Anti-EGFRvIII antibodies and uses thereof

Monoclonal antibodies and CARs targeting EGFRvIII address the lack of effective treatments by specifically binding and disrupting EGFRvIII signaling, inducing cancer cell death and providing therapeutic options for diverse diseases.

JP2025535476APending Publication Date: 2025-10-24PHANES THERAPEUTICS INC
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Patent Information

Application Number
JP2025523530
Authority / Receiving Office
JP · JP
Patent Type
Applications
Current Assignee / Owner
Priority Date
2023-08-03
Filing Date
2023-10-23
Publication Date
2025-10-24

AI Technical Summary

Technical Problem

Current treatments for EGFRvIII-associated cancers lack effective antibodies that can specifically target and inhibit the constitutively activated EGFRvIII receptor, which is prevalent in various cancer types and contributes to tumorigenesis.

Method used

Development of monoclonal antibodies and chimeric antigen receptors (CARs) that specifically bind to EGFRvIII, inhibiting its signaling and inducing tumor cell lysis through mechanisms like ADCC, ADCP, and CDC, and recruiting conjugated drugs, with sequences defined by specific CDRs.

Benefits of technology

The antibodies and CARs effectively target and kill cancer cells expressing EGFRvIII, offering therapeutic potential for a range of cancers and inflammatory/autoimmune diseases by specifically binding and disrupting EGFRvIII signaling pathways.

✦ Generated by Eureka AI based on patent content.

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Abstract

Anti-EGFRvIII antibodies and antigen-binding fragments thereof, and chimeric antigen receptors (CARs) comprising the anti-EGFRvIII antigen-binding domain are described. Also described are nucleic acids encoding the antibodies and CARs, compositions comprising the antibodies and host cells comprising the CARs, methods of producing the antibodies and CARs, and methods of using the antibodies and CARs to treat or prevent diseases, such as cancer and / or inflammatory diseases and / or autoimmune diseases.
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Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 380,614, filed October 24, 2022, U.S. Provisional Patent Application No. 63 / 486,261, filed February 22, 2023, and U.S. Provisional Patent Application No. 63 / 517,461, filed August 3, 2023, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] FIELD OF THE INVENTION The present invention relates to monoclonal anti-EGFRvIII antibodies and chimeric antigen receptors (CARs) comprising an anti-EGFRvIII antigen-binding domain, nucleic acids and expression vectors encoding the antibodies and CARs, recombinant cells containing the vectors and CARs, and compositions comprising the antibodies, CARs, and recombinant cells expressing the CARs. Methods of making the antibodies and CARs, and methods of using the antibodies and CARs to treat diseases, including cancer, inflammatory diseases, autoimmune diseases, and / or related complications, are also provided.

[0003] Reference to an electronically submitted sequence listing This application contains an electronically submitted Sequence Listing, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0004] The epidermal growth factor receptor (EGFR), also known as EGFR, HER1, or ERBB1, is a transmembrane receptor tyrosine kinase that, upon binding to one of its potential ligands, induces the formation of EGFR homodimers, phosphorylation of the C-terminus, and subsequent downstream signaling (An, Z. et al., Oncogene 2018, (37): 1561-1575). The EGFR splice variant, EGFRvIII, contains a deletion of exons 2-7 in the extracellular domain and has been identified in various cancer types, primarily glioblastoma multiforme (GBM), where it is found in 40-50% of high-grade gliomas in which EGFR is upregulated (Sugawa, N. et al., Proc Natl Acad Sci USA. 1990, (87): 8602-8606; Nishikawa, R. et al., Proc Natl Acad Sci USA. 1994, (91): 7727-7731). At the same time, EGFRvIII has not been detected in normal tissues in numerous studies, making it an ideal tumor-specific antigen for anti-cancer biologics targeting EGFRvIII-positive tumors (Gan, H. et al, FEBS Journal. 2013, (280): 5350-5370).

[0005] Unlike the parent EGFR, EGFRvIII is constitutively activated. Although its signaling activity is weaker than that of ligand-bound EGFR, activation and phosphorylation of EGFRvIII can be induced by wild-type EGFR and may function cooperatively to enhance downstream signaling (Fan, Q. et al., Cancer Cell. 2013, (24): 438-439). This activity remains highly tumorigenic, and EGFRvIII alone has been reported to be sufficient to confer a growth advantage over tumor cells expressing only the mutant form (Nishikawa, R. et al., Proc Natl Acad Sci USA. 1994, (91): 7727-7731; Fan, Q. et al., Cancer Cell. 2013, (24): 438-439; Batra, S. et al., Cell Growth Differ. 1995, (6): 1251-1259). EGFRvIII has been reported to exhibit reduced internalization and degradation compared to EGFR, which may play a role in its enhanced tumorigenicity (Grandal, M. et al., Carcinogenesis. 2007, (28): 1408-1417). Because EGFRvIII shares the same cytoplasmic domain as EGFR, it can enhance the same signaling pathways. In the case of membrane-bound EGFRvIII, these include the MAPK, ERK, PI3K / AKT, JAK / STAT, and PKC pathways, which together constitute a global activation of cell survival and proliferation pathways, increasing tumorigenesis (An, Z. et al., Oncogene 2018, (37): 1561-1575; Padfield, E. et al., Frontiers in Oncology. 2015, (5): 1-8). Antibodies that inhibit EGFRvIII signaling, either by preventing dimerization or by inducing cell surface turnover, have great potential for treating EGFRvIII-associated cancers. Additionally, antibodies that bind to EGFRvIII can help the immune system kill cancer cells. Summary of the Invention [Means for solving the problem]

[0006] In one general aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII.

[0007] An isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having the following polypeptide sequence: (1) SEQ ID NOs: 3, 4, 5, 6, 7, and 8, respectively, or SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively; (2) SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively, or SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively; (3) SEQ ID NOs: 31, 32, 33, 34, 35, and 36, respectively, or SEQ ID NOs: 37, 38, 39, 40, 41, and 42, respectively; (4) SEQ ID NOs: 45, 46, 47, 48, 49, and 50, respectively, or SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively; (5) SEQ ID NOs: 59, 60, 61, 62, 63, and 64, respectively, or SEQ ID NOs: 65, 66, 67, 68, 69, and 70, respectively; (6) SEQ ID NOs: 73, 74, 75, 76, 77, and 78, or SEQ ID NOs: 79, 80, 81, 82, 83, and 84, respectively; (7) SEQ ID NOs: 87, 88, 89, 90, 91, and 92, respectively, or SEQ ID NOs: 93, 94, 95, 96, 97, and 98, respectively; (8) SEQ ID NOs: 101, 102, 103, 104, 105, and 106, respectively, or SEQ ID NOs: 107, 108, 109, 110, 111, and 112, respectively; (9) SEQ ID NOs: 115, 116, 117, 118, 119, and 120, respectively, or SEQ ID NOs: 121, 122, 123, 124, 125, and 126, respectively; (10) SEQ ID NOs: 129, 130, 131, 132, 133, and 134, respectively, or SEQ ID NOs: 135, 136, 137, 138, 139, and 140, respectively; (11) SEQ ID NOs: 143, 144, 145, 146, 147, and 148, respectively, or SEQ ID NOs: 149, 150, 151, 152, 153, and 154, respectively; (12) SEQ ID NOs: 157, 158, 159, 160, 161, and 162, respectively, or SEQ ID NOs: 163, 164, 165, 166, 167, and 168, respectively; (13) SEQ ID NOs: 171, 172, 173, 174, 175, and 176, respectively, or SEQ ID NOs: 177, 178, 179, 180, 181, and 182, respectively; (14) SEQ ID NOs: 185, 186, 187, 188, 189, and 190, respectively, or SEQ ID NOs: 191, 192, 193, 194, 195, and 196, respectively; (15) SEQ ID NOs: 199, 200, 201, 202, 203, and 204, respectively, or SEQ ID NOs: 205, 206, 207, 208, 209, and 210, respectively; (16) SEQ ID NOs: 213, 214, 215, 216, 217, and 218, respectively, or SEQ ID NOs: 219, 220, 221, 222, 223, and 224, respectively; (17) SEQ ID NOs: 227, 228, 229, 230, 231, and 232, respectively, or SEQ ID NOs: 233, 234, 235, 236, 237, and 238, respectively; (18) SEQ ID NOs: 241, 242, 243, 244, 245, and 246, respectively, or SEQ ID NOs: 247, 248, 249, 250, 251, and 252, respectively; (19) SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively, or SEQ ID NOs: 261, 262, 263, 264, 265, and 266, respectively; (20) SEQ ID NOs: 269, 270, 271, 272, 273, and 274, respectively, or SEQ ID NOs: 275, 276, 277, 278, 279, and 280, respectively; (21) SEQ ID NOs: 283, 284, 285, 286, 287, and 288, or SEQ ID NOs: 289, 290, 291, 292, 293, and 294, respectively; (22) SEQ ID NOs: 297, 298, 299, 300, 301, and 302, respectively, or SEQ ID NOs: 303, 304, 305, 306, 307, and 308, respectively; (23) SEQ ID NOs: 311, 312, 313, 314, 315, and 316, respectively, or SEQ ID NOs: 317, 318, 319, 320, 321, and 322, respectively; (24) SEQ ID NOs: 325, 326, 327, 328, 329, and 330, respectively, or SEQ ID NOs: 331, 332, 333, 334, 335, and 336, respectively; or (25) SEQ ID NOs: 341, 342, 343, 344, 345, and 346, respectively, or SEQ ID NOs: 347, 348, 349, 350, 351, and 352, respectively. Including, An isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, preferably human EGFRvIII, is provided.

