Antibodies against CTLA-4 and methods of use thereof

By developing CTLA-4-binding monoclonal antibody fragments with specific amino acid sequences, the efficiency and specificity of CTLA-4 antibodies in the regulation of immune cells in the prior art have been solved, and more efficient immunotherapy effects have been achieved.

CN119278213APending Publication Date: 2025-01-07DANA FARBER CANCER INSTITUTE INC
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Patent Information

Application Number
CN202280089946.2
Authority / Receiving Office
CN · China
Patent Type
Applications(China)
Current Assignee / Owner
Priority Date
2021-11-24
Filing Date
2022-11-23
Publication Date
2025-01-07

AI Technical Summary

Technical Problem

The prior art is difficult to effectively utilize CTLA-4 antibodies to achieve their potential in therapy, especially with regard to the efficiency and specificity of the regulation of immune cells.

Method used

A monoclonal antibody or fragment thereof containing a specific amino acid sequence was developed to bind to the CTLA-4 protein, thereby enhancing its role on immune cells and binding to other antigen-binding fragments to improve specificity .

Benefits of technology

Through this method, the binding ability and therapeutic efficiency of CTLA-4 antibodies on immune cells is significantly improved, the regulatory function of CTLA-4 is enhanced, and a more efficient immunotherapy regimen is provided.

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Abstract

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

[0001] This application claims priority to U.S. Provisional Application No. 63 / 282,887, filed on November 24, 2021, the entire contents of which are incorporated herein by reference.

[0002] All patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications are hereby incorporated by reference in their entirety into this application in order to more fully describe the state of the art as 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 files and records, but otherwise reserves any and all copyrights whatsoever.

[0004] Sequence Listing

[0005] This application contains a sequence listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on [], is named [] and is [] bytes in size. Technical Field

[0006] The present invention relates to antibodies against human cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and methods of using the same. Background Art

[0007] CTLA-4 is an inhibitory receptor that acts as a major negative regulator of T cell responses. The affinity of CTLA4 for its natural B7 family ligands CD80 and CD86 is stronger than that for their cognate stimulatory coreceptor CD28. Summary of the invention

[0008] The present invention provides human cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and methods of using the same.

[0009] Embodiments relate to an isolated antibody or fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated antibody or fragment thereof 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 VHCDR1 comprising the amino acid sequence of SEQ ID NO: 28, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 29, a VHCDR3 comprising the amino acid sequence of SEQ ID NO: 30, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 64, a VLCDR2 comprising the amino acid sequence of SEQ ID NO: 65, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 66. CDR3; 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 VL CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 40. NO: 38, 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 VLCDR1 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 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 CDR2 comprising the amino acid sequence of SEQ ID NO:88; CDR1, 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: 424, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 430, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 441. NO:414, a VH CDR2 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 VL CDR2 comprising the amino acid sequence of SEQ ID NO:421, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:423. NO:421, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:421, a VLCDR1 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 CDR1, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or (s) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 432, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 433, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 473, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 474, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 475; or (t) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 434, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 435 CDR2, 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 VLCDR1 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 CDR1, 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 CDR2, 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 VLCDR1 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 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 900. CDR2, VHCDR3 comprising the amino acid sequence of SEQ ID NO: 868, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 901, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 902, and 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 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 CDR2, 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 VLCDR2 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, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927.;

[0010] Aspects of the invention also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody 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 VHCDR1 comprising the amino acid sequence of SEQ ID NO:28, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:29, a VHCDR3 comprising the amino acid sequence of SEQ ID NO:30, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:64, a VLCDR2 comprising the amino acid sequence of SEQ ID NO:65, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:66. CDR3; 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 VL CDR2 comprising the amino acid sequence of SEQ ID NO: 39, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 40. NO: 38, 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 VLCDR1 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 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 CDR2 comprising the amino acid sequence of SEQ ID NO:88; CDR1, 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: 424, a VL CDR2 comprising the amino acid sequence of SEQ ID NO: 430, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 441. NO:414, a VH CDR2 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 VL CDR2 comprising the amino acid sequence of SEQ ID NO:421, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:423. NO:421, a VH CDR3 comprising the amino acid sequence of SEQ ID NO:421, a VLCDR1 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 CDR1, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or (s) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 432, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 433, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 473, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 474, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 475; or (t) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 434, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 435 CDR2, 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 VLCDR1 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 CDR1, 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 CDR2, 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 VLCDR1 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 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 900. CDR2, VHCDR3 comprising the amino acid sequence of SEQ ID NO: 868, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 901, VLCDR2 comprising the amino acid sequence of SEQ ID NO: 902, and 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 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 CDR2, 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 VLCDR2 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, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927.;

[0011] Still further, aspects of the invention relate to an isolated antibody or fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated antibody or fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 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 NO: NO: 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] In addition, aspects of the invention relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 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 NO: NO: 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.

[0013] For example, 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:1, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:2.

[0014] Another embodiment relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:1, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:2.

[0015] Another embodiment relates to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof 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] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:3, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:4.

[0017] An embodiment relates to an isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:5, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:6.

[0018] Additionally, an embodiment relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:5, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:6.

[0019] Still further, the embodiments relate to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:7, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:8.

[0020] In addition, the embodiments relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:7, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:8.

[0021] Additional embodiments relate to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof 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] In addition, the embodiments relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:9, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:10.

[0023] An embodiment relates to an isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 11, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 12.

[0024] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:11, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:12.

[0025] In addition, the embodiments relate to an isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or the antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 13, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 14.

[0026] Still further, the embodiments relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 13, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 14.

[0027] In addition, the embodiments relate to an isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or the antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:15, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:16.

[0028] Still further, the embodiments relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:15, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:16.

[0029] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 17, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 18.

[0030] In addition, the embodiments relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 17, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 18.

[0031] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 19, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 20.

[0032] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 19, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 20.

[0033] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:21, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:22.

[0034] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:21, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:22.

[0035] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:23, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:24.

[0036] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:23, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:24.

[0037] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:385, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:386.

[0038] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:385, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:386.

[0039] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 387, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 388.

[0040] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:387, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:388.

[0041] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 389, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 390.

[0042] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 389, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 390.

[0043] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 391, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 392.

[0044] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 391, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 392.

[0045] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 393, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 394.

[0046] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 393, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 394.

[0047] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:395, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:396.

[0048] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:395, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:396.

[0049] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:397, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:398.

[0050] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:397, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:398.

[0051] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:399, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:400.

[0052] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:399, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:400.

[0053] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:401, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:402.

[0054] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:401, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:402.

[0055] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:403, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:404.

[0056] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:403, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:404.

[0057] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:405, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:406.

[0058] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:405, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:406.

[0059] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:407, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:408.

[0060] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:407, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:408.

[0061] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:409, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:410.

[0062] Embodiments also relate to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:409, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:410.

[0063] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:411, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:412.

[0064] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:411, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:412.

[0065] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1001, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1002.

[0066] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1001, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1002.

[0067] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1003, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1004.

[0068] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1003, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1004.

[0069] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1005, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1006.

[0070] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:1005, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:1006.

[0071] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1007, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1008.

[0072] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1007, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1008.

[0073] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1009, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1010.

[0074] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1009, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1010.

[0075] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1011, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1012.

[0076] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:1011, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:1012.

[0077] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1013, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1014.

[0078] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1013, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1014.

[0079] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1015, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1016.

[0080] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:1015, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:1016.

[0081] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1017, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1018.

[0082] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:1017, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:1018.

[0083] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1019, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1020.

[0084] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1019, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1020.

[0085] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1021, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1022.

[0086] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1021, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1022.

[0087] 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), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1023, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1024.

[0088] An embodiment also relates to an isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1023, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1024.

[0089] Aspects of the invention also relate to the isolated monoclonal antibody or antigen binding fragment of claims 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 or 79, wherein the isolated monoclonal antibody or antigen binding fragment comprises a wild-type Fc or a modified Fc.

[0090] In addition, aspects of the present invention relate to a separated bispecific antibody comprising an antibody or fragment as described herein and a second antigen binding fragment specific 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.

[0091] In an embodiment, the fragment and the second fragment are each independently selected from a Fab fragment, a single chain variable fragment (scFv) or a single domain antibody.

[0092] Embodiments may also include an Fc fragment. For example, the Fc fragment includes a wild-type Fc fragment or a modified Fc fragment.

[0093] Aspects of the invention relate to a nucleic acid encoding an antibody as described herein.

[0094] Still further, aspects of the invention relate to a nucleic acid encoding a bispecific antibody as described herein.

[0095] Additionally, aspects of the present invention relate to a pharmaceutical composition comprising an antibody or fragment thereof as described herein and a pharmaceutically acceptable carrier or excipient.

[0096] Embodiments may also include at least one additional therapeutic agent. For example, the therapeutic agent may be a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine.

[0097] Aspects of the invention relate to a pharmaceutical composition comprising a bispecific antibody as described herein and a pharmaceutically acceptable carrier or excipient.

[0098] In an embodiment, the pharmaceutical composition may further comprise at least one additional therapeutic agent. For example, the therapeutic agent may be a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine.

[0099] Additionally, aspects of the invention relate to an isolated cell comprising one or more polynucleotides encoding an antibody or fragment as described herein.

[0100] Additionally, aspects of the invention relate to an isolated cell comprising one or more polynucleotides encoding a bispecific antibody or fragment thereof as described herein.

[0101] Additionally, aspects of the invention relate to a vector comprising a nucleic acid as described herein.

[0102] Additionally, aspects of the invention relate to a cell comprising a vector as described herein.

[0103] Aspects of the invention relate to methods of treating cancer in a subject. For example, the method comprises administering to the subject a therapeutically effective amount of a CTLA-4 antibody or fragment thereof as described herein. For example, the method comprises administering to the subject a therapeutically effective amount of a CTLA-4 bispecific antibody as described herein.

[0104] Aspects of the invention also relate to a chimeric antigen receptor (CAR). In an embodiment, the CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain, wherein the extracellular domain is an isolated monoclonal antibody or an 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 VH CDR4 comprising the amino acid sequence of SEQ ID NO: 31 NO:64, a VL CDR1 comprising the amino acid sequence of SEQ ID NO:65, a VL CDR2 comprising the amino acid sequence of SEQ ID NO:66, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:67; 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. VL CDR3 of the amino acid sequence of 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 VLCDR3 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 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 CDR2 comprising the amino acid sequence of SEQ ID NO:52, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:53. CDR1, 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 VHCDR1 comprising the amino acid sequence of SEQ ID NO: 55, a VH CDR2 comprising the amino acid sequence of SEQ ID NO: 56, a VHCDR3 comprising the amino acid sequence of SEQ ID NO: 57, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 91, a VLCDR2 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, 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 VH CDR3 comprising the amino acid sequence of SEQ ID NO: 419; 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 CDR1, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 426, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 427, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 467, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 468, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 469; or (r) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 428, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or (s) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, VH comprising the amino acid sequence of SEQ ID NO: 432 CDR2, 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 VLCDR1 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 CDR1, 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 VLCDR1 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 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 865. CDR2, a VHCDR3 comprising the amino acid sequence of SEQ ID NO: 865, a VL CDR1 comprising the amino acid sequence of SEQ ID NO: 898, a VLCDR2 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 CDR1, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 873, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 874, VLCDR1 comprising the amino acid sequence of SEQ ID NO: 907, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 908, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 909; or (gg) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 875, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 876, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 877, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 910, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 911, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 912; or (hh) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 878, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 1000 CDR2, 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 CDR1, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 887, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 888, VLCDR1 comprising the amino acid sequence of SEQ ID NO: 922, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 923, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 924; or (ll) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927. For example, the transmembrane domain comprises CD28. ;

[0105] In an embodiment, the transmembrane domain further comprises a stalk region positioned between the extracellular domain and the transmembrane domain.

[0106] Embodiments may also include one or more additional co-stimulatory molecules positioned between the transmembrane domain and the intracellular signaling domain. For example, the co-stimulatory molecule is CD28, 4-1BB, ICOS or OX40. For example, the intracellular signaling domain includes the CD3 zeta chain.

[0107] In an embodiment, the antibody is a Fab or a scFV.

[0108] Aspects of the present invention also relate to a nucleic acid encoding a CAR as described herein. In an embodiment, the nucleic acid may also include a nucleic acid encoding a polypeptide positioned after the intracellular signaling domain. For example, the polypeptide is an antibody or a cytokine, such as a scFV.

[0109] Aspects of the invention relate to a nucleic acid encoding a CAR, wherein the CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain, the nucleic acid further comprising a nucleic acid encoding a polypeptide positioned after the intracellular signaling domain, wherein the polypeptide comprises an isolated monoclonal antibody or an 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 30. CDR2, 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 CDR3 comprising the amino acid sequence of SEQ ID NO: 70 CDR1, 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 VLCDR2 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, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 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 CDR2 comprising the amino acid sequence of SEQ ID NO:52, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:53. CDR1, 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 VLCDR1 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, 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 VH CDR3 comprising the amino acid sequence of SEQ ID NO: 419; 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 CDR1, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 426, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 427, VLCDR1 comprising the amino acid sequence of SEQ ID NO: 467, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 468, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 469; or (r) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 428, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or (s) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, VH comprising the amino acid sequence of SEQ ID NO: 432 CDR2, 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 CDR1, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 441, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 442, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 482, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 483, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 484; or (w) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 443, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 444, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 445, VLCDR1 comprising the amino acid sequence of SEQ ID NO: 485, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 486, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 487; or (x) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 446, 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 comprising the amino acid sequence of SEQ ID NO: 857 CDR1, 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 VL CDR2 comprising the amino acid sequence of SEQ ID NO: 864, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 865. 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 VLCDR1 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: 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 NO: 872, a VH CDR1 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 VLCDR2 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: 879, a VL CDR2 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. 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 VLCDR3 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 CDR1, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 887, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 888, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 922, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 923, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 924; or (ll) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927. . ;

[0110] Aspects of the invention relate to a vector comprising a nucleic acid as described herein.

[0111] Aspects of the invention also relate to a cell comprising a vector as described herein.

[0112] Aspects of the present invention relate to a genetically engineered cell that expresses and carries a chimeric antigen receptor as described herein on the cell surface membrane. For example, the cell is a T cell or a NK cell. For example, the T cell is CD4+ or CD8+.

[0113] Embodiments may also include mixed populations of CD4+ and CD8+ cells.

[0114] Aspects of the invention also relate to a genetically engineered cell that expresses and carries a chimeric antigen receptor on the cell surface membrane, and the genetically engineered cell is also engineered to express and secrete a polypeptide, wherein the polypeptide is an isolated monoclonal antibody or an 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 NO:30, a VH CDR3 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 VLCDR3 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 VH CDR2 comprising the amino acid sequence of SEQ ID NO:71 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 VL CDR2 comprising the amino acid sequence of SEQ ID NO: 44, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 45. ID NO:44, a VH CDR2 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 VH CDR3 comprising the amino acid sequence of SEQ ID NO:52 a VL CDR1 comprising the amino acid sequence of SEQ ID NO:85, a VLCDR2 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 VL CDR2 comprising the amino acid sequence of SEQ ID NO: 59, a VL CDR3 comprising the amino acid sequence of SEQ ID NO: 60 NO:59, a VH CDR2 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 VLCDR3 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 VL CDR2 comprising the amino acid sequence of SEQ ID NO:418, and a VL CDR3 comprising the amino acid sequence of SEQ ID NO:419. 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 CDR1, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 426, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 427, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 467, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 468, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 469; or (r) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 428, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 429, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 430, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 470, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 471, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 472; or (s) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 431, VH comprising the amino acid sequence of SEQ ID NO: 432 CDR2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 433, a VLCDR1 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 CDR1, 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 VLCDR1 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 comprising the amino acid sequence of SEQ ID NO: 857 CDR1, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 858, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 859, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 892, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 893, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 894; or (bb) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 860, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 861, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 862, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 895, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 896, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 897; or (cc) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 863, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 864, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 865 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 VLCDR1 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 CDR1, 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 CDR2, 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 CDR1, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 887, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 888, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 922, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 923, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 924; or (ll) VH CDR1 comprising the amino acid sequence of SEQ ID NO: 889, VH CDR2 comprising the amino acid sequence of SEQ ID NO: 890, VH CDR3 comprising the amino acid sequence of SEQ ID NO: 891, VL CDR1 comprising the amino acid sequence of SEQ ID NO: 925, VL CDR2 comprising the amino acid sequence of SEQ ID NO: 926, and VL CDR3 comprising the amino acid sequence of SEQ ID NO: 927.;

[0115] Other objects and advantages of the present invention will become apparent from the ensuing description. BRIEF DESCRIPTION OF THE DRAWINGS

[0116] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0117] Figure 1 Shown is the ELISA combined with PEG-purified phage at varying dilutions.

[0118] Figure 2 FACS confirmation of CTLA-4 phage binding to 1G5 Jurkat cells expressing CTLA-4 is shown.

[0119] Figure 3 Anti-CTLA-4 antibody amino acid (AA) and nucleotide (Nuc) sequences are provided."HC" is the heavy chain, and "LC" is the light chain.

[0120] Figures 4 to 8 Anti-CTLA-4 antibody amino acid (AA) and nucleotide (Nuc) sequences are provided.

[0121] Figures 9 to 11 Anti-CTLA-4 antibody amino acid (AA) and nucleotide (Nuc) sequences are provided.

[0122] Fig.12 The percentage of 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 soluble antibodies at the indicated concentrations, washed, and then incubated with labeled CD80-Fc. Samples were read on a BD FACSCanto II. A reduction in fluorescence was used to indicate a blockade of binding of CD80 to CTLA4 on the cell surface.

[0123] Fig.13 A ribbon diagram adapted from Metzler, William J. et al., “Solution Structure of Human CTLA-4 and Delineation of a CD80 / CD86 Binding Site Conserved in CD28” is shown.

[0124] Fig.14 Shown is a ribbon diagram adapted from Stamper, Carin C. et al. "Crystal Structure of the B7-1 / CTLA-4 Complex That Inhibits Human Immune Responses." Nature.

[0125] Fig.15 Schematic diagram showing the mechanism of suppression of immune response.

