows ELISA binding curves to show the binding affinity of anti-CTLA4 candidates to hCTLA4. Panel B shows ELISA binding curves to show the binding affinity of anti-CTLA4 candidates to mCTLA4. Panel C shows ELISA binding curves to show the binding affinity of anti-CTLA4 candidates to BSA (negative control). [Figure 56-2] A continuation of Figure 56-1 is shown.

[0166] [Figure 57] 1 shows a non-limiting exemplary study design for determining the kinetic properties of antibody candidates using BLI-Octet.

[0167] [Figure 58] Non-limiting data showing the kinetic properties of anti-CTLA4 candidates are shown. All tested candidates have high dissociation rates compared to ipilimumab, even when they have high binding responses. Among the candidates, C, B, and G appear to have the lowest dissociation rates. KD-dissociation constant, Kon-binding rate, Kon-binding rate.

[0168] [Figure 59] FIG. 1 shows a schematic diagram of a non-limiting exemplary study design to determine the ability of anti-CTLA4 antibodies to block CD80 / CD86 / ipilimumab binding and to determine the similarity of antibody and ligand localization on the surface of CTLA4.

[0169] [Figure 60] Shown is a chart of percentage blockade of CD80 / CD86 / ipilimumab binding to CTLA4 by anti-CTLA4 candidates. CD80: Ipilimumab blocks CD80 to the highest extent. Candidates showing highest CD80 blockade: I, E, J. CD86: All candidates show 100% blockade of CD86 binding. This may be due to the demonstrated low affinity of CD86 binding to CTLA4. Ipilimumab: All candidates block ipilimumab binding to CTLA4 to some degree, even those that cross-react with mCTLA4.

[0170] [Figure 61]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 with contact residues highlighted. Panel B shows ipilimumab complexed with -mCTLA4 with aligned epitopes highlighted.

[0171] [Figure 62] 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):608-11. doi:10.1038 / 35069118. Erratum in: Nature 2001 May 31;411(6837):617. PMID:11279502. Panel A shows CD80 complexed with -hCTLA4 with contact residues highlighted. Panel B shows CD80 complexed with -mCTLA4 with aligned epitopes highlighted.

[0172] [Figure 63] FIG. 1 shows a schematic diagram of a non-limiting exemplary assay design for determining the binding affinity of a candidate antibody to Jurkat cells expressing CTLA4.

[0173] [Figure 64] 1 shows non-limiting exemplary binding curves of binding affinity of anti-CTLA4 candidates to CTLA4+ Jurkat cells.

[0174] [Figure 65] FIG. 1 shows a schematic diagram of a non-limiting exemplary study design for evaluating the biological significance of antibody binding in promoting CD28 signaling.

[0175] [Figure 66] 1 shows a non-limiting exemplary graph of fold induction of luciferase versus concentration of anti-CTLA4 candidates. Across both plates, ipilimumab shows the highest induction of luciferase expression. Five of the eight candidates tested: E, H, J, F, and I show similar responses.

[0176] [Figure 67-1] 1 shows the germline alignment of anti-CTLA-4 antibodies. [Figure 67-2] A continuation of Figure 67-1 is shown. [Figure 67-3] Continuation of Figure 67-2 is shown. [Figure 67-4] Continuation of Figure 67-3 is shown. [Figure 67-5] Continuation of Figure 67-4 is shown. [Figure 67-6] Continuation of Figure 67-5 is shown. [Figure 67-7] Continuation of Figure 67-6 is shown. [Figure 67-8] Continuation of Figure 67-7. [Figure 67-9] Continuation of Figure 67-8. [Figure 67-10] Continuation of Figure 67-9. [Figure 67-11] Continuation of Figure 67-10. [Figure 67-12] Continuation of Figure 67-11 is shown.

[0177] [Figure 68-1] The amino acid sequence of the anti-CTLA-4 antibody is shown. [Figure 68-2] A continuation of Figure 68-1 is shown. [Figure 68-3] Continuation of Figure 68-2 is shown. [Figure 68-4] Continuation of Figure 68-3 is shown. [Figure 68-5] Continuation of Figure 68-4 is shown. [Figure 68-6] Continuation of Figure 68-5 is shown.

[0178] [Figure 69-1] 1 shows the nucleic acid sequence of an anti-CTLA-4 antibody. [Figure 69-2] A continuation of Figure 69-1 is shown. [Figure 69-3] Continuation of Figure 69-2 is shown. [Figure 69-4] Continuation of Figure 69-3 is shown. [Figure 69-5] Continuation of Figure 69-4 is shown. [Figure 69-6] Continuation of Figure 69-5 is shown. [Figure 69-7] Continuation of Figure 69-6 is shown. [Figure 69-8] Continuation of Figure 69-7. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0179] Detailed Description Abbreviations and Definitions

[0180] Detailed description of one or more embodiments is provided herein. However, it is understood that the present invention can be embodied in various forms. Therefore, the specific details disclosed herein should not be interpreted as limiting, but as a basis for the claims and as a representative basis for teaching a person skilled in the art to use the present invention in any suitable manner.

[0181] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. The use of the words "a" or "an" when used in conjunction with the term "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0182] Whenever any of the phrases "for example," "such as," "including," and the like are used herein, unless expressly stated otherwise, it is understood that the phrase "without limitation" is accompanying. Similarly, "an example," "exemplary," and the like are understood to be non-limiting.

[0183] The term "substantially" permits deviations from the descriptors that do not adversely affect the intended purpose. It is understood that a descriptor is modified by the term "substantially" even if the word "substantially" is not expressly recited.

[0184] Terms such as "comprising," "including," "having," and "involving" (and similarly "comprises," "includes," "has," and "involves") are used interchangeably and have the same meaning. Specifically, each of the terms is defined consistent with the general U.S. patent law definition of "comprising," and therefore is to be construed as an open term meaning "at least the following," and not excluding additional features, limitations, aspects, etc. Thus, for example, "a process comprising steps a, b, and c" means that the process includes at least steps a, b, and c. Whenever the terms "a" or "an" are used, they are to be understood as "one or more," unless such an interpretation is insignificant in the context.

[0185] As used herein, the term "about" is used herein to mean approximately, roughly, approximately, or within the region. 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 numerical values ​​above and below the set forth value by a variance of 20 percent above or below (higher or lower).

[0186] CTLA-4

[0187] Human antibody-phage screening was performed against human CTLA-4 (rounds 1-3) or murine CTLA-4 (round 3). The first set of antibodies was discovered by panning against the soluble protein and the second set by PMPL panning.

[0188] Binding activity has 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 immuno-oncology. The anti-CTLA-4 mAbs described herein may have therapeutic potential. CTLA-4 antibodies with various affinities to CTLA-4 have been identified.

[0189] An aspect of the invention provides isolated antibodies specific for CTLA-4. When used herein with respect to nucleic acids such as cells, DNA or RNA, the term "isolated" refers to a molecule that is separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. The term "isolated" may also refer to a nucleic acid or peptide that is substantially free of cellular material, viral material or medium, if produced by recombinant DNA technology, or chemical precursors or other chemicals, if chemically synthesized. For example, an "isolated nucleic acid" can include a nucleic acid fragment that is not naturally occurring as a fragment and would not be found in the natural state. "Isolated" may also refer to a cell or polypeptide that is isolated from other cellular proteins or tissues. An isolated polypeptide can encompass 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 that displayed CTLA-4 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 multispecific antibodies or as payloads for engineered cells such as CAR-T cells, CAR-NK cells, or engineered B cells.

[0190] Unique recombinant monoclonal CTLA-4 antibodies are described herein. "Recombinant" in reference to a polypeptide (such as an antibody) or polynucleotide refers to a form of a polypeptide or polynucleotide that is not naturally occurring, a non-limiting example of which can be made by combining polynucleotides or polypeptides that are not normally together.

[0191] The amino acid sequences of monoclonal CTLA-4 antibodies are provided herein (see Tables 1-26). The amino acid sequences of the heavy and light chain complementarity determining regions CDRs of the CTLA-4 antibodies are below underlined (CDR1), underlined and bold (CDR2), or underlined and bold italic (CDR3). Table 1. Amino acid sequences of the CTLA-4-PMPL-Rd2-A4 (E1-A4) Ab variable regions TIFF2024541476000002.tif39152 Table 2: Amino acid sequences of the CTLA-4-PMPL-Rd2-A8 (E1-A8) Ab variable regions TIFF2024541476000003.tif39152 Table 3: Amino acid sequences of the CTLA-4-PMPL-Rd2-A9 (E1-A9) Ab variable regions TIFF2024541476000004.tif39152 Table 4: Amino acid sequences of the CTLA-4-PMPL-Rd2-A10 (E1-A10) Ab variable regions TIFF2024541476000005.tif39152 Table 5: Amino acid sequences of the CTLA-4-PMPL-Rd2-A12 (E1-A12) Ab variable regions TIFF2024541476000006.tif39152 Table 6: Amino acid sequences of the CTLA-4-PMPL-Rd2-B6 (E1-B6) Ab variable regions TIFF2024541476000007.tif39152 Table 7: Amino acid sequences of the CTLA-4-PMPL-Rd2-B10 (E1-B10) Ab variable regions TIFF2024541476000008.tif39152 Table 8. Amino acid sequences of the CTLA-4-PMPL-Rd2-B11 (E1-B11) Ab variable regions TIFF2024541476000009.tif39152 Table 9. Amino acid sequences of the CTLA-4-PMPL-Rd2-B12 (E1-B12) Ab variable regions TIFF2024541476000010.tif39152 Table 10: Amino acid sequences of the aCTLA-4_T2-C10 (T2-C10) Ab variable regions TIFF2024541476000011.tif39152 Table 11: Amino acid sequences of the CTLA-4-PMPL-Rd2-C12 (E1-C12) Ab variable regions TIFF2024541476000012.tif39152 Table 12. Amino acid sequences of the aCTLA-4_E1-D7 (E1-D7) Ab variable regions TIFF2024541476000013.tif39152 Table 13: Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-A4 (E2-A4) Ab variable regions TIFF2024541476000014.tif43152 Table 14: Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-A7 (E2-A7) Ab variable regions TIFF2024541476000015.tif39152 Table 15: Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-A10 (E2-A10) Ab variable regions TIFF2024541476000016.tif39152 Table 16: Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-A12 (E2-A12) Ab variable regions TIFF2024541476000017.tif39152 Table 17: Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-C4 (E2-C4) Ab variable regions TIFF2024541476000018.tif39152 Table 18. Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-D4 (E2-D4) Ab variable regions TIFF2024541476000019.tif39152 Table 19: Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-E5 (E2-E5) Ab variable regions TIFF2024541476000020.tif39152 Table 20: Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-F1 (E2-F1) Ab variable regions TIFF2024541476000021.tif39152 Table 21. Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-G5 (E2-G5) Ab variable regions TIFF2024541476000022.tif39152 Table 22: Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-G9 (E2-G9) Ab variable regions TIFF2024541476000023.tif39152 Table 23: Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-H5 (E2-H5) Ab variable regions TIFF2024541476000024.tif39152 Table 24: Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-H8 (E2-H8) Ab variable regions TIFF2024541476000025.tif39152 Table 25. Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-H9 (E2-H9) Ab variable regions TIFF2024541476000026.tif39152 Table 26: Amino acid sequences of the CTLA-4-PMPL-Rd2-E2-H10 (E2-H10) Ab variable regions TIFF2024541476000027.tif39152 Table 57. Amino acid sequences of the hCTLA4-Rd3b-T1-E6 (T1-E6) Ab variable regions TIFF2024541476000028.tif45152 Table 58. Amino acid sequences of the hCTLA4-Rd3b-E4-F2 (E4-F2) Ab variable regions TIFF2024541476000029.tif41152 Table 59. Amino acid sequences of the hCTLA4-Rd3b-T1-A3 (T1-A3) Ab variable regions TIFF2024541476000030.tif46152 Table 60. Amino acid sequences of the hCTLA4-T1-A6 (T1-A6) Ab variable regions TIFF2024541476000031.tif45152 Table 61. Amino acid sequences of the hCTLA4-Rd3b-T1-A10 (T1-A10) Ab variable regions TIFF2024541476000032.tif46152 Table 62: Amino acid sequences of the hCTLA4-Rd3b-E2-D5 (E2-D5) Ab variable regions TIFF2024541476000033.tif46152 Table 63: Amino acid sequences of the hCTLA4-Rd3b-E2-G6 (E2-G6) Ab variable regions TIFF2024541476000034.tif46152 Table 64. Amino acid sequences of the hCTLA4-Rd3b-E2-H3 (E2-H3) Ab variable regions TIFF2024541476000035.tif45152 Table 65. Amino acid sequences of the CTLA4pmpl-Rd2-E2-H12 (E2-H12) Ab variable regions TIFF2024541476000036.tif46152 Table 66: Amino acid sequences of the CTLA4pmpl-Rd2-E2-H2 (E2-H2) Ab variable regions TIFF2024541476000037.tif46152 Table 67: Amino acid sequences of the CTLA4pmpl-Rd2-E2-D10 (E2-D10) Ab variable regions TIFF2024541476000038.tif46152 Table 68: Amino acid sequences of the CTLA4pmpl-Rd2-E2-D11 (E2-D11) Ab variable regions TIFF2024541476000039.tif46152