[0008] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof has an identity at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 169, 183, 197, 211, 225, 239, 253, 267, 281, 295, 309, 323, 339, 353, 354, 355, 356, 360, 361, 362, 363, 366, 367, 368, 369, 374, 375, 376, 377, 382, ​​383, 384, or 385. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22, 24, 25, 26, 28, 29, 31, 32, 34, 35, 35, 35, 35, 35, 36, 36, 37, 37, 37, 37, 37, 38, 38, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 114, 128, 142, 156, 170, 184, 198, 212, 226, 240, 254, 268, 282, 296, 310, 324, 340, 357,

[0009] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 29 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 30; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 43 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 44; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 57 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 58; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 71 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 72; (7) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 85 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 86; (8) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 99 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 100; (9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 113 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 114; (10) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 127 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 128; (11) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 141 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 142; (12) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 156; (13) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 170; (14) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 184; (15) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 197 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 198; (16) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 211 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 212; (17) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 225 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 226. (18) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 239 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 240; (19) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 253 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 254. (20) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 267 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 268; (21) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 282; (22) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 295 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 296; (23) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 309 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 310; (24) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 323 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 324. (25) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 339 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 340; (26) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (27) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (28) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359; (29) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (30) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358; (31) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (32) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357; (33) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (34) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (35) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (36) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (37) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (38) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 360 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (39) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 360 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (40) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 361 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (41) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 361 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (42) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 362 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (43) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 362 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (44) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 363 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (45) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 363 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (46) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (47) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (48) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (49) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373. (50) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370; (51) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371; (52) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372; (53) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (54) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (55) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (56) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (57) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (58) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (59) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (60) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (61) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (62) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (63) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (64) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (65) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (66) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (67) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (68) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (69) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (70) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (71) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (72) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (73) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (74) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (75) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (76) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (77) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (78) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (79) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (80) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (81) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (82) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (83) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (84) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (85) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (86) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (87) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (88) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387; or (89) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388.

[0010] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof binds to EGFRvIII and is capable of inducing effector-mediated tumor cell lysis via antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC), and / or mediating the recruitment of a conjugated drug, and / or forming a bispecific antibody with another monoclonal antibody or antigen-binding fragment thereof that has cancer-killing activity.

[0011] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is chimeric.

[0012] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof is human or humanized.

[0013] Also provided is an isolated bispecific antibody or antigen-binding fragment thereof comprising the monoclonal antibody or antigen-binding fragment thereof of the invention.

[0014] Also provided is an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the invention.

[0015] Also provided is a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the invention.

[0016] Host cells containing vectors containing isolated nucleic acids encoding the monoclonal antibodies or antigen-binding fragments thereof, or bispecific antibodies or antigen-binding fragments thereof of the invention are also provided.

[0017] In certain embodiments, a pharmaceutical composition is provided comprising an isolated monoclonal antibody or antigen-binding fragment thereof, or an isolated bispecific antibody or antigen-binding fragment thereof of the invention and a pharmaceutically acceptable carrier.

[0018] Also provided is a method for specifically targeting EGFRvIII on the surface of cancer cells in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the present invention.

[0019] Also provided is a method for treating cancer in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the present invention. The cancer may be any liquid or solid cancer, for example, but not limited to, lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma, and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0020] Also provided is a method of treating an inflammatory disease and / or an autoimmune disease in a subject in need thereof, comprising administering to the subject a pharmaceutical composition of the present invention.

[0021] Also provided is a method for producing a monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the invention, comprising culturing cells containing nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof, under conditions for production of the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof, and recovering the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof from the cells or culture.

[0022] Also provided is a method for producing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof, or a bispecific antibody or antigen-binding fragment thereof of the invention, comprising combining the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0023] Also provided is a method for determining the level of EGFRvIII in a subject. The method includes (a) obtaining a sample from the subject, (b) contacting the sample with an antibody or antigen-binding fragment thereof of the present invention, and (c) determining the level of EGFRvIII in the subject. In certain embodiments, the sample is a tissue sample. The tissue sample may be, for example, a cancer tissue sample. In certain embodiments, the sample is a blood sample.

[0024] In another general aspect, the invention relates to a chimeric antigen receptor (CAR) construct that induces T cell-mediated cancer killing, the CAR construct comprising at least one antigen-binding domain that specifically binds human EGFRvIII, a hinge region, a transmembrane region, and an intracellular signaling domain.

[0025] An isolated polynucleotide comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) is provided. The CAR can comprise: (a) an extracellular domain comprising at least one antigen-binding domain that specifically binds to EGFRvIII, preferably human EGFRvIII; (b) a hinge region; (c) a transmembrane region; and (d) an intracellular signaling domain.