[0126] Fig.16The ribbon diagrams of existing therapies: Ipilimumab-IgG1 and Tremelimumab-IgG2 are shown. Panels A and B show ribbon diagrams adapted from Ramagopal, Udupi A. et al. "Structural Basis for Cancer Immunotherapy by the First-in-Class Checkpoint Inhibitor Ipilimumab". Panel C shows a ribbon diagram adapted from He, Mengnan et al. "Remarkably Similar CTLA-4 Binding Properties of Therapeutic Ipilimumab and Tremelimumab Antibodies".

[0127] Fig.17 A graph showing exemplary panning results for anti-CTLA4 antibodies. Rounds: 1, 1b, 2, 2b, 3, 3b. Mouse CTLA4 was introduced in rounds 3 and 3b. The result was 10 unique antibodies.

[0128] Fig.18 A graph showing an exemplary kinetic analysis of aCTLA4 antibodies.

[0129] Fig.19 A graph showing an exemplary competition analysis of aCTLA4 antibodies is shown.

[0130] Fig. 20 Figures showing FACS of aCTLA4 antibodies bound to T cells. E1-D7 binds significantly more CTLA4 on T cells than T2-C10. Anti-CTLA4ab from biolegend was used for control at a single point (recommended staining concentration). Ipi IgG is an antibody cloned in the laboratory to represent ipilimumab, it is not a commercial preparation or biosimilar.

[0131] Fig.21 An exemplary FACS of aCTLA4 Ab binding to T cells is shown.

[0132] Fig. 22 An exemplary graph showing ELISA results for mouse / human CTLA4 cross-reactivity. E1-D7 cross-reacts with mouse CTLA4.

[0133] Fig.23 A schematic diagram of an exemplary mechanism of T cells adapted from P. Ott et al. 2014 is shown.

[0134] Fig.24A schematic diagram of the MLR assay is shown. Schematic diagram provided by Explicyte. Scheme: Co-culture of CD4+ T cells with monocyte-derived DCs, each from a different donor. This indicates that allorecognition by dendritic cells leads to activation of T cells. DCs expressing PDL1 can inhibit T cell activation. Addition of immune checkpoint inhibitors can recover T cells from inhibition. We investigated whether anti-PD1 enhances T cell activation. The measured IFNy and IL2 cytokine production is an indicator of T cell activation.

[0135] Fig.25 Data from the immunophenotyping of monocytes, imMo-DC and mMo-DC adapted from Miltenyi are shown. In this scheme, the separation CD14+ monocytes using Miltenyi CD14+ microbeads are cultured and cultivated for 5 days in Miltenyi Mo-DC culture medium (pre-prepared culture medium with GM-CSF+IL4). Then, TNF-α (1000U / ml), IL-1β (5ng / ml), IL-6 (10ng / ml) and prostaglandin E2 (PGE2) (1 μM) are added and cultivated for 2 days to make DC mature. mMo-DC expresses various DC markers involved in the formation of the immunological synapse between DC and the original T cells, including CD80, CD86 and MHC II (HLA-DR). Mature Mo-DC also expresses DC activation markers CD83, CD40 and CCR7.

[0136] Fig.26 Data and analysis of AD staining of DC donors are shown.

[0137] Fig. 27 Exemplary MLR1 data are shown: a graph measuring IFNγ production. We investigated whether E1-D7 inhibits T cell activation. A decrease in IFNγ was observed in wells treated with E1-D7 compared to T cells + DC alone, anti-PD1 treatment, and irrelevant Ab controls. An absorbance value of 4 was used to indicate saturation.

[0138] Fig.28 Exemplary data for the combination of aCTLA4 / aPD-1 MLR: graph of IFNγ production is shown.

[0139] Fig.29 An exemplary schematic diagram of the second panning using CTLA4-PMPL is shown.

[0140] Fig.30 Shown are exemplary graphs of data from combining ELISA with PEG-purified phage at varying dilutions.

[0141] Fig.31An exemplary FACS confirmation that our CTLA4 phage can bind to 1G5 Jurkat cells expressing CTLA-4 is shown.

[0142] Fig.32 Graph showing exemplary kinetic screening data of aCTLA4 scFv-Fc supernatants.

[0143] Fig.33 An exemplary binding curve for hCTLA4 is shown.

[0144] Fig.34 An exemplary binding curve for mCTLA4 is shown.

[0145] Fig.35 BSA negative control binding curve is shown.

[0146] Fig.36 A graph of the data and observations is shown. The data are arranged in descending order of the association response at 25 nM concentration of the antibody. All candidate antibodies have higher dissociation rates than Ipilimumab and Biolegenda CTLA4 (commercial control). Most candidates appear to have higher association responses than the commercial control. Four candidates (highlighted in the table) have lower dissociation constants than E1-D7 (Theiler Antibodies—Laboratory Control). E2-D10 and E1-A12 have the same heavy chain but different light chain 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.

[0147] Fig.37 A graph showing exemplary data from a competition assay is shown. CD80 blocking: All candidate antibodies blocked CD80 binding to some extent, but not more than ipilimumab, so I selected candidates with higher blocking (lower second association values) than E1-D7 (lab control, Taylor candidate). CD86 blocking: All candidates had negative second association values, indicating that they all completely blocked CD86 (normalized value = 0). I selected candidates with negative values ​​closer to zero when compared to ipilimumab, as higher negative values ​​can indicate antibody dissociation. E1-A8 and E2-A4 belong to the same germline family. E1-D7 blocking: The highlighted candidates blocked E1-D7 binding better than ipilimumab. Ipilimumab blocking: The highlighted candidates blocked ipilimumab binding better than E1-D7. All candidates that blocked CD80 and CD86 performed relatively poorly in terms of kinetics.

[0148] Fig.38 An exemplary graph of CTLA4 binding is shown. Jurkat cells were transduced with hCTLA4 and used to generate binding curves.

[0149] Fig.39 Shown is a schematic diagram 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.

[0150] Fig.40 Figures of exemplary Promega CTLA4 bioassay data are shown. Anti-CTLA4 scFv-Fc was tested for Ipi biosimilar IgG. Plates were read at intervals of 1 second (left column) or 10 seconds (right column) using BMG polarstar omega. E1-B10, E1-A8, E2-A4 and E1-D7 showed similar activity at about 40% of Ipi. On plate 2, E2-G9 showed activity comparable to ipi, but E2-H10, E1-A4 and E2-H12 showed minimal improvement.

[0151] Fig.41 The diagram of bispecific design is shown.When expressing, the BsAb constructed with IgG1 hinge shows a mixture of monomer and dimer, thus increasing the dimerization frequency that we convert to IgG3 hinge.We initially set up two constructs, one with (G4S)5 joints before IgG3 hinge, and the other without leading joint.The second joint is maintained at 5 repetitions, thinking that the 2nd scFv provides sufficient space to reach the binding site without interference from hinge.

[0152] Fig.42 The sequence of the IgG3 hinge linker sequence is shown.

[0153] Fig.43 Pictures of exemplary labeled SDS Page Gels for non-reduced and reduced E1D7-IgG3 hinge-P4B3M3 and P4B3M3-IgG3 hinge-E1D7 bsAbs are shown.

[0154] Fig.44 Graphs of exemplary data are shown. SA sensors were loaded with biotinylated CTLA4 or PD1 and allowed to associate / dissociate as indicated. Dissociation rates for sensors loaded with PD1 were unreliable due to sensor drift.

[0155] Fig.45 Data from an exemplary dual binding assay are shown. CTLA4+, PD1+, or CTLA4+PD1+ Jurkat cells were first coated with the indicated amount of single or dual specific protein. Next, soluble protein was added and allowed to bind to the free arms. As shown here, the monospecific ab cannot bind to significant amounts of soluble antigen, yet when bound to cells expressing PD1, the bsAb is able to capture soluble CTLA4 from solution.

[0156] Fig.46 Non-limiting exemplary data from competition assays for Abs (including combination mAbs) versus CD80 hFc for CTLA4+ / PD1+1G5Jurkat are shown. BsAbs exhibited significantly improved competition for CD80 binding to PD1+CTLA4+jurkat cells when compared to monospecific antibodies and mixtures.

[0157] Fig.47 Non-limiting exemplary data from a % competition assay for binding to CTLA4 on 1G5 Jurkat with CD80 hFc are shown. CD80 competition assay using CTLA4-transduced Jurkat cells: Four bispecific constructs were tested for CD80 competition. Two had a complete 5,5 linker conformation, and the other two had an abbreviated 0,5 linker. We also switched the order of the aPD1 / aCTLA4 abs in the final construct. As shown here, E1-D7 and E1-D7+P4B3m3 combination therapies caused moderate competition, however the formation of bsAbs showed a significant increase in CD80 competition, which may be due to the additional binding potential generated by the anti-PD1 domain.

[0158] Fig.48 Panel (A) Characteristics of clear cell renal cell carcinoma (adapted from Li et al., 2019) and panel (B) model for cell differentiation (adapted from Tun et al., 2010) are shown.

[0159] Fig.49 A graph showing OS of ccRCC patients adapted from Feng et al., 2019 is shown.

[0160] Fig.50 A diagram showing the representation of immune populations of ccRCC adapted from Chevrier et al., 2017 is shown.

[0161] Fig.51Diagrams and graphs of the CTLA4 signaling pathway are shown. Panel A shows a diagram of the CTLA4 signaling pathway. Panel B shows a graph of CTLA4 overexpression in ccRCC (adapted from Lie et al., 2020). Panel C shows a diagram of ipilimumab and CTLA4 blockade.

[0162] Fig.52 Figures and diagrams of the PD1 signaling pathway adapted from Kim et al., 2021 are shown. Panel A shows the PD1-PDL1 signaling pathway. Panel B shows histological images of PD1 overexpression in ccRCC. Panel C shows a diagram of PD1 blockade and non-restrictive effects.

[0163] Fig.53 A diagram of dual checkpoint blockade of a bispecific antibody is shown. Panel A shows a schematic diagram of dual CTLA4 / PDL checkpoint blockade. Panel B shows a non-limiting exemplary bispecific antibody format.

[0164] Fig.54 Table showing exemplary anti-CTLA4 / anti-PD1 bispecific antibodies used in the clinic.

[0165] Fig.55 A schematic diagram of a non-limiting exemplary experimental design for evaluating the binding affinity of anti-CTLA4 candidates to human and mouse CTLA4 at varying concentrations using ELISA is shown.

[0166] Fig.56 Non-limiting data and results of ELISA binding curves are shown. Panel A shows an ELISA binding curve to indicate the binding affinity of anti-CTLA4 candidates to hCTLA4. Panel B shows an ELISA binding curve to indicate the binding affinity of anti-CTLA4 candidates to mCTLA4. Panel C shows an ELISA binding curve to indicate the binding affinity of anti-CTLA4 candidates to BSA (negative control).

[0167] Fig.57 A non-limiting exemplary experimental design for characterizing the kinetic properties of antibody candidates using BLI-Octet is shown.

[0168] Fig.58 Non-limiting data indicating the kinetic properties of anti-CTLA4 candidates are shown. All candidates tested had higher off-rates when compared to Ipilimumab, even when they had higher on-responses. Among the candidates, C, B, and G appear to have the lowest off-rates. KD - dissociation constant, Kon - on-rate, Kon - on-rate.

[0169] Fig.59 Schematic representation of a non-limiting exemplary experimental design for determining the ability of anti-CTLA4 antibodies to block CD80 / CD86 / ipilimumab binding and determining similarities in the localization of the antibodies and ligands on the surface of CTLA4 is shown.

[0170] Fig.60 A graph showing the percent blockade of CD80 / CD86 / Ipilimumab binding to CTLA4 by anti-CTLA4 candidates. CD80: Ipilimumab blocks CD80 to the highest extent. Candidates showing the highest CD80 blockade: I, E, J. CD86: All candidates showed 100% blockade of CD86 binding. This may be due to the documented lower affinity of CD86 binding to CTLA4. Ipilimumab: All candidates blocked Ipilimumab binding to CTLA4 to some extent, even those that cross-reacted with mCTLA4.

[0171] Fig.61 Figure 2 shows a ribbon diagram of ipilimumab complex adapted from He M, Chai Y, Qi J, Zhang CWH, Tong Z, Shi Y, Yan J, Tan S, Gao GF. Remarkably similar CTLA-4 binding properties of therapeutic ipilimumab and tremelimumab antibodies. Oncotarget. 2017 May 19; 8(40): 67129-67139. doi: 10.18632 / oncotarget.18004. PMID: 28978021; PMCID: PMC5620161. Panel A shows ipilimumab complexed with the highlighted hCTLA4-contact residues. Panel B shows ipilimumab complexed with the highlighted mCTLA4-aligned epitopes.

[0172] Fig.62Shown is a ribbon diagram of the CD80 complex adapted from Stamper CC, Zhang Y, Tobin JF, Erbe DV, Ikemizu S, Davis SJ, Stahl ML, Seehra J, Somers WS, Mosyak L. Crystal structure of the B7-1 / CTLA-4 complex that inhibits human immune responses. Nature. 2001 Mar 29; 410(6828): 608-11. doi: 10.1038 / 35069118. Errata in Nature 2001 May 31; 411(6837): 617. PMID: 11279502. Panel A shows CD80 in complex with the highlighted hCTLA4-contact residues. Panel B shows CD80 in complex with the highlighted mCTLA4-aligned epitope.

[0173] Fig.63 A non-limiting exemplary schematic diagram of the experimental design for determining the binding affinity of candidate antibodies to Jurkat cells expressing CTLA4 is shown.

[0174] Fig.64 Non-limiting exemplary binding curves of the binding affinity of anti-CTLA4 candidates to CTLA4+ Jurkat cells are shown.

[0175] Fig.65 A non-limiting exemplary schematic diagram of the experimental design used to assess the biological significance of antibody binding in promoting CD28 signaling is shown.

[0176] Fig.66 A non-limiting exemplary graph of fold induction of luciferase versus concentration of anti-CTLA4 candidates is shown. Across both panels, Ipilimumab showed the highest induction of luciferase expression. Five of the eight tested candidates: E, H, J, F, and I showed similar responses.

[0177] Fig.67 Anti-CTLA-4 antibody germline alignment is shown.

[0178] Fig.68 Anti-CTLA-4 antibody amino acid sequences are shown.

[0179] Fig.69 Anti-CTLA-4 antibody nucleic acid sequences are shown. DETAILED DESCRIPTION

[0180] Abbreviations and definitions

[0181] The specific implementation of one or more embodiments is provided herein. However, it should be 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 the basis of the claims and as a representative basis for teaching those skilled in the art to adopt the present invention in any appropriate manner.

[0182] Unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. In the claims and / or specification, when used in conjunction with the term "comprising," the use of the word "a" or "an" may mean "one," but is also consistent with "one or more," "at least one," and "one or more than one."

[0183] Wherever the phrases "for example," "such as," "including," etc. are used herein, unless expressly stated otherwise, they should be understood to be followed by the phrase "and but not limited to." Similarly, "example," "exemplary," etc. should be understood to be non-limiting.

[0184] The term "substantially" allows deviations from the descriptor that do not adversely affect the intended purpose. Even if the word "substantially" is not explicitly recited, the descriptive term should be understood to be modified by the term "substantially".

[0185] The terms "comprising" and "including" and "having" and "involving" (and similarly "comprises," "includes," "has," and "involves") and the like are used interchangeably and have the same meaning. Specifically, each of these terms is defined to be consistent with the common U.S. patent law definition of "comprising," and thus should be interpreted as open terms meaning "at least the following," and should also be interpreted as not excluding additional features, limitations, aspects, etc. Thus, for example, "a process involving steps a, b, and c" means a process including at least steps a, b, and c. Whenever the term "a" or "an" is used, it should be understood to mean "one or more / one or more" unless such an interpretation does not make sense in the context.

[0186] The term "about" is used herein to mean approximately, roughly, around, or in the region thereof. When the term "about" is used in conjunction with a numerical range, the term modifies the range by extending the limits above and below the numerical values ​​set forth. Typically, the term "about" is used herein to modify the numerical values ​​above and below the stated value by a change upward or downward (higher or lower), such as 20%.

[0187] CTLA-4

[0188] Human antibody-phage screening was performed against human CTLA-4 (rounds 1 to 3) or murine CTLA-4 (round 3). The first set of antibodies was discovered via panning against soluble protein, and the second set was discovered by PMPL panning.

[0189] The binding activity of a panel of human monoclonal antibodies against human CTLA-4 has been characterized. Anti-CTLA-4 immunotherapy is being evaluated as part of the emerging field of immuno-oncology. The anti-CTLA-4 mAbs described herein may have therapeutic potential. CTLA-4 antibodies have been identified as having varying affinities for CTLA-4.

[0190] Aspects of the present invention provide isolated antibodies specific for CTLA-4. As used herein, the term "isolated" with respect to cells, nucleic acids (such as DNA or RNA) refers to molecules separated from other DNA or RNA present in the natural source of the macromolecule, respectively. The term "isolated" may also refer to nucleic acids or peptides that are substantially free of cellular material, viral material or culture medium when produced by recombinant DNA technology or substantially free of chemical precursors or other chemicals when chemically synthesized. For example, "isolated nucleic acid" may include nucleic acid fragments that are not naturally present as fragments and are not found in the natural state. "Isolated" may also refer to cells or polypeptides separated from other cellular proteins or tissues. Isolated polypeptides may include both purified polypeptides and recombinant polypeptides. Isolated antibodies are identified by using a 27 billion member human single-chain antibody (scFv) phage library that is panned against soluble or paramagnetic proteoliposomes that display CTLA4 as a 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 to construct multispecific antibodies or as payloads for genetically engineered cells such as CAR-T cells, CAR-NK cells, or genetically engineered B cells.

[0191] Described herein are unique recombinant monoclonal CTLA-4 antibodies. "Recombinant" as it relates to a polypeptide (such as an antibody) or a polynucleotide refers to a form of the polypeptide or polynucleotide that does not exist in nature, a non-limiting example of which can be formed by combining polynucleotides or polypeptides that do not normally occur together.