[0192] The nucleic acid sequences of monoclonal CTLA-4 antibodies are provided herein (see Tables 27-52). Table 27. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-A4 (E1-A4) Ab variable regions TIFF2024541476000040.tif68152 Table 28. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-A8 (E1-A8) Ab variable regions TIFF2024541476000041.tif68152 Table 29. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-A9 (E1-A9) Ab variable regions TIFF2024541476000042.tif68152 Table 30: Nucleic acid sequences of the CTLA-4-PMPL-Rd2-A10 (E1-A10) Ab variable regions TIFF2024541476000043.tif68152 Table 31. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-A12 (E1-A12) Ab variable regions TIFF2024541476000044.tif68152 Table 32. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-B6 (E1-B6) Ab variable regions TIFF2024541476000045.tif68152 Table 33. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-B10 (E1-B10) Ab variable regions TIFF2024541476000046.tif68152 Table 34. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-B11 (E1-B11) Ab variable regions TIFF2024541476000047.tif68152 Table 35. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-B12 (E1-B12) Ab variable regions TIFF2024541476000048.tif68152 Table 36: Nucleic acid sequences of the aCTLA-4_T2-C10 (T2-C10) Ab variable regions TIFF2024541476000049.tif72152 Table 37. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-C12 (E1-C12) Ab variable regions TIFF2024541476000050.tif68152 Table 38. Nucleic acid sequences of the aCTLA-4_E1-D7 (E1-D7) Ab variable regions TIFF2024541476000051.tif68152 Table 39. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-A4 (E2-A4) Ab variable regions TIFF2024541476000052.tif68152 Table 40. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-A7 (E2-A7) Ab variable regions TIFF2024541476000053.tif68152 Table 41. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-A10 (E2-A10) Ab variable regions TIFF2024541476000054.tif68152 Table 42: Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-A12 (E2-A12) Ab variable regions TIFF2024541476000055.tif68152 Table 43: Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-C4 (E2-C4) Ab variable regions TIFF2024541476000056.tif68152 Table 44. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-D4 (E2-D4) Ab variable regions TIFF2024541476000057.tif68152 Table 45. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-E5 (E2-E5) Ab variable regions TIFF2024541476000058.tif68152 Table 46: Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-F1 (E2-F1) Ab variable regions TIFF2024541476000059.tif68152 Table 47. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-G5 (E2-G5) Ab variable regions TIFF2024541476000060.tif68152 Table 48. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-G9 (E2-G9) Ab variable regions TIFF2024541476000061.tif68152 Table 49. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-H5 (E2-H5) Ab variable regions TIFF2024541476000062.tif68152 Table 50. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-H8 (E2-H8) Ab variable regions TIFF2024541476000063.tif68152 Table 51. Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-H9 (E2-H9) Ab variable regions TIFF2024541476000064.tif78152 Table 52: Nucleic acid sequences of the CTLA-4-PMPL-Rd2-E2-H10 (E2-H10) Ab variable regions TIFF2024541476000065.tif68152 Table 69. Nucleic acid sequence of hCTLA4-Rd3b-T1-E6_PelB-F_2021-02-10_E05(T1-E6) Ab variable region TIFF2024541476000066.tif68152 Table 70: Nucleic acid sequence of hCTLA4-Rd3b-E4-F2_PelB-F_2021-02-10_F06(E4-F2) Ab variable region TIFF2024541476000067.tif68152 Table 71 Nucleic acid sequence of hCTLA4-Rd3b-T1-A3_PelB-F_2021-02-10_B01(T1-A3) Ab variable region TIFF2024541476000068.tif68152 Table 72: Nucleic acid sequence of hCTLA4-T1-A6_PelB-F_2021-02-05_B04(T1-A6) Ab variable region TIFF2024541476000069.tif68152 Table 73: Nucleic acid sequence of hCTLA4-Rd3b-T1-A10_PelB-F_2021-02-10_F01(T1-A10) Ab variable region TIFF2024541476000070.tif68152 Table 74: Nucleic acid sequence of hCTLA4-Rd3b-E2-D5_PelB-F_2021-02-10_B05(E2-D5) Ab variable region TIFF2024541476000071.tif68152 Table 75. Nucleic acid sequence of hCTLA4-Rd3b-E2-G6_PelB-F_2021-02-10_C08(E2-G6) Ab variable region TIFF2024541476000072.tif68152 (Table 76) Nucleic acid sequence of hCTLA4-Rd3b-E2-H3_PelB-F_2021-02-10_H08(E2-H3) Ab variable region TIFF2024541476000073.tif68152 Table 77: Nucleic acid sequence of the CTLA4pmpl-Rd2-E2-H12_PelB-F_2021-08-06_H06 (E2-H12) Ab variable region TIFF2024541476000074.tif68152 Table 78: Nucleic acid sequence of the CTLA4pmpl-Rd2-E2-H2_PelB-F_2021-08-06_H05 (E2-H2) Ab variable region TIFF2024541476000075.tif68152 Table 79: Nucleic acid sequence of the CTLA4pmpl-Rd2-E2-D10_PelB-F_2021-08-06_H02(E2-D10) Ab variable region TIFF2024541476000076.tif68152 Table 80: Nucleic acid sequence of the CTLA4pmpl-Rd2-E2-D11_PelB-F_2021-08-06_A03 (E2-D11) Ab variable region TIFF2024541476000077.tif68152

[0193] The amino acid sequences of the heavy and light chain complementarity determining regions of the CTLA-4 antibody are shown below in Tables 53A-B. Table 53A: Complementarity determining regions (CDRs) of the heavy chain (VH) of the CTLA-4 antibody TIFF2024541476000078.tif192152TIFF2024541476000079.tif192152 Table 53B. Complementarity determining regions (CDRs) of the light chain (VL) of the CTLA-4 antibody TIFF2024541476000080.tif20152TIFF2024541476000081.tif230152TIFF2024541476000082.tif144152

[0194] The nucleic acid sequences of the heavy and light chain complementarity determining regions of the CTLA-4 antibody are shown below in Tables 54A-B. Table 54A: Complementarity determining regions (CDRs) of the heavy chain (VH) of the CTLA-4 antibody TIFF2024541476000083.tif39152TIFF2024541476000084.tif230152TIFF2024541476000085.tif229152TIFF2024541476000086.tif146152 Table 54B. Complementarity determining regions (CDRs) of the light chain (VL) of the CTLA-4 antibody TIFF2024541476000087.tif67152TIFF2024541476000088.tif231152TIFF2024541476000089.tif227152TIFF2024541476000090.tif53152

[0195] The amino acid sequences of the heavy and light chain framework regions of the CTLA4 antibody are shown below in Tables 55A-B. Table 55A: Heavy chain (VH) framework regions (FR) of CTLA-4 antibodies TIFF2024541476000091.tif142146TIFF2024541476000092.tif227146TIFF2024541476 000093.tif231146TIFF2024541476000094.tif228146TIFF2024541476000095.tif98146 Table 55B: Light chain (VL) framework regions (FR) of CTLA-4 antibodies TIFF2024541476000096.tif119144TIFF2024541476000097.tif227144TIFF2024541476 000098.tif231144TIFF2024541476000099.tif230144TIFF2024541476000100.tif72144

[0196] The nucleic acid sequences of the heavy and light chain framework regions of the CTLA-4 antibody are shown below in Tables 56A-B. Table 56A: Heavy chain (VH) framework regions (FR) of CTLA-4 antibodies TIFF2024541476000101.tif125146TIFF2024541476000102.tif230146TIFF202 4541476000103.tif221146TIFF2024541476000104.tif230146TIFF20245414760 00105.tif229146TIFF2024541476000106.tif230146TIFF2024541476000107.t if225146TIFF2024541476000108.tif230146TIFF2024541476000109.tif248146 Table 56B: Light chain (VL) framework regions (FR) of CTLA-4 antibodies TIFF2024541476000110.tif189146TIFF2024541476000111.tif227146TIFF2024541476000112.tif231146TIFF2024541476000113.tif226146TIFF2024541476000114.tif232146TIFF2024541476000115.tif231146TIFF2024541476000116.tif227146TIFF2024541476000117.tif230146TIFF2024541476000118.tif185146

[0197] 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 certain percentage of 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 the sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing the position of each sequence, which may be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. For example, an antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity when compared to a specific region or the full length of any one of the anti-CTLA-4 antibodies described herein. For example, an antibody can have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acid sequence identity when compared to a specific region or the full length of any one of the anti-CTLA-4 antibodies described herein. Sequence identity or similarity for the nucleic acids and proteins of the 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 Current Protocols in Molecular Biology, eds. Ausubel et al. (2007). For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, or visual inspection can be used to determine percent sequence identity or similarity for the nucleic acids and proteins of the invention.

[0198] As used herein, "polypeptide" can encompass a single "polypeptide" as well as multiple "polypeptides" and refers to a molecule composed of monomers (amino acids) linked in a linear chain by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain of two or more amino acids can refer to a "polypeptide" herein, and the term "polypeptide" can be used in place of or interchangeably with any of these terms. "Polypeptide" can also refer to post-expression modified products of a polypeptide, such as, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced by recombinant technology and is not necessarily translated from a specific nucleic acid sequence. It can be generated in any manner, including chemical synthesis. With respect 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 that modify, add, delete, or replace a single amino acid or a small percentage of amino acids in the encoded sequence are collectively referred to herein as "conservatively modified variants." In some embodiments, the modification results in the replacement 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 compared to unmodified CTLA-4 antibodies.

[0199] For example, a "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are defined in the art as: 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 non-essential amino acid residue in an immunoglobulin polypeptide is replaced with another amino acid residue from the same side chain family. In another embodiment, an amino acid chain can be replaced with a structurally similar chain that differs in the order and / or composition of the side chain family members.

[0200] antibody

[0201] As used herein, an "antibody" or an "antigen-binding polypeptide" can refer to a polypeptide or polypeptide complex that specifically recognizes and binds an antigen. An antibody can be a whole antibody and any antigen-binding fragment, or a single chain thereof. For example, an "antibody" can include any protein or peptide-containing molecule that contains at least a portion of an immunoglobulin molecule that has the biological activity of binding to an antigen. Non-limiting examples include the complementarity determining regions (CDRs) of a heavy or light chain or a ligand-binding portion thereof, the variable region of a heavy or light chain, the constant region of a heavy or light chain, the framework (FR) region, or any portion thereof, or at least a portion of a binding protein. As used herein, the term "antibody" can refer to immunoglobulin molecules and immunoglobulin (Ig) molecules, i.e., immunologically active portions of molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen. "Specifically binds" or "immunoreacts" means that the antibody reacts with one or more antigenic determinants of a desired antigen and not with other polypeptides.

[0202] As used herein, the term "antibody fragment" or "antigen-binding fragment" refers to (ab’)2 , F (ab)2 , F ab ', F ab"Antibody fragment" refers to a portion of an antibody, such as a Fv, scFv, etc. Antibody fragments, regardless of structure, bind to the same antigen recognized by the complete 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 such as Fab, Fab', F(ab')2, Fd, Fvs, single chain Fv (scFv), single chain antibodies, dAbs (domain antibodies), minibodies, disulfide-linked Fv (sdFv), fragments comprising either a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies.

[0203] A "single-chain variable fragment" or "scFv" is a fragment of an immunoglobulin heavy chain (V H ) and light chain (V L Single-chain Fv ("scFv") polypeptide molecules are covalently linked VH:VL heterodimers that can be expressed from gene fusions containing 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 embodiments, the domains are linked by a short linker peptide of 10 to about 25 amino acids. The linker can be rich in glycine for flexibility and serine or threonine for solubility, and can be rich in the VH:VL heterodimers. H N-terminus of V LThe C-terminus of the scFv molecule may be linked to the C-terminus of either the IgG1A or the IgG2A or vice versa. The protein retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker. Many methods have been described for identifying chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to the structure of the antigen binding site. See, for example, U.S. Patent Nos. 5,091,513, 5,892,019, 5,132,405, and 4,946,778, each of which is incorporated herein by reference in its entirety.

[0204] Very large naive human scFv libraries have been and can be made to provide a large source of rearranged antibody genes against a large number of target molecules. Smaller libraries can be constructed from individuals with infectious diseases to isolate disease-specific 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)).

[0205] The antibodies of the present invention can also be modified to produce mosaic antibodies. Mosaic antibodies are antibodies in which the external amino acid residues of an antibody of one species are rationally replaced or "mosaicized" by the external amino acid residues of an antibody of a second species, so 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 characteristics, the immunogenicity of an antibody can be reduced by substituting exposed residues that are different from those typically found in antibodies of another mammalian species. This reasonable substitution of external residues should have little or no effect on the internal domains or interdomain contacts. Thus, the ligand binding properties should be unaffected, since the changes are limited to the variable region framework residues. This process is called "mosaicism" because only the outer surface or skin of the antibody is altered, while the supporting residues remain unaffected.

[0206] The "mosaicization" process utilizes sequence data for human antibody variable domains compiled by 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 those described in EP 519596, U.S. Patent 6,797,492, and in Padlan et al., 1991.

[0207] Antibody molecules obtained from humans are associated with one of five classes: IgG, IgM, IgA, IgE, and IgD, which differ from each other in 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 (γ, μ, α, δ, ε), with several subclasses within them (e.g., γ1-γ4). Certain classes also have subclasses, such as IgG1, IgG2, IgG3, and IgG4. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, are well characterized and are known to confer functional specialization. With respect to IgG, a standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of about 23,000 daltons and two identical heavy chain polypeptides with a molecular weight of 53,000-70,000. The four chains are typically joined by disulfide bonds in a "Y" configuration, with the light chains surrounding the heavy chains, which begin at the mouth of the "Y" and continue through the variable region. The immunoglobulin or antibody molecules described herein can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.

[0208] Light chains are classified as either kappa or lambda (κ, λ). Each heavy chain class can be associated with either a kappa or lambda light chain. Generally, light and heavy chains are covalently linked to each other, and the "tails" of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the immunoglobulin is produced by either a hybridoma, a B cell, or a genetically engineered host cell. In the heavy chains, the amino acid sequence runs from the N-terminus at the forked end of the Y-shape to the C-terminus at the bottom of each chain.

[0209] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in a functional sense. The variable domains (VL and VH) of both the light and heavy chain portions determine antigen recognition and specificity. Conversely, the constant domains (CL, and CH1, CH2 or CH3) of the light and heavy chains confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, and complement binding. The term "antigen-binding site" or "binding portion" can refer to the portion of an immunoglobulin molecule involved 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 sections within the V regions of the heavy and light chains, called "hypervariable regions", are interposed between more conserved adjacent sections known as "framework regions" or "FRs". Thus, the term "FR" can refer to the amino acid sequences naturally found between and adjacent to the hypervariable regions of immunoglobulins. In an antibody molecule, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged 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 called "complementarity-determining regions" or "CDRs." The VH and VL regions, containing the CDRs and frameworks (FRs) of the CTLA-4 antibody, are shown in Tables 1A to 12B.

[0210] The six CDRs present in each antigen-binding domain are short non-contiguous sequences of amino acids that are specifically arranged to form the antigen-binding domain when the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids of the antigen-binding domain, the FR regions, show less intermolecular variation. The framework regions adopt a predominantly beta-sheet conformation, and the CDRs form loops that connect and in some cases form part of the beta-sheet structure. The framework regions function to form a scaffold for positioning the CDRs in the correct orientation by non-covalent interactions between the chains. The antigen-binding domain formed by the positioned CDRs provides a surface complementary to the epitope on the antigen in the immune response, facilitating non-covalent binding of the antibody to its cognate epitope. The amino acids which comprise the CDRs and framework regions, respectively, have been previously identified (see "Sequences of Proteins of Immunological Interest," Kabat, E., et al., USDepartment of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)) and can be readily identified for heavy or light chain variable regions by one of ordinary skill in the art.