[0026] In certain embodiments, the antigen-binding domain comprises a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, and a light chain variable region comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having the following polypeptide sequences: (1) SEQ ID NOs: 3, 4, 5, 6, 7, and 8, respectively, or SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively; (2) SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively, or SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively; (3) SEQ ID NOs: 31, 32, 33, 34, 35, and 36, respectively, or SEQ ID NOs: 37, 38, 39, 40, 41, and 42, respectively; (4) SEQ ID NOs: 45, 46, 47, 48, 49, and 50, respectively, or SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively; (5) SEQ ID NOs: 59, 60, 61, 62, 63, and 64, respectively, or SEQ ID NOs: 65, 66, 67, 68, 69, and 70, respectively; (6) SEQ ID NOs: 73, 74, 75, 76, 77, and 78, or SEQ ID NOs: 79, 80, 81, 82, 83, and 84, respectively; (7) SEQ ID NOs: 87, 88, 89, 90, 91, and 92, respectively, or SEQ ID NOs: 93, 94, 95, 96, 97, and 98, respectively; (8) SEQ ID NOs: 101, 102, 103, 104, 105, and 106, respectively, or SEQ ID NOs: 107, 108, 109, 110, 111, and 112, respectively; (9) SEQ ID NOs: 115, 116, 117, 118, 119, and 120, respectively, or SEQ ID NOs: 121, 122, 123, 124, 125, and 126, respectively; (10) SEQ ID NOs: 129, 130, 131, 132, 133, and 134, respectively, or SEQ ID NOs: 135, 136, 137, 138, 139, and 140, respectively; (11) SEQ ID NOs: 143, 144, 145, 146, 147, and 148, respectively, or SEQ ID NOs: 149, 150, 151, 152, 153, and 154, respectively; (12) SEQ ID NOs: 157, 158, 159, 160, 161, and 162, respectively, or SEQ ID NOs: 163, 164, 165, 166, 167, and 168, respectively; (13) SEQ ID NOs: 171, 172, 173, 174, 175, and 176, respectively, or SEQ ID NOs: 177, 178, 179, 180, 181, and 182, respectively; (14) SEQ ID NOs: 185, 186, 187, 188, 189, and 190, respectively, or SEQ ID NOs: 191, 192, 193, 194, 195, and 196, respectively; (15) SEQ ID NOs: 199, 200, 201, 202, 203, and 204, respectively, or SEQ ID NOs: 205, 206, 207, 208, 209, and 210, respectively; (16) SEQ ID NOs: 213, 214, 215, 216, 217, and 218, respectively, or SEQ ID NOs: 219, 220, 221, 222, 223, and 224, respectively; (17) SEQ ID NOs: 227, 228, 229, 230, 231, and 232, respectively, or SEQ ID NOs: 233, 234, 235, 236, 237, and 238, respectively; (18) SEQ ID NOs: 241, 242, 243, 244, 245, and 246, respectively, or SEQ ID NOs: 247, 248, 249, 250, 251, and 252, respectively; (19) SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively, or SEQ ID NOs: 261, 262, 263, 264, 265, and 266, respectively; (20) SEQ ID NOs: 269, 270, 271, 272, 273, and 274, respectively, or SEQ ID NOs: 275, 276, 277, 278, 279, and 280, respectively; (21) SEQ ID NOs: 283, 284, 285, 286, 287, and 288, or SEQ ID NOs: 289, 290, 291, 292, 293, and 294, respectively; (22) SEQ ID NOs: 297, 298, 299, 300, 301, and 302, respectively, or SEQ ID NOs: 303, 304, 305, 306, 307, and 308, respectively; (23) SEQ ID NOs: 311, 312, 313, 314, 315, and 316, respectively, or SEQ ID NOs: 317, 318, 319, 320, 321, and 322, respectively; (24) SEQ ID NOs: 325, 326, 327, 328, 329, and 330, respectively, or SEQ ID NOs: 331, 332, 333, 334, 335, and 336, respectively; or (25) SEQ ID NOs: 341, 342, 343, 344, 345, and 346, respectively, or SEQ ID NOs: 347, 348, 349, 350, 351, and 352, respectively.

[0027] In certain embodiments, the antigen binding domain is a polynucleotide that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 169, 183, 197, 211, 225, 239, 253, 267, 281, 295, 309, 323, 339, 353, 354, 355, 356, 360, 361, 362, 363, 366, 367, 368, 369, 374, 375, 376, 377, 382, ​​383, 384, or 385. and a heavy chain variable region having a peptide sequence, or a light chain variable region having a polypeptide sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2, 16, 30, 44, 58, 72, 86, 100, 114, 128, 142, 156, 170, 184, 198, 212, 226, 240, 254, 268, 282, 296, 310, 324, 340, 357, 358, 359, 364, 365, 370, 371, 372, 373, 378, 379, 380, 381, 386, 387, or 388.

[0028] In certain embodiments, the antigen-binding domain comprises: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 29 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 30; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 43 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 44; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 57 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 58; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 71 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 72; (7) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 85 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 86; (8) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 99 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 100; (9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 113 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 114; (10) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 127 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 128; (11) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 141 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 142; (12) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 156; (13) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 170; (14) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 184; (15) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 197 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 198; (16) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 211 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 212; (17) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 225 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 226. (18) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 239 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 240; (19) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 253 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 254. (20) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 267 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 268; (21) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 282; (22) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 295 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 296; (23) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 309 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 310; (24) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 323 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 324. (25) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 339 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 340; (26) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (27) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (28) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359; (29) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (30) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358; (31) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (32) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357; (33) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (34) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (35) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (36) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (37) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (38) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 360 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (39) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 360 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (40) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 361 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (41) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 361 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (42) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 362 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (43) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 362 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (44) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 363 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (45) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 363 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (46) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (47) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (48) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (49) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373. (50) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370; (51) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371; (52) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372; (53) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (54) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (55) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (56) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (57) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (58) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (59) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (60) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (61) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (62) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (63) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (64) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (65) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (66) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (67) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (68) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (69) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (70) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (71) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (72) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (73) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (74) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (75) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (76) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (77) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (78) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (79) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (80) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (81) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (82) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (83) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (84) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (85) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (86) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (87) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (88) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387; or (89) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388.

[0029] In certain embodiments, the antigen-binding domain is humanized.

[0030] In certain embodiments, the antigen-binding domain is a single-chain variable fragment (scFv).

[0031] In certain embodiments, the antigen-binding domain is a humanized single-chain variable fragment (scFv).

[0032] In certain embodiments, a chimeric antigen receptor (CAR) comprises one or more antigen-binding domains.

[0033] In certain embodiments, the intracellular signaling domain comprises one or more costimulatory domains and one or more activation domains.

[0034] Also provided is a chimeric antigen receptor (CAR) encoded by the isolated polynucleotide of the invention.

[0035] Also provided is a vector comprising an isolated polynucleotide comprising a nucleic acid encoding a CAR of the invention.

[0036] Host cells comprising the vectors of the invention are also provided.

[0037] In certain embodiments, the host cell is a T cell, preferably a human T cell. In certain embodiments, the host cell is a NK cell, preferably a human NK cell. The T cell or NK cell can be engineered to express a CAR of the present invention to treat a disease such as, for example, cancer.

[0038] Also provided is a method of making a host cell expressing a chimeric antigen receptor (CAR) of the invention, the method comprising transducing a T cell or NK cell with a vector comprising an isolated nucleic acid encoding a CAR of the invention.

[0039] Also provided is a method for producing CAR-T cells or CAR-NK cells of the invention, comprising culturing T cells or NK cells containing an isolated polynucleotide comprising a nucleic acid encoding a chimeric antigen receptor (CAR) of the invention under CAR-T cell or CAR-NK cell-producing conditions, and recovering the CAR-T cells or CAR-NK cells.

[0040] Also provided is a method for generating a population of RNA engineered cells comprising a chimeric antigen receptor (CAR) of the invention, the method comprising contacting the cells with an isolated polynucleotide comprising a nucleic acid encoding a chimeric antigen receptor (CAR) of the invention, wherein the isolated polynucleotide is an in vitro transcribed RNA or a synthetic RNA.

[0041] Also provided are methods of treating cancer in a subject in need thereof, comprising administering to the subject the CAR-T cells and / or CAR-NK cells of the invention. The cancer can be any liquid or solid cancer, including, but not limited to, lung cancer, gastric cancer, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma, and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0042] In certain embodiments, the method of treating cancer in a subject in need thereof further comprises administering to the subject in need thereof an agent that increases the efficacy of cells expressing a CAR molecule.

[0043] In certain embodiments, the method of treating cancer in a subject in need thereof further comprises administering to the subject in need thereof an agent that ameliorates one or more side effects associated with administration of cells expressing a CAR molecule.