[0192] The amino acid sequences of monoclonal CTLA-4 antibodies are provided herein (see Tables 1 to 26); the amino acid sequences of the heavy and light chain complementary determining regions (CDRs) of the CTLA-4 antibodies are underlined below (CDR1), Underlined and bold ( CDR2 ) or underlined, italicized, and bolded ( CDR3 ):

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[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239] The nucleic acid sequences of monoclonal CTLA-4 antibodies are provided herein (see Tables 27 to 52) ：

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290] 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:

[0291] Table 53A. Heavy chain of CTLA-4 antibody (V H ) Complementarity Determining Region (CDR)

[0292]

[0293]

[0294]

[0295] Table 53B. Light chain of CTLA-4 antibody (V L ) Complementarity Determining Region (CDR)

[0296]

[0297]

[0298]

[0299] 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:

[0300] Table 54A. Heavy chain of CTLA-4 antibody (V H ) Complementarity Determining Region (CDR)

[0301]

[0302]

[0303]

[0304] Table 54B. Light chain of CTLA-4 antibody (V L ) Complementarity Determining Region (CDR)

[0305]

[0306]

[0307]

[0308] The amino acid sequences of the heavy and light chain framework regions of the CTLA4 antibody are shown below in Tables 55A-B:

[0309] Table 55A. Heavy chain of CTLA-4 antibody (V H ) Framework region (FR)

[0310]

[0311]

[0312]

[0313]

[0314]

[0315] Table 55B. Light chain of CTLA-4 antibody (V L ) Framework region (FR)

[0316]

[0317]

[0318]

[0319]

[0320] The nucleic acid sequences of the heavy and light chain framework regions of the CTLA-4 antibody are shown below in Tables 56A-B:

[0321] Table 56A. Heavy chain of CTLA-4 antibody (V H ) Framework region (FR)

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329] Table 56B. Light chain of CTLA-4 antibody (V L ) Framework region (FR)

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337] The CTLA-4 antibodies described herein bind to CTLA-4. In one embodiment, the CTLA-4 antibody has a high affinity and high specificity for CTLA-4. Some embodiments are also characterized by antibodies having a specified percentage of identity or similarity to the amino acid sequence or nucleotide sequence of the anti-CTLA-4 antibodies described herein. For example, "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing the positions in each sequence, and for the purpose of comparison, each sequence can be aligned. When 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, the antibody may 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 specified region or the full length of any of the anti-CTLA-4 antibodies described herein. For example, the antibody may have 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more nucleic acid identity when compared to a specified region or the full length of any of the anti-CTLA-4 antibodies described herein. Sequence identity or similarity to nucleic acids and proteins of the present invention can be determined by sequence comparison and / or alignment by methods known in the art, for example, using software programs known in the art, such as those described in Ausubel et al., (2007) Current Protocols in Molecular Biology. For example, sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignments, or visual inspection can be used to determine the percent sequence identity or similarity to nucleic acids and proteins of the present invention.

[0338] As used herein, "polypeptide" may encompass the singular "polypeptide" as well as the plural "polypeptides", and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also called peptide bonds). The term "polypeptide" refers to any one or more chains of two or more amino acids, rather than a specific length of the product. Therefore, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains" or any other terms used to refer to one or more chains of two or more amino acids may refer to "polypeptides" herein, and the term "polypeptide" may replace any of these terms or be used interchangeably with any of these terms. "Polypeptide" may also refer to the product of post-expression modification of a polypeptide (including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage or modification by non-naturally occurring amino acids through known protecting / blocking groups). A polypeptide may be derived from a natural biological source or produced by recombinant technology, but need not be translated from a specified nucleic acid sequence. It may be generated in any manner, including by chemical synthesis. With respect to amino acid sequences, those skilled in the art will readily recognize that individual substitutions, deletions or additions to a nucleic acid sequence, peptide sequence, polypeptide sequence or protein sequence that alter, add, delete or substitute 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 alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants of the anti-CTLA-4 antibodies disclosed herein may exhibit increased cross-reactivity to CTLA-4 compared to unmodified CTLA-4 antibodies.

[0339] For example, a "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-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 by another amino acid residue from the same side chain family. In another embodiment, an amino acid string can be replaced by a structurally similar string that differs in the order and / or composition of the side chain family members.

[0340] Antibody

[0341] As used herein, "antibody" or "antigen-binding polypeptide" may refer to a polypeptide or polypeptide complex that specifically recognizes an antigen and binds to the antigen. The antibody may be a whole antibody and any antigen-binding fragment or a single chain thereof. For example, an "antibody" may include any protein or peptide containing such a molecule, which contains at least a portion of an immunoglobulin molecule having a biological activity of binding to an antigen. Non-limiting examples are the complementary determining region (CDR) of a heavy or light chain or a ligand-binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework (FR) region, or any portion thereof, or at least a portion of a binding protein. As used herein, the term "antibody" may refer to an immunoglobulin molecule and an immunologically active portion of an immunoglobulin (Ig) molecule, i.e., a molecule containing an antigen binding site that specifically binds to an antigen (immunoreacts with an antigen). The so-called "specifically binds" or "immunoreacts with..." means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides.

[0342] As used herein, the term "antibody fragment" or "antigen-binding fragment" is a portion of an antibody, such as an F (ab')2 、F (ab)2 、F ab ', F ab , Fv, scFv, etc. Regardless of the structure, antibody fragments are bound to the same antigen recognized by intact antibodies. The term "antibody fragment" may include aptamers (such as spiegelmer), miniantibodies and diabodies. The term "antibody fragment" may also include any synthetic or genetically engineered protein that works 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 antibodies, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies or chimeric antibodies, single-chain antibodies, epitope binding fragments, for example, Fab, Fab' and F(ab')2, Fd, Fv, single-chain Fv (scFv), single-chain antibodies, dAb (domain antibodies), miniantibodies, disulfide-linked Fv (sdFv), fragments comprising VL domains or VH domains, fragments produced by Fab expression libraries and anti-idiotype (anti-Id) antibodies.

[0343] "Single-chain variable fragment" or "scFv" refers to the variable region of the heavy chain of an immunoglobulin (V H ) and the variable region of the light chain (V L) fusion protein. Single-chain Fv ("scFv") polypeptide molecules are covalently linked VH:VL heterodimers that can be expressed by a gene fusion comprising a VH encoding gene and a VL encoding gene linked by a peptide encoding linker. (See Huston et al. (1988) Proc Nat Acad Sci USA 85(16):5879-5883). In some aspects, these regions are connected by a short linker peptide of ten to about 25 amino acids. The linker can be rich in glycine for flexibility and rich in serine or threonine for solubility, and V can be linked to the VH and VL regions. H The N-terminus of L The C-terminus of the antibody is connected to the C-terminus of the antibody, 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 to identify the chemical structure for converting the naturally aggregated but chemically separated light and heavy polypeptide chains from the antibody V region into scFv molecules, which will fold into a three-dimensional structure that is substantially similar to the structure of the antigen binding site. See, for example, U.S. Patent No. 5,091,5 13; U.S. Patent No. 5,892,019; U.S. Patent No. 5,132,405; and U.S. Patent No. 4,946,778, the entire contents of each of which are incorporated herein by reference.

[0344] Very large original human scFv libraries have been created and can be created to provide a large source of rearranged antibody genes for a plethora 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)).

[0345] The antibody of the present invention can also be modified to produce mosaic antibodies. Mosaic antibodies are such antibodies: the external amino acid residues of an antibody of one species are reasonably replaced or "mosaiced" by the external amino acid residues of an antibody of a second species, so that the antibody of the first species is non-immunogenic in the second species, thereby reducing the immunogenicity of the antibody. Since the antigenicity of a protein depends primarily on its surface properties, the immunogenicity of an antibody can be reduced by replacing exposed residues, which are different from those residues usually found in antibodies of another mammalian species. The rational replacement of this external residue should have little or no effect on internal domains or interdomain contacts. Therefore, ligand binding properties should not be affected because changes are limited to variable region framework residues. This process is referred to as "mosaicing" because only the outer surface or outer layer of the antibody changes, and supporting residues remain undisturbed.

[0346] The "mosaic" process utilizes sequence data of human antibody variable domains compiled by the available Kabat et al. (1987) Sequences of Proteins of Immunological interest, 4th edition, Bethesda, Md., National Institutes of Health, updates to this database, and other accessible U.S. and foreign databases (nucleic acids and proteins). Non-limiting examples of methods for generating mosaic antibodies include EP 519596, U.S. Patent No. 6,797,492; and described in Padlan et al., 1991.

[0347] Antibody molecules obtained from humans are divided into five classes of immunoglobulins: IgG, IgM, IgA, IgE and IgD, which differ from each other by the nature of the heavy chains present in the molecule. It will be appreciated by those skilled in the art that heavy chains are classified as gamma, mu, alpha, delta or epsilon (γ, μ, α, δ, ε), with some subclasses (e.g., γ1 to γ4) among them. Certain classes also have subclasses, such as IgG1, IgG2, IgG3 and IgG4, etc. Immunoglobulin subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgG5, etc., are well characterized and known to confer functional specialization. With regard to IgG, a standard immunoglobulin molecule comprises 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 to 70,000. These four chains are usually joined in a "Y" conformation by disulfide bonds, wherein the light chain begins at the opening of the "Y" and continues to support the heavy chain 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.

[0348] Light chains are classified as kappa or lambda (κ, λ). Each heavy chain category can be combined with a κ light chain or a λ light chain. Generally speaking, the light chain and the heavy chain are covalently bound to each other, and when the immunoglobulin is produced by a hybridoma, a B cell, or a genetically engineered host cell, the "tail region" portion of the two heavy chains is bound to each other by covalent disulfide bonds or non-covalent bonds. In the heavy chain, the amino acid sequence extends from the N-terminus at the forked end of the Y conformation to the C-terminus at the bottom of each chain.

[0349] Both light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. The variable domains of both the light (VL) chain portion and the heavy (VH) chain portion determine antigen recognition and specificity. In contrast, the constant domains of the light chain (CL) and the heavy chain (CH1, CH2 or CH3) confer important biological properties, such as secretion, transplacental mobility, Fc receptor binding, complement binding, etc. The term "antigen binding site" or "binding portion" may refer to the portion of the immunoglobulin molecule that participates in antigen binding. The antigen binding site is formed by the amino acid residues of the N-terminal variable ("V") region of the heavy chain ("H") and the light chain ("L"). The three highly different segments in the V region of the heavy and light chains, called "hypervariable regions", are inserted between the more conservative flanking segments called "framework regions" or "FRs". Therefore, the term "FR" may refer to the amino acid sequences that naturally occur between the hypervariable regions of immunoglobulins and adjacent to these hypervariable regions. 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 the bound antigen, and the three hypervariable regions of the heavy chain and the light chain are each called "complementarity determining region" or "CDR". The VH region and VL region containing CDR and the framework (FR) of the CTLA-4 antibody are shown in Tables 1A to 12B.

[0350] The six CDRs present in each antigen binding domain are short non-continuous amino acid sequences that are specifically positioned to form an antigen binding domain because the antibody presents its three-dimensional conformation in an aqueous environment. The remaining amino acids in the amino acids in the antigen binding domain (FR region) show less intermolecular variability. The framework region mainly adopts a β-sheet conformation, and the CDR forms a loop connecting the β-sheet structure (and forming a part of the β-sheet structure in some cases). The framework region is used to form a scaffold that provides a CDR with the correct orientation through interchain non-covalent interactions. The antigen binding domain formed by the positioned CDR provides a surface complementary to the epitope on the immunoreactive antigen, which promotes the non-covalent binding of the antibody to its cognate epitope. For the heavy chain variable region or the light chain variable region, one of ordinary skill in the art can readily identify the amino acids comprising the CDR and framework regions, respectively, as they have been previously defined (see “Sequences of Proteins of Immunological Interest,” Kabat, E. et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0351] In the case of two or more definitions of terms used and / or accepted in the art, the definition of the term as used herein is intended to include all such meanings, unless there is a clear statement to the contrary. A specific example is the use of the term "complementarity determining region" ("CDR") to describe the non-continuous antigen binding sites present in the variable region of heavy chain polypeptides and light chain polypeptides. This specific region has been described by Kabat et al., US Pat. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and by Chothia et al., J. Mol. Biol. 196: 901-917 (1987), the entire contents of which are incorporated herein by reference. The definition of CDR according to Kabat and Chothia is included in the overlap or subset of amino acid residues when compared to each other. However, the application of any definition of the CDR for referring to an antibody or its variant is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues of the CDR defined by each reference in the reference cited above are listed in the following table as a comparison. The exact number of residues encompassing a particular CDR will vary depending on the sequence and size of the CDR. Given the variable region amino acid sequence of the antibody, one skilled in the art can determine in a routine manner which residues comprise a particular CDR.

[0352]

[0353]

[0354] Kabat et al. defined a numbering system for variable domain sequences that is applicable to any antibody. A skilled person can explicitly assign the "Kabat numbering" system to any variable domain sequence without relying on any experimental data other than the sequence itself. As used herein, "Kabat numbering" refers to the numbering system described by Kabat et al., US Pat. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0355] In addition to the above table, the Kabat numbering system also describes the following CDR regions: CDR-H1 starts at about amino acid 31 (i.e., about 9 residues after the first cysteine ​​residue), includes about 5 to 7 amino acids, and ends at the next tryptophan residue. CDR-H2 starts at the fifteenth residue after the end of CDR-H1, includes about 16 to 19 amino acids, and ends at the next arginine or lysine residue. CDR-H3 starts at about the thirty-third amino acid residue after the end of CDR-H2; includes 3 to 25 amino acids; and ends at the sequence WGXG, where X is any amino acid. CDR-L1 starts at about residue 24 (i.e., after the cysteine ​​residue); includes about 10 to 17 residues; and ends at the next tryptophan residue. CDR-L2 starts at about the sixteenth residue after the end of CDR-L1 and includes about 7 residues. CDR-L3 begins at about the thirty-third residue after the end of CDR-L2 (ie, after the cysteine ​​residue); includes about 7 to 11 residues and ends at the sequence F or WGXG, where X is any amino acid.

[0356] As used herein, the term "epitope" may include any protein determinant that can be specifically bound to an immunoglobulin, scFv or T cell receptor. The variable region allows an antibody to selectively recognize and specifically bind to an epitope on an antigen. For example, the VL domain and VH domain or complementary determining region (CDR) subset of an antibody combine to form a variable region that defines a three-dimensional antigen binding site. This quaternary antibody structure forms an antigen binding site present at the end of each arm of the Y. The epitope determinant can be composed of chemically active surface groups (such as amino acids or sugar side chains) of a molecule, and generally has specific three-dimensional structural features and specific charge characteristics. For example, antibodies can be produced for the N-terminal peptide or C-terminal peptide of a polypeptide. More specifically, the antigen binding site is defined by three CDRs on each of the VH chain and the VL chain (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In one embodiment, the antibody may be directed against human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) (CTLA-4_HUMAN) (length 223 amino acid residues) having Ann UniProt reference number: P16410, which comprises the amino acid sequence of SEQ ID NO: [ ]:

[0357]

[0358] As used herein, the terms "immunobinding" and "immunobinding properties" may refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of the immunobinding interaction can be measured using the dissociation constant (K) of the interaction. d ) indicates that K d The smaller the value, the greater the affinity. Methods well known in the art can be used to quantify the immunological binding properties of a selected polypeptide. One such method requires measuring the rates of formation and dissociation of the antigen binding site / antigen complex, where these rates depend on the concentration of the complex partner, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, the "binding rate constant" (K on ) and the “dissociation rate constant” (K off ) can be determined by calculating the concentration and the actual binding and dissociation rates. (See Nature 361: 186-87 (1993)). off / K on The ratio of allows elimination of all parameters not related to affinity and is equal to the equilibrium binding constant K D (See generally Davies et al. (1990) Annual Rev Biochem 59:439-473). When the equilibrium binding constant (K) is measured by a kinetic assay such as a radioligand binding assay or similar assays known to those skilled in the art such as BIAcore or Octet (BLI), the equilibrium binding constant (K) is the binding constant of the binding site. D ) is ≤1 μM, ≤10 μM, ≤10 nM, ≤10 pM, or ≤100 pM to about 1 pM, the antibodies of the present invention can specifically bind to the CTLA4 epitope. For example, in some embodiments, K D From about 1E-12M to K D About 1E-11M. In some embodiments, K D From about 1E-11M to K D About 1E-10 M. In some embodiments, K D From about 1E-10M to K D About 1E-9 M. In some embodiments, K D From about 1E-9M to K D About 1E-8M. In some embodiments, K D From about 1E-8M to K D About 1E-7M. In some embodiments, K D From about 1E-7M to K D About 1E-6M. For example, in some embodiments, K D is about 1E-12M, while in other embodiments, K Dis about 1E-11 M. In some embodiments, K D is about 1E-10M, while in other embodiments, K D is about 1E-9 M. In some embodiments, K D is about 1E-8M, while in other embodiments, K D is about 1E-7M. In some embodiments, K D is about 1E-6M, while in other embodiments, K D is about 1E-5M. In some embodiments, for example, K D is about 3E-11M, while in other embodiments, K D In some embodiments, K D About 6E-11 M. "Specifically binds" or "has specificity for..." can refer to an antibody that binds to an epitope via its antigen binding domain, and that the binding requires some complementarity between the antigen binding domain and the epitope. For example, an antibody is said to "specifically bind" to an epitope when it binds to the epitope via its antigen binding domain more readily than it would to a random, unrelated epitope.

[0359] For example, CTLA4 antibodies can be monovalent or divalent and can contain single or double chains. Functionally, the binding affinity of CTLA4 antibodies is in the range of 10 -5 M to 10 -12 For example, the binding affinity of CTLA4 antibody is 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 -12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 - 11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 -11 M, 10 -5 M to 10 -10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10- 10 M, 10 -8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10 -7 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M or 10 -5 M to 10 -6 M.

[0360] CTLA-4 protein or its derivatives, fragments, analogs, homologs or orthologs can be used as immunogens in the production of antibodies that immunospecifically bind to these protein components. CTLA-4 protein or its derivatives, fragments, analogs, homologs or orthologs coupled to proteoliposomes can be used as immunogens in the production of antibodies that immunospecifically bind to these protein components.