[0211] Where there are more than one definition for a term used and / or accepted in the art, the definition of the term as used herein is intended to encompass all such meanings unless expressly stated to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-contiguous antigen binding sites found within the variable regions of both heavy and light chain polypeptides. This particular region is described by Kabat et al., USDept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., Mol. Biol. 196:901-917 (1987), which are incorporated herein by reference in their entireties. The definitions of CDR by Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nevertheless, it is intended that the application of either definition to refer to the CDR of an antibody or variants thereof is within the scope of the term as defined and used herein. The appropriate amino acid residues encompassing the CDRs defined by each of the above cited references are set forth in the following table for comparison. The exact residue numbers which comprise a particular CDR will vary depending on the sequence and size of the CDR, and one of skill in the art can routinely determine which residues constitute a particular CDR given the variable region amino acid sequence of an antibody. TIFF2024541476000119.tif48128

[0212] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. One of skill in the art can unambiguously assign this "Kabat numbering" system to any variable domain sequence, without relying on other experimental data of the sequence itself. As used herein, "Kabat numbering" refers to the numbering system set forth by Kabat et al., USDept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0213] In addition to the above table, the Kabat numbering system describes the CDR regions as follows: CDR-H1 begins about amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), includes about 5-7 amino acids, and ends at the next tryptophan residue. CDR-H2 begins about the 15th residue after the end of CDR-H1, includes about 16-19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 begins about the 33rd amino acid residue after the end of CDR-H2; includes 3-25 amino acids, and ends with the sequence WGXG (X is any amino acid). CDR-L1 begins about residue 24 (i.e., after the cysteine ​​residue), includes about 10-17 residues, and ends at the next tryptophan residue. CDR-L2 begins about the 16th residue after the end of CDR-L1 and includes about 7 residues. CDR-L3 begins approximately 33 residues after the end of CDR-L2 (i.e., after the cysteine ​​residue), includes about 7-11 residues, and ends with the sequence F or WGXG (where X is any amino acid).

[0214] As used herein, the term "epitope" can include any protein determinant capable of specifically binding to an immunoglobulin, scFv, or T-cell receptor. The variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, the VL and VH domains of an antibody, or a subset of the complementarity determining regions (CDRs), combine to form the variable regions that define a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of a Y. Epitope 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 (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) on each of the VH and VL chains. In one embodiment, the antibody may be directed to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) (CTLA-4_HUMAN) (223 amino acid residues in length) having Ann UniProt accession number P16410, which comprises the amino acid sequence of SEQ ID NO: []. TIFF2024541476000120.tif57160

[0215] As used herein, the terms "immunological binding" and "immunological binding properties" can refer to the type of non-covalent interactions that occur between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength, or affinity, of an immunological binding interaction is determined by the dissociation constant (K d ), and K dA smaller K represents a greater affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of antigen-binding site / antigen complex formation and dissociation, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, both "on-rate constants" (K on ) and "off rate constant" (K off ) can be determined by calculation of the concentrations and actual rates of association and dissociation. (See Nature 361:186-87 (1993)). K off / K on The ratio of D (See generally, Davies et al. (1990) Annual Rev Biochem 59:439-473.) The antibodies of the present invention may have an equilibrium binding constant (K) as measured by a kinetic assay, e.g., a radioligand binding assay or similar assay known to those of skill in the art, e.g., BIAcore or Octet (BLI). D ) is ≦1 μM, ≦10 μM, ≦10 nM, ≦10 pM, or ≦100 pM to about 1 pM. For example, in some embodiments, D is between about 1E-12M and about 1E-11M. D In some embodiments, K D is between about 1E-11M and about 1E-10M. D In some embodiments, K D is between about 1E-10M and about 1E-9M. D In some embodiments, K D is between about 1E-9M and about 1E-8M. D In some embodiments, K D is between about 1E-8M and about 1E-7M D In some embodiments, K D is between about 1E-7M and about 1E-6M DFor example, in some embodiments, K D is about 1E-12M, and in other embodiments, K D is about 1E-11M. In some embodiments, K D is about 1E-10M, and in other embodiments, K D is about 1E-9M. In some embodiments, K D is about 1E-8M, and in other embodiments, K D is about 1E-7M. In some embodiments, K D is about 1E-6M, and in other embodiments, K D is about 1E-5M. In some embodiments, for example, K D is about 3E-11M, and in other embodiments, K D is about 3E-12M. In some embodiments, K D is about 6E-11M. "Specifically binds" or "having specificity" can refer to an antibody that binds to an epitope through the antigen-binding domain of the antibody, where the binding involves some complementarity between the antigen-binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope if it binds to that epitope through its antigen-binding domain more readily than it binds to a random, unrelated epitope.

[0216] For example, CTLA4 antibodies can be monovalent or bivalent and can comprise single or double chains. Functionally, the binding affinity of a CTLA4 antibody is greater than or equal to 10 -5 M~10 -12 For example, the binding affinity of the CTLA4 antibody is in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10 -9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10-8 M~10 -11 M, 10 -9 M~10 -11 M, 10 -10 M~10 -11 M, 10 -5 M~10 -10 M, 10 -6 M~10 -10 M, 10 -7 M~10 -10 M, 10 -8 M~10 -10 M, 10 -9 M~10 -10 M, 10 -5 M~10 -9 M, 10 -6 M~10 -9 M, 10 -7 M~10 -9 M, 10 -8 M~10 -9 M, 10 -5 M~10 -8 M, 10 -6 M~10 -8 M, 10 -7 M~10 -8 M, 10 -5 M~10 -7 M, 10 -6 M~10 -7 M, 10 -5 M~10 -6 It's M.

[0217] CTLA-4 protein, or derivatives, fragments, analogs, homologs or orthologs thereof, can be used as immunogens in the generation of antibodies that immunologically specifically bind to these protein components. CTLA-4 protein coupled to proteoliposomes, or derivatives, fragments, analogs, homologs or orthologs thereof, can be used as immunogens in the generation of antibodies that immunologically specifically bind to these protein components.

[0218] One of ordinary skill in the art will recognize that one can determine, without undue experimentation, whether a human monoclonal antibody has the same specificity as a human monoclonal antibody of the invention by determining whether the former prevents the latter from binding to CTLA-4. For example, if the human monoclonal antibody being tested exhibits reduced binding by and competes with a human monoclonal antibody of the invention, then it is likely that the two monoclonal antibodies bind to the same or closely related epitopes.

[0219] Another method of determining whether a human monoclonal antibody has the specificity of the human monoclonal antibody of the present invention is to preincubate the human monoclonal antibody of the present invention with the CTLA-4 protein with which it is normally reactive, then add the human monoclonal antibody to be tested and determine whether the human monoclonal antibody to be tested is inhibited in its ability to bind to CTLA-4. If the human monoclonal antibody to be tested is inhibited, then it likely has the same, or a functionally equivalent, epitopic specificity as the monoclonal antibody of the present invention. Screening of the human monoclonal antibodies of the present invention can also be performed using CTLA-4 to determine whether the monoclonal antibody to be tested can neutralize CTLA-4.

[0220] Various procedures known in the art can be used for the production of polyclonal or monoclonal antibodies directed against the proteins of the invention or against their derivatives, fragments, analogs, homologs, or orthologs (see, e.g., Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, incorporated herein by reference).

[0221] 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 that is the target of the desired immunoglobulin, 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).

[0222] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" or "monoclonal antibody composition" 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 a monoclonal antibody are identical in all molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of an antigen characterized by a unique binding affinity for it.

[0223] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent to induce lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro.

[0224] The immunizing agent may contain a protein antigen, a fragment thereof, or a fusion protein thereof. For example, peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if a non-human mammalian source is 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). The immortalized cell line may be a transformed mammalian cell, particularly a myeloma cell of rodent, bovine, and human origin. For example, a rat or mouse myeloma cell line is used. The hybridoma cells may be cultured in a suitable medium containing one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent growth of HGPRT-deficient cells.

[0225] Useful immortalized cell lines are those that fuse efficiently, maintain stable high-level expression of antibody by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Immortalized cell lines are, for example, mouse myeloma lines available 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 have also 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).

[0226] The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies against the antigen. For example, the binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis as described in Munson and Pollard, Anal. Biochem., 107:220 (1980). Furthermore, in therapeutic applications of monoclonal antibodies, it is important to identify antibodies that have a high degree of specificity and high binding affinity for the target antigen.

[0227] 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.

[0228] 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.

[0229] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Patent No. 4,816,567, which is 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 capable of specifically binding to genes encoding the heavy and light chains of a mouse antibody). The hybridoma cells of the invention serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain the synthesis of the monoclonal antibody in the recombinant host cells. The DNA can also 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. Pat. No. 4,816,567; Morrison, Nature 368,812-13 (1994)), or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be used in place of the constant domains of an antibody of the invention, or in place of the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0230] A fully human antibody is an antibody molecule in which the entire sequences of both the light and heavy chains, including, for example, the CDRs, arise from human genes. Such antibodies are referred to herein as "humanized antibodies" or "fully human antibodies." Human monoclonal antibodies, such as fully human antibodies and humanized antibodies, can be prepared by using trioma technology, human B-cell hybridoma technology (see Kozbor, et al, 1983 Immunol Today 4:72), and EBV hybridoma technology 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 are available 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 "MONOCLONAL ANTIBODIES AND CANCER THERAPY", Alan R. Liss, Inc., pp. 77-96).

[0231] A "humanized antibody" may be an antibody derived from a non-human species (such as a mouse) whose amino acid sequence (e.g., in the CDR regions) has been modified to increase 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. Furthermore, humanized antibodies can be produced in transgenic plants as a cheaper alternative to existing mammalian systems. For example, the transgenic plants can be tobacco plants, i.e., Nicotiania benthamiana, and Nicotiana tabaccum. Antibodies are purified from plant leaves. Stable transformation of plants can be achieved using Agrobacterium tumefaciens or particle gun technology. For example, a nucleic acid expression vector containing at least the heavy and light chain sequences is expressed via transformation in bacterial cultures, i.e., A. tumefaciens strain BLA4404. Infiltration of plants can be achieved by injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and centrifugation. Isolation and purification of antibodies can be easily performed by many methods known to those skilled in the art. Other methods of antibody production in plants are described, for example, in Fischer et al., Vaccine, 2003, 21:820-5, and Ko et al, Current Topics in Microbiology and Immunology, Vol. 332, 2009, pp. 55-78. Thus, the present invention further provides any cell or plant comprising a vector encoding or producing an antibody of the present invention.

[0232] Antibodies can be modified by, for example, CDR grafting (EP 239,400; 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. Patent 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 those skilled in the art to reduce the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (generally rodents) and improve activation of the human immune system (see, e.g., 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).

[0233] 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. After 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. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as 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).

[0234] Human antibodies can additionally be produced using transgenic non-human animals that are modified to produce fully human antibodies in response to challenge with an antigen rather than the animal's endogenous antibodies. (See WO 94 / 02602 and U.S. Pat. No. 6,673,986). The endogenous genes encoding heavy and light immunoglobulin chains in the non-human host are disabled, and active loci encoding human heavy and light immunoglobulin chains are inserted into the host's genome. Human genes are incorporated, for example, using yeast artificial chromosomes that contain the necessary human DNA segments. Animals that provide all the desired modifications are then obtained as progeny by mating intermediate transgenic animals that contain less than the full complement of modifications. An unrealistic example of such a non-human animal is the mouse, called Xenomouse™, as disclosed in WO 96 / 33735 and WO 96 / 34096. This animal produces B cells that secrete fully human immunoglobulins. Antibodies can be obtained directly from the animal after immunization with an immunogen of interest, e.g., as a polyclonal antibody preparation, or alternatively, from immortalized B cells derived from the animal, such as hybridomas that produce monoclonal antibodies. In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly or further modified to obtain antibody analogs, such as, for example, single chain Fv (scFv) molecules.

[0235] Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. 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., WO 98 / 24893, WO 96 / 34096, WO 96 / 33735, U.S. Patent 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 herein by reference in their entireties. Additionally, companies such as Creative BioLabs (Shirley, NY) can offer to provide human antibodies against a selected antigen using technology similar to that described above.

[0236] An example of a method for producing a non-human host, exemplified as a mouse, lacking expression of endogenous immunoglobulin heavy chains is disclosed in U.S. Patent No. 5,939,598. This can be obtained by a method comprising deleting a J segment gene from at least one endogenous heavy chain locus in an embryonic stem cell to prevent rearrangement of the locus and to prevent the formation of a transcript of the rearranged immunoglobulin heavy chain locus, the deletion being performed by a targeting vector containing a gene encoding a selectable marker, and producing a transgenic mouse from the embryonic stem cell, the somatic and germ cells of which contain a gene encoding a selectable marker.

[0237] One method for producing an antibody of interest, such as a human antibody, is disclosed in U.S. Patent No. 5,916,771. The method involves introducing an expression vector containing 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 the antibody containing the heavy and light chains.

[0238] In a further improvement of this procedure, methods for identifying clinically relevant epitopes on immunogens and correlative methods for selecting antibodies that immunospecifically bind with high affinity to the relevant epitopes are disclosed in WO 99 / 53049.

[0239] 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 those described in WO 93 / 64701, which have a targeting moiety (e.g., a ligand for a cell surface receptor) and a nucleic acid binding moiety (e.g., polylysine), viral vectors (e.g., DNA or RNA viral vectors), fusion proteins such as those described in International Application PCT / US95 / 02140 (WO 95 / 22618), which are fusion proteins containing a targeting moiety (e.g., an antibody specific for a target cell) and a nucleic acid binding moiety (e.g., protamine), plasmids, phages, viral vectors, and the like. Vectors can be chromosomal, non-chromosomal, or synthetic. Retroviral vectors can also be used, including Moloney murine leukemia virus. DNA viral vectors can also be used, including pox vectors such as orthopox or avipox vectors, herpes viral vectors such as herpes simplex I virus (HSV) vectors (see Geller, AI et al, J. Neurochem, 64:487 (1995); Lim, F., et al, DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, AI et al, Proc Natl. Acad. Sci.: USA 90, 7603 (1993); Geller, AI et al, Proc Natl. Acad. Sci USA 87:1149 (1990)), adenoviral vectors (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 viral vectors (Kaplitt, MG. et al, Nat. Genet. 8:148 (1994)).