[0044] In certain embodiments, the method of treating cancer in a subject in need thereof further comprises administering to the subject in need thereof an agent that treats an EGFRvIII-associated disease.

[0045] The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the application is not limited to the precise embodiments shown in the drawings. [Brief explanation of the drawings]

[0046] [Figure 1A-B] Figures 1A-G show the binding of purified chimeric anti-EGFRvIII mAb (rabbit mAb VH and VL regions fused to human IgG1 heavy and kappa light chain constant regions, respectively) to immobilized recombinant EGFRvIII in an ELISA assay. The antigen was C-terminally 6xHis-tagged human EGFRvIII(25-378) (ACROBiosystems, CAT#: EGI-H52H4). An isotype control (IgG1) was used as a negative control. [Figure 1C-D] Figures 1A-G show the binding of purified chimeric anti-EGFRvIII mAb (rabbit mAb VH and VL regions fused to human IgG1 heavy and kappa light chain constant regions, respectively) to immobilized recombinant EGFRvIII in an ELISA assay. The antigen was C-terminally 6xHis-tagged human EGFRvIII(25-378) (ACROBiosystems, CAT#: EGI-H52H4). An isotype control (IgG1) was used as a negative control. [Figure 1E-F] Figures 1A-G show the binding of purified chimeric anti-EGFRvIII mAb (rabbit mAb VH and VL regions fused to human IgG1 heavy and kappa light chain constant regions, respectively) to immobilized recombinant EGFRvIII in an ELISA assay. The antigen was C-terminally 6xHis-tagged human EGFRvIII(25-378) (ACROBiosystems, CAT#: EGI-H52H4). An isotype control (IgG1) was used as a negative control. [Figure 1G]Figures 1A-G show the binding of purified chimeric anti-EGFRvIII mAb (rabbit mAb VH and VL regions fused to human IgG1 heavy and kappa light chain constant regions, respectively) to immobilized recombinant EGFRvIII in an ELISA assay. The antigen was C-terminally 6xHis-tagged human EGFRvIII(25-378) (ACROBiosystems, CAT#: EGI-H52H4). An isotype control (IgG1) was used as a negative control. [Figure 2A] 2A-G are graphs showing binding of anti-EGFRvIII mAb to ExpiCHO-S cells transiently expressing EGFRvIII, analyzed by FACS. An isotype control (IgG1) was used as a negative control. [Figure 2B-C] 2A-G are graphs showing binding of anti-EGFRvIII mAb to ExpiCHO-S cells transiently expressing EGFRvIII, analyzed by FACS. An isotype control (IgG1) was used as a negative control. [Figure 2D-E] 2A-G are graphs showing binding of anti-EGFRvIII mAb to ExpiCHO-S cells transiently expressing EGFRvIII, analyzed by FACS. An isotype control (IgG1) was used as a negative control. [Figure 2F-G] 2A-G are graphs showing binding of anti-EGFRvIII mAb to ExpiCHO-S cells transiently expressing EGFRvIII, analyzed by FACS. An isotype control (IgG1) was used as a negative control. [Figure 3A-B] 3A to 3D show the binding activity of anti-EGFRvIII chimeric mAb to wild-type EGFR evaluated by ELISA. [Figure 3C-D] 3A to 3D show the binding activity of anti-EGFRvIII chimeric mAb to wild-type EGFR evaluated by ELISA. [Figure 4A-B]Figures 4A-D are graphs showing the binding of humanized anti-EGFRvIII mAbs to immobilized recombinant EGFRvIII in an ELISA assay. The antigen was C-terminally 6xHis-tagged human EGFRvIII(25-378) (ACROBiosystems, CAT#: EGI-H52H4). 16C11-H1L1 refers to a humanized mAb constructed using the 16C11-H1 heavy chain and 16C11-L1 light chain shown in Table 7; other humanized clones follow the same naming convention. An isotype control (IgG1) was used as a negative control. [Figure 4C-D] Figures 4A-D are graphs showing the binding of humanized anti-EGFRvIII mAbs to immobilized recombinant EGFRvIII in an ELISA assay. The antigen was C-terminally 6xHis-tagged human EGFRvIII(25-378) (ACROBiosystems, CAT#: EGI-H52H4). 16C11-H1L1 refers to a humanized mAb constructed using the 16C11-H1 heavy chain and 16C11-L1 light chain shown in Table 7; other humanized clones follow the same naming convention. An isotype control (IgG1) was used as a negative control. [Figure 5A-B] Figures 5A-5D show the binding of humanized anti-EGFRvIII mAbs to ExpiCHO-S cells transiently expressing EGFRvIII, analyzed by FACS. 16C11-H1L1 refers to a humanized mAb constructed using the 16C11-H1 heavy chain and 16C11-L1 light chain shown in Table 7; other humanized clones follow the same naming convention. An isotype control (IgG1) was used as a negative control. [Figure 5C-D] Figures 5A-5D show the binding of humanized anti-EGFRvIII mAbs to ExpiCHO-S cells transiently expressing EGFRvIII, analyzed by FACS. 16C11-H1L1 refers to a humanized mAb constructed using the 16C11-H1 heavy chain and 16C11-L1 light chain shown in Table 7; other humanized clones follow the same naming convention. An isotype control (IgG1) was used as a negative control. DETAILED DESCRIPTION OF THE INVENTION

[0047] Throughout the Background and specification, various publications, articles, and patents are cited or described. Each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles and the like which has been included in the specification is for the purpose of providing a context for the present invention. Such discussion is not an admission that any or all of these matters form part of the prior art with respect to any invention(s) disclosed or claimed.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, certain terms used herein have the meanings given herein.

[0049] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0050] Unless otherwise specified, any numerical values, e.g., concentrations or concentration ranges, described herein should be understood to be modified in all instances by the term "about." Thus, numerical values ​​typically include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, unless the context clearly dictates otherwise, the use of numerical ranges explicitly includes all individual numerical values ​​within that range, including all possible subranges, integers, and fractions of values ​​within such ranges.

[0051] Unless otherwise indicated, the term "at least" before a series of elements should be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0052] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," "contains," or "containing," or any other variation thereof, are understood to imply that the specified integer or group of integers is inclusive, but not the exclusion of any other integer or group of integers, and are non-exclusive or open-ended. For example, a device, composition, mixture, process, method, or article that includes a list of elements is not necessarily limited to only those elements, but may include other elements not expressly listed or inherent in such composition, mixture, process, method, article, or device. Furthermore, unless expressly stated otherwise, "or" refers to an inclusive or, not an exclusive or. For example, condition A or B is satisfied by any one of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), and both A and B are true (or exist).

[0053] As used herein, the conjunction "and / or" between multiple listed elements is understood to encompass the individual and combined options. For example, when two elements are joined by "and / or," the first option refers to the application of the first element without the second element. The second option refers to the application of the second element without the first element. The third option refers to the application of the first and second elements together. Any one of these options is understood to fall within this meaning and therefore meet the requirements of the term "and / or" as used herein. The simultaneous application of more than one of these options is also understood to fall within this meaning and therefore meet the requirements of the term "and / or."

[0054] As used herein, the term "consists of" or variations such as "consist of" or "consisting of," as used throughout the specification and claims, indicates that any recited integer or group of integers is inclusive, but that no additional integers or groups of integers can be added to the specified method, structure, or composition.

[0055] As used herein, the term "consists essentially of" or variations such as "consist essentially of" or "consisting essentially of," as used throughout the specification and claims, indicates the inclusion of any recited integer or group of integers, and any inclusion of any recited integer or group of integers that does not materially alter the basic or novel characteristics of the specified method, structure, or composition. See MPEP § 2111.03.