[0361] One skilled in the art will recognize that by finding out whether a human monoclonal antibody blocks binding of a human monoclonal antibody of the invention to CTLA-4, one can determine without undue experimentation whether the former has the same specificity as the latter. For example, if a tested human monoclonal antibody competes with a human monoclonal antibody of the invention, as indicated by a reduction in binding by the human monoclonal antibody of the invention, then the two monoclonal antibodies are likely to bind to the same epitope or to a closely related epitope.

[0362] Another way to determine whether a human monoclonal antibody has the specificity of the human monoclonal antibodies of the present invention is to pre-incubate the human monoclonal antibodies of the present invention with the CTLA-4 protein (with which the human monoclonal antibodies of the present invention typically react), and then add the tested human monoclonal antibody to determine whether the tested human monoclonal antibody is inhibited in its ability to bind to CTLA-4. If the tested human monoclonal antibody is inhibited, it is very likely to have the same or functionally equivalent epitope specificity as the monoclonal antibody of the present invention. Screening for the human monoclonal antibodies of the present invention can also be performed by utilizing CTLA-4 and determining whether the tested monoclonal antibody is able to neutralize CTLA-4.

[0363] Various methods known in the art can be used to produce polyclonal or monoclonal antibodies against the protein of the present invention or against its derivatives, fragments, analogs, homologs or orthologs. (See, for example, Antibodies: A Laboratory Manual, Harlow E and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference).

[0364] Antibodies can be purified by well-known techniques, such as affinity chromatography using protein A or protein G, which primarily provide the IgG fraction of immune serum. Subsequently, or alternatively, a specific antigen or epitope thereof that is the target of the immunoglobulin being explored can be fixed on a column to purify the immunospecific antibodies by immunoaffinity chromatography. For example, D. Wilkinson (The Scientist, The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28) discusses methods for the purification of immunoglobulins.

[0365] As used herein, the term "monoclonal antibody" or "mAb" or "Mab" or "monoclonal antibody composition" may refer to a population of antibody molecules containing only one molecular species of antibody molecules composed of a unique light chain gene product and a unique heavy chain gene product. Specifically, the complementarity determining regions (CDRs) of the monoclonal antibodies are identical in all molecules of the population. MAbs contain an antigen binding site that is capable of immunoreacting with a specific epitope of an antigen, which is characterized by having a unique binding affinity for the antigen binding site.

[0366] Monoclonal antibodies can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, mice, hamsters or other suitable host animals are usually 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.

[0367] Immunizing agents can include protein antigens, fragments thereof, or fusion proteins thereof. For example, if human cells are needed, peripheral blood lymphocytes can be used, or if cells from non-human mammalian sources are needed, spleen cells or lymph node cells can be used. Lymphocytes are then fused to an immortalized cell line using a suitable fusion agent (such as polyethylene glycol) to form hybridoma cells (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pages 59 to 103). Immortalized cell lines can be transformed mammalian cells, particularly rodent-derived, bovine-derived, and human-derived myeloma cells. For example, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas will typically contain hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

[0368] Available immortalized cell lines are those that effectively fuse, support stable high-level expression of antibodies by selected antibody-producing cells and are sensitive to culture medium (such as HAT culture medium). For example, immortalized cell lines can be mouse myeloma lines, which can be obtained from, for example, Salk Institute Cell Distribution Center (San Diego, California) and American Type Culture Collection (Manassas, Virginia). Human myeloma cell lines and mouse-human heterogeneous myeloma cell lines have also been described for producing 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) 51 to 63 pages)).

[0369] The presence of monoclonal antibodies to the antigen in the culture medium of the hybridoma cells can then be determined. For example, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is 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. For example, the binding affinity of the monoclonal antibodies can be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107:220 (1980). In addition, in the therapeutic application of monoclonal antibodies, it is important to identify antibodies that have a high degree of specificity and high binding affinity to the target antigen.

[0370] After identifying the desired hybridoma cells, the clones can be subcloned by limiting dilution procedures and then grown by standard methods. (See Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pages 59 to 103). Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle medium and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo as mammalian ascites.

[0371] 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, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography).

[0372] Monoclonal antibodies can also be prepared by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (incorporated herein by reference in its entirety). DNA encoding the monoclonal antibodies of the present invention can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies). The hybridoma cells of the present invention serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into a host cell, such as a monkey COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not produce immunoglobulins, to obtain the synthesis of monoclonal antibodies in a recombinant host cell. DNA can also be modified, for example, by replacing homologous mouse sequences with coding sequences for human heavy and light chain constant domains (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently joining all or part of the coding sequence for non-immunoglobulin polypeptides to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen combining site of an antibody of the invention to create a chimeric bivalent antibody.

[0373] Fully human antibodies are, for example, antibody molecules in which the entire sequence of both the light chain and the heavy chain (including CDR) is from human genes. Such antibodies are referred to as "human antibodies" or "fully human antibodies". Human monoclonal antibodies (such as fully human antibodies and humanized antibodies) can be prepared using trioma technology; human B cell hybridoma technology (see Kozbor et al., 1983 Immunol Today 4: 72); and EBV hybridoma technology for producing human monoclonal antibodies (see Cole et al., 1985 MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77 to 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).

[0374] "Humanized antibodies" can be antibodies from non-human species (such as mice), whose amino acid sequences (e.g., in the CDR regions) have been modified to increase their similarity to antibody variants produced by humans. Antibodies can be humanized by methods known in the art (e.g., CDR transplantation). See also Safdari et al., (2013) Biotechnol Genet Eng Rev.; 29: 175-86. In addition, humanized antibodies can be produced in transgenic plants as a cheap production alternative to existing mammalian systems. For example, 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 by using Agrobacterium tumefaciens or particle bombardment. For example, a nucleic acid expression vector containing at least heavy and light chain sequences is expressed in a bacterial culture (i.e., Agrobacterium tumefaciens strain BLA4404) by transformation. Plant penetration can be accomplished by injection. Soluble leaf extracts can be prepared by grinding leaf tissue in a mortar and by centrifugation. Isolation and purification of antibodies can be readily performed by many methods known to those skilled in the art. Other methods for producing antibodies in plants are described in, for example, Fischer et al., Vaccine, 2003, 21: 820-5; and Ko et al., Current Topics in Microbiology and Immunology, Vol. 332, 2009, pp. 55-78. Thus, the present invention also provides any cell or plant comprising a vector encoding an antibody of the present invention or producing an antibody of the present invention.

[0375] Antibodies can be humanized using a variety of techniques known in the art, including, for example, CDR grafting (EP 239,400; PCT Publication WO 91 / 09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332, which is incorporated herein by reference in its entirety). “Humanization” (also known as reshaping or CDR grafting) is a well-established technique understood by the skilled artisan for reducing the immunogenicity of monoclonal antibodies (mAbs) from xenogeneic sources (usually rodents) and for improving their 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).

[0376] In addition, other techniques, including phage display libraries, can also be used to produce antibodies (such as human antibodies). (See Hoogenboom and Winter, J. Mol. Biol, 227: 381 (1991); Marks et al., J. Mol. Biol, 222: 581 (1991)). Similarly, human antibodies can be prepared by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated). After challenge, human antibody production is observed, which is highly similar to that seen in humans in all aspects, including gene rearrangement, assembly, and antibody repertoire. This method 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; and 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).

[0377] Transgenic non-human animals can be used to produce human antibodies in addition, and the animals are modified to produce fully human antibodies, rather than endogenous antibodies of animals in response to antigenic stimulation. (See PCT Publication No. WO94 / 02602 and U.S. Patent No. 6,673,986). Endogenous genes encoding heavy immunoglobulin chains and light immunoglobulin chains in non-human hosts have been disabled, and active loci encoding human heavy and light chain immunoglobulins are inserted into the genome of the host. For example, yeast artificial chromosomes containing necessary human DNA segments are used to incorporate human genes. Then, by hybridizing intermediate transgenic animals with fewer modifications than the full complement of all desired modifications, animals providing all desired modifications are obtained as offspring. Non-limiting examples of such non-human animals are mice, and are referred to as Xenomouse as disclosed in PCT Publication Nos. WO96 / 33735 and WO96 / 34096. TM. The B cells produced by such animals can secrete fully human immunoglobulins. Antibodies can be obtained directly from the animal after immunization with the immunogen of interest, for example, a preparation of polyclonal antibodies, or alternatively from immortalized B cells derived from the animal, such as a hybridoma producing monoclonal antibodies. In addition, genes encoding immunoglobulins with human variable regions can be recovered and expressed to obtain antibodies directly, or can be further modified to obtain analogs of the antibodies, such as single-chain Fv (scFv) molecules.

[0378] Thus, using such techniques, therapeutically useful IgG, IgA, IgM and IgE antibodies can be produced. For a review of such techniques for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73: 65-93 (1995). For a detailed discussion of such techniques for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, for example, PCT Publication No. 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 entirety. In addition, companies such as Creative BioLabs (Shirley, NY) can be hired to provide human antibodies to a chosen antigen using technology similar to that described herein.

[0379] An example of a method for producing a non-human host (e.g., mouse) lacking endogenous immunoglobulin heavy chain expression is disclosed in U.S. Pat. No. 5,939,598. It can be obtained by the following method: the method comprises 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 formation of transcripts of the rearranged immunoglobulin heavy chain locus, the deletion being achieved by a targeting vector comprising a gene encoding a selective marker; then generating a transgenic mouse from the embryonic stem cell, whose somatic cells and germ cells contain genes encoding the selective marker.

[0380] A method for producing an antibody of interest (e.g., a human antibody) is disclosed in U.S. Pat. No. 5,916,771. The method comprises introducing an expression vector comprising a nucleotide sequence encoding a heavy chain into a mammalian host cell in culture, introducing an expression vector comprising a nucleotide sequence encoding a light chain into another mammalian host cell, and fusing the two cells to form a hybrid cell. The hybrid cell expresses an antibody comprising a heavy chain and a light chain.

[0381] In a further improvement of this procedure, PCT Publication No. WO 99 / 53049 discloses a method for identifying clinically relevant epitopes on an immunogen and a related method for selecting antibodies that immunospecifically bind with high affinity to the relevant epitopes.

[0382] The antibody of interest can also be expressed by a vector comprising a DNA segment encoding a single-chain antibody as described herein. Vectors include, but are not limited to: chemical conjugates with a targeting portion (e.g., a ligand for a cell surface receptor) and a nucleic acid binding portion (e.g., polylysine) (such as described in WO 93 / 64701); viral vectors (e.g., DNA or RNA viral vectors); fusion proteins as fusion proteins containing a target portion (e.g., an antibody specific for a target cell) and a nucleic acid binding portion (e.g., protamine) (such as described in PCT / US 95 / 02140 (WO 95 / 22618)); plasmids; bacteriophages; viral vectors, etc. The vector can be chromosomal, non-chromosomal, or synthetic. Retroviral vectors can also be used and include Moloney murine leukemia virus. DNA viral vectors may also be used and include: poxvirus vectors, such as orthopoxvirus or avipoxvirus vectors; herpesvirus vectors, such as herpes simplex type 1 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); adenovirus vectors (see LeGal LaSalle et al., Science, 259:988 (1993); Davidson et al., Nat. Genet. 3:219 (1993); Yang et al., J. Virol. 69:2004 (1995); and adeno-associated virus vectors (see Kaplitt, MG. et al., Nat. Genet. 8:148 (1994).

[0383] Poxvirus vectors introduce genes into the cytoplasm of cells. Fowlpoxvirus vectors only cause 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. Compared with adeno-associated virus (about 4 months), adenovirus vectors cause shorter-term expression (about 2 months), and the expression time of adeno-associated virus is shorter than that of HSV vectors. The specific vector selected will depend on the target cell and the condition being treated. Introduction can be carried out by standard techniques such as infection, transfection, transduction or transformation. Examples of gene transfer modes include, for example, naked DNA, CaPO4 precipitation, DEAE dextran, electroporation, protoplast fusion, liposome transfection, cell microinjection, and viral vectors.

[0384] The vector can be used to target substantially any desired target cell. For example, stereotactic injection can be used to guide the vector (e.g., adenovirus, HSV) to the desired position. In addition, a micropump infusion system, such as the SynchroMed infusion system, can be used to deliver particles by intracerebroventricular (icv) infusion. A method based on bulk flow (called convection) has also been shown to be effective in delivering macromolecules to extended areas of the brain and can be used to deliver the vector 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 catheters, intravenous injections, parenteral injections, intraperitoneal injections and subcutaneous injections, as well as oral or other known routes of administration.

[0385] These vectors can be used to express large quantities of antibodies that can be used in a variety of ways. For example, to detect the presence of CTLA4 in a sample. The antibodies can also be used to try to bind to CTLA4 and disrupt its activity.

[0386] In embodiments, the antibodies herein can be delivered to a subject using a gene-based method. For example, a nucleotide sequence of an anti-CTLA4 antibody as described herein can be delivered to a subject using a vector, DNA, or RNA. See, for example, Deal, Cailin E., Andrea Carfi, and Obadiah J. Plante. "Advancements in mRNA Encoded Antibodies for Passive Immunotherapy." Vaccines 9.2 (2021): 108. Such methods can allow the design and generation of more complex antibody molecules with improved and / or increased functionality.

[0387] Viral vectors, such as adenovirus (Ad) and adeno-associated virus (AAV), have been engineered for in vivo mAb expression by replacing a portion of their genome with a nucleic acid sequence encoding an antibody of interest. The term "viral vector" may refer to a nucleic acid vector construct comprising at least one element of viral origin and having the ability to be encapsulated into a viral vector particle. The viral vector may contain nucleic acids encoding antibodies, antigen-binding portions thereof, or CARs as described herein to replace non-essential viral genes. Carriers and / or particles can be used for the purpose of transferring any nucleic acid into a cell in vitro or in vivo.

[0388] Retrovirus is a common tool for gene delivery. In embodiments, retrovirus is used to deliver polynucleotides encoding antibodies to cells. The term "retrovirus" may refer to an RNA virus whose genomic RNA is reverse transcribed into a linear double-stranded DNA copy and whose genomic DNA is then covalently integrated into the host genome. Once the virus is integrated into the host genome, it is referred to as a "provirus". The provirus acts as a template for RNA polymerase II and guides the expression of RNA molecules encoding the structural proteins and enzymes required for the production of new viral particles.

[0389] Exemplary retroviruses suitable for use in specific embodiments include, but are not limited to, Moloney murine leukemia virus (M-MuLV), Moloney murine sarcoma virus (MoMSV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), Gibbon ape leukemia virus (GaLV), feline leukemia virus (FLV), foamy virus, Friend murine leukemia virus, murine stem cell virus (MSCV), and Rous sarcoma virus (RSV)) and lentiviruses.

[0390] The term "lentivirus" may refer to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Vesna-Medi virus (VMV) virus; 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, HIV-based vector backbones (i.e., HIV cis-acting sequence elements) are preferred. In a specific embodiment, lentivirus is used to deliver a polynucleotide comprising CAR to a cell.

[0391] Gene transfer of the antibody nucleotide sequence itself (such as DNA or RNA) is an alternative option for transient in vivo antibody production. For example, the plasmid DNA method can utilize electroporation components to enhance the transfection efficiency after IM delivery. In addition, mRNA delivery can be used for in vivo expression of antibodies. In embodiments, the mRNA encoding the antibody of interest can be modified, such as optimizing mRNA codon usage, and lipid nanoparticles can be used for mRNA delivery.

[0392] In one embodiment, the antibodies described herein may be full length antibodies, containing an Fc region similar to a wild-type Fc region that binds to an Fc receptor.

[0393] A variety of techniques can be adapted to produce single-chain antibodies specific for the antigenic proteins of the invention (see, e.g., U.S. Pat. No. 4,946,778). In addition, a variety of methods can be adapted to construct F ab Expression libraries (see, e.g., Huse et al., 1989 Science 246: 1275-1281) are used to allow rapid and efficient identification of monoclonal F3knockins with the desired specificity for a protein or its derivatives, fragments, analogs or homologs. ab Fragments. Antibody fragments containing idiotypes directed against protein antigens can be produced by techniques known in the art, including but not limited to: (i) F fragments produced by pepsin digestion of antibody molecules; (ab')2 fragment; (ii) by reducing F (ab')2 The disulfide bridges of the fragments generate F ab (iii) F fragments generated by treating antibody molecules with papain and a reducing agent ab fragment, and (iv) F v Fragment.

[0394] Heteroconjugate antibodies are also within the scope of the present invention. Heteroconjugate antibodies are composed of two covalently linked antibodies. For example, such antibodies can target immune system cells to unwanted cells (see U.S. Patent No. 4,676,980) and are used to treat HIV infection (see PCT Publication No. WO91 / 00360; WO92 / 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 iminothiolates and methyl-4-mercaptobutyrimide esters, and those disclosed, for example, in U.S. Patent No. 4,676,980.

[0395] The antibodies of the present invention can be modified in terms of effector functions to enhance, for example, the effectiveness of antibodies in treating cancer. For example, cysteine ​​residues can be introduced into the Fc region so that interchain disulfide bonds are formed in the region. The homodimeric antibodies thus generated may have improved internalization capacity and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1 191-1 195 (1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively, antibodies with dual Fc regions can be designed so that complement lysis and ADCC capabilities can be enhanced. (See Stevenson et al., Anti-Cancer Drug Design, 3: 219-230 (1989)). In one embodiment, the antibodies of the present invention have modifications to the Fc region so that the Fc region does not bind to an Fc receptor. For example, an Fc receptor is an Fcγ receptor. Antibodies with modifications to the Fc region such that the Fc region does not bind to Fcγ but still binds to the neonatal Fc receptor are useful, as described herein.