[0240] Poxvirus vectors introduce genes into the cytoplasm of cells. Avipoxvirus vectors provide only short-term expression of nucleic acids. Adenovirus vectors, adeno-associated virus vectors, and herpes simplex virus (HSV) vectors can be used to introduce nucleic acids into neural cells. Adenovirus vectors provide shorter-term expression (about 2 months) than adeno-associated virus (about 4 months), which in turn is shorter than HSV vectors. The particular vector selected will depend on the target cell and the condition being treated. Introduction can be by standard techniques (e.g., infection, transfection, transduction, or transformation). Examples of modes of gene introduction include, for example, naked DNA, CaP04 precipitation, DEAE-dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

[0241] Vectors can be used to target essentially any desired target cell. For example, stereotactic injection can be used to direct vectors (e.g., adenovirus, HSV) to the desired location. In addition, particles can be delivered by intracerebroventricular (icv) injection using a minipump infusion system such as the SynchroMed Infusion System. A method based on bulk flow, called convection, has also proven effective in delivering large molecules to extended regions of the brain and may be useful for delivering vectors to target cells. (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 catheter, intravenous, parenteral, intraperitoneal, and subcutaneous injection, as well as oral or other known routes of administration.

[0242] These vectors can be used to express large amounts of antibodies that can be used in a variety of ways, for example to detect the presence of CTLA4 in a sample. The antibodies can also be used to attempt to bind and destroy CTLA4 activity.

[0243] In embodiments, the antibodies herein can be delivered to a subject using gene-based approaches. For example, the nucleotide sequences of the anti-CTLA4 antibodies described herein can be delivered to a subject using vectors, DNA or RNA. See, e.g., 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 improve and / or add functionality to allow for the design and generation of more complex antibody molecules.

[0244] Viral vectors such as adenovirus (Ad) and adeno-associated virus (AAV) have been engineered to express mAbs in vivo by replacing parts of their genome with nucleic acid sequences encoding antibodies of interest. The term "viral vector" can refer to a nucleic acid vector construct that contains at least one element of viral origin and has the ability to be packaged into a viral vector particle. The viral vector can contain a nucleic acid encoding an antibody, antigen-binding portion thereof, or CAR as described herein in place of a non-essential viral gene. The vector and / or particle can be utilized for the purpose of introducing any nucleic acid into cells, either in vitro or in vivo.

[0245] Retroviruses are common tools for gene delivery. In embodiments, retroviruses are used to deliver polynucleotides encoding antibodies to cells. The term "retrovirus" can refer to an RNA virus that reverse transcribes its genomic RNA into a linear double-stranded DNA copy and then covalently integrates its genomic DNA into the host genome. Once a virus is integrated into the host genome, it is called a "provirus." The provirus serves as a template for RNA polymerase II to direct the expression of RNA molecules that code for structural proteins and enzymes required to produce new viral particles.

[0246] Exemplary retroviruses suitable for use in certain embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend murine leukemia virus, murine stem cell virus (MSCV), Rous sarcoma virus (RSV), and lentiviruses.

[0247] The term "lentivirus" can refer to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (including human immunodeficiency virus, HIV type 1 and HIV type 2), Visna-Maedi virus (VMV), Caprine Arthritis Encephalitis Virus (CAEV), Equine Infectious Anemia Virus (EIAV), Feline Immunodeficiency Virus (FIV), Bovine Immunodeficiency Virus (BIV), and Simian Immunodeficiency Virus (SIV). In one embodiment, an HIV-based vector backbone (i.e., HIV cis-acting sequence elements) is preferred. In certain embodiments, lentiviruses are used to deliver polynucleotides, including CAR, to cells.

[0248] Genetic transfer of the antibody nucleotide sequence itself as DNA or RNA is an alternative option for transient in vivo antibody production. For example, the plasmid DNA approach can utilize electroporation components to increase transfection efficiency after IM delivery. Also, mRNA delivery can be used for in vivo expression of the antibody. In embodiments, the mRNA encoding the antibody of interest can be modified, such as optimizing mRNA codon usage, and lipid nanoparticles can be used for mRNA delivery.

[0249] In one embodiment, the antibodies described herein may be full-length antibodies that include an Fc region similar to a wild-type Fc region that binds to an Fc receptor.

[0250] Techniques can be adapted for the production of single chain antibodies specific for antigenic proteins of the present invention (see, e.g., U.S. Pat. No. 4,946,778). In addition, methods can be used to produce monoclonal antibodies with the desired specificity for a protein or a derivative, fragment, analog or homolog thereof. ab To allow for rapid and effective identification of fragments, ab Methods can be adapted for the construction of expression libraries (see, e.g., Huse, et al, 1989 Science 246:1275-1281). Antibody fragments containing the idiotype to a protein antigen can be produced by techniques known in the art, including, but not limited to: (i) F produced by pepsin digestion of the antibody molecule. (ab’)2 Fragment, (ii)F (ab’)2 F produced by reducing the disulfide bridges of the fragment ab (iii) F fragments produced by treating antibody molecules with papain and a reducing agent. ab fragments, and (iv) F v piece.

[0251] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. Such antibodies can, for example, target immune system cells to unwanted cells (see U.S. Pat. No. 4,676,980) and can be for the treatment of HIV infection (see WO 91 / 00360, WO 92 / 20373). Antibodies can be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using disulfide exchange reactions or by forming thioether bonds. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate, and those disclosed, for example, in U.S. Pat. No. 4,676,980.

[0252] The antibodies of the invention can be modified with respect to effector function, for example, to enhance the effectiveness of the antibody in treating cancer. For example, cysteine ​​residues 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 antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Alternatively, antibodies can be engineered with dual Fc regions, thereby having enhanced complement lysis and ADCC capabilities. (See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989)). In one embodiment, the antibodies of the invention have a modification of the Fc region such that the Fc region does not bind to Fc receptors. For example, the Fc receptor is an Fcγ receptor. Antibodies with modifications of the Fc region such that the Fc region does not bind Fcγ but still binds to neonatal Fc receptors are useful as described herein.

[0253] In certain embodiments, the antibodies of the present invention may include Fc variants that contain amino acid substitutions that modify the antigen-independent effector functions of the antibody, in particular the circulating half-life of the antibody. Such antibodies, when compared to antibodies lacking these substitutions, exhibit either increased or decreased binding to FcRn and therefore have increased or decreased serum half-life, respectively. Fc variants with improved affinity for FcRn are expected to have longer serum half-lives, and such molecules have useful applications in methods of treating mammals where a long half-life of the administered antibody is desired, for example, to treat chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity are expected to have shorter resting periods, and such molecules may also be useful, for example, for administration to mammals where a shortened circulation time may be advantageous, for example, in in vivo imaging, or in situations where the starting antibody has toxic side effects if present in the circulation for an extended period of time. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta, and thus are also useful in treating diseases or disorders in pregnant women. In addition, other applications in which reduced FcRn binding affinity may be desirable include applications in which localization to the brain, kidney, and / or liver is desirable. In one embodiment, the Fc variant-containing antibody may exhibit reduced transport from the vasculature across the epithelium of the renal glomerulus. In another embodiment, the Fc variant-containing antibody may exhibit reduced transport from the brain across the blood-brain barrier (BBB) ​​into the vascular space. In one embodiment, the antibody with altered FcRn binding comprises an Fc domain with one or more amino acid substitutions in the "FcRn binding loop" of the Fc domain. The FcRn binding loop is composed of amino acid residues 280-299 (according to EU numbering). Exemplary amino acid substitutions with altered FcRn binding activity are disclosed in WO 05 / 047327, which is incorporated herein by reference. In certain exemplary embodiments, an antibody of the invention, or a fragment thereof, comprises an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0254] In some embodiments, mutations are introduced into the constant region of the mAb such that the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the mAb is modified. For example, the mutation is a LALA mutation in the CH2 domain. In one embodiment, the antibody (e.g., a human mAb, or a bispecific Ab) contains a mutation on one scFv unit of the heterodimeric mAb that reduces the ADCC activity. In another embodiment, the mAb contains mutations on both chains of the heterodimeric mAb that completely eliminates the ADCC activity. For example, the mutation introduced into one or both scFv units of the mAb is a LALA mutation in the CH2 domain. These mAbs with variable ADCC activity can be optimized such that the mAb shows maximum selective killing towards cells expressing one antigen recognized by the mAb, but minimal killing towards a second antigen recognized by the mAb.

[0255] In other embodiments, the antibodies of the invention for use in the diagnostic and therapeutic methods described herein have a constant region, such as, for example, an IgG1, IgG2, or IgG4 heavy chain constant region, which may be altered to reduce or eliminate glycosylation. For example, the antibodies of the invention may also include Fc variants comprising amino acid substitutions that alter the glycosylation of the antibody. For example, the Fc variants may have reduced glycosylation (e.g., N-linked or O-linked glycosylation). In some embodiments, the Fc variants have reduced glycosylation of the N-linked glycan normally found at amino acid position 297 (EU numbering). In another embodiment, the antibody comprises an amino acid substitution near or within a glycosylation motif, such as an N-linked glycosylation motif comprising the amino acid sequence NXT or NXS. In a specific embodiment, the antibody comprises an Fc variant comprising an amino acid substitution at amino acid position 228 or 299 (EU numbering). In a more specific embodiment, the antibody comprises an IgG1 or IgG4 constant region comprising S228P and T299A mutations (EU numbering).

[0256] Exemplary amino acid substitutions that reduce or alter glycosylation are disclosed in WO 05 / 018572, which is incorporated herein by reference. In some embodiments, the antibodies or fragments thereof of the invention are modified to eliminate glycosylation. Such antibodies or fragments thereof may be referred to as "agly" antibodies or fragments thereof (e.g., "agly" antibodies). Without wishing to be bound by theory, "agly" antibodies or fragments thereof may have improved safety and stability profiles in vivo. An exemplary agly antibody, or fragment thereof, comprises a deglycosylated Fc region of an IgG4 antibody that lacks Fc effector functions, thereby eliminating the possibility of Fc-mediated toxicity to normal living tissues and cells expressing CTLA4. In yet other embodiments, the antibodies or fragments thereof of the invention comprise altered glycans. For example, the antibodies may have a reduced number of fucose residues on the N-glycan at Asn297 of the Fc region, i.e., are defucosylated. In another embodiment, the antibody can have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.

[0257] The present invention is also directed to immunoconjugates comprising antibodies conjugated to cytotoxic agents such as toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or to radioisotopes (the latter being radioconjugates).

[0258] 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, mitgellin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Non-limiting examples include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Re is an example.

[0259] Conjugates of antibodies and cytotoxic agents 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 triene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al, Science 238:1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotides to antibodies (see WO 94 / 11026 and U.S. Pat. No. 5,736,137).

[0260] Those of skill in the art will recognize that a wide variety of possible moieties may be attached to a given antibody or other molecule of the invention (see, e.g., "Conjugate Vaccines," Contributions to Microbiology and Immunology, JM Cruse and RE Lewis, Jr (eds), Carger Press, New York, (1989), incorporated herein by reference in its entirety.

[0261] The binding can be achieved by any chemical reaction that will link the two molecules, so long as the antibody and the other moiety retain their respective activities. The binding can include many chemical mechanisms, such as covalent binding, affinity binding, intercalation, coordinate binding, complex formation. In one embodiment, the binding is a covalent bond. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of an external cross-linking molecule. Many bivalent or multivalent binding agents are useful for linking protein molecules, such as the antibodies of the present invention, to other molecules. For example, representative binding agents can include organic compounds such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene, and hexamethylenediamine. This list is not intended to be exhaustive of the various classes of binding agents known in the art, but rather is illustrative of the more common binding 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. (For example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describes the use of MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester).) See also U.S. Pat. No. 5,030,719, which describes the use of halogenated acetylhydrazide derivatives coupled to antibodies via oligopeptide linkers.Non-limiting examples of useful linkers that can be used with the antibodies of the invention include (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride, (ii) SMPT (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog (21558G), (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., catalog number 21651G), (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propianamide]hexanoate (Pierce Chem. Co., catalog number 2165-G), and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co., catalog number 2165-G) conjugated to EDC. Chem. Co., catalog number 24510).

[0262] The linkers described herein contain components with different attributes, thus resulting in conjugates with different physicochemical 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. Furthermore, the linker SMPT can contain sterically hindered disulfide bonds to form conjugates with improved stability. Disulfide bonds are generally less stable than other bonds, because they are cleaved in vitro, resulting in fewer available conjugates. In particular, sulfo-NHS can enhance the stability of carbodiimide bonds. Carbodiimide bonds (such as EDC) when used in combination with sulfo-NHS form esters that are more resistant to hydrolysis than carbodiimide bond reactions alone.

[0263] The antibody disclosed herein can also be formulated as an immunoliposome.The liposome containing the antibody is prepared by methods known in the art, such as those 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. Patent Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[0264] 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 generate liposomes with the desired diameter. Fab' fragments of the antibody of the invention can be coupled to liposomes via a disulfide exchange reaction as described in Martin et al., J.Biol.Chem.,257:286-288 (1982).