[0056] As used herein, "subject" refers to any animal, preferably a mammal, and most preferably a human. As used herein, the term "mammal" encompasses any mammal. Examples of mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc., more preferably humans.

[0057] The words "right", "left", "bottom" and "top" designate directions in the figures to which reference is made.

[0058] It should also be understood that the terms "about," "approximately," "generally," "substantially," and similar terms used herein when referring to dimensions or characteristics of preferred inventive components indicate that the described dimensions / characteristics are not precise boundaries or parameters, as understood by those skilled in the art, and do not exclude minor variations therefrom that are functionally the same or similar. At a minimum, such references involving numerical parameters are expected to include variations that are not expected to alter the number of least significant digits, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0059] The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences (e.g., anti-EGFRvIII antibodies and the polynucleotides encoding them, chimeric antigen receptors (CARs) comprising an antigen-binding domain specific for EGFRvIII and the polynucleotides encoding them), refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, when compared and aligned for maximum correspondence as determined using one of the following sequence comparison algorithms or by visual inspection:

[0060] For sequence comparison, typically one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence, based on the designated program parameters.

[0061] Optimal alignment of sequences for comparison can be determined, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 1981; 2:482, by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 1970; 48:443, by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 1988; 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by visual inspection (generally, see Current Protocols in Molecular Biology, F.M. Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., 1995 Supplement (See Ausubel)

[0062] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., J. Mol. Biol. 1990; 215: 403-410 and Altschul et al., Nucleic Acids Res. 1997; 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0063] Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of word hits in each direction is halted if: the cumulative alignment score falls by an amount X from its maximum achieved value; the accumulation of one or more negative-scoring residue alignments causes the cumulative score to fall below zero; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 1989;89:10915).

[0064] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 1993; 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0065] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, for example, a polypeptide will generally be substantially identical to a second polypeptide where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0066] As used herein, the term "isolated" means that a biological component (e.g., a nucleic acid, peptide, or protein) has been substantially separated, produced separately, or purified from other biological components of the organism in which it naturally occurs, i.e., from other chromosomal and extrachromosomal DNA and RNA, and proteins. Thus, "isolated" nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. "Isolated" nucleic acids, peptides, and proteins can be part of a composition and still be isolated, if the composition is not part of the nucleic acid, peptide, or protein's natural environment. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids.

[0067] As used herein, the term "polynucleotide," which may be interchangeably referred to as "nucleic acid molecule," "nucleotide," or "nucleic acid," refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, and hybrid molecules containing DNA and RNA that may be single-stranded or, more commonly, double-stranded, or a mixture of single- and double-stranded regions. Furthermore, "polynucleotide" refers to triple-stranded regions containing RNA or DNA, or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases and DNA or RNA with backbones modified for stability or other reasons. "Modified" bases include, for example, tritylated bases and unusual bases, such as inosine. Various modifications can be made to DNA and RNA. Thus, "polynucleotide" encompasses chemically, enzymatically, or metabolically modified forms of polynucleotides typically found in nature, as well as the DNA and RNA chemical forms characteristic of viruses and cells. "Polynucleotide" also encompasses relatively short nucleic acid strands often referred to as oligonucleotides.

[0068] As used herein, the term "vector" is a replicon into which another nucleic acid segment may be operatively inserted so as to bring about the replication or expression of the segment.

[0069] As used herein, the term "host cell" refers to a cell that contains a nucleic acid molecule of the invention. A "host cell" can be any cell type, e.g., a primary cell, a cultured cell, or a cell from a cell line. In one embodiment, a "host cell" is a cell that has been transfected with a nucleic acid molecule of the invention. In another embodiment, a "host cell" is the progeny or potential progeny of such a transfected cell. The progeny of a cell may or may not be identical to the parent cell due, for example, to mutations or environmental influences that may occur in subsequent generations, or to integration of the nucleic acid molecule into the host cell genome.

[0070] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term includes transcription of a gene into RNA. The term also includes translation of RNA into one or more polypeptides, and further includes all naturally occurring post-transcriptional and post-translational modifications. The expressed antibody may be in the cytoplasm of a host cell, in an extracellular environment, such as the growth medium of a cell culture, or anchored to the cell membrane.

[0071] As used herein, the terms "peptide," "polypeptide," or "protein" can refer to a molecule composed of amino acids and recognized as a protein by those skilled in the art. Conventional one-letter or three-letter codes for amino acid residues are used herein. The terms "peptide," "polypeptide," and "protein" can be used interchangeably herein to refer to polymers of amino acids of any length. The polymers can be linear or branched, can contain modified amino acids, and can be interrupted by non-amino acids. The terms also encompass amino acid polymers that are modified naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

[0072] The peptide sequences described herein are written according to the usual convention whereby the N-terminal region of the peptide is on the left and the C-terminal region is on the right. Although isomeric forms of amino acids are known, unless expressly indicated otherwise, the amino acids represented are in the L-form.

[0073] Chimeric antigen receptor (CAR) As used herein, the term "chimeric antigen receptor" (CAR) refers to a recombinant polypeptide comprising at least an extracellular domain that specifically binds to an antigen or target, a transmembrane domain, and an intracellular T cell receptor activation signaling domain. When the extracellular domain of the CAR binds to a target antigen on the surface of a target cell, clustering of the CAR occurs, providing an activating stimulus to the cell containing the CAR. The CAR redirects the specificity of immune effector cells, triggering proliferation, cytokine production, phagocytosis, and / or the production of molecules that can mediate cell death of target antigen-expressing cells in a major histocompatibility (MHC)-independent manner.

[0074] In one embodiment, a CAR comprises an antigen-binding domain, a hinge region, a costimulatory domain, an activation domain, and a transmembrane region. In one embodiment, a CAR comprises an antigen-binding domain, a hinge region, two costimulatory domains, an activation domain, and a transmembrane region. In one embodiment, a CAR comprises two antigen-binding domains, a hinge region, a costimulatory domain, an activation domain, and a transmembrane region. In one embodiment, a CAR comprises two antigen-binding domains, a hinge region, two costimulatory domains, an activation domain, and a transmembrane region.

[0075] As used herein, the term "signal peptide" refers to a leader sequence at the amino-terminus (N-terminus) of a nascent CAR protein that co- or post-translationally targets the nascent protein to the endoplasmic reticulum, leading to subsequent surface expression.

[0076] As used herein, the term "extracellular antigen-binding domain," "extracellular domain," or "extracellular ligand-binding domain" refers to the portion of a CAR that is located on the outside of the cell membrane and is capable of binding to an antigen, target, or ligand.

[0077] As used herein, the term "hinge region" refers to the portion of a CAR that connects two adjacent domains of the CAR protein, e.g., the extracellular domain and the transmembrane domain.

[0078] As used herein, the term "transmembrane domain" refers to the portion of a CAR that extends across the cell membrane and anchors the CAR to the cell membrane. This is sometimes referred to as the "transmembrane region."

[0079] Costimulatory domain As used herein, chimeric antigen receptors can incorporate a costimulatory (signaling) domain to enhance their efficacy. The costimulatory (signaling) domain can be derived from a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient immune response.The costimulatory domain can be derived from costimulatory molecules, including CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, programmed death-1 (PD-1), inducible T cell costimulatory factor (ICOS), lymphocyte function-associated antigen-1 (LFA-1; CD11a and CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP10, Fc gamma receptor, MHC class I molecule, TNFR, integrin, signaling lymphocyte activation molecule, BTLA, Toll ligand receptor, ICAM-1, CDS, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGAL, CD1a, CD1b, CD1c, CD1d, ITGAM, ITGAX, ITGB1, CD29, ITGB 2(CD18), ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, cytokine receptor, activating NK cell receptor, or any combination thereof.