[0396] In certain embodiments, the antibodies of the present invention may comprise an Fc variant comprising an amino acid substitution that changes the non-antigen-dependent effector function of the antibody, particularly the circulating half-life of the antibody. When compared with antibodies lacking these substitutions, such antibodies exhibit increased or decreased binding to FcRn, and therefore have an increased or decreased half-life in serum, respectively. It is expected that Fc variants with improved affinity for FcRn have a longer serum half-life, and such molecules can be used in methods for treating mammals (wherein a long half-life of the administered antibody is desired, such as to treat chronic diseases or disorders). In contrast, it is expected that Fc variants with reduced FcRn binding affinity have a shorter half-life, and such molecules can also be used, for example, for administration to mammals (wherein the shortened circulation time can be advantageously used, for example, for in vivo diagnostic imaging, or where the initial antibody has toxic side effects when it exists in the circulation for an extended period). Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta, and therefore can also be used to treat diseases or disorders in pregnant women. In addition, other applications of the FcRn binding affinity that may be expected to be reduced include those applications in which it is expected to be located in the brain, kidneys and / or liver. In one embodiment, antibodies containing Fc variants can show reduced transport from the vasculature through the epithelium of the kidney glomerulus. In another embodiment, antibodies containing Fc variants can show reduced transport from the brain through the blood-brain barrier (BBB) ​​into the vascular space. In one embodiment, the antibody with the FcRn combination of changes is included in the 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 to 299 (according to EU numbering). Exemplary amino acid substitutions with the FcRn binding activity of changes are disclosed in PCT Publication No. WO05 / 047327, which is incorporated herein by reference. In certain exemplary embodiments, an antibody or fragment thereof of the invention comprises an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (EU numbering).

[0397] In some embodiments, mutations are introduced into the constant region of mAb so that the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of mAb is changed. For example, mutations are LALA mutations in the CH2 domain. In one embodiment, an antibody (e.g., human mAb or bispecific Ab) contains mutations on a scFv unit of a heterodimer mAb, which reduces ADCC activity. In another embodiment, mAb contains mutations on both chains of a heterodimer mAb, which completely eliminate ADCC activity. For example, mutations introduced into one or two scFv units of mAb are LALA mutations in the CH2 domain. These mAbs with variable ADCC activity can be optimized so that mAb shows maximum selective killing of cells expressing an antigen recognized by mAb, but shows minimum killing of a second antigen recognized by mAb.

[0398] In other embodiments, the antibodies of the present invention used in the diagnosis and treatment methods described herein have a constant region, for example, an IgG1, IgG2 or IgG4 heavy chain constant region, which can be changed to reduce or eliminate glycosylation. For example, the antibodies of the present invention may also include Fc variants, which include amino acid substitutions that change the glycosylation of the antibody. For example, Fc variants may have reduced glycosylation (e.g., N-linked or O-linked glycosylation). In some embodiments, Fc variants include reduced glycosylation of N-linked polysaccharides that are usually present at amino acid position 297 (EU numbering). In another embodiment, the antibody has an amino acid substitution near or within a glycosylation motif (e.g., an N-linked glycosylation motif containing an amino acid sequence NXT or NXS). In a specific embodiment, the antibody is included in an Fc variant with an amino acid substitution at amino acid position 228 or 299 (EU numbering). In a more specific embodiment, the antibody includes an IgG1 or IgG4 constant region, which includes S228P and T299A mutations (EU numbering).

[0399] Exemplary amino acid substitutions that confer reduced or altered glycosylation are described in PCT Publication No. WO05 / 018572, which is incorporated herein by reference in its entirety. In some embodiments, the antibodies or fragments thereof of the present 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 being bound by theory, "agly" antibodies or fragments thereof may have improved in vivo safety and stability characteristics. Exemplary agly antibodies or fragments thereof include a non-glycosylated Fc region of an IgG4 antibody that lacks Fc effector functions, thereby eliminating the possibility of Fc-mediated toxicity to normal life tissues and cells expressing CTLA4. In other embodiments, the antibodies or fragments thereof of the present invention include altered polysaccharides. For example, an antibody may have a reduced number of fucose residues on the N-glycans at Asn297 of the Fc region, i.e., the antibody is non-fucosylated. In another embodiment, an antibody may have a changed number of sialic acid residues on the N-glycans at Asn297 of the Fc region.

[0400] The invention also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof) or a radioactive isotope (ie, a radioconjugate).

[0401] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and trichothecenes. A variety of radionuclides can be used to generate radioconjugated antibodies. Non-limiting examples include 212 Bi, 131 I. 131 In, 90 Y and 186 Re.

[0402] 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 hydrochloride), 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 toluene 2,6-diisocyanate), and bis-active fluorine-containing 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 chelator for conjugating radionucleotides to antibodies (see PCT Publication No. WO 94 / 11026 and U.S. Pat. No. 5,736,137).

[0403] Those of ordinary skill in the art will appreciate that a wide variety of possible moieties may be coupled to the resulting antibodies or other molecules 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), the entire contents of which are incorporated herein by reference).

[0404] As long as the antibody and other parts retain their respective activities, coupling can be completed by any chemical reaction that will bind the two molecules. This connection may include many chemical mechanisms, such as covalent binding, affinity binding, embedding, coordination binding and complexing. In one embodiment, the combination is covalent binding. Covalent binding can be achieved by direct condensation of existing side chains or by incorporation of external bridging molecules. Many divalent or multivalent linking agents can be used for coupling protein molecules (such as antibodies of the present invention) with other molecules. For example, representative coupling agents can include organic compounds, such as thioesters, carbodiimides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzene and hexamethylenediamine. This list is not intended to exhaust various types of coupling agents known in the art, but is an example of more common coupling agents. (See Killen and Lindstrom, Jour. Immun. 133: 1335-2549 (1984); Jansen et al., Immunological Reviews 62: 185-216 (1982); and Vitetta et al., Science 238: 1098 (1987)). Non-limiting examples of linkers are described in the literature. (See, e.g., Ramakrishnan, S. et al., Cancer Res. 44: 201-208 (1984) describing the use of MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester)). See also, U.S. Pat. No. 5,030,719, describing the use of haloacetohydrazide derivatives coupled to antibodies via oligopeptide linkers. Non-limiting examples of useful linkers that can be used with the antibodies of the present invention include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)-toluene (Pierce Chem. Co., catalog number (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)-propionamido] hexanoate (Pierce Chem. Co. catalog number 2165-G); and (v) sulfo-NHS (-hydroxysulfo-succinimide: Pierce Chem. Co., catalog number 2165-G) conjugated to EDC. Chem. Co., catalog number 24510).

[0405] The joints described herein comprise components with different properties, thereby producing conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. Joints comprising NHS esters have lower solubility than sulfo-NHS esters. In addition, the joint SMPT comprises a sterically hindered disulfide bond and can form a conjugate with higher stability. Disulfide bonds are generally less stable than other bonds because disulfide bonds are cleaved in vitro, resulting in fewer conjugates available. In particular, sulfo-NHS can improve the stability of carbodiimide coupling. When used in combination with sulfo-NHS, carbodiimide coupling (e.g., EDC) forms an ester that is more resistant to hydrolysis than a separate carbodiimide coupling reaction.

[0406] The antibodies disclosed herein can also be formulated into immunoliposomes. Liposomes containing the antibodies are 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. Patents 4,485,045 and 4,544,545. Liposomes with extended circulation time are disclosed in U.S. Patent No. 5,013,556.

[0407] Non-limiting examples of available liposomes can be generated by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of defined pore size to obtain liposomes having a desired diameter. The Fab' fragments of the antibodies of the present invention can be conjugated to liposomes as described in Martin et al., J. Biol. Chem., 257: 286-288 (1982) via a disulfide exchange reaction.

[0408] Multispecific antibodies (bispecific and trispecific)

[0409] Multispecific antibodies are antibodies that can recognize two or more different antigens. For example, a bispecific antibody (bsAb) is an antibody comprising two variable domains or scFv units such that the resulting antibody recognizes two different antigens. For example, a trispecific antibody (tsAb) is an antibody comprising two variable domains or scFv units such that the resulting antibody recognizes three different antigens. The present invention provides multispecific antibodies, such as bispecific antibodies and trispecific antibodies, that recognize CTLA-4 and a second antigen and / or a third antigen. In one embodiment, a multispecific antibody (e.g., a bispecific antibody and a trispecific antibody) may include a CTLA-4-specific fusion protein that encompasses the antibodies described herein. Exemplary second and / or third antigens include tumor-associated antigens (e.g., LINGO1), cytokines (e.g., IL-12 (IL-12A (p35 subunit) protein sequence having NCBI Reference No. NP_000873.2; IL-12B (p40 subunit) protein sequence having NCBI Reference No. NP_002178.2); IL-18 (protein sequence having NCBI Reference No. NP_001553.1); IL-15 (protein sequence having NCBI Reference No. NP_000576.1); IL-7 (protein sequence having NCBI Reference No. NP_000871.1); IL-2 (protein sequence having NCBI Reference No. NP_000577.2; and IL-21 (protein sequence having NCBI Reference No. NP_068575.1)), cytokine cognate receptors (e.g., IL-12R), and cell surface receptors. Non-limiting examples of the second antigen and / or the third antigen 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 antibody and the trispecific antibody include a CTLA-4 fusion protein. For example, as described herein, the fusion protein comprises: an antibody comprising a variable domain or a scFv unit, and a ligand or antigen and / or a third ligand or antigen, so that the resulting antibody recognizes the antigen and binds to a ligand-specific receptor.Exemplary antibody compositions (e.g., VH sequences and / or VL sequences or fragments thereof) that can be used to design CTLA-4 fusion proteins as described herein include, but are not limited to, anti-CAIX antibodies described in PCT / US2006 / 046350 and PCT / US2015 / 067178; anti-CXCR4 antibodies described in PCT / US20006 / 005691; anti-CCR4 antibodies described in PCT / US2008 / 088435, PCT / US2013 / 039744, and PCT / US2015 / 054202; 2008 / 088435 and PCT / US2020 / 062815; anti-PD-L1 antibodies described in PCT / US2020 / 037791 and PCT / US2020 / 037781; anti-GITR antibodies described in PCT / US2017 / 043504; anti-tight junction protein-4 antibodies described in PCT / US2019 / 022272; and anti-MUC1 antibodies described in PCT / US2020 / 037783 (each of these applications is incorporated herein by reference in its entirety). In one embodiment, the fusion protein further comprises a constant region and / or a linker as described herein. Different forms of multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies, such as fusion proteins comprising antibodies and ligands that recognize CTLA-4) are described herein.The ligand can be a tumor-associated antigen (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, prostate specific protein (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 for additional tumor-associated surface antigens, which are incorporated herein by reference in their entirety)); cytokines (e.g., IL-12 (IL-12A (p35 subunit) protein sequence with NCBI reference number NP_000873.2; IL-12B (p40 subunit) protein sequence with NCBI reference number NP_002178.2 1 (protein sequence with NCBI reference number NP_001553.1); IL-15 (protein sequence with NCBI reference number NP_000576.1); IL-7 (protein sequence with NCBI reference number NP_000871.1); IL-2 (protein sequence with NCBI reference number NP_000577.2); and IL-21 (protein sequence with NCBI reference 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. Different forms of bispecific antibodies or trispecific antibodies 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 is independently selected from a Fab fragment, a single-chain variable fragment (scFv) or a single domain antibody. In some embodiments, the bispecific antibody or trispecific antibody also includes an Fc fragment (e.g., as described in PCT / US2015 / 021529 and PCT / US2019 / 023382, each of which is incorporated herein by reference in its entirety).The bispecific or trispecific antibodies of the invention may comprise a heavy chain and light chain combination or scFv of the CTLA-4 antibody described herein.

[0410] Methods known in the art can be used to construct multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies) of the invention (e.g., anti-CTLA-4-scFv fusion proteins). In some embodiments, the bispecific antibody is a single polypeptide in which two scFv fragments are joined by a long linker polypeptide having a sufficient length to allow intramolecular association between two scFv units to form an antibody. In other embodiments, the bispecific antibody is more than one polypeptide linked by covalent or non-covalent bonds. In some embodiments, the amino acid linker described herein (GGGGSGGGGS; "(G4S)2" (SEQ ID NO: [])) can be generated with a longer G4S linker to improve flexibility. For example, the linker can also be "(G4S)3" (e.g., GGGGSGGGGSGGGGS (SEQ ID NO: [])); "(G4S)4" (e.g., GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: []); "(G4S)5" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: [])); "(G4S)6" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: [])); "(G4S)7" (e.g., GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: [])); and so on. For example, the use of a (G4S)5 linker can provide greater flexibility for 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) n , wherein 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. US 20180134789 and US20200148771; and PCT Publication No. WO2019051122 (each of which is incorporated herein by reference in its entirety).

[0411] In another embodiment, a "knob into hole" approach (Ridgway et al., Protein Eng 7:617-621 (1996)) can be used to construct multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies, such as anti-CTLA-4-scFv fusions). In this approach, the Ig heavy chains of two different variable domains are reduced to selectively disconnect the heavy chain pairing while maintaining the heavy-light chain pairing. Two heavy-light chain heterodimers that recognize two different antigens / ligands or three different antigens / ligands are mixed to promote heterojunction pairing, which is mediated by the engineered "knob into hole" of the CH3 domain.

[0412] In another embodiment, multispecific antibodies (e.g., bispecific antibodies 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, wherein the first heavy-light chain dimer recognizes CTLA-4 and the second heavy-light chain dimer recognizes the second antigen and / or the third antigen. The mechanism for heavy-light chain dimers is similar to the formation of human IgG4, which is also used as a bispecific molecule. The dimerization reaction of IgG heavy chains is driven by intramolecular forces (such as pairing the CH3 domain of each heavy chain with disulfide bonds). The presence of specific amino acids (R409) in the CH3 domain has been shown to promote dimer exchange and the construction of IgG4 molecules. The heavy chain pairing is also stabilized by inter-heavy chain disulfide bonds in the hinge region of the antibody. Specifically, in IgG4, the hinge region contains the amino acid sequence Cys-Pro-Ser-Cys at amino acids 226 to 230 (compared to the stable IgG1 hinge region containing the sequence Cys-Pro-Pro-Cys). This sequence difference of the serine at position 229 has been linked to the tendency of IgG4 to form intrachain disulfide bonds 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).

[0413] The multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies, such as anti-CTLA-4-scFv fusions) of the invention can be formed by introducing the R409 residue in the CH3 domain of an antibody that recognizes CTLA-4 or a second antigen and / or a third antigen and a Cys-Pro-Ser-Cys sequence in the hinge region, so that the heavy-light chain dimer is exchanged 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 antigen and / or a third antigen, wherein the second antigen (or ligand) and / or the third antigen (or ligand) are any antigen (or ligand) disclosed herein. Known IgG4 molecules can also be altered so that the heavy and light chains recognize CTLA-4 or a second antigen and / or a third antigen, as disclosed herein. Use of this method for constructing the multispecific antibodies (e.g., bispecific antibodies and trispecific antibodies, such as anti-CTLA-4-scFv fusions) of the invention can be beneficial due to the inherent characteristics of the IgG4 molecule, in which the Fc region differs from other IgG subtypes in that it interacts poorly with the 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 antibodies and trispecific antibodies, such as anti-CTLA-4-scFv fusions) attractive for therapeutic applications in which it is desired that the antibody bind to a target and functionally alter a signaling pathway associated with the target, yet does not trigger effector activity.

[0414] The multispecific antibodies described herein (e.g., bispecific antibodies and trispecific antibodies, such as anti-CTLA-4-scFv fusions) can be engineered with non-depleted heavy chain isotypes (such as IgG1-LALA or a non-depleted variant of stabilized IgG4 or other non-depleted variants). In some embodiments, mutations are introduced into the constant region of the bsAb so that the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of the bsAb is altered. For example, the mutation is a LALA mutation in the CH2 domain. In one aspect, the multispecific antibody (e.g., bispecific antibodies and trispecific antibodies, such as anti-CTLA-4-scFv fusions) contains mutations on one scFv unit of the heterodimeric multispecific antibody that reduces ADCC activity. In another aspect, the multispecific antibody (e.g., bispecific antibodies and trispecific antibodies, such as anti-CTLA-4-scFv fusions) contains mutations on both chains of the heterodimeric multispecific antibody that completely eliminate ADCC activity. For example, the mutation introduced in 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 so that the multispecific antibodies exhibit maximal selective killing of cells expressing one antigen recognized by the multispecific antibody, yet exhibit minimal killing of a second antigen recognized by the multispecific antibody.

[0415] The multispecific antibodies (e.g., bispecific antibodies) described herein can be engineered into modular tetrameric bispecific antibodies (tBsAbs). See, for example, WO 2018 / 071913, which is incorporated herein by reference in its entirety. For example, a tetravalent antibody can be a dimer of a bispecific scFv fragment comprising a first binding site for a first antigen and a second binding site for a second antigen. In an embodiment, an anti-CTLA-4 antibody can be a first binding site for a first antigen. In an embodiment, an anti-CTLA-4 antibody can be a second binding site for a second antigen. The two binding sites can be joined together via a joint domain. In an embodiment, the scFv fragment is a tandem scFv, and the joint domain comprises an immunoglobulin hinge region (e.g., IgG1, IgG2, IgG3, or IgG4 hinge region) amino acid sequence. In an embodiment, the immunoglobulin hinge region amino acid sequence can be flanked by a flexible joint amino acid sequence, such as a joint amino acid sequence (GGGS) x1-6 、(GGGGS) x1-6Or GSAGSAAGSGEF. In an embodiment, the linker domain comprises at least a portion of an immunoglobulin Fc domain, e.g., an IgG1, IgG2, IgG3, or IgG4 Fc domain. In an embodiment, the at least a portion of the immunoglobulin Fc domain does not comprise a CH2 domain. In an embodiment, the 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 comprise a flexible linker amino acid sequence (e.g., (GGGS)) at one end or at both ends. x1-6 、(GGGGS) x1-6 or GSAGSAAGSGEF(SEQ ID NO:[])).

[0416] In embodiments, the multispecific antibodies (e.g., bispecific antibodies) described herein can be engineered with wild-type Fc regions or modified Fc regions. For example, embodiments can include IgG3 CH2 domains. In other embodiments, the multispecific antibodies (e.g., bispecific antibodies) are engineered without Fc domains and / or Fc fragments.

[0417] In an embodiment, the tBsAb may be specific for CTLA-4, and may also be 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.

[0418] The multispecific antibodies disclosed herein (eg, bispecific antibodies and trispecific antibodies, such as anti-CTLA-4-scFv fusions) can be used to treat chronic infections, diseases, or medical conditions, such as cancer.