[0265] Multispecific antibodies (bispecific and trispecific)

[0266] A multispecific antibody is an antibody that can recognize two or more different antigens. For example, a bispecific antibody (bsAb) is an antibody that contains two variable domains or scFv units such that the resulting antibody recognizes two different antigens. For example, a trispecific antibody (tsAb) is an antibody that contains two variable domains or scFv units such that the resulting antibody recognizes three different antigens. The present invention provides multispecific antibodies, such as bispecific and trispecific antibodies, that recognize CTLA-4 and a second and / or third antigen. In one embodiment, the multispecific antibodies (e.g., bispecific and trispecific antibodies) can include a CTLA-4 specific fusion protein that includes 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 Accession No. NP_000873.2; IL-12B (p40 subunit) protein sequence having NCBI Accession No. NP_002178.2; IL-18 (protein sequence having NCBI Accession No. NP_001553.1); IL-15 (protein sequence having NCBI Accession No. NP_000576.1); IL-7 (protein sequence having NCBI Accession No. NP_000871.1); IL-2 (protein sequence having NCBI Accession No. NP_000577.2); and IL-21 (protein sequence having NCBI Accession No. NP_068575.1), cytokine cognate receptors (e.g., 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 a CTLA-4 fusion protein.For example, the fusion protein can include an antibody comprising a variable domain or scFv unit as described herein, and a ligand or antigen and / or a third ligand or antigen, such that the resulting antibody recognizes the antigen and binds to a ligand-specific receptor. Exemplary antibody compositions (e.g., VH and / or VL sequences or fragments thereof) that are useful in designing the CTLA-4 fusion proteins described herein include the anti-CAIX antibodies described in International Applications PCT / US2006 / 046350 and PCT / US2015 / 067178, the anti-CXCR4 antibodies described in PCT / US20006 / 005691, the anti-CCR4 antibodies described in PCT / US2008 / 088435, PCT / US2013 / 039744, and PCT / US2015 / 054202, the anti-TCR4 antibodies described in PCT / US2008 / 088435, PCT / US2013 / 039744, and PCT / US2015 / 054202, and the anti-TCR4 antibodies described in PCT / US2008 / 088435, PCT / US2013 / 039744, and PCT / US2015 / 054202. 435 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-MUC1 antibodies described in PCT / US2020 / 037783 (each of which applications is incorporated by reference in its entirety). 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 and trispecific antibodies, such as fusion proteins comprising an antibody and a ligand that recognize CTLA-4) are described herein.Ligands include tumor associated antigens (e.g., LINGO1, ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), MUC1, MSLN, CD19, 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 cancer tumor antigen-1 (PCTA-1), GAGE, MAGE, mesothelin, β-catenin, TGF-βRII, BRCA1 / 2, SAP-1, HPV-E6, HPV-E7 (and for additional tumor associated surface antigens see International Application PCT Publication No. PCT-2003 / 013393, which is incorporated by reference in its entirety). cytokines (e.g., IL-12 (IL-12A (p35 subunit) protein sequence having NCBI Accession No. NP_000873.2; IL-12B (p40 subunit) protein sequence having NCBI Accession No. NP_002178.2; IL-18 (IL-18A (p35 subunit) protein sequence having NCBI Accession No. NP_002178.2; IL-18B (p40 subunit) protein sequence having NCBI Accession No. NP_001 553.1); IL-15 (protein sequence having NCBI accession number NP_000576.1); IL-7 (protein sequence having NCBI accession number NP_000871.1); IL-2 (protein sequence having NCBI accession number NP_000577.2); and IL-21 (protein sequence having NCBI accession number 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. Bispecific or trispecific antibodies in various formats are also provided herein.In some embodiments, each of the anti-CTLA-4 fragment and the second antigen-specific fragment and / or the third antigen-specific fragment are 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 comprises an Fc fragment (e.g., as described in PCT / US2015 / 021529 and PCT / US2019 / 023382, each of which is incorporated by reference in its entirety). The bispecific or trispecific antibody of the invention can comprise a combination of heavy and light chains or scFvs of the CTLA-4 antibodies described herein.

[0267] Multispecific antibodies (e.g., bispecific and trispecific antibodies) of the invention (e.g., anti-CTLA-4-scFv fusion proteins) can be constructed using methods known in the art. In some embodiments, bispecific antibodies are single polypeptides in which two scFv fragments are linked by a long linker polypeptide, of sufficient length to allow intramolecular association between the two scFv units to form the antibody. In other embodiments, bispecific antibodies are two or more polypeptides linked by covalent or non-covalent bonds. In some embodiments, the amino acid linker shown 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., GGGGSGGGGSGGGGGS (SEQ ID NO: [])), "(G4S)4" (e.g., GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: []), "(G4S)5" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGGS (SEQ ID NO: [])), "(G4S)6" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: [])), "(G4S)7" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: [])), etc. For example, the use of a (G4S)5 linker can provide greater flexibility to the ligands 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) nwhere n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Non-limiting examples of linkers known to those of skill in the art that can be used to construct the fusions described herein can be found in U.S. Pat. No. 9,708,412, U.S. Patent Application Publication Nos. 20180134789 and 20200148771, and WO 2019051122, each of which is incorporated by reference in its entirety.

[0268] In another embodiment, multispecific antibodies (e.g., bispecifics and trispecifics 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, Ig heavy chains of two different variable domains are reduced to selectively cleave heavy chain pairing while preserving the heavy-light chain pairing. Two heavy-light chain heterodimers recognizing two or three different antigens / ligands are mixed to promote heterologous ligand binding pairing mediated through engineered "knob-in-hole" of the CH3 domain.

[0269] In another embodiment, multispecific antibodies (e.g., bispecific and trispecific antibodies, such as anti-CTLA-4-scFv fusions) can be constructed by exchanging heavy-light chain dimers from two or more different antibodies to generate hybrid antibodies, where a first heavy-light chain dimer recognizes CTLA-4 and a second heavy-light chain dimer recognizes a second antigen and / or a third antigen. The mechanism of heavy-light chain dimerization is similar to the formation of human IgG4, which also functions as a bispecific molecule. Dimerization of IgG heavy chains is driven by intramolecular forces, such as pairing of the CH3 domains of each heavy chain with disulfide bridges. The presence of a specific amino acid in the CH3 domain (R409) has been shown to promote dimer exchange and assembly of IgG4 molecules. Heavy chain pairing is also further stabilized by inter-heavy 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 (compared to the stable IgG1 hinge region which contains the sequence Cys-Pro-Pro-Cys) at amino acids 226 to 230. This difference in the sequence of serine at position 229 has been linked to the propensity of IgG4 to form intrachain disulfides in the hinge region (Van der Neut Kolfschoten, M. et al, 2007, Science 317:1554-1557 and Labrijn, AF et al, 2011, Journal of Immunol 187:3238-3246).

[0270] Multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-CTLA-4-scFv fusions) of the invention can be made through the introduction of the R409 residue in the CH3 domain and a Cys-Pro-Ser-Cys sequence in the hinge region of an antibody that recognizes CTLA-4 or a second and / or third antigen, such that the heavy-light chain dimers are swapped to produce an antibody molecule with one heavy-light chain dimer that recognizes CTLA-4 and a second heavy-light chain dimer that recognizes a second and / or third antigen, the second and / or third antigen (or ligand) being any antigen (or ligand) disclosed herein. Known IgG4 molecules can also be modified such that the heavy and light chains recognize CTLA-4 or a second and / or third antigen as disclosed herein. The use of this method to construct the multispecific antibodies of the invention (e.g., bispecific and trispecific antibodies such as anti-CTLA-4-scFv fusions) can be beneficial due to the unique characteristics of the IgG4 molecule, whose 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 particular property makes these IgG4-based multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-CTLA-4-scFv fusions) attractive for therapeutic applications, where the antibody is required to bind to a target and functionally modify a signaling pathway associated with the target, but does not induce effector activity.

[0271] The multispecific antibodies described herein (e.g., bispecific and trispecific antibodies such as anti-CTLA-4-scFv fusions) can be engineered with a non-depleting heavy chain isotype, such as IgG1-LALA or stabilized IgG4 or one of the other non-depleting variants. In some embodiments, mutations are introduced into the constant region of the bsAb such that the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the bsAb is modified. For example, the mutation is a LALA mutation in the CH2 domain. In one aspect, the multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-CTLA-4-scFv fusions) contain a mutation on one scFv unit of the heterodimeric multispecific antibody that reduces the ADCC activity. In another aspect, the multispecific antibodies (e.g., bispecific and trispecific antibodies such as anti-CTLA-4-scFv fusions) contain a mutation on both chains of the heterodimeric multispecific antibody that completely eliminates the ADCC activity. For example, the mutation introduced into one or both scFv units of a multispecific antibody (e.g., bispecific antibodies and trispecific antibodies such as anti-CTLA-4-scFv fusions) is a LALA mutation 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 antibody shows maximum selective killing towards cells expressing one antigen recognized by the multispecific antibody, but minimum killing towards the second antigen recognized by the multispecific antibody.

[0272] The multispecific antibodies (e.g., bispecific antibodies) described herein can be engineered as modular tetrameric bispecific antibodies (tBsAbs). See, e.g., WO2018 / 071913, which is incorporated herein by reference in its entirety. For example, the tetravalent antibody can be a dimer of bispecific scFv fragments comprising 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 the first antigen. In embodiments, the anti-CTLA-4 antibody can be the second binding site for the second antigen. The two binding sites can be linked together via a linker domain. In embodiments, the scFv fragment is a tandem scFv, and the linker domain comprises an immunoglobulin hinge region (e.g., an IgG1, IgG2, IgG3, or IgG4 hinge region) amino acid sequence. In embodiments, the immunoglobulin hinge region amino acid sequence can be, for example, the linker amino acid sequence (GGGS) x1-6 , (GGGGS) x1-6 , or GSAGSAAGSGEF. In embodiments, the linker domain comprises at least a portion of an immunoglobulin Fc domain, such as an IgG1, IgG2, IgG3, or IgG4 Fc domain. In embodiments, at least a portion of the immunoglobulin Fc domain does not comprise a CH2 domain. In embodiments, at least a portion of the immunoglobulin Fc domain can be a CH2 domain. An exemplary CH2 domain amino acid sequence comprises APELLGGPDVFLF (SEQ ID NO: [ ]). The Fc domain can be linked to the C-terminus of an immunoglobulin hinge region (e.g., an IgG1, IgG2, IgG3, or IgG4 hinge region) amino acid sequence. The linker domain can be flanked at one or both ends by a flexible linker amino acid sequence (e.g., (GGGS) x1-6 , (GGGGS) x1-6 , or GSAGSAAGSGEF (SEQ ID NO:[])).

[0273] 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 include an IgG3CH2 domain. In other embodiments, the multispecific antibodies (e.g., bispecific antibodies) are engineered without an Fc domain and / or Fc fragment.

[0274] In embodiments, the tBsAb is specific for CTLA-4 and may also be specific for 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.

[0275] The multispecific antibodies disclosed herein (e.g., bispecific and trispecific antibodies such as anti-CTLA-4-scFv fusions) may be useful in the treatment of chronic infections, diseases, or medical conditions, e.g., cancer.

[0276] Use of antibodies against CTLA-4

[0277] The antibodies of the present invention that specifically bind to CTLA-4 protein or fragments thereof can be administered for the treatment of CTLA-4-related diseases or disorders. "CTLA-4-related diseases or disorders" include disease states and / or symptoms associated with disease states in which there is an increase in the level of CTLA-4 and / or activation of cell signaling pathways involving CTLA-4. 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, squamous cell carcinoma of the skin, melanoma, renal cancer, e.g., clear cell renal cell carcinoma, or gastric cancer.

[0278] The antibodies of the present invention, such as bispecific, polyclonal, monoclonal, humanized and fully human antibodies, can be used as therapeutic agents. Such agents will generally be used to treat or prevent cancer in subjects, improve vaccine efficiency, or enhance natural immune response. Antibody preparations, for example, those with high specificity and high affinity for their target antigens, will generally be administered to subjects to produce effects due to binding with the target. Administration of the antibody can neutralize, inhibit, or interfere with the activity of CTLA-4 protein.

[0279] Pharmaceutical Compositions

[0280] The antibody specifically binding to the CTLA-4 protein or its fragment of the present invention can be administered for the treatment of cancer in the form of a pharmaceutical composition.The principles and considerations involved in preparing therapeutic pharmaceutical compositions containing antibodies, as well as guidance in the selection of ingredients, 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.

[0281] The specific dosage and treatment regimen for a particular patient will depend on a variety of factors, such as the specific antibody, variant or derivative thereof used, the patient's age, weight, general health, sex, and diet, as well as the time of administration, frequency of excretion, drug combinations, and the severity of the particular disease being treated. The judgment of such factors by a medical practitioner is within the skill of the art. The amount will also depend on the individual patient being 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 pharmaceutical and pharmacokinetic principles well known in the art.

[0282] A therapeutically effective amount of an antibody of the invention can be the amount necessary to achieve a therapeutic goal. As mentioned above, this can be a binding interaction between the antibody and its target antigen, which in certain cases interferes with the function of the target. The amount that needs to be administered further depends on the binding affinity of the antibody for its specific antigen, and also on the rate at which the administered antibody is depleted from the free volume of the other subject to which it is administered. The dosage of an antigen-binding polypeptide described herein administered to a subject (e.g., a patient) is typically 0.1 mg / kg to 100 mg / kg of patient body weight, 0.1 mg / kg to 20 mg / kg of patient body weight, or 1 mg / kg to 10 mg / kg of patient body weight. Human antibodies have a longer half-life in the human body than antibodies from other species due to the immune response to the foreign polypeptide. Thus, it is often possible to administer human antibodies at lower dosages and less frequently. Additionally, the dosage and frequency of administration of the antibodies of the present disclosure can be reduced by enhancing antibody uptake and tissue (e.g., brain) penetration by modifications such as, for example, lipidation. A typical range for therapeutically effective administration of an antibody or antibody fragment of the invention can be, by way of non-limiting example, from about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical administration frequency can range, for example, from twice daily to once a week.

[0283] When an antibody fragment is used, the smallest inhibitory fragment that specifically binds to the binding domain of the target protein is preferred. For example, peptide molecules that retain the ability to bind to the target protein sequence can be designed based on the variable region sequence of the antibody. Such peptides can be chemically synthesized and / or produced by recombinant DNA technology. (See, for example, Marasco et al, Proc. Natl. Acad. Sci. USA, 90:7889-7893 (1993)). The formulation can also contain two or more active compounds as necessary for the specific indication to be treated, e.g., those with complementary activities that do not adversely affect each other. Alternatively, or in addition, the composition can contain an agent that enhances its function, such as a cytotoxic agent, a cytokine (e.g., IL-15), a chemotherapeutic agent, or a growth inhibitory agent. Such molecules are suitably present in combination in an amount effective for the intended purpose.

[0284] The active ingredients can also be encapsulated in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions, respectively.

[0285] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0286] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing antibodies, which matrices are in the form of shaped articles, such as films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow the release of molecules for over 100 days, while certain hydrogels release proteins for shorter periods of time.