[0080] Activation domain As used herein, chimeric antigen receptor can comprise an activation domain.The activation domain can include, but is not limited to, CD3.CD3 is a component of the T cell receptor on natural T cells and has been shown to be an important intracellular activation component in CAR.In a preferred embodiment, CD3 is CD3 zeta.

[0081] Hinge Area As described herein, a chimeric antigen receptor can comprise a hinge region, which is part of the extracellular domain and is sometimes referred to as a "spacer" region. A variety of hinges can be used in accordance with the present invention, including the costimulatory molecules described above, immunoglobulin (Ig) sequences, or other appropriate molecules to achieve a desired special distance from the target cell. In some embodiments, the entire extracellular domain comprises the hinge region.

[0082] transmembrane region As used herein, a chimeric antigen receptor (CAR) can include a transmembrane region / domain. A CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. Similarly, this can be fused to the intracellular domain of the CAR. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used. In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or a different surface membrane protein to minimize interaction with other members of the receptor complex. The transmembrane domain can be derived from either a natural or synthetic source. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions that are particularly useful in the present invention include CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, programmed death-1 (PD-1), inducible T-cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1; CD11a and CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14;TNFSF14), NKG2C, Ig alpha (CD79a), DAP10, Fc gamma receptor, MHC class I molecule, TNFR, integrin, signaling lymphocyte activation molecule, BTLA, Toll ligand receptor, ICAM-1, CDS, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGAL, CD1a, CD1b, CD1c, CD1d, ITGAM, ITGAX, ITGB1, CD29, ITGB 2(CD18), ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD 160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, cytokine receptor, activating NK cell receptor, immunoglobulin protein, or a fragment or any combination thereof;

[0083] immune cells According to certain aspects, the present invention provides a cell that is an immune cell comprising an isolated polynucleotide comprising a nucleotide sequence encoding a CAR provided herein or a vector comprising the isolated polynucleotide. Immune cells comprising the isolated polynucleotide and / or vector of the present invention can be referred to as "engineered immune cells." Preferably, engineered immune cells are derived from a human (are cells of human origin prior to being recombined).

[0084] The engineered immune cells can be, for example, of the lymphoid lineage. Non-limiting examples of lymphoid lineage cells include T cells and natural killer (NK) cells. T cells express the T cell receptor (TCR), with most cells expressing α and β chains, and a smaller population expressing γ and δ chains. T cells useful as engineered immune cells of the present invention include CD4 + or CD8 + T helper cells (CD4 + ), cytotoxic T cells (also called cytotoxic T lymphocytes, CTLs; CD8 + These immune cells include, but are not limited to, immune cells (e.g., immune cells), as well as memory T cells, including central memory T cells, stem-like memory T cells, and effector memory T cells, natural killer T cells, mucosal-associated invariant T cells, and γδ T cells. Other exemplary immune cells include, but are not limited to, macrophages, antigen-presenting cells (APCs), or any immune cell that expresses an inhibitor of a cell-mediated immune response, such as an immune checkpoint inhibitor pathway receptor (e.g., PD-1). Precursor cells of immune cells that can be used in accordance with the present invention include hematopoietic stem and / or progenitor cells. Hematopoietic stem and / or progenitor cells can be derived from bone marrow, umbilical cord blood, adult peripheral blood after cytokine mobilization, etc., by methods known in the art. The immune cells are engineered to recombinantly express the CAR of the present invention.

[0085] Immune cells and their precursor cells can be isolated by methods known in the art, including commercially available methods (see, e.g., Rowland Jones et al., Lymphocytes: A Practical Approach, Oxford University Press, NY 1999). Sources of immune cells or their precursors include, but are not limited to, peripheral blood, umbilical cord blood, bone marrow, or other sources of hematopoietic cells. Various techniques can be used to separate cells to isolate or enrich for desired immune cells. For example, negative selection methods can be used to remove cells that are not the desired immune cells. Additionally, positive selection methods can be used to isolate or enrich for desired immune cells or their precursors, or positive and negative selection methods can be used in combination. When isolating a specific type of cell, such as a specific T cell, various cell surface markers or combinations of markers (e.g., CD3, CD4, CD8, CD34) can be used to separate the cells.

[0086] Immune cells or their precursor cells can be autologous (autologous) or non-autologous (non-autologous) to the subject to whom they are administered in the treatment methods of the present invention. Autologous cells are isolated from the subject to whom engineered immune cells recombinantly expressing a CAR are administered. Optionally, cells can be obtained by leukapheresis, in which leukocytes are selectively removed from drawn blood, recombined, and then reinfused back into the donor. Alternatively, allogeneic cells from a non-autologous donor can be used. In the case of a non-autologous donor, the cells are sorted and matched with human leukocyte antigens (HLA) to determine the appropriate level of compatibility. For both autologous and non-autologous cells, the cells can be cryopreserved, if necessary, until ready for use.

[0087] Various methods for isolating immune cells that can be used for recombinant expression of the CARs of the invention have been previously described and can be used, including using peripheral blood donor lymphocytes (Sadelain et al., Nat. Rev. Cancer 2003; 3:35-45; Morgan et al., Science 2006; 314:126-9), using lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) in tumor biopsies (Panelli et al., J. Immunol. 2000; 164:495-504; Panelli et al., J. Immunol. 2000; 164:4382-92), and using antigen-specific peripheral blood leukocytes selectively expanded in vitro using artificial antigen-presenting cells (AAPCs) or dendritic cells (Dupont et al., Cancer Res. 2005; 65:5417-427; Papanicolaou et al., Blood 2003; 102:2498-505). When using stem cells, the cells can be isolated by methods well known in the art (see, for example, Klug et al., Hematopoietic Stem Cell Protocols, Humana Press, NJ 2002; Freshney et al., Culture of Human Stem Cells, John Wiley & Sons 2007).

[0088] According to certain embodiments, a method of generating engineered immune cells comprises transfecting or transducing immune effector cells isolated from an individual to express one or more CARs according to embodiments of the present invention. Methods of preparing immune cells for immunotherapy are described, for example, in WO2014 / 130635, WO2013 / 176916, and WO2013 / 176915, which are incorporated herein by reference. Individual steps that can be used in the preparation of engineered immune cells are disclosed, for example, in WO2014 / 039523, WO2014 / 184741, WO2014 / 191128, WO2014 / 184744, and WO2014 / 184143, which are incorporated herein by reference.

[0089] In one specific embodiment, immune effector cells, e.g., T cells, are genetically modified with a CAR of the invention (e.g., transduced with a viral vector comprising a nucleic acid encoding the CAR), and then activated and expanded in vitro. In various embodiments, T cells can be activated and expanded before or after genetic modification to express a CAR using, for example, methods described in US 6,352,694, US 6,534,055, US 6,905,680, US 6,692,964, US 5,858,358, US 6,887,466, US 6,905,681, US 7,144,575, US 7,067,318, US 7,172,869, US 7,232,566, US 7,175,843, US 5,883,223, US 6,905,874, US 6,797,514, US 6,867,041, US 2006 / 121005, which are incorporated herein by reference. T cells can be expanded in vitro or in vivo. Generally, T cells of the present invention can be expanded by contact with a surface bearing an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. As a non-limiting example, a population of T cells can be stimulated, as described herein, by contact with, for example, an anti-CD3 antibody, or an antigen-binding fragment thereof, or an anti-CD3 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore, or by activation of the CAR itself. To costimulate accessory molecules on the surface of T cells, a ligand that binds to the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate T cell proliferation.Suitable conditions for T cell culture include, for example, an appropriate medium (e.g., Minimum Essential Medium or RPMI Media 1640 or X-vivo 5 (Lonza)) that may contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine serum or human serum), cytokines such as IL-2, IL-7, IL-15, and / or IL-21, insulin, IFN-γ, GM-CSF, TGFβ, and / or other additives for cell growth known to those of skill in the art. In other embodiments, T cells can be activated and stimulated with feeder cells and appropriate antibodies and cytokines for expansion using methods such as those described in U.S. Pat. No. 6,040,177, U.S. Pat. No. 5,827,642, and WO2012129514, which are incorporated herein by reference.