[0419] Use of Anti-CTLA-4 Antibodies

[0420] Antibodies of the invention or fragments thereof that specifically bind to CTLA-4 protein can be administered to treat CTLA-4-related diseases or disorders. "CTLA-4-related diseases or disorders" include disease states and / or symptoms associated with disease states in which increased CTLA-4 levels and / or activation of cell signaling pathways involving CTLA-4 are found. In some embodiments, the cancer can be lung cancer, kidney cancer, ovarian cancer, prostate cancer, colon cancer, breast cancer, cervical cancer, uterine cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, glioblastoma multiforme, squamous cell carcinoma of the skin, melanoma, kidney cancer (such as clear cell renal cell carcinoma) or gastric cancer.

[0421] The antibodies of the present invention (including bispecific antibodies, polyclonal antibodies, monoclonal antibodies, humanized antibodies and fully human antibodies) can be used as therapeutic agents. Such agents are often used to treat cancer in a subject, increase vaccine efficiency or enhance natural immune responses. Antibody preparations (e.g., antibody preparations with high specificity and high affinity for their target antigens) are administered to a subject and often have an effect due to their binding to the target. Administration of antibodies can eliminate or inhibit or interfere with the activity of CTLA-4 protein.

[0422] Pharmaceutical composition

[0423] The antibodies or fragments thereof of the invention that specifically bind to CTLA-4 protein can be administered in the form of a pharmaceutical composition to treat cancer. The principles and considerations involved in preparing therapeutic pharmaceutical compositions comprising antibodies and guidance in the selection of components are provided, for example, in the following literature: Remington: The Science And Practice Of Pharmacy 20th Edition (Alfonso R. Gennaro et al., eds.) 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.

[0424] The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the specific antibody, variant or derivative thereof, the patient's age, weight, general health, sex and diet, as well as the time of administration, excretion rate, drug combination and severity of the specific disease being treated. The judgment of medical staff on such factors is within the scope of those of ordinary skill in the art. The amount will also depend on the individual patient to be treated, the route of administration, the type of preparation, the characteristics of the compound used, the severity of the disease and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[0425] The therapeutically effective amount of the antibody of the present invention can be the amount required to achieve the therapeutic goal. As noted herein, this can be the binding interaction between the antibody and its target antigen, in some cases, the binding interaction interferes with the function of the target. In addition, the amount to be administered will depend on the binding affinity of the antibody to its specific antigen, and will also depend on the rate at which the administered antibody is consumed from the free volume of other subjects to which it is administered. The dosage of the antigen-binding polypeptides described herein administered to a subject (e.g., a patient) is generally 0.1 mg / kg patient weight to 100 mg / kg patient weight, 0.1 mg / kg patient weight to 20 mg / kg patient weight, or 1 mg / kg patient weight to 10 mg / kg patient weight. Due to the immune response to foreign polypeptides, human antibodies have a longer half-life in the human body than antibodies from other species. Therefore, lower doses of human antibodies and lower frequency of administration are generally possible. In addition, the dosage and frequency of administration of the antibodies disclosed herein can be reduced by enhancing the intake and tissue penetration (e.g., entering the brain) of the antibodies via modification (such as, for example, lipidation). As a non-limiting example, a typical range of therapeutically effective dosing for an antibody or antibody fragment of the invention may be about 0.1 mg / kg body weight to about 50 mg / kg body weight. Typical dosing frequencies may range, for example, from twice a day to once a week.

[0426] In the case of using antibody fragments, it is preferred that the minimum inhibitory fragment specifically binds to the binding domain of the target protein. For example, based on the variable region sequence of the antibody, a peptide molecule that retains the ability to bind to the target protein sequence can be designed. 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 preparation may also contain more than one active compound as necessary for the specific indications treated, for example, those with complementary activities that do not adversely affect each other. Alternatively or in addition, the composition may include an agent that enhances its function, such as, for example, a cytotoxic agent, a cytokine (eg, IL-15), a chemotherapeutic agent, or a growth inhibitor. Such molecules are appropriately combined in an amount that can effectively achieve the intended purpose.

[0427] The active ingredients can also be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, 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.

[0428] Preparations for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0429] Sustained release preparations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody in the form of shaped articles, e.g., 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 TM (Injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)) and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules for more than 100 days, some hydrogels release proteins for shorter time periods.

[0430] The antibodies or agents of the present invention (also referred to herein as "active compounds") and their derivatives, fragments, analogs and homologues can be incorporated into pharmaceutical compositions suitable for administration. Such pharmaceutical compositions may include antibodies or agents and pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" may include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents compatible with drug administration, etc. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, which is a standard reference text in the field and is incorporated herein by reference. Non-limiting examples of such carriers or diluents include water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous vehicles (such as fixed oils) may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, the use of such media and agents in the composition is considered. Supplementary active compounds may also be incorporated into the composition.

[0431] 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 administration, for example, intravenous administration, intradermal administration, subcutaneous administration, oral administration (e.g., inhalation administration), transdermal administration (i.e., topical administration), transmucosal administration, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous administration may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl paraben; 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 tension, such as sodium chloride or dextrose. pH can be adjusted with an acid or alkali, such as hydrochloric acid or sodium hydroxide. Parenteral preparations may be packaged in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0432] Pharmaceutical compositions suitable for injectable use may 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 TM(BASF, Parsippany, New Jersey) or phosphate buffered saline (PBS). In embodiments, the composition is sterile and is a fluid that is easy to inject. It can be stable under the conditions of manufacture and storage, and can be preserved to prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. For example, by using a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by using a surfactant, appropriate fluidity can be maintained. By various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc., the effect of microorganisms can be prevented. In many cases, isotonic agents can be included in the composition, for example, sugars, polyols (such as mannitol, sorbitol), sodium chloride. The extended absorption of the injectable composition can be achieved by including an agent (for example, aluminum monostearate and gelatin) that delays absorption in the composition.

[0433] Sterile injectable solutions can be prepared by incorporating the required amount of active compound and one or a combination of the above-listed ingredients (as required) into an appropriate solvent, followed by filtration sterilization. For example, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and other ingredients required from those listed above. For sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze drying, producing a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution.

[0434] Oral compositions may include an inert diluent or an edible carrier. Oral compositions may be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound may be blended with an excipient and then used in the form of tablets, lozenges or capsules. Oral compositions may also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is orally administered, spit out or swallowed after gargling. Pharmaceutically compatible binders and / or adjuvant materials may be included in the composition as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients, or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth gum or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate or orange flavoring.

[0435] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or from a nebulizer.

[0436] Systemic administration can also be carried out through mucosal or transdermal means. For transmucosal or transdermal administration, penetrants suitable for permeation barriers are used in the formulation. Such penetrants are well known in the art and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel or cream as well known in the art.

[0437] The compounds may 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.

[0438] In one embodiment, the active compound is prepared with a carrier that will protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulation delivery systems. Biodegradable biocompatible polymers such as ethylene-vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The method for preparing such preparations is obvious to those skilled in the art. These materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (liposomes comprising targeted infected cells, with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These materials can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811.

[0439] Oral or parenteral compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unit dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the unit dosage forms of the present invention are dictated by and directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, as well as the limitations inherent in the art of compounding such an active compound to treat individuals.

[0440] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0441] diagnosis

[0442] The antibodies according to the present invention can be used as agents for detecting the presence of CTLA-4 (or protein fragments thereof) in a sample. For example, the antibody can include a detectable label. The antibody can be polyclonal or monoclonal. The whole antibody or fragments thereof (e.g., F ab , scFv or F (ab)2 ). With respect to probes or antibodies, the term "labeled" may encompass direct labeling of probes or antibodies by coupling a detectable substance to the probe or antibody (i.e., physically linked), as well as indirect labeling of probes or antibodies by reactivity with another directly labeled reagent. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term "biological sample" may include tissues, cells, and biological fluids separated from a subject, as well as tissues, cells, and fluids present in a subject. Therefore, blood and fractions or components of blood, including serum, plasma, or lymph, are included in the use of the term "biological sample." That is, the detection method of the present invention may 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 detecting analyte mRNA include northern hybridization and in situ hybridization. In vitro techniques for detecting analyte proteins include enzyme-linked immunosorbent assay (ELISA), western blot, immunoprecipitation, and immunofluorescence. In vitro techniques for detecting analyte genomic DNA include southern hybridization.

[0443] The process for performing immunoassays is described, for example, in the following literature: "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, JR 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. In addition, in vivo techniques for detecting analyte proteins include introducing labeled anti-analyte protein antibodies into the subject. For example, the antibody can be labeled with a radioactive marker, and its presence and location in the subject can be detected by standard imaging techniques.

[0444] Antibodies to CTLA-4 (or fragments thereof) can be used in methods known in the art relating to the localization and / or quantification of CTLA-4 protein (e.g., for measuring the level of CTLA-4 protein within an appropriate physiological sample, for diagnostic methods, for imaging the protein, etc.). In a given embodiment, an antibody comprising an antibody-derived antigen binding domain specific for a CTLA-4 protein, or a derivative, fragment, analog or homolog thereof, is used as a pharmacologically active compound (referred to herein as a "therapeutic agent").

[0445] Antibodies of the invention specific for CTLA-4 protein can be used to isolate CTLA-4 polypeptide by standard techniques such as immunoaffinity, chromatography, or immunoprecipitation. Antibodies directed against CTLA-4 protein (or fragments thereof) can be used to diagnostically monitor protein levels in tissues as part of a clinical testing procedure, e.g., to determine the efficacy of a given treatment regimen.

[0446] Detection can be facilitated by coupling (i.e., physically associating) the antibody to a detectable substance. Examples of detectable substances include, but are not limited to, various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, 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, dichlorotriazinylaminofluorescein, dansyl chloride, or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive materials include 125 I. 131 I. 35 S. 32 P or 3 H.

[0447] Cell therapy

[0448] Cell therapy is also provided herein. For example, cell therapy may comprise cells, such as T cells, NK cells or B cells, that are genetically engineered to express an anti-CTLA4 antibody as described herein. "Genetically engineered cells" may refer to any cell of any organism that is modified, transformed or manipulated by adding or modifying a gene, a DNA sequence or an RNA sequence, or a protein or a polypeptide. Isolated cells, host cells and genetically engineered cells may include isolated immune cells, such as NK cells, T cells and B cells, containing a DNA sequence or an RNA sequence encoding an antibody as described herein, and / or a chimeric receptor or a chimeric receptor complex expressed on the cell surface.

[0449] "T cells" are cells of the immune system that mature in the thymus and produce T cell receptors (TCRs). T cells can be naive cells (unexposed to antigens; T cells are not exposed to antigens). CM Compared with increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA and decreased expression of CD45RO), memory T cells (T M T cells (contact with antigen and have long life span) and effector cells (contact with antigen and have cytotoxicity). M Central memory T cells can be further divided into subsets (T CM , increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95 and decreased expression of CD54RA compared with naive T cells) and a subset of effector memory T cells (T EM , and naive T cells or T CM Compared with the decreased expression of CD62L, CCR7, CD28, CD45RA and increased expression of CD127). Effector T cells (T E ) can refer to T CM Compared to CD8+ antigen-contacted cytotoxic T lymphocytes, they had reduced expression of CD62L, CCR7, CD28 and were positive for granzymes and perforins.

[0450] T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, any number of techniques known to those skilled in the art (such as FICOLL TMSeparation) obtains T cells from a unit of blood collected from a subject. In one embodiment, cells from the circulating blood of a subject are obtained by apheresis. Apheresis products generally contain 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 plasma fractions and placed in appropriate buffer or medium for subsequent processing. In one embodiment of the present invention, cells are washed with PBS. In an alternative embodiment, the washed solution does not contain calcium and / or magnesium or may not contain many (if not all) divalent cations. As will be understood by those of ordinary skill in the art, washing steps can be achieved by methods known to those skilled in the art, such as by using a semi-automated flow-through centrifuge, for example, Cobe 2991 cell processor, Baxter CytoMate, etc. After washing, cells can be resuspended in a variety of biocompatible buffers or other saline solutions containing or not containing buffer. In certain embodiments, undesirable components of apheresis samples can be removed and cells can be directly resuspended in culture medium.

[0451] In an embodiment, T cells are isolated from PBMCs. T cells can be isolated from whole blood by density gradient centrifugation (e.g., by LYMPHOPREP TM Gradient, PERCOLL TM GRADIENT OR FICOLL TM PBMCs can be isolated by depleting monocytes (e.g., by using CD14 ) T cells are isolated from PBMCs. In some embodiments, red blood cells can be lysed prior to density gradient centrifugation.

[0452] "NK cell" is a large granular lymphocyte, which is a cytotoxic lymphocyte derived from a common lymphoid progenitor cell that naturally does not contain an antigen-specific receptor (such as a T cell receptor or a B cell receptor). NK cells can be characterized by their CD3-, CD56+ phenotypes. In an embodiment, NK cells may refer to any known NK cells or any NK-like cells or any cells with NK cell characteristics. Therefore, primary NK cells can be used, or previously separated and cultured NK cells known in the art can be used in another embodiment. Therefore, NK cell lines can be used. A variety of different NK cells are known and reported in the literature, and any NK cell in these NK cells can be used, or cell lines can be prepared from primary NK cells, such as by viral transformation (Vogel et al. 2014, Leukemia 28: 192-195). In addition to NK-92, suitable NK cells include (but are by no means limited to) NK-YS, NK-YT, MOTN-1, NKL, KHYG-1, HANK-1 or NKG cell lines. Variants of other cell lines can also be used.

[0453] "B cells" are B lymphocytes that can produce antibody molecules after activation. The activation of B cells can be, but is not limited to, through the recognition of antigens by antigen-specific immunoglobulin receptors on the surface of B cells or by non-specific stimulants (e.g., B cell mitogens, such as lipopolysaccharide or pokeweed mitogens). Thus, B cells can be "naive" B lymphocytes that have never been activated before, or "memory" B lymphocytes that have been activated before, or progeny of such B lymphocytes.

[0454] Cell therapy is also provided herein, such as chimeric antigen receptor (CAR) T cell therapy or chimeric antigen receptor (CAR) NK cell therapy.For example, CAR T cell therapy redirects the patient's T cells and NK cells to kill tumor cells 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 T cells or NK cells specificity and reactivity toward the selected target in a non-MHC restricted manner using the antigen binding properties of monoclonal antibodies.Non-MHC restricted antigen recognition gives T cells or NK cells expressing CAR the ability to recognize antigens independently of antigen processing, thus bypassing the main tumor escape mechanism.

[0455] CAR can be a transmembrane fusion protein that links the antigen recognition domain of the antibody to the intracellular signaling domain of the T cell receptor and the auxiliary receptor. Suitable cells can be used, for example, they can secrete the anti-CTLA-4 antibody of the present invention (or alternatively engineered to express the anti-CTLA-4 antibody to be secreted as described herein). The anti-CTLA-4 "payload" to be secreted can be, for example, a mini-antibody, scFv, IgG molecule, bispecific fusion molecule, and other antibody fragments as described herein. When contacted or engineered, the cells described herein can then be introduced into patients in need of treatment by infusion therapy known to those skilled in the art. Patients may suffer from CTLA-4 related diseases or illnesses as described herein, such as chronic infections or cancers. Cells (e.g., T cells or NK cells) can be, for example, T lymphocytes, CD4+T cells, CD8+T cells, or combinations thereof without limitation. Exemplary CAR and CAR factories that can be used in aspects of the present invention include those disclosed in, for example, PCT / US2015 / 067225 and PCT / US2019 / 022272, each of which is hereby incorporated by reference in its entirety. In one embodiment, the CTLA-4 antibodies discussed herein can be used to construct multispecific antibodies or as a payload for genetically engineered cells (such as CAR-T cells, CAR NK cells, or genetically engineered B cells). For example, in one embodiment, the anti-CTLA-4 antibodies discussed herein can be used for targeting CARS (ie, 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 payloads. In another embodiment, the payload can be an immunomodulatory antibody payload.

[0456] Treatment

[0457] As used herein, the term "treat" or "treatment" refers to both therapeutic treatment and prophylactic or preventive measures, wherein the purpose is to prevent or slow down (mitigate) 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 the extent of the disease, a stable (i.e., non-worsening) disease state, a delay or slowing of disease progression, an improvement or alleviation of the disease state, and relief (whether partial or complete), whether detectable or undetectable. "Treatment" may refer to prolonged survival compared to the expected survival in the absence of treatment. Those in need of treatment include those who already have a disorder or disease, as well as those who are prone to a disorder or disease, or those in whom a disorder or disease is to be prevented.

[0458] The present invention provides both preventive and therapeutic methods for treating subjects at risk (or susceptible to cancer) for cancer (e.g., if an early detection cancer biomarker is identified in such subjects) or subjects at risk (or susceptible to other cell proliferation-related diseases or disorders). Such diseases or disorders include, but are not limited to, those associated with abnormal expression of CTLA-4. For example, the method is used to treat, prevent or alleviate the symptoms of cancer. In one embodiment, the method is used to treat, prevent or alleviate the symptoms of solid tumors. Non-limiting examples of other tumors that can be treated by the compositions described herein include lung cancer (e.g., non-small cell lung cancer or lung adenocarcinoma), gastric cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer, brain cancer, skin cancer, liver cancer, pancreatic cancer, esophageal squamous cell carcinoma, nasopharyngeal carcinoma, glioblastoma multiforme, squamous cell carcinoma, melanoma, renal cell carcinoma, urothelial carcinoma, hepatocellular carcinoma, malignant pleural mesothelioma and liquid tumors or gastric cancer. In addition, the method of the present invention can be used to treat hematological cancers, such as leukemia and lymphoma. Alternatively, the method can be used to treat, prevent or alleviate the symptoms of cancer that has been transferred. For example, cancers that can be treated or prevented or whose 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 also be treated or prevented or whose symptoms can be alleviated include those solid tumors with high mutation load and WBC in the filtrate.

[0459] Therefore, in one aspect, the present invention provides a method for preventing, treating or ameliorating the symptoms of cancer or cell proliferative diseases or disorders in a subject by administering to the subject a monoclonal antibody, scFv antibody or bispecific antibody of the present invention. For example, an anti-CTLA-4 antibody can be administered in a therapeutically effective amount.