[0287] The antibody or agent of the present invention (also referred to herein as "active compound"), as well as their derivatives, fragments, analogs, and homologs, can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include the antibody or agent and a pharma- ceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles, such as liposomes and fixed oils, can also be used. The use of such media and agents for pharma- ceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, its use in the composition is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0288] The pharmaceutical composition of the present 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 contain the following components: a sterile diluent, e.g., water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparabens, 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 adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0289] 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 dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In embodiments, the composition is sterile and fluid to the extent that easy syringability 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. 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 dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, sodium chloride, can be included in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0290] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent with one or combination of the ingredients listed above as necessary, followed by filtration sterilization.For example, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion solvent and other necessary ingredients listed above.In the case of sterile powder for preparing sterile injectable solution, the preparation method is vacuum drying and freeze-drying, which produces powder of active ingredient and any additional desired ingredients from the solution that has been previously sterile filtered.

[0291] Oral compositions include inert diluents or edible carriers. 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, where the compound in the fluid carrier is applied orally, swirled in the mouth, 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 may contain any of the following: 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 corn starch; a lubricant such as magnesium stearate or sterote; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring ingredient such as peppermint, methyl salicylate, or orange flavoring, or compounds of a similar nature.

[0292] 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.

[0293] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is 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 achieved through the use of nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams generally known in the art.

[0294] The compounds can also be prepared in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0295] In one embodiment, the active compound is prepared in a carrier 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 such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are clear to those skilled in the art. Materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharma-ceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0296] For the convenience of administration and uniformity of dosage, oral or parenteral compositions can be formulated in dosage unit form.The dosage unit form used herein refers to a physically separate unit suitable as a unitary dose for the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with the required pharmaceutical carrier.The specification of the dosage unit form of the present invention is determined and directly depends on the specific characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the inherent limitations of the technology of compounding such active compound for the treatment of an individual.

[0297] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0298] diagnosis

[0299] The antibodies according to the invention can be used as agents for detecting the presence of CTLA-4 (or a protein fragment thereof) in a sample. For example, the antibody can include a detectable label. The antibody can be polyclonal or monoclonal. The intact antibody, or a fragment thereof (e.g., F ab , scFv, or F (ab)2 ) can be used. With respect to a probe or antibody, the term "labeled" can encompass direct labeling of the probe or antibody by binding (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 a DNA probe with biotin so 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 biological fluids present within a subject. Thus, the use of the term "biological sample" includes blood, and fractions or components of blood, including serum, plasma, or lymph. That is, the detection method of the present invention can be used to detect analyte mRNA, protein, or genomic DNA in a biological sample in vitro and in vivo. For example, in vitro techniques for detection of analyte mRNA include Northern hybridization and in situ hybridization. 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.

[0300] Procedures for performing 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. Additionally, in vivo techniques for detecting 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 the subject can be detected by standard imaging techniques.

[0301] Antibodies against the CTLA-4 protein (or fragments thereof) can be used in methods known in the art relating to localization and / or quantification of the CTLA-4 protein (e.g., for use in measuring levels of CTLA-4 protein in an appropriate physiological sample, for use in diagnostic methods, for use in protein imaging). In certain embodiments, antibodies specific for the CTLA-4 protein, or derivatives, fragments, analogs, or homologs thereof, that contain an antigen-binding domain derived from the antibody, are utilized as pharma- ceutical active compounds (hereinafter referred to as "therapeutic agents").

[0302] Antibodies specific for the CTLA-4 protein of the invention can be used to isolate CTLA-4 polypeptides by standard techniques, such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies to the CTLA-4 protein (or fragments thereof) can be used diagnostically to monitor protein levels in tissues as part of a clinical trial procedure, for example, to determine the effectiveness of a given therapeutic regimen.

[0303] Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-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; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include, but are not limited to, 125 I, 131 I, 35 S, 32 P, or 3 Examples include H.

[0304] cell therapy

[0305] Cell therapy is also provided herein. For example, cell therapy can include cells, such as T cells, NK cells, or B cells, that are genetically engineered to express the anti-CTLA4 antibodies described herein. "Genetically engineered cells" can refer to any cell of any organism that has been modified, transformed, or engineered by the addition or modification of a gene, 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 DNA or RNA sequences encoding the antibodies described herein, and / or chimeric receptors or chimeric receptor complexes expressed on the cell surface.

[0306] A "T cell" is a cell of the immune system that matures in the thymus and produces a T cell receptor (TCR). T cells are naive cells (those that have not been exposed to an antigen; TCM increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA and decreased expression of CD45RO compared to normal T cells), and memory T cells (T M ) (contacted with antigen, long-lived), and effector cells (contacted with antigen, cytotoxic). M There is a further subgroup of central memory T cells (T CM , which increase expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95 and decrease expression of CD54RA compared to naive T cells) and effector memory T cells (T EM , naive T cells or T CM They can be subdivided into effector T cells (T E ) is T CM CD8+ can refer to cytotoxic T lymphocytes that have been in contact with an antigen and have reduced expression of CD62L, CCR7, and CD28 compared to CD8+, and are positive for granzymes and perforin.

[0307] T cells can be obtained from many sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural fluid, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a volume of blood drawn from a subject using many techniques known to those skilled in the art, such as FICOLL™ separation. In one embodiment, cells are obtained from the circulating blood of a subject by apheresis. The product of apheresis typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and placed in an appropriate buffer or medium for further processing. In one embodiment of the present invention, the cells are washed with PBS. In alternative embodiments, the wash solution may be free of calcium and / or magnesium, or free of many, if not all, divalent cations. As will be appreciated by those of skill in the art, the washing step may be accomplished by methods known to those of skill in the art, for example, by using a semi-automated flow-through centrifuge, such as a Cobe 2991 cell processor, a Baxter CytoMate, etc. After washing, the cells may be resuspended in a variety of biocompatible buffers or other saline solutions with or without buffers. In certain embodiments, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0308] In embodiments, T cells are isolated from PBMCs. PBMCs can be isolated from buffy coats obtained by density gradient centrifugation of whole blood, for example, centrifugation through a LYMPHOPREP™ gradient, a PERCOLL™ gradient, or a FICOLL™ gradient. T cells can be isolated from PBMCs by depleting monocytes, for example, using CD14 DYNABEADS®. In some embodiments, red blood cells can be lysed prior to density gradient centrifugation.

[0309] "NK cells" are large granular lymphocytes and cytotoxic lymphocytes derived from a common lymphocyte precursor that does not naturally contain an antigen-specific receptor (e.g., T cell receptor or B cell receptor). NK cells may be characterized by their CD3-, CD56+ phenotype. In embodiments, NK cells may refer to any known NK cell or any NK-like cell, or any cell having characteristics of an NK cell. Thus, primary NK cells may be used, or in another embodiment, previously isolated and cultured NK cells known in the art may be used. Thus, NK cell lines may be used. A number of different NK cells are known and reported in the literature, any of which may be used, or cell lines may be prepared from primary NK cells, for example, by viral transformation (Vogel et al. 2014, Leukemia 28:192-195). Suitable NK cells include, but are in no way limited to, NK-YS, NK-YT, MOTN-1, NKL, KHYG-1, HANK-1, or NKG cell lines in addition to NK-92. Variants of other cell lines can also be used.

[0310] A "B cell" is a B lymphocyte that can produce antibody molecules after activation. Activation of a B cell can be, but is not limited to, 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 been previously activated, or a "memory" B lymphocyte that has been previously activated, or the progeny of such a B lymphocyte.

[0311] Also provided herein are cell therapies such as chimeric antigen receptor (CAR) T cell therapy or chimeric antigen receptor (CAR) NK cell therapy. CAR T cell therapy redirects a patient's T cells and NK cells to kill tumor cells, for example, by exogenous expression of CAR on T cells or NK cells. For example, when expressed in T cells or NK cells, CAR has the ability to redirect the specificity and reactivity of T cells or NK cells to non-MHC-restricted selected targets, utilizing the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition gives CAR-expressing T cells or NK cells the ability to recognize antigens independent of antigen processing, thus avoiding a major mechanism of tumor evasion.

[0312] CARs can be transmembrane fusion proteins linking the antigen recognition domain of an antibody to the intracellular signaling domains of a T cell receptor and co-receptor. For example, suitable cells capable of secreting the anti-CTLA-4 antibodies of the invention (or alternatively engineered to express the secreted anti-CTLA-4 antibodies described herein) can be used. The secreted anti-CTLA-4 "payload" can be, for example, a minibody, scFv, IgG molecule, bispecific fusion molecule, and other antibody fragments described herein. After contact or engineering, the cells described herein can be introduced into a patient in need of treatment by infusion therapy known to those skilled in the art. The patient can have a CTLA-4-related disease or disorder described herein, such as a chronic infection or cancer. The cells (e.g., T cells or NK cells) can be, for example, but not limited to, T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof. Exemplary CARs and CAR factories useful in aspects of the invention include, for example, those disclosed in PCT / US2015 / 067225 and PCT / US2019 / 022272, each of which is incorporated herein by reference in its entirety. In one embodiment, the CTLA-4 antibodies discussed herein can be used in the construction of multispecific antibodies or as a payload for engineered cells such as CAR-T cells, CAR NK cells, or engineered B cells. For example, in one embodiment, the anti-CTLA-4 antibodies discussed herein can be used for targeting CARS (i.e., as a targeting moiety). In another embodiment, the anti-CTLA-4 antibodies discussed herein can be used as a targeting moiety and different CTLA-4 antibodies targeting different epitopes can be used as a payload. In another embodiment, the payload can be an immunomodulatory antibody payload.

[0313] Treatment method

[0314] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or slow down (alleviate) undesirable physiological changes or disorders, such as the progression of cancer. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction in extent of disease, stable (i.e., not worsening) state of disease, delay or slowing of disease progression, improvement or alleviation of disease symptoms, remission (partial or total), whether detectable or not. "Treatment" can refer to prolonging survival compared to expected survival in the absence of treatment. Those in need of treatment include those already suffering from a disease condition or disorder, as well as those susceptible to a disease condition or disorder, or those in need of prevention of a disease condition or disorder.

[0315] The present invention provides both preventative and therapeutic methods of treating subjects at risk (or susceptible) for cancer (e.g., when an early detection cancer biomarker is identified in such subjects), or other cell proliferation-related diseases or disorders. Such diseases or disorders include, but are not limited to, diseases or disorders associated with aberrant expression of CTLA-4. For example, the methods are used to treat, prevent, or alleviate the symptoms of cancer. In one embodiment, the methods are used to treat, prevent, or alleviate the symptoms of solid tumors. Non-limiting examples of other tumors that may be treated by the compositions described herein include lung cancer (e.g., non-small cell lung cancer or lung adenocarcinoma), gastric cancer, colon cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain tumor, 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 gastric cancer. Furthermore, the method of the present invention can be used to treat blood cancers such as leukemia and lymphoma. Alternatively, the method can be used to treat, prevent, or alleviate the symptoms of metastasized cancer. For example, cancers that can be treated or prevented or symptoms can be alleviated include B-cell chronic lymphocytic leukemia (CLL), non-small cell lung cancer, melanoma, ovarian cancer, lymphoma, or renal cell carcinoma. Cancers that can be treated or prevented or symptoms can be alleviated also include solid tumors with high mutation burden and WBC in filtrate.

[0316] Thus, in one aspect, the invention provides a method for preventing, treating, or alleviating a symptomatic cancer or cell proliferative disease or disorder in a patient by administering to the patient a monoclonal antibody, scFv antibody, or bispecific antibody of the invention. For example, an anti-CTLA-4 antibody can be administered in a therapeutically effective amount.

[0317] Subjects at risk for cancer or a cell proliferation-related disease or disorder can include patients with a family history of cancer or subjects who have been exposed to agents known or suspected of causing cancer. Administration of a prophylactic agent can occur prior to the manifestation of cancer, such that the disease is prevented or, alternatively, its progression is delayed.

[0318] In another embodiment, tumor cell growth is inhibited by contacting the cell with an anti-CTLA4 antibody of the invention. The cell can be any cell that expresses CTLA-4.

[0319] The present invention also includes a method of increasing or enhancing an immune response to an antigen. The immune response is increased or enhanced by administering to a subject a monoclonal antibody, scFv antibody, or bispecific antibody of the present invention. The immune response is enhanced, for example, by enhancing 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 the result of an infection. The infection is a chronic infection. The increase or enhancement of the immune response to an antigen can be measured by many methods known in the art. For example, the 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 by vaccination.

[0320] Thus, in another aspect, the invention provides a method of increasing vaccine efficacy by administering to a subject a monoclonal or scFv antibody of the invention and a vaccine. The antibody and vaccine are administered sequentially or simultaneously. The vaccine is a tumor vaccine, a bacterial vaccine, or a viral vaccine.

[0321] Combination Method

[0322] The compositions of the invention described herein can be administered in combination with chemotherapeutic agents. Chemotherapeutic agents that can be administered with the compositions of the present disclosure include 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, cisplatin, and vincristine sulfate); hormones (e.g., medroxyglucose, cyclosporine ... These include, but are not limited to, progesterone, estramustine sodium phosphate, 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., betamethasone sodium phosphate); and others (e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

[0323] In additional embodiments, the compositions of the invention described herein can be administered in combination with cytokines, including but 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-α.

[0324] In additional embodiments, the compositions described herein can be administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.

[0325] In some embodiments, the compositions described herein can be administered in combination with other immunotherapeutic agents, including, but not limited to, simtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab, altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab, bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab, catumaxomab, cetuximab, sitatuzumab, cixutumumab, clivatuzumab, conatumumab, daratumumab, drozitumab, dutuzumab, tsg ... Rigotumab, Dusigitumab, Detumomab, Dacetuzumab, Dalotuzumab, Eclomeximab, Elotuzumab, Ensituximab, Ertumaxomab, Etaracizumab, Farletuzumab, Ficlatuzumab, Fizitumumab, Franvotumab, Futuximab, Ganitumab, Gemtuzumab, Girentuximab, Glembatumumab, Ibritumomab, Igovomab, Imgatuzumab, Indatuximab, Inotuzumab, Intetumumab, Ipilimumab Mab, iratumumab, labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab, mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab, moxetumomab, narnatumumab, naptumomab, necitumumab, nimotuzumab, nofetumomab, ocaratuzumab, ofatumumab, olaratuzumab, onartuzumab, oportuzumab, oregovomab, panitumumab, palsatuzumab, patritumab, pemtumomab Examples of such agents include, but are not limited to, pertuzumab, pintumomab, pritumumab, racotumomab, radletumumab, rilotumumab, rituximab, lobatumumab, satumomab, sibrotuzumab, siltuximab, solitomab, tacatuzumab, taplitumomab, tenatumomab, teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab, ublituximab, veltuzumab, borsetuzumab, votumumab, zalutumumab, CC49, and 3F8.