[0090] Antibodies and antigen-binding domains The present invention generally relates to isolated anti-EGFRvIII antibodies, chimeric antigen receptors (CARs), nucleic acids and expression vectors encoding the antibodies and CARs, recombinant cells containing the vectors, and compositions comprising the antibodies, CARs, and recombinant cells expressing the CARs. Methods for making the antibodies and CARs, and methods for using the antibodies and CARs to treat diseases, including cancer, inflammatory diseases, and autoimmune diseases. The antibodies and antigen-binding domains of the CARs of the present invention possess one or more desirable functional properties, including, but not limited to, high-affinity binding to EGFRvIII, high specificity for EGFRvIII, the ability to stimulate complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and / or antibody-dependent cell-mediated cytotoxicity (ADCC) against cells expressing EGFRvIII, and the ability to inhibit tumor growth in subjects and animal models when administered alone or in combination with other anti-cancer therapies.

[0091] In a general aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof that binds to EGFRvIII.

[0092] As used herein, the term "antibody" is used broadly to include immunoglobulins or antibody molecules, including human antibodies, humanized antibodies, composite antibodies, and chimeric antibodies, as well as antibody fragments, whether monoclonal or polyclonal. Generally, antibodies are proteins or peptide chains that exhibit binding specificity to a specific antigen. Antibody structure is well known. Immunoglobulins can be assigned to five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) depending on the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into isotypes IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4. Thus, antibodies of the present invention can be of any of the five major classes or corresponding subclasses. Preferably, antibodies of the present invention are IgG1, IgG2, IgG3, or IgG4. Antibody light chains of vertebrate species can be assigned to one of two clearly distinct types, kappa and lambda, based on the amino acid sequence of their constant domains. Thus, antibodies of the present invention can contain kappa or lambda light chain constant domains. According to certain embodiments, the antibodies of the present invention comprise heavy and / or light chain constant regions of a rat or human antibody. In addition to the heavy and light chain constant domains, the antibodies contain an antigen-binding region composed of a light chain variable region and a heavy chain variable region, each of which contains three domains (i.e., complementarity-determining regions 1 to 3; CDR1, CDR2, and CDR3). The light chain variable region domains are alternatively referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains are alternatively referred to as HCDR1, HCDR2, and HCDR3.

[0093] "Complementarity-determining regions" (CDRs) are antibody regions that bind antigens. CDRs can be defined using various schemes, such as Kabat (Wu et al., J Exp Med 132: 211-50, 1970) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia (Chothia et al., J Mol Biol 196: 901-17, 1987), IMGT (Lefranc et al., Dev Comp Immunol 27: 55-77, 2003), and AbM (Martin and Thornton J Bmol Biol 263: 800-15, 1996). The correspondence between various outlines and variable region numbering has been described (see, e.g., Lefranc et al., Dev Comp Immunol 27: 55-77, 2003; Honegger and Pluckthun, J Mol Biol 309:657-70, 2001; the International ImmunoGeneTics (IMGT) database, a web resource, http: / / www_imgt_org). Available programs, such as abYsis by UCL Business PLC, can be used to delineate the CDRs. The terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3", as used herein, include CDRs as defined by any of the above methods, Kabat, Chothia, IMGT or AbM, unless otherwise expressly stated herein. For example, the correspondence between numbering systems, including Kabat numbering and the IMGT specific numbering system, is well known to those of skill in the art (see, e.g., Kabat, Chothia, Martin, Lefranc et al.).

[0094] [Table 1-1]

[0095] As used herein, the term "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to EGFRvIII is substantially free of antibodies that do not bind to the same EGFRvIII). Moreover, an isolated antibody is substantially free of other cellular material and / or chemicals.

[0096] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies of the invention can be produced by hybridoma methods, phage display techniques, single lymphocyte gene cloning techniques, or by recombinant DNA methods. For example, monoclonal antibodies can be produced by hybridomas comprising B cells obtained from a transgenic non-human animal, e.g., a transgenic mouse or rat, whose genome comprises a human heavy chain transgene and a light chain transgene.

[0097] As used herein, the terms "antigen-binding fragment" and / or "antigen-binding domain" refer to antibody fragments such as, for example, diabodies, Fab, Fab', F(ab'), Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (ds diabodies), single-chain antibody molecules (scFv), single-domain antibodies (sdab), scFv dimers (bivalent diabodies), multispecific antibodies formed from portions of an antibody comprising one or more CDRs, camelized single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment that binds to an antigen but does not comprise the complete antibody structure. An antigen-binding fragment can bind to the same antigen as the parent antibody or parent antibody fragment. According to certain embodiments, the antigen-binding fragment comprises a light chain variable region, a light chain constant region, and the Fd segment of a heavy chain. According to other particular embodiments, the antigen-binding fragment comprises Fab and F(ab'). The antigen-binding domain is capable of binding to the same antigen as the parent antibody. According to particular embodiments, the antigen-binding domain comprises a single-chain antibody molecule (scFv).

[0098] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art, which comprises a heavy chain variable region and a light chain variable region linked by a short peptide of about 15 to about 20 amino acids. As used herein, the term "single-domain antibody" refers to a conventional single-domain antibody in the art, which comprises a heavy chain variable region and a heavy chain constant region, or comprises only a heavy chain variable region.

[0099] As used herein, the term "human antibody" refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any technique known in the art. This definition of a human antibody includes intact or full-length antibodies, fragments thereof, and / or antibodies comprising at least one human heavy and / or light chain polypeptide.

[0100] As used herein, the terms "humanized antibody" and / or "humanized antigen-binding domain" refer to a non-human antibody and / or non-human antigen-binding domain that has been modified to increase sequence homology with a human antibody and / or human antigen-binding domain, such that the antigen-binding properties of the antibody and / or antigen-binding domain are retained but its antigenicity in the human body is reduced.

[0101] As used herein, the terms "chimeric antibody" and / or "chimeric antigen-binding domain" refer to antibodies and / or antigen-binding domains in which the amino acid sequences of the immunoglobulin molecule are derived from two or more species. The variable regions of both the light and heavy chains often correspond to the variable regions of antibodies and / or antigen-binding domains derived from one species of mammal (e.g., mouse, rat, rabbit, etc.) having the desired specificity, affinity, and capacity, while the constant regions correspond to the sequences of antibodies and / or antigen-binding domains derived from another species of mammal (e.g., human) to avoid eliciting an immune response in that species.

[0102] As used herein, the term "multispecific antibody" refers to an antibody comprising a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes do not overlap or substantially do not overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody comprises a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific, trispecific, or tetraspecific antibody molecule.