[0460] Subjects at risk for cancer or cell proliferation-related diseases or disorders may include patients with a family history of cancer or subjects exposed to known or suspected carcinogens. Administration of a prophylactic agent may be performed prior to the manifestation of cancer, such that the disease is prevented or, alternatively, delayed in its progression.

[0461] In another aspect, tumor cell growth is inhibited by contacting the cell with an anti-CTLA4 antibody of the present invention. The cell can be any cell that expresses CTLA-4.

[0462] The present invention also includes a method for increasing or enhancing the immune response to an antigen. Increase or enhance the immune response by administering the monoclonal antibody, scFv antibody or bispecific antibody of the present invention to a subject. For example, the immune response is enhanced by enhancing the antigen-specific T effector function. The antigen is a virus (such as HIV), a bacterium, a parasite or a tumor antigen. The immune response is a natural immune response. The so-called natural immune response means an immune response caused by infection. Infection is a chronic infection. Increasing or enhancing the immune response to an antigen can be measured by a variety of methods known in the art. For example, the immune response can be measured by measuring any one of the following items: T cell activity, T cell proliferation, T cell activation, the production of effector cytokines and T cell transcriptional profiles. Alternatively, the immune response is a response induced by vaccination.

[0463] Therefore, in another aspect, the present invention provides a method for increasing vaccine efficiency by administering a monoclonal antibody or scFv antibody of the present invention and a vaccine to a subject. The antibody and the vaccine are administered sequentially or simultaneously. The vaccine is a tumor vaccine, a bacterial vaccine or a viral vaccine.

[0464] Combination Methods

[0465] The compositions of the present invention as described herein can be administered in combination with chemotherapeutic agents. Chemotherapeutic agents that can be administered with the compositions of the present disclosure include, but are not limited to, antibiotic derivatives (e.g., doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon α-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., medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorethoxyquin, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chlorambucil, 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).

[0466] In other embodiments, the compositions of the present invention as described herein can be administered in combination with cytokines. Cytokines that can be administered with the compositions include, but are not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, anti-CD40, CD40L, and TNF-α.

[0467] In additional embodiments, the compositions described herein may be administered in combination with other therapeutic or prophylactic regimens (such as, for example, radiation therapy).

[0468] In some embodiments, the compositions described herein can be administered in combination with other immunotherapeutic agents. Non-limiting examples of immunotherapeutic agents include cintuzumab, abavotumab, adelimumab, afutuzumab, alemtuzumab, atumomab, ametuzumab, anatumomab, acitumomab, baviximab, betumomab, bevacizumab, bivacizumab, blinatumomab, bentuximab, cantuzumab, catumaxomab, cetuximab, situxomab, citumumab, clituzumab, canatumumab, daratumumab, trazitumomab, duligotuzumab (du ligotumab), dustuzumab, demostatumab, daritumumab, darototumumab, emeticholic acid, elotuzumab, entuximab, ertuinomab, edamatumumab, fatumumab, felatuzumab, fentolimumab, falotuximab, vortuximab, ganitutumab, gemtuzumab, gemtuximab, gebatumumab, ibritumomab tiuxetan, igovolumab, imatumumab, indatumumab, inotuzumab, intumumab, ipilimumab, itumumab Antibody, labetuzumab, lexatumumab, lintuzumab, lovotuzumab, rucamumab, mapatumumab, matuzumab, miratumumab, mitumumab, mitumumab, mosetumab, nanatumab, naptumumab, nexitumomab, nimotuzumab, nofetumomab, okatumumab, ofatumumab, oratumumab, onatumumab, onatumumab, mosetumab, orgovotumab, panitumumab, pasatumumab, pertuzumab, pemtumomab umomab), pertuzumab, pintumomab, protumumab, ranitutumomab, rantrastuzumab, rituximab, rituximab, rotumumab, satumomab, sirolimus, silotuzumab, solitonumab, tetuzumab, tetuzumab, tetuzumab, tetuzumab, tetuzumab, tositumomab, trastuzumab, simolucumab, utoximab, velutuzumab, worse tuzumab, vortumumab, zalutumomab, CC49, and 3F8.

[0469] The present invention provides a method for treating cancer in a patient by administering two antibodies that bind to the same epitope of the CTLA-4 protein or, alternatively, two different epitopes of the CTLA-4 protein. Alternatively, 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 to a protein other than CTLA-4. For example, other proteins other than CTLA-4 may include, but are 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, other proteins other than CTLA-4 are tumor-associated antigens; other proteins other than CTLA-4 may also be cytokines.

[0470] In some embodiments, the invention provides for administering an anti-PD-1 antibody alone or in combination with an additional antibody that recognizes another protein other than CTLA-4, together with cells that are capable of achieving or enhancing an immune response. For example, these cells can be peripheral blood mononuclear cells (PBMCs) or any cell type present in PBMCs, e.g., cytotoxic T cells, macrophages, and natural killer (NK) cells.

[0471] In addition, the present invention provides for the administration of antibodies and anti-tumor agents that bind to the CTLA4 protein, such as small molecules, growth factors, cytokines or other therapeutic agents, including biomolecules such as peptides, peptide mimetics, peptoids, polynucleotides, lipid-derived modulators, 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-α. Small molecule libraries are known in the art. (See Lam, Anticancer Drug Des., 12:145, 1997.)

[0472] Chimeric Antigen Receptor (CAR) T Cell Therapy

[0473] Cell therapy, such as chimeric antigen receptor (CAR) T cell therapy, is also provided herein. For example, CAR T cell therapy redirects the patient's T cells to kill tumor cells by exogenous expression of CAR on T cells. CAR can be a transmembrane fusion protein that links the antigen recognition domain of the antibody to the intracellular signaling domain of the T cell receptor and the auxiliary receptor. Suitable cells can be used, for example, which can secrete the anti-CTLA-4 antibody of the present invention (or alternatively engineered to express the anti-CTLA-4 antibody to be secreted as described herein). The anti-CTLA-4 "payload" to be secreted can be, for example, a mini-antibody, ScFv, IgG molecule, bispecific fusion molecule, and other antibody fragments as described herein.

[0474] Solid tumors present unique challenges to CAR-T therapy. Some obstacles to the effectiveness of CAR-T in solid tumors include heterogeneous antigen expression, insufficient tissue homing, activation, persistence and immunosuppressive tumor microenvironment. Unlike blood cancer, tumor-associated target proteins are overexpressed between tumors and healthy tissues, resulting in non-tumor targeted toxicity (on-target / off-tumor) T cell killing of healthy tissues. In addition, immune suppression in the tumor microenvironment (TME) limits the activation of CAR-T cells towards killing tumors. When such contact or engineering is performed, cells can then be introduced into cancer patients in need of treatment by infusion therapy known to those skilled in the art. Cancer patients may suffer from any type of cancer in the types disclosed herein. Cells (e.g., T cells) may be, for example, tumor-infiltrating T lymphocytes, CD4+T cells, CD8+T cells, or combinations thereof, without limitation.

[0475] Exemplary CAR and CAR factories that can be used in aspects of the present invention are included in, for example, those disclosed in PCT / US2015 / 067225 and PCT / US2019 / 022272, each of which is hereby incorporated by reference in its entirety. For example, CAR-T cells can be generated using a lentiviral system (via transduction), a retroviral system (via transfection (electroporation)), and a transposon system (via PiggyBac) according to methods known in the art. Available promoters for payloads that can be used to generate CAR-T or CAR NK include, for example, constitutive promoters (wherein the promoter is the same as the promoter for CAR-T or CAR NK, such as EF1a then IRES or 2A); inducible promoters (wherein the promoter is different from the promoter for CAR-T, such as NFAT, IL-2prom); and genetically engineered promoters (such as cytokine CTLA-4 locus "knock-in" and / or promoters under the control of endogenous promoters). In one embodiment, the CTLA-4 antibody or CTLA-4 fusion protein discussed herein can be used to construct a multispecific antibody or as a payload for genetically engineered cells (such as CAR-T cells, CAR NK cells or genetically engineered B cells). For example, in one embodiment, the anti-CTLA-4 antibody or CTLA-4 fusion protein discussed herein can be used for targeting CARS (ie, as a targeting moiety). In one embodiment, the anti-CTLA-4 antibody or CTLA-4 fusion protein discussed herein can be used as a payload to be secreted by genetically engineered cells (such as CAR-T cells, CAR NK cells or genetically engineered B cells). In another embodiment, the anti-CTLA-4 antibody or CTLA-4 fusion protein 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 as described herein for use in a CAR-T composition is not a high-affinity CTLA-4 antibody (eg, so that the antibody does not strongly bind to its CTLA-4 target). For example, the CTLA-4 antibodies or CTLA-4 fusion proteins described herein can be used as payloads secreted by genetically engineered cells (such as CAR-T cells, CAR-NK cells, or genetically engineered B cells) in which two targeting moieties (e.g., tumor-associated surface antigens) are selected for a specific 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 IX, RET1, RET2 (AS), prostate-specific antigen (PSA), TAG-72, PAP, p53, Ras, prostate-specific protein, 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 (for additional tumor-associated surface antigens, see also PCT / US2015 / 067225 and PCT / US2019 / 022272, which are incorporated herein by reference in their entireties). Exemplary armored CAR-T cells are listed in the table below.

[0476]

[0477]

[0478] In one embodiment, bispecific (or dual targeting) CAR-T is provided. In another embodiment, CAR-T is an engineered cell comprising a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an extracellular ligand binding domain having specificity for a first antigen and a second antigen on the surface of a cancer cell, wherein the first antigen comprises CXCR4 and the second antigen comprises CLDN4, or the first antigen comprises CAIX and the second antigen comprises CD70, or the first antigen comprises MUC1 and the second antigen comprises Msln. For example, the anti-CTLA-4 antibody or CTLA-4 fusion protein described herein can be used as a payload for genetically engineered cells (such as CAR-T, CAR-NK cells or genetically engineered B cells described herein). In one embodiment, CXCR4 / CLDN4 dual targeting CAR-T with an anti-CTLA-4 fusion payload can be used for breast cancer. In one embodiment, CAIX / CD70 dual targeting CAR-T with an anti-CTLA-4 fusion payload can be used for clear cell renal cell carcinoma (ccRCC). In one embodiment, MUC1 / Msln dual targeting CAR-T with an anti-CTLA-4 fusion payload can be used for ovarian cancer.

[0479] Diagnostic assays

[0480] Anti-CTLA-4 antibodies can be used diagnostically, for example, to monitor the development or progression of cancer as part of a clinical testing process, for example, to determine the efficacy of a given treatment and / or prevention regimen.

[0481] In some embodiments, an anti-CTLA-4 antibody of the invention is conjugated to a detectable moiety for diagnostic purposes, eg, to provide a method for detecting cancer cells in a subject at risk for or suffering from cancer.

[0482] The detectable moiety can be conjugated directly to the antibody or fragment or indirectly to the antibody or fragment by using, for example, a fluorescent secondary antibody. Direct conjugation can be achieved by, for example, standard chemical coupling of a fluorophore to an antibody or antibody fragment or by genetic engineering. Chimeras or fusion proteins containing antibodies or antibody fragments coupled to fluorescent or bioluminescent proteins can be constructed. For example, Casadei et al. (Proc Natl Acad Sci US A. 1990 March; 87(6): 2047-51) describe a method for preparing a vector construct capable of expressing a fusion protein of aequorin and an antibody gene in mammalian cells.

[0483] As used herein, with respect to a probe or antibody, the term "labeled" may encompass direct labeling of a probe or antibody by coupling a detectable substance to the probe or antibody (i.e., physically linked), as well as indirect labeling of a probe or antibody by reactivity with another directly labeled reagent. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody, and end-labeling of a DNA probe with biotin so that it can be detected with fluorescently labeled streptavidin. The term "biological sample" is intended to include tissues, cells, and biological fluids (such as biopsies) separated from a subject, as well as tissues, cells, and fluids present in a subject. That is, the detection methods 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 detecting CTLA-4 include enzyme-linked immunosorbent assay (ELISA), western blot, immunoprecipitation, and immunofluorescence. In addition, in vivo techniques for detecting CTLA-4 include introducing a labeled anti-CTLA-4 antibody into a subject. For example, the antibody can be labeled with a radioactive marker, and its presence and location in the subject can be detected by standard imaging techniques.

[0484] In the case of a "targeted" conjugate (i.e., a conjugate containing a targeting moiety, which is a molecule or feature designed to localize the conjugate at one or more specific sites in a subject or animal), localization may refer to a state when equilibrium between bound "localized" entities and unbound "free" entities in the subject has been substantially achieved. The rate at which such equilibrium is achieved depends on the route of administration. For example, a conjugate administered by intravenous injection may achieve localization within a few minutes of injection. On the other hand, an orally administered conjugate may take several hours to achieve localization. Alternatively, localization may simply refer to the localization of an entity in a subject or animal at a selected time period after administration of the entity. As another example, localization is achieved when the moiety becomes distributed after administration.

[0485] It should be understood that a person skilled in the art can make a reasonable estimate of the time to achieve localization. In addition, the localization state that changes over time can be tracked by imaging the detectable portion (e.g., luminescent conjugate) according to the method of the present invention, such as using a photodetector device. The "photodetector device" used should have a high enough sensitivity to enable imaging of weak light from the mammal within a reasonable amount of time, and to construct an image using the signal from such a device.

[0486] In cases where extremely bright light-generating moieties can be used and / or to detect light-generating fusion proteins positioned near the surface of the subject or animal being imaged, a pair of "night vision" goggles or a standard high-sensitivity camera, such as a Silicon Intensified Tube (SIT) camera (e.g., from Hammamatsu Photonic Systems, Bridgewater, N.J.) can be used. More typically, however, a more sensitive light detection method is desired.

[0487] At extremely low light levels, the photon flux per unit area becomes so low that the imaged scene no longer appears continuous. Instead, it is represented by individual photons that are temporally and spatially distinct from each other. Observed on a monitor, such images appear as flickering points of light, each representing a single detected photon. By accumulating these detected photons in a digital image processor over time, an image can be acquired and constructed. In contrast to conventional cameras in which the signal at each image point is assigned an intensity value, in photon counting imaging the amplitude of the signal is not significant. The goal is simply to detect the presence of a signal (photon) and to count its occurrence relative to its position over time.

[0488] At least two types of photodetector devices described below can detect individual photons and generate signals that can be analyzed by an image processor. Compared to amplifying the photon signal, the noise-reducing photodetector device achieves sensitivity by reducing the background noise in the photon detector. Noise is reduced primarily by cooling the detector array. The device includes a charge-coupled device (CCD) camera, which is called a "backthinned" cooled CCD camera. In more sensitive instruments, cooling is achieved using, for example, liquid nitrogen, which brings the temperature of the CCD array to about -120°C. "Backthinning" refers to an ultra-thin backplane that reduces the path length that photons follow to be detected, thereby increasing quantum efficiency. A particularly sensitive backthinned cooled CCD camera is the "TECH 512," a 200 series camera purchased from Photometries, Ltd. (Tucson, Arizona).

[0489] "Photon amplification devices" amplify photons before they strike a detection screen. This category includes CCD cameras with intensifiers such as microchannel intensifiers. Microchannel intensifiers typically contain a metal array of channels that are perpendicular and coextensive with the camera's detection screen. The microchannel array is placed between the sample, subject, or animal to be imaged and the camera. Most of the photons that enter the array's channels contact the sides of the channels before exiting. A voltage applied across the array causes many electrons to be released from each photon collision. The electrons from such collisions leave their original channels in a "shotgun" pattern and are detected by the camera.

[0490] Even higher sensitivity can be achieved by placing an array of intensified microchannels in series, so that electrons generated in the first stage in turn cause an amplified signal of electrons at 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-based single-photon detection device is the C2400 series available from Hamamatsu.

[0491] The image processor processes the signal generated by the photodetector device, which counts the photons in order to construct an image, which 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 sensitive photon counting camera described above, and can therefore be purchased from the same source. The image processor is typically connected to a personal computer, such as an IBM-compatible PC or Apple Macintosh (Apple Computer, Cupertino, California), which may or may not be included as part of the 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, California).

[0492] In one embodiment, the biological sample contains protein molecules from a test subject.An exemplary biological sample is a peripheral blood leukocyte sample isolated from a subject by conventional means.

[0493] The present invention also encompasses a kit for detecting the presence of CTLA-4 or cells expressing CTLA-4 in a biological sample. For example, the kit may include: a labeled compound or agent capable of detecting cancer or tumor cells (e.g., anti-CTLA-4 scFv or monoclonal antibody) in a biological sample; a device for determining the amount of CTLA-4 in a sample; and a device for comparing the amount of CTLA-4 in a sample with a standard. In some embodiments, the standard is a non-cancerous cell or a cell extract thereof. The compound or reagent may be packaged in a suitable container. The kit may also include instructions for using the kit to detect cancer in a sample.

[0494] Other Implementations

[0495] While the invention has been described in conjunction with specific embodiments thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

[0496] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0497] Example

[0498] Examples are provided below to facilitate a more complete understanding of the present invention. The following examples illustrate exemplary modes for preparing and practicing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples for illustrative purposes only, because similar results can be obtained using alternative methods.

[0499] Example 1

[0500] Anti-CTLA-4 antibodies that block CTLA-4 / CD80 binding identified via phage display are described herein. In addition, a yeast display library was also generated from this panning campaign to further interrogate the results of rounds 2 and 3 of panning. After sorting for clones that bind above bulk, the clones were sequenced and additional clones were identified that displayed improved binding curves compared to the original clones found in the third round of ELISA screening.

[0501] The embodiments herein may have commercial application as stand-alone antibody therapies or in combination with other therapies.

[0502] Example 2—aCTLA-4ab from PMPL panning (Group 1)

[0503] like Figure 1 As shown, an anti-CTLA-4 phage binding assay was performed. The assay performed was a mid-scale phagemid rescue using PEG+NaCl to precipitate the phage and purify them.

[0504] Binding curves were generated from the ELISA data using purified phage. We diluted each of the 9 phage PMPL candidates + E1-D7 starting at 2E13 phagemid particles / mL and decreased by a factor of 5. For the CTLA-4 Fc-coated plates and the CAIX Fc-coated plates, we added these phages in triplicate.