[0326] The present invention provides a method of treating cancer in a patient by administering two antibodies that bind to the same epitope of the CTLA-4 protein, or alternatively, to two different epitopes of the CTLA-4 protein. Alternatively, 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, cancer can be treated by administering a bispecific antibody that binds to CTLA-4 and also to a protein other than CTLA-4. For example, the 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 protein other than CTLA-4 can be a tumor-associated antigen, and the protein other than CTLA-4 can be a cytokine.

[0327] In some embodiments, the invention provides for the administration of anti-PD-1 antibodies, alone or in combination with an additional antibody that recognizes another protein other than CTLA-4, together with cells capable of achieving or enhancing an immune response. For example, these cells can be peripheral blood mononuclear cells (PBMCs), or any cell type found in PBMCs, such as cytotoxic T cells, macrophages, and natural killer (NK) cells.

[0328] In addition, the present invention provides for the administration of antibodies that bind to CTLA4 protein and other therapeutic agents, including anti-neoplastic agents such as small molecules, growth factors, cytokines, or 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 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.)

[0329] Chimeric antigen receptor (CAR) T cell therapy

[0330] Also provided herein are cell therapies, such as chimeric antigen receptor (CAR) T cell therapy. CAR T cell therapy redirects a patient's T cells to kill tumor cells, for example, by exogenous expression of a CAR on the T cells. A CAR can be a transmembrane fusion protein that links the antigen recognition domain of an antibody to the intracellular signaling domain of a T cell receptor and co-receptor. For example, suitable cells can be used that are capable of secreting (or alternatively engineered to express) an anti-CTLA-4 antibody described herein that secretes the anti-CTLA-4 antibody of the present invention. The secreted anti-CTLA-4 "payload" can be, for example, a minibody, ScFv, IgG molecule, bispecific fusion molecule, and other antibody fragments described herein.

[0331] Solid tumors offer unique challenges for CAR-T therapy. Some barriers to CAR-T efficacy in solid tumors include heterogeneous antigen expression, poor tissue homing, activation, persistence, and immunosuppressive tumor microenvironment. Unlike hematological cancers, tumor-associated target proteins are overexpressed between tumors and healthy tissues, resulting in T cell killing of on-target / off-tumor healthy tissues. Furthermore, immunosuppression in the tumor microenvironment (TME) limits the activation of CAR-T cells toward tumor killing. After such contact or manipulation, the cells can be introduced into a cancer patient in need of treatment. The cancer patient can have any of the types of cancer as disclosed herein. The cells (e.g., T cells) can be, for example, but not limited to, tumor-infiltrating T lymphocytes, CD4+ T cells, CD8+ T cells, or combinations thereof.

[0332] Exemplary CARs and CAR factories useful in embodiments of the invention include, for example, those disclosed in PCT / US2015 / 067225 and PCT / US2019 / 022272, each of which is incorporated herein by reference in its entirety. For example, CAR-T cells can be generated according to methods known in the art using lentiviral systems (via transduction), retroviral systems (via transfection (electroporation)), and transposon systems (via PiggyBac). Useful promoters of payloads that can be used to generate CAR-T or CAR NK include, for example, constitutive promoters (the promoter is the same as that of CAR-T or CAR NK, e.g., EF1a, then IRES or 2A); inducible promoters (the promoter is different from that of CAR-T, e.g., NFAT, IL-2 promoter); and engineered promoters (such as CTLA-4 locus "knock-in" of cytokines and / or promoters under the control of endogenous promoters). In one embodiment, the CTLA-4 antibodies or CTLA-4 fusion proteins discussed herein can be used in the construction of multispecific antibodies or as a payload for engineered cells such as CAR-T cells, CAR NK cells, or engineered B cells. For example, in one embodiment, the anti-CTLA-4 antibodies or CTLA-4 fusion proteins discussed herein can be used for targeting CARS (i.e., as a targeting moiety). In one embodiment, the anti-CTLA-4 antibodies or CTLA-4 fusion proteins discussed herein can be used as a payload secreted by engineered cells such as CAR-T cells, CAR NK cells, or engineered B cells. In another embodiment, the anti-CTLA-4 antibodies or CTLA-4 fusion proteins discussed herein can be used as a targeting moiety and different CTLA-4 antibodies targeting different epitopes can be used as payloads. In another embodiment, the payload can be an immunomodulatory antibody payload.In some embodiments, the CTLA-4 antibody or CTLA-4 fusion protein described herein for use in a CAR-T composition is not a high affinity CTLA-4 antibody (e.g., such that the antibody does not bind tightly to its CTLA-4 target). For example, the CTLA-4 antibody or CTLA-4 fusion protein described herein can be used as a payload secreted by engineered cells, such as CAR-T cells, CAR-NK cells, or engineered B cells, with two targeting moieties (e.g., tumor-associated surface antigens) selected for a particular cancer (i.e., MSLN and MUC1 for ovarian cancer). Non-limiting examples of tumor-associated surface antigens include ErbB2 (HER2 / neu), carcinoembryonic antigen (CEA), epithelial cell adhesion molecule (EpCAM), epidermal growth factor receptor (EGFR), MUC1, MSLN, CD19, CD20, CD30, CD40, CD22, RAGE-1, MN-CA, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-38, IL-39, IL-40, IL-51, IL-52, IL-53, IL-61, IL-72, IL-83, IL-84, IL-85, IL-86, IL-87, IL-88, IL-99, IL-190, IL-19 IX, RET1, RET2 (AS), prostate specific antigen (PSA), TAG-72, PAP, p53, Ras, prostein, PSMA, survivin, 9D7, prostate cancer tumor antigen-1 (PCTA-1), GAGE, MAGE, mesothelin, β-catenin, TGF-βRII, BRCA1 / 2, SAP-1, HPV-E6, HPV-E7 (see also PCT / US2015 / 067225 and PCT / US2019 / 022272, which are incorporated by reference in their entireties for additional tumor-associated surface antigens). Exemplary armored CAR-T cells are listed in the table below. TIFF2024541476000121.tif132154

[0333] In one embodiment, a bispecific (or dual-targeted) CAR-T is provided. In another embodiment, the CAR-T is an engineered cell comprising a chimeric antigen receptor comprising an extracellular ligand-binding domain specific for a first antigen and a second antigen on the surface of a cancer cell, the first antigen comprising CXCR4 and the second antigen comprising CLDN4, or the first antigen comprising CAIX and the second antigen comprising CD70, or the first antigen comprising MUC1 and the second antigen comprising Msln. For example, the anti-CTLA-4 antibody or CTLA-4 fusion protein described herein can be used as a payload for engineered cells, such as CAR-T, CAR-NK cells, or engineered B cells, described herein. In one embodiment, a CXCR4 / CLDN4 dual-targeted CAR-T with an anti-CTLA-4 fusion payload can be used for breast cancer. In one embodiment, a CAIX / CD70 dual-targeted CAR-T with an anti-CTLA-4 fusion payload can be used for clear cell renal cell carcinoma (ccRCC). In one embodiment, a MUC1 / Msln dual-targeting CAR-T with an anti-CTLA-4 fusion payload can be used for ovarian cancer.

[0334] Diagnostic Assays

[0335] Anti-CTLA-4 antibodies can be used diagnostically, for example, to monitor the development or progression of cancer, e.g., as part of a clinical trial procedure to determine the effectiveness of a given therapeutic and / or prophylactic regimen.

[0336] In some embodiments, for diagnostic purposes, the anti-CTLA-4 antibodies of the invention are linked to a detectable moiety, e.g., to provide a method for detecting cancer cells in a subject at risk for or afflicted with cancer.

[0337] The detectable moiety can be conjugated directly to the antibody or fragment, or indirectly, for example, by using a fluorescent secondary antibody. Direct conjugation can be achieved, for example, by standard chemical coupling of a fluorophore to the antibody or antibody fragment, or through genetic engineering. Chimeras, or fusion proteins containing an antibody or antibody fragment linked to a fluorescent or bioluminescent protein, can be constructed. For example, Casadei et al. (Proc Natl Acad Sci USA. 1990 Mar;87(6):2047-51) describe a method for making a vector construct capable of expressing aequorin and antibody fusion protein genes in mammalian cells.

[0338] As used herein, the term "labeled" with respect to a probe or antibody can encompass direct labeling of the probe or antibody by binding (i.e., physically linking) a detectable substance to the probe or antibody, and 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 a DNA probe with biotin so 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 (e.g., biopsy material), as well as tissues, cells, and biological fluids present within a subject. That is, the detection method of the present invention can be used to detect cells expressing CTLA-4 in a biological sample in vitro and in vivo. For example, in vitro techniques for detection of CTLA-4 include enzyme-linked immunosorbent assay (ELISA), Western blotting, immunoprecipitation, and immunofluorescence. Additionally, in vivo techniques for detection of CTLA-4 include introducing a labeled anti-CTLA-4 antibody into a subject. 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.

[0339] In the case of "targeted" conjugates, i.e., conjugates that include a targeting moiety, which is a molecule or feature designed to localize the conjugate within a subject or animal at a specific site or sites, localization can refer to a state in which an equilibrium between the bound "localized" entity and the unbound "free" entity within the subject is essentially achieved. The rate at which such equilibrium is achieved depends on the route of administration. For example, a conjugate administered by intravenous injection can achieve localization within minutes of injection. On the other hand, an orally administered conjugate may take several hours to achieve localization. Alternatively, localization can simply refer to the location of the entity within a subject or animal at a selected period of time after the entity is administered. As another example, localization is achieved when the moiety becomes distributed after administration.

[0340] 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 tracked by imaging the detectable moiety (e.g., luminescent conjugate) according to the method of the present invention, such as with a photodetector device. The "photodetector device" used should be sensitive enough to allow imaging of weak light from within the mammal in a reasonable time and to use the signal from such a device to construct an image.

[0341] In cases where it is possible to use extremely bright light-generating moieties and / or detect light-generating fusion proteins localized near the surface of the object or animal being imaged, "night vision" goggles or standard highly sensitive video cameras such as Silicon Intensified Tube (SIT) cameras (e.g., from Hammamatsu Photonic Systems, Bridgewater, NJ) can be used. More typically, however, more sensitive light detection methods are required.

[0342] At very low light levels, the photon flux per unit area becomes so low that the scene being imaged does not appear continuous. Instead, it is represented by individual photons that differ from each other both in time and space. When viewed on a monitor, such an image appears as sparkling points of light, each representing a single detected photon. By accumulating these detected photons over time in a digital image processor, 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 is of no importance. The objective is to simply detect the presence of a signal (photon) and count the occurrences of the signal relative to its location over time.

[0343] At least two types of photodetector devices, described below, are capable of detecting individual photons and generating a signal that can be analyzed by an image processor. Noise-reduced photodetection devices achieve sensitivity by reducing the background noise of the photon detector rather than amplifying the photon signal. Noise is primarily reduced by cooling the detector array. Devices include charge-coupled device (CCD) cameras called "back-thinned" cooled CCD cameras. In more sensitive instruments, cooling is achieved using, for example, liquid nitrogen, which brings the temperature of the CCD array to about -120°C. "Back-thinned" refers to an ultra-thin backplate that reduces the path length that photons must travel before being detected, thereby increasing quantum efficiency. A particularly sensitive back-thinned cryogenic CCD camera is the "TECH 512" series 200 camera available from Photometries, Ltd. (Tucson, Ariz.).

[0344] "Photon amplification devices" amplify photons before they hit the detection screen. This class includes CCD cameras equipped with intensifier tubes, such as microchannel intensifier tubes. Microchannel intensifier tubes typically contain a metal array of channels perpendicular to and coextensive 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 that enter the channels of the array contact the side of the channel before exiting. A voltage applied across the array results in the ejection of many electrons from each photon collision. Electrons from such collisions exit their channel of origin in a "shotgun" pattern and are detected by the camera.

[0345] Even higher 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 in the second stage. However, the increase in sensitivity is achieved at the expense of spatial resolution, which decreases with each additional stage of amplification. An exemplary microchannel intensifier tube-based single-photon detection device is the C2400 series available from Hamamatsu.

[0346] Image processors process signals generated by photodetector devices that count photons to construct an image that can be displayed on a monitor or printed on a video printer, for example. Such image processors are typically sold as part of a system that includes the highly sensitive photon-counting camera described above, and are therefore available from the same sources. 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 and printed by a variety of image processing programs (such as "ADOBE PHOTOSHOP", Adobe Systems, Adobe Systems, Mt. View, Calif., etc.).

[0347] In one 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.

[0348] The present invention also encompasses kits for detecting the presence of CTLA-4 or CTLA-4 expressing cells in a biological sample. For example, the kit can include a labeled compound or agent (e.g., an anti-CTLA-4 scFv or monoclonal antibody) capable of detecting cancer or tumor cells in a biological sample, a means for determining the amount of CTLA-4 in the sample, and a means for comparing the amount of CTLA-4 in the sample with a standard. The standard, in some embodiments, is a non-cancerous cell or a cell extract thereof. The compound or agent can be packaged in a suitable container. The kit can further include instructions for using the kit to detect cancer in a sample.

[0349] Other embodiments

[0350] Although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to be illustrative, but not limiting, of the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0351] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims. EXAMPLES

[0352] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes of making and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, and are for illustrative purposes only, since alternative methods can be used to obtain similar results.

[0353] Example 1 Described herein are anti-CTLA-4 antibodies identified by phage display that block CTLA-4 / CD80 binding. Additionally, a yeast display library was also generated from this panning campaign to further explore the results of the second and third rounds of panning. After selecting clones that bound higher than bulk, the clones were sequenced to identify additional clones that showed improved binding curves compared to the original ones found in the third round of ELISA screening.

[0354] Embodiments herein may have commercial applications as stand-alone antibody therapies or in combination with other therapies.

[0355] Example 2 - aCTLA-4 antibodies obtained from PMPL panning (Group 1) An anti-CTLA-4 phage binding assay was performed as shown in Figure 1. The assay performed was a mid-scale phagemid rescue using PEG+NaCl to precipitate the phages and purify them.

[0356] Binding curves were generated from the ELISA data using purified phages. We diluted each of the nine phage PMPL candidates + E1-D7 in 5-fold increments starting at 2E13 phagemid particles / mL. We added these phages in triplicate to CTLA-4 Fc-coated and CAIX Fc-coated plates.

[0357] FACS confirms that the CTLA-4 phage can bind to CTLA-4 expressing 1G5 Jurkat cells as shown in Figure 2. The negative control was cells + secondary antibody (red box) and the positive comparison / control was the current E1-D7 candidate (green box).

[0358] For the purpose of filtering the candidates, we consider CTLA-4 phage candidates with more than 25% positive cells as positive. Of our nine possible phage candidates, there was only one equivalent candidate, B10 (circled in blue), as it was more than 75% positive. There were also some partial positives, these being B6 and C12, circled in orange, with more than 25% positive.

[0359] Example 3 Soluble proteins utilized herein include the following:

[0360] Human CTLA-4 Human CTLA-4, His tag (CT4-H5229) is expressed from human 293 cells (HEK293). It contains the amino acids Ala37-Phe162 (Accession number NP_005205.2). TIFF2024541476000122.tif16150

[0361] Mouse CTLA-4, His tagged (CT4-M52H5) is expressed from human 293 cells (HEK293) and contains the amino acids Glu36-Phe162 (Accession number NP_033973). TIFF2024541476000123.tif16150

[0362] Full-length CTLA4 was cloned using cDNA obtained from Transomic (BC074893).

[0363] Example 4 - aCTLA-4 Overview of CTLA4: - Protein receptors that act as immune checkpoints - Overall structure: extracellular V domain, transmembrane domain, cytoplasmic tail - Homodimer in membrane-bound form; monomer in soluble form

[0364] CTLA4 Binding: - CD28 homolog - Binds to CD-80 / 86 (B7-1 / 2) - Highly conserved MYPPY motif involved in binding

[0365] Mechanism for suppressing immune response (Figure 15) - Still much debate: ligand competition, negative signaling, ligand removal, effects on T cell motility

[0366] Background (Figure 23): - T cells are key mediators of antitumor immunity - They express inhibitory immune checkpoints: these immune checkpoints act to limit T cell activation, preventing collateral damage to the body from unchecked T cell activity - PD1 protein is expressed on the surface of T cells; when ligated to its ligands PDL-1 and PDL-2, it promotes negative pathways and inhibits T cell function - When ligated to its ligands PDL-1 and PDL-2, it promotes negative pathways and inhibits T cell function: tumor cells also express PDL1, which may promote tumor evasion.

[0367] MLR Assay (Figure 24): - Co-culture of CD4+ T cells from different donors with monocyte-derived DCs: Allorecognition of dendritic cells leads to T cell activation - DCs expressing PDL1 can suppress T cell activation: Addition of immune checkpoint inhibitors can rescue T cells from suppression. - To investigate whether anti-PD1 enhances T cell activation - Measure production of IFNγ and IL2 cytokines, indicators of T cell activation

[0368] protocol: - Isolate CD14+ monocytes using Miltenyi CD14+ MicroBeads - Culture in Miltenyi Mo-DC medium (pre-prepared medium containing GM-CSF + IL4) - Culture for 5 days, then add TNF-α (1000 U / ml), IL-1β (5 ng / ml), IL-6 (10 ng / ml), and prostaglandin E2 (PGE2) (1 μM) and culture for 2 days to mature DCs. - mMo-DCs express various DC markers involved in the formation of immune synapses between DCs and naive T cells, including CD80, CD86, and MHC II (HLA-DR). Mature Mo-DCs also expressed the DC activation markers CD83, CD40, and CCR7.

[0369] Protocol continued: - Isolate T cells on the day of the MLR test (CD4+ negative selection kit StemCell) - 100,000 T cells and 10,000 MoDC cells per well in the MLR - Add antibody at the desired concentration and incubate the cultures for 5 days - Save the supernatant for ELISA screening (IL2 and IFNγ)

[0370] aCTLA4 antibody MLR1 and 2: MLR1: - 2 T cell donors, 2 DC donors - DCs were cultured for 7 days - secondary cytokines were added on day 5 - Cells were combined and cultured for 4 days. - Positive control: aPD-1 antibody - Negative control: aSARS2 antibody

[0371] MLR2: - Testing two aCTLA4 antibodies (D7, C10) in combination with an aPD-1 antibody. - 4 plates: 2 T cell donors, 2 concentrations of aPD1 - Antibody dilutions were made using aPD-1 medium (starting at 100 nM, 5 x 4 dilutions): 5 nM aPD-1 medium and 100 nM aPD-1 medium - 100000 T cells + 10000 MoDCs per well - First, 50 μl of antibody was combined with 50 μl of T cells. - Ab / T cells were then added to DCs - Control: aPD-1+ cells, high aPD-1+ cells, DC+T cells - Incubate for 5 days

[0372] FACS to confirm that our CTLA4 phage can bind to CTLA-4 expressing 1G5 Jurkat cells (Figure 31). - To ensure that we had found suitable candidates, we retroactively screened a second plate of CTLA4 PMPL phage colonies and combined them with the data from plate E1. All unique colonies were cloned into scFv-Fc expression vectors and transfected into Expi293F cells for high-throughput screening by Octet. - SA sensors were loaded with biotinylated CTLA4. The scFv-Fc concentration in expi293 supernatants was measured by IgG quantification ELISA (Bethyl) and used to normalize the samples. Binding of scFv-Fc in the supernatants was measured using OctetRed96.

[0373] Non-limiting examples of categories considered for data analysis here: Very important 1. CD80 Blockade 2.CD86 Blockade 3. Dissociation constant (Kd) Moderately important 1. Mouse cross-reactivity low importance 1. Response

[0374] Example selections: E1-A8

[0375] Non-exclusive reasons for candidate selection: Shows the highest CD80 blockade among selected candidates CD86 blockade is equivalent to ipilimumab Shows significant cross-reactivity with mouse CTLA4 Higher response than both ipilimumab and E1-D7

[0376] Cons: Relatively high dissociation rates compared to most other antibodies tested

[0377] Note: E1-A8 has the same heavy chain as E2-A4, which exhibits the highest response rate and the lowest dissociation constant. E1-A8 is derived from the same V gene (IGHV3) as ipilimumab, but with a different allele

[0378] Example 5 - Engineering and characterization of anti-PD1 / anti-CTLA4 bispecific antibodies Dual checkpoint blockade with bispecific antibodies (Figure 53): Without wishing to be bound by theory, advantages may include lower therapeutic doses, cooperative binding, targeted therapy, and reduced manufacturing costs.

[0379] A non-limiting summary of the objects described herein: 1. Discovery and characterization of novel anti-CTLA4 antibodies: 1A: Kinetic screening and characterization 1B: Epitope binning 1C: Cell binding assay 1D: Biological assay 2. Engineering and Characterization of Anti-PD1 / Anti-CTLA4 Bispecific Antibodies 3. Characterization of anti-PD1 / anti-CTLA4 bispecific antibodies as CAR T payloads

[0380] hCTLA2 ELISA binding curve data and results (Figure 56 Panel A): - ELISA binding curves to show the binding affinity of anti-CTLA4 candidates to hCTLA4. - All candidates have similar binding affinity for hCTLA4 at the highest concentration. - Most candidates show binding affinity similar to ipilimumab - Top 3: I, J, H - A, E, I have the best saturation concentration

[0381] mCTLA4 ELISA Binding Curve Data and Results (Figure 56 Panel B) - Candidates showing high cross-reactivity: E, F, A - Candidates showing moderate cross-reactivity: J, H - Ipilimumab and Biolegend's anti-CTLA4 do not show cross-reactivity consistent with literature

[0382] BSA negative curve (Figure 56 Panel C) - Demonstrate the binding affinity of anti-CTLA4 candidates to BSA (negative control)

[0383] Binding affinity of anti-CTLA4 candidates to CTLA4+ Jurkat cells (Figure 64) - A clear mismatch between the binding affinity of the antibody to cells and to soluble proteins. - J and H have much higher binding affinity and lower saturation concentration than ipilimumab - E and F have binding properties similar to ipilimumab

[0384] Non-limiting exemplary conclusions that can be drawn from the data presented herein: Five anti-CTLA4 antibodies screened from different candidate libraries showed the following: A significant binding affinity to CTLA4 b. Ability to block the interaction of CTLA4 with CD80 / 86 c. Biofunctional relevance by promoting CD80 / CD28 signaling in effector cells.

[0385] Without wishing to be bound by theory, the following studies may supplement the data presented herein. - Further testing using a bispecific format to determine the most suitable anti-CTLA4 antibody for anti-PD1 / anti-CTLA4 bispecificity - In vitro assays for characterizing bispecific antibodies - Characterization and efficacy testing of bispecific antibodies as payloads in CAR T cell therapy.

[0386] Equivalent 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 specifically described herein which equivalents are considered to be within the scope of this invention and are covered by the following claims.

Claims

1. 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: 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 (b) 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 (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 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. An isolated antibody or fragment thereof comprising:

2. 2. The isolated antibody or fragment thereof of claim 1, which is an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

3. A heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 1, 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 heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 2, 6, 8 2. The isolated antibody or fragment thereof of claim 1, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of: 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.

4. 2. The isolated antibody or fragment thereof of claim 1, which is an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), and is selected from the group consisting of SEQ ID NOs: 3, 1, 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: 4, 2, 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.

5. 2. The isolated antibody or fragment thereof of claim 1, which is 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 is selected from the group consisting of SEQ ID NOs: 3, 1, 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, or 1023, and the light chain comprises an amino acid sequence about 95% identical to SEQ ID NO: 4, 2, 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, or 1024.

6. 6. The isolated monoclonal antibody or antigen-binding fragment of claim 5, comprising a wild-type Fc or a modified Fc.

7. 10. An isolated bispecific antibody comprising the antibody or fragment of claim 1 and a second antigen-binding fragment having specificity for a molecule on an immune cell.

8. 8. The bispecific antibody of claim 7, 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, 4-1BB, TIGIT, VISTA, HEVM, BTLA, CD47, PDL1, MICA, MICB, and KIR.

9. 8. The bispecific antibody of claim 7, wherein each of the fragment and the second fragment is independently selected from a Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody.

10. The bispecific antibody of claim 7 further comprising an Fc fragment.

11. 11. The bispecific antibody of claim 10, wherein the Fc fragment comprises a wild-type Fc fragment or a modified Fc fragment.

12. A nucleic acid encoding the antibody of claim 1.

13. A nucleic acid encoding the bispecific antibody of claim 7.

14. A pharmaceutical composition comprising the antibody or fragment thereof according to any one of claims 1 to 6 and a pharmaceutically acceptable carrier or excipient.

15. 15. The pharmaceutical composition of claim 14, further comprising at least one additional therapeutic agent.

16. 16. The pharmaceutical composition of claim 15, wherein the therapeutic agent is a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine.

17. A pharmaceutical composition comprising the bispecific antibody of claim 7 and a pharmaceutically acceptable carrier or excipient.

18. 18. The pharmaceutical composition of claim 17, further comprising at least one additional therapeutic agent.

19. 19. The pharmaceutical composition of claim 18, wherein the therapeutic agent is a toxin, a radiolabel, an siRNA, a small molecule, or a cytokine.

20. An isolated cell comprising one or more polynucleotides encoding the antibody or fragment thereof of claim 1.

21. 8. An isolated cell comprising one or more polynucleotides encoding the bispecific antibody or fragment thereof of claim 7.

22. 22. The isolated cell of claim 20 or 21, which is an immune cell.

23. 23. The isolated cell of claim 22, wherein the immune cell is a T cell, an NK cell, or a B cell.

24. A vector comprising the nucleic acid of claim 12 or 13.

25. A cell comprising the vector of claim 24.

26. The cell of claim 25, which is an immune cell.

27. 27. The cell of claim 26, wherein the immune cell is a T cell, an NK cell, or a B cell.

28. A pharmaceutical composition for treating cancer in a subject in need thereof, comprising a therapeutically effective amount of a CTLA-4 antibody or fragment thereof described in any one of claims 1 to 6.

29. 12. A pharmaceutical composition for treating cancer in a subject in need thereof, comprising a therapeutically effective amount of a CTLA-4 bispecific antibody of any one of claims 7 to 11.

30. 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 is: (a) 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 (b) 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 (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 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. Including, CAR.

31. 31. The CAR of claim 30, wherein the transmembrane domain further comprises a stalk region disposed between the extracellular domain and the transmembrane domain.

32. The CAR of claim 30, wherein the transmembrane domain comprises CD28.

33. The CAR of claim 30, further comprising one or more additional costimulatory molecules disposed between the transmembrane domain and the intracellular signaling domain.

34. The CAR of claim 33, wherein the costimulatory molecule is CD28, 4-1BB, ICOS, or OX40.

35. The CAR of claim 30, wherein the intracellular signaling domain comprises a CD3 zeta chain.

36. The CAR of claim 30, wherein the antibody is a Fab or scFv.

37. A nucleic acid encoding the CAR of claim 30.

38. 38. The nucleic acid of Claim 37, further comprising a nucleic acid encoding a polypeptide disposed after the intracellular signaling domain.

39. 39. The nucleic acid of claim 38, wherein the polypeptide is an antibody or a cytokine.

40. 40. The nucleic acid of claim 39, wherein the antibody is an scFv.

41. a nucleic acid encoding a CAR, the CAR comprising an intracellular signaling domain, a transmembrane domain, and an extracellular domain, and further comprising a nucleic acid encoding a polypeptide disposed after the intracellular signaling domain, the polypeptide comprising an isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the monoclonal antibody or the fragment thereof comprising: (a) 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 (b) 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 (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 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. A nucleic acid comprising:

42. A vector comprising the nucleic acid of any one of claims 37 to 41.

43. A cell comprising the vector of claim 42.

44. 44. The cell of claim 43, which is an immune cell.

45. A genetically engineered cell that expresses and retains the chimeric antigen receptor according to any one of claims 30 to 36 on its cell surface membrane.

46. 45. The cell of claim 44, wherein the immune cell is a T cell, an NK cell, or a B cell.

47. 47. The cell of claim 46, wherein the T cell is CD4+ or CD8+.

48. 48. The genetically engineered cell of claim 47, comprising a mixed population of CD4+ and CD8+ cells.

49. A genetically engineered cell that expresses and retains a chimeric antigen receptor on its cell surface membrane and is further engineered to express and secrete a polypeptide, wherein the 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 is: (a) 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 (b) 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 (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 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. Genetically engineered cells, including

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