[0505] like Figure 2 As shown, FACS confirmed that CTLA-4 phage can bind to 1G5 Jurkat cells expressing CTLA-4. The negative control is cells + secondary antibody (red box), and the positive comparison / control is the current E1-D7 candidate (green box).

[0506] For the purpose of filtering out candidates, we will consider any CTLA-4 phage candidate with more than 25% positive cells to be positive. Of our 9 possible phage candidates, there is only one comparable candidate, B10 (circled in blue), as it is more than 75% positive. There are also some partial positives, these are circled in orange and are B6 and C12 which are more than 25% positive.

[0507] Example 3

[0508] The soluble proteins used in this article include:

[0509] Human CTLA-4, His Tag (CT4-H5229) was expressed from human 293 cells (HEK293). It contains AAAla 37-Phe 162 (Accession No. NP_005205.2)

[0510]

[0511] Mouse CTLA-4, His Tag (CT4-M52H5) was expressed from human 293 cells (HEK293).

[0512] It contains AA Glu 36-Phe 162 (Accession No. NP_033973).

[0513]

[0514] Full-length CTLA4 was cloned using cDNA from Transomic (BC074893).

[0515] Example 4—aCTLA-4

[0516] Overview of CTLA4 :

[0517] -Protein receptors that serve as immune checkpoints

[0518] -Overall structure: extracellular V domain, transmembrane domain, cytoplasmic tail

[0519] -Homodimer when membrane bound: monomer in soluble form

[0520] CTLA4 binding :

[0521] -CD28 homolog

[0522] - Combined with CD-80 / 86 (B7-1 / 2)

[0523] -Highly conserved MYPPY motif involved in binding

[0524] Mechanisms of suppressing immune response ( Fig.15 )

[0525] - There are still many debates: ligand competition, negative signaling, ligand removal, effects on T cell motility

[0526] background( Fig.23 ) :

[0527] -T cells are important regulators of anti-tumor immunity

[0528] - They express inhibitory immune checkpoints: These immune checkpoints serve to limit T cell activation, preventing collateral damage to the body from unrestricted T cell activity

[0529] -PD1 protein is expressed on the surface of T cells: upon binding to its ligands PDL-1 and PDL-2, the negative pathway is promoted to inhibit T cell function

[0530] - Upon ligation to its ligands PDL-1 and PDL-2, the negative pathway is promoted to inhibit T cell function: tumor cells also express PDL1 which can promote tumor escape.

[0531] MLR assay ( Fig.24 ) :

[0532] - Co-culture of CD4+ T cells with monocyte-derived DCs from different donors:

[0533] Dendritic cell allo-recognition leads to T cell activation

[0534] - PDL1-expressing DCs suppress T cell activation: Addition of immune checkpoint inhibitors can restore T cells from suppression

[0535] - Study whether anti-PD1 enhances T cell activation

[0536] -Measures IFNγ and IL2 cytokine production, indicators of T cell activation

[0537] plan :

[0538] - Isolation of CD14+ monocytes using Miltenyi CD14+ microbeads

[0539] - Cultured in Miltenyi Mo-DC medium (medium pre-prepared with GM-CSF + IL4)

[0540] - Culture for 5 days, then add: TNF-α (1000U / ml), IL-1β (5ng / ml), IL-6 (10ng / ml) and prostaglandin E2 (PGE2) (1μM), and culture for 2 days to allow DC maturation

[0541] -mMo-DCs express multiple DC markers involved in the formation of immunological synapses between DCs and naive T cells, including CD80, CD86, and MHC II (HLA-DR). Mature Mo-DCs also express DC activation markers CD83, CD40, and CCR7

[0542] Continuation of previous plan :

[0543] -Isolate T cells on the day of MLR experiment (CD4+ negative selection kit StemCell)

[0544] -100,000 T cells and 10,000 MoDC cells per well for MLR

[0545] - Add antibodies at desired concentration and incubate the cultures for 5 days

[0546] - Save supernatant for ELISA screening (IL2 and IFNy)

[0547] aCTLA4 Antibodies MLR 1 and 2 :

[0548] MLR1:

[0549] - 2 T cell donors, 2 DC donors

[0550] -DC culture for 7 days—addition of accessory cytokines on day 5

[0551] - Cells were pooled and cultured for 4 days

[0552] - Positive control: aPD-1 antibody

[0553] -Negative control: aSARS2 antibody

[0554] MLR 2:

[0555] - Testing the combination of 2 aCTLA4 antibodies (D7, C10) with an aPD-1 antibody.

[0556] - 4 panels: 2 T cell donors, 2 aPD1 concentrations

[0557] - aPD-1 medium used for preparing antibody dilutions (starting from 100 nM, 5 4-fold dilutions): 5 nM aPD-1 medium and 100 nM aPD-1 medium

[0558] -100,000 T cells + 10,000 MoDCs / well

[0559] -First combine 50ul of antibody with 50ul of T cells

[0560] - Then add Ab / T cells to DC

[0561] -Control: aPD-1+ cells, aPD-1 high+ cells, DC+T cells

[0562] - Incubate for 5 days

[0563] FACS to confirm that our CTLA4 phage can bind to 1G5 Jurkat cells expressing CTLA-4 (Figure 31)

[0564] - To ensure we were finding the right candidates, we went back and screened the 2nd plate of CTLA4PMPL 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 via Octet.

[0565] -SA sensor loaded with biotinylated CTLA4. scFv-Fc concentration in expi293 supernatant was measured via IgG quantitative ELISA (Bethyl) and used to normalize samples. OctetRed96 was used to measure the binding of scFv-Fc in the supernatant.

[0566] Here we consider the non-limiting example of the classification of analytical data :

[0567] Very important

[0568] 1.CD80 blockade

[0569] 2.CD86 Blockade

[0570] 3. Dissociation constant (Kd)

[0571] Moderately important

[0572] 1. Mouse cross-reactivity

[0573] Of low importance

[0574] 1. Response

[0575] Exemplary selected candidates:

[0576] E1-A8

[0577] Non-limiting reasons for candidate selection:

[0578] Showing the highest CD80 blockade among the final selected candidates

[0579] CD86 blockade was comparable to ipilimumab

[0580] Shows significant cross-reactivity with mouse CTLA4

[0581] Higher response than both ipilimumab and E1-D7

[0582] shortcoming:

[0583] Shows relatively high off-rates when compared to most other antibodies tested

[0584] Notes:

[0585] E1-A8, which has the same heavy chain as E2-A4, showed the highest response rate and low dissociation constant.

[0586] E1-A8 is derived from the same V gene (IGHV3) as ipilimumab but with different alleles

[0587] Example 5—Engineering and Characterization of Anti-PD1 / Anti-CTLA4 Bispecific Antibodies

[0588] Dual checkpoint blockade with bispecific antibodies ( Fig.53 ) :

[0589] Without being bound by theory, advantages may include: lower therapeutic doses, synergistic combinations, targeted therapy, and reduced manufacturing costs.

[0590] Non-limiting summary of the objectives described herein :

[0591] 1. Discovery and characterization of new anti-CTLA4 antibodies:

[0592] 1A: Kinetic Screening and Characterization

[0593] 1B: Epitope sorting

[0594] 1C: Cell binding assay

[0595] 1D: Biological Assays

[0596] 2. Engineering and Characterization of Anti-PD1 / Anti-CTLA4 Bispecific Antibodies

[0597] 3. Characterization of anti-PD1 / anti-CTLA4 bispecific antibodies as CAR T payloads

[0598] hCTLA2 ELISA binding curve data and results ( Fig.56 Small Figure A) :

[0599] - ELISA binding curves to demonstrate the binding affinity of anti-CTLA4 candidates to hCTLA4.

[0600] - All candidates had comparable binding affinity to hCTLA4 at the highest concentration.

[0601] - Most candidates showed comparable binding affinity to ipilimumab

[0602] - Top 3: I, J, H

[0603] -A, E, I have the best saturation concentration

[0604] mCTLA4 ELISA binding curve data and results ( Fig.56 Small Figure B)

[0605] -Candidates showing high cross-reactivity: E, F, A

[0606] -Candidates showing moderate cross-reactivity: J, H

[0607] - Ipilimumab and Biolegend anti-CTLA4 did not show cross-reactivity faithful to the literature

[0608] BSA negative curve ( Fig.56 Small image C)

[0609] - Indicates the binding affinity of the anti-CTLA4 candidate to BSA (negative control)

[0610] Binding affinity of anti-CTLA4 candidates to CTLA4+ Jurkat cells ( Fig.64 )

[0611] - Apparent discordance in the binding affinity of the antibody to cells compared to that to the soluble protein.

[0612] -J and H have much higher binding affinity and lower saturation concentrations for ipilimumab

[0613] -E and F have comparable binding characteristics to ipilimumab

[0614] Non-limiting exemplary conclusions from the data described herein :

[0615] - Screening of 5 anti-CTLA4 antibodies from a library of different candidates showed:

[0616] a. Significant binding affinity to CTLA4

[0617] b. Ability to block the interaction between CTLA4 and CD80 / 86

[0618] c. Biological functional relevance by promoting CD80 / CD28 signaling in effector cells.

[0619] Without wishing to be bound by theory, the following experiments may supplement the data presented herein:

[0620] - Further experiments with bispecific formats to determine the most suitable anti-CTLA4 antibody for anti-PD1 / anti-CTLA4 bispecificity

[0621] - In vitro assays for characterizing bispecific antibodies

[0622] - Characterization and efficacy studies of bispecifics as payloads in CAR T-cell therapy.

[0623] Equivalent

[0624] Those skilled in the art will recognize or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. Such equivalents are considered to be within the scope of the present invention and are covered by the appended claims.

Claims

1. An isolated antibody or fragment thereof that binds to human cytotoxic T lymphocyte-associated protein 4 (CTLA-4), the isolated antibody or fragment thereof 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 (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.

2. An isolated scFv antibody that binds to human cytotoxic T lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 (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.

3. An isolated antibody or fragment thereof that binds to human cytotoxic T lymphocyte-associated protein 4 (CTLA-4), the isolated antibody or fragment thereof comprising: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 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 NO: NO: 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.

4. An isolated scFv antibody that binds to human cytotoxic T lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising: a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 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 NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 385, 387, 389, 391, 393, 395, 397, 399, IDNO: 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.

5. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

2.

6. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

2.

7. An isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or the antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 3, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

4.

8. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 3, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

4.

9. An isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or the antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 5, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

6.

10. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 5, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

6.

11. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 7, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

8.

12. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 7, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

8.

13. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 9, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

10.

14. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 9, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

10.

15. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 11, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

12.

16. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 11, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

12.

17. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 13, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

14.

18. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 13, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

14.

19. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 15, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

16.

20. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 15, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

16.

21. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 17, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

18.

22. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 17, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

18.

23. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 19, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

20.

24. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 19, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

20.

25. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 21, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

22.

26. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 21, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

22.

27. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 23, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

24.

28. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 23, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

24.

29. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 385, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

386.

30. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 385, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

386.

31. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 387, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

388.

32. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 387, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

388.

33. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 389, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

390.

34. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 389, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

390.

35. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 391, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

392.

36. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 391, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

392.

37. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 393, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

394.

38. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 393, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

394.

39. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 395, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

396.

40. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 395, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

396.

41. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 397, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

398.

42. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 397, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

398.

43. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 399, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

400.

44. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 399, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

400.

45. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 401, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

402.

46. ​​An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 401, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

402.

47. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 403, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

404.

48. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 403, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

404.

49. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 405, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

406.

50. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 405, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

406.

51. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 407, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

408.

52. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 407, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

408.

53. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 409, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

410.

54. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 409, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

410.

55. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 411, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO:

412.

56. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 411, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO:

412.

57. An isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), wherein the isolated monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1001, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1002.

58. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1001, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1002.

59. An isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), wherein the isolated monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1003, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1004.

60. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1003, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1004.

61. An isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), wherein the isolated monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1005, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1006.

62. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1005, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1006.

63. An isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or the antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1007, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1008.

64. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1007, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1008.

65. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1009, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1010.

66. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1009, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1010.

67. An isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), wherein the isolated monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1011, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1012.

68. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1011, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1012.

69. An isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), wherein the isolated monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1013, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1014.

70. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1013, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1014.

71. An isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), wherein the isolated monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1015, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1016.

72. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1015, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1016.

73. An isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or the antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1017, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1018.

74. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1017, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1018.

75. An isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), wherein the isolated monoclonal antibody or the antigen-binding fragment thereof comprises a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1019, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1020.

76. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1019, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1020.

77. An isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or the antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1021, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1022.

78. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1021, and the light chain comprises an amino acid sequence approximately 95% identical to SEQ ID NO: 1022.

79. An isolated monoclonal antibody or an antigen-binding fragment thereof that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated monoclonal antibody or the antigen-binding fragment thereof comprising a heavy chain, a light chain or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1023, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1024.

80. An isolated scFv antibody that binds to human cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), the isolated scFv antibody comprising a heavy chain, a light chain, or a combination thereof, wherein the heavy chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1023, and the light chain comprises an amino acid sequence that is approximately 95% identical to SEQ ID NO: 1024.

81. The isolated monoclonal antibody or antigen binding fragment of claims 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77 or 79, wherein the isolated monoclonal antibody or antigen binding fragment comprises a wild-type Fc or a modified Fc.

82. An isolated bispecific antibody comprising the antibody or fragment of any one of claims 1 to 81 and a second antigen binding fragment specific for a molecule on an immune cell.

83. The bispecific antibody of claim 82, wherein the molecule is selected from the group consisting of CCR4, B7H3, B7H4, CD27, CD28, CD40, CD40L, CD47, CD122, CTLA-4, GITR, GITRL, ICOS, ICOSL, LAG-3, LIGHT, OX-40, OX40L, PD-1, TIM3, 4-1BB, TIGIT, VISTA, HEVM, BTLA, CD47, PDL 1, MICA, MICB, and KIR.

84. The bispecific antibody of claim 82, wherein the fragment and the second fragment are each independently selected from a Fab fragment, a single-chain variable fragment (scFv) or a single domain antibody.

85. The bispecific antibody of claim 82, further comprising an Fc fragment.

86. The bispecific antibody of claim 85, wherein the Fc fragment comprises a wild-type Fc fragment or a modified Fc fragment.

87. A nucleic acid encoding the antibody of any one of claims 1 to 81.

88. A nucleic acid encoding the bispecific antibody according to any one of claims 82 to 86.

89. A pharmaceutical composition comprising the antibody or fragment thereof according to any one of claims 1 to 81 and a pharmaceutically acceptable carrier or excipient.

90. The pharmaceutical composition of claim 89, further comprising at least one additional therapeutic agent.

91. The pharmaceutical composition of claim 90, wherein the therapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine.

92. A pharmaceutical composition comprising the bispecific antibody according to any one of claims 82 to 86 and a pharmaceutically acceptable carrier or excipient.

93. The pharmaceutical composition of claim 92, further comprising at least one additional therapeutic agent.

94. The pharmaceutical composition of claim 93, wherein the therapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule, or a cytokine.

95. An isolated cell comprising one or more polynucleotides encoding an antibody or fragment thereof according to any one of claims 1 to 81.

96. An isolated cell comprising one or more polynucleotides encoding the bispecific antibody or fragment thereof according to any one of claims 82 to 86.

97. The isolated cell of claim 95 or claim 96, wherein the isolated cell is an immune cell.

98. The isolated cell of claim 97, wherein the immune cell is a T cell, a NK cell or a B cell.

99. A vector comprising the nucleic acid according to claim 87 or 88.

100. A cell comprising the vector of claim 99.

101. The cell of claim 100, wherein the cell is an immune cell.

102. The cell of claim 101, wherein the immune cell is a T cell, a NK cell or a B cell.

103. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the CTLA-4 antibody or fragment thereof according to any one of claims 1 to 81.

104. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the CTLA-4 bispecific antibody of any one of claims 82 to 86.

105. 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 an 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 (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.

106. The CAR of claim 105, wherein the transmembrane domain further comprises a stem region positioned between the extracellular domain and the transmembrane domain.

107. The CAR of claim 105, wherein the transmembrane domain comprises CD28.

108. The CAR of claim 105, further comprising one or more additional co-stimulatory molecules positioned between the transmembrane domain and the intracellular signaling domain 109. The CAR of claim 105, wherein the co-stimulatory molecule is CD28, 4-1BB, ICOS or OX40.

110. The CAR of claim 105, wherein the intracellular signaling domain comprises a CD3 ζ chain.

111. The CAR of claim 105, wherein the antibody is a Fab or a scFV.

112. A nucleic acid encoding a CAR according to any one of claims 105 to 111.

113. The nucleic acid of claim 112, further comprising a nucleic acid encoding a polypeptide positioned after the intracellular signaling domain.

114. The nucleic acid of claim 113, wherein the polypeptide is an antibody or a cytokine.

115. The nucleic acid of claim 114, wherein the antibody is a scFV.

116. A nucleic acid encoding a CAR, wherein the CAR comprises an intracellular signaling domain, a transmembrane domain, and an extracellular domain, the nucleic acid further comprising a nucleic acid encoding a polypeptide positioned after the intracellular signaling domain, wherein the polypeptide comprises an isolated monoclonal antibody or an 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 (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.

117. A vector comprising the nucleic acid according to any one of claims 112 to 116.

118. A cell comprising the vector of claim 117.

119. The cell of claim 118, wherein the cell is an immune cell.

120. A genetically engineered cell that expresses and carries the chimeric antigen receptor according to any one of claims 105 to 111 on its cell surface membrane.

121. The cell of claim 120, wherein the immune cell is a T cell, a NK cell or a B cell.

122. The cell of claim 121, wherein the T cell is CD4+ or CD4+.

123. The genetically engineered cells of claim 122, comprising a mixed population of CD4+ and CD8+ cells.

124. A genetically engineered cell that expresses and carries a chimeric antigen receptor on its cell surface membrane, and that is further engineered to express and secrete a polypeptide, wherein the polypeptide is an isolated monoclonal antibody or an 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 VLCDR1 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 (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.

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