[0103] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds to no more than two epitopes or two antigens. A bispecific antibody is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. In one embodiment, the first and second epitopes are on the same antigen, e.g., the same protein (or subunit of a multimeric protein). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes are on different antigens, e.g., different proteins (or different subunits of a multimeric protein). In one embodiment, a bispecific antibody comprises heavy and light chain variable domain sequences that have binding specificity for a first epitope and heavy and light chain variable domain sequences that have binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises a half antibody or fragment thereof that has binding specificity for a first epitope and a half antibody or fragment thereof that has binding specificity for a second epitope, hi one embodiment, a bispecific antibody comprises an scFv or fragment thereof that has binding specificity for a first epitope and an scFv or fragment thereof that has binding specificity for a second epitope. In one embodiment, the first epitope is located on an EGFRvIII of the present invention, and the second epitope is located on PD-1, PD-L1, TIM-3, LAG-3, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD3, CD73, CD47, TIP-1, GPC3, apelin, DLL3, folate receptor alpha, Claudin18.2, MUC16, mesothelin, IL13Ra2, PSCA, p95HER2, ROR1, ROR2, NKp46, and / or other tumor-associated immunosuppressive factors or surface antigens. In one embodiment, the first and second epitopes are located on the same EGFRvIII of the present invention.

[0104] As used herein, an antibody and / or antigen-binding domain that "specifically binds to EGFRvIII" has a KD of 1 x 10 -7 M or less, preferably 1 × 10 -8 M or less, preferably 5×10 -9 M or less, 1×10 -9 M or less, 5×10 -10 M or less, or 1 x 10 -10 The term "KD" refers to an antibody and / or antigen-binding domain that binds to EGFRvIII, preferably human EGFRvIII, with a molar concentration (M) or less. The term "KD" refers to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). KD values ​​for antibodies and / or antigen-binding domains can be determined in light of the present disclosure using methods in the art. For example, the KD of an antibody and / or antigen-binding domain can be determined by using surface plasmon resonance, e.g., by using a biosensor system, e.g., a Biacore® system, or by using biolayer interferometry technology, e.g., an Octet RED96 system.

[0105] The smaller the KD value of an antibody and / or antigen-binding domain, the higher the affinity with which the antibody and / or antigen-binding domain binds to a target antigen.

[0106] As used herein, the term "IC 50 " refers to the half-maximal inhibitory concentration of a monoclonal or bispecific antibody or antigen-binding fragment thereof of the present invention. 50 is a measure of the potency of a monoclonal or bispecific antibody or antigen-binding fragment thereof of the invention to inhibit binding of a target antigen (i.e., receptor or ligand) to its natural ligand or receptor, or to inhibit the function of the target antigen in a cell. In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof or bispecific antibody or antigen-binding fragment thereof has an activity of about 10 -7 Less than M, about 10 -8 Less than M, about 10 -9 Less than M, about 10 -10 Less than M, about 10-11 Less than M, about 10 -12 Less than M or about 10 -13 Has a KD of less than M.

[0107] As used herein, "EC 50 " refers to the half-maximal effective concentration of a monoclonal or bispecific antibody of the present invention or an antigen-binding fragment thereof, or a CAR comprising the antigen-binding domain. EC 50 refers to the concentration of a monoclonal or bispecific antibody or antigen-binding fragment thereof, or a CAR comprising an antigen-binding domain, to induce a biological response (i.e., cell death) halfway between baseline and peak over a specific exposure period. In certain embodiments, a monoclonal antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, or a CAR comprising an antigen-binding domain has an EC of less than about 1 μM, about 1000 nM to about 100 nM, about 100 nM to about 10 nM, about 10 nM to about 1 nM, about 1000 pM to about 500 pM, about 500 pM to about 200 pM, less than about 200 pM, about 200 pM to about 150 pM, about 200 pM to about 100 pM, about 100 pM to about 10 pM, or about 10 pM to about 1 pM. 50 It has.

[0108] According to a particular aspect, the present invention provides an isolated monoclonal antibody or antigen-binding fragment thereof, or chimeric antigen receptor (CAR), comprising an antigen-binding domain, wherein the monoclonal antibody or antigen-binding fragment thereof, or the antigen-binding domain comprises a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, and a light chain variable region comprising light chain complementarity determining region 1 (LCDR1), LCDR2, LCDR3, having the following polypeptide sequences: (1) SEQ ID NOs: 3, 4, 5, 6, 7, and 8, respectively, or SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively; (2) SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively, or SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively; (3) SEQ ID NOs: 31, 32, 33, 34, 35, and 36, respectively, or SEQ ID NOs: 37, 38, 39, 40, 41, and 42, respectively; (4) SEQ ID NOs: 45, 46, 47, 48, 49, and 50, respectively, or SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively; (5) SEQ ID NOs: 59, 60, 61, 62, 63, and 64, respectively, or SEQ ID NOs: 65, 66, 67, 68, 69, and 70, respectively; (6) SEQ ID NOs: 73, 74, 75, 76, 77, and 78, or SEQ ID NOs: 79, 80, 81, 82, 83, and 84, respectively; (7) SEQ ID NOs: 87, 88, 89, 90, 91, and 92, respectively, or SEQ ID NOs: 93, 94, 95, 96, 97, and 98, respectively; (8) SEQ ID NOs: 101, 102, 103, 104, 105, and 106, respectively, or SEQ ID NOs: 107, 108, 109, 110, 111, and 112, respectively; (9) SEQ ID NOs: 115, 116, 117, 118, 119, and 120, respectively, or SEQ ID NOs: 121, 122, 123, 124, 125, and 126, respectively; (10) SEQ ID NOs: 129, 130, 131, 132, 133, and 134, respectively, or SEQ ID NOs: 135, 136, 137, 138, 139, and 140, respectively; (11) SEQ ID NOs: 143, 144, 145, 146, 147, and 148, respectively, or SEQ ID NOs: 149, 150, 151, 152, 153, and 154, respectively; (12) SEQ ID NOs: 157, 158, 159, 160, 161, and 162, respectively, or SEQ ID NOs: 163, 164, 165, 166, 167, and 168, respectively; (13) SEQ ID NOs: 171, 172, 173, 174, 175, and 176, respectively, or SEQ ID NOs: 177, 178, 179, 180, 181, and 182, respectively; (14) SEQ ID NOs: 185, 186, 187, 188, 189, and 190, respectively, or SEQ ID NOs: 191, 192, 193, 194, 195, and 196, respectively; (15) SEQ ID NOs: 199, 200, 201, 202, 203, and 204, respectively, or SEQ ID NOs: 205, 206, 207, 208, 209, and 210, respectively; (16) SEQ ID NOs: 213, 214, 215, 216, 217, and 218, respectively, or SEQ ID NOs: 219, 220, 221, 222, 223, and 224, respectively; (17) SEQ ID NOs: 227, 228, 229, 230, 231, and 232, respectively, or SEQ ID NOs: 233, 234, 235, 236, 237, and 238, respectively; (18) SEQ ID NOs: 241, 242, 243, 244, 245, and 246, respectively, or SEQ ID NOs: 247, 248, 249, 250, 251, and 252, respectively; (19) SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively, or SEQ ID NOs: 261, 262, 263, 264, 265, and 266, respectively; (20) SEQ ID NOs: 269, 270, 271, 272, 273, and 274, respectively, or SEQ ID NOs: 275, 276, 277, 278, 279, and 280, respectively; (21) SEQ ID NOs: 283, 284, 285, 286, 287, and 288, or SEQ ID NOs: 289, 290, 291, 292, 293, and 294, respectively; (22) SEQ ID NOs: 297, 298, 299, 300, 301, and 302, respectively, or SEQ ID NOs: 303, 304, 305, 306, 307, and 308, respectively; (23) SEQ ID NOs: 311, 312, 313, 314, 315, and 316, respectively, or SEQ ID NOs: 317, 318, 319, 320, 321, and 322, respectively; (24) SEQ ID NOs: 325, 326, 327, 328, 329, and 330, respectively, or SEQ ID NOs: 331, 332, 333, 334, 335, and 336, respectively; or (25) SEQ ID NOs: 341, 342, 343, 344, 345, and 346, respectively, or SEQ ID NOs: 347, 348, 349, 350, 351, and 352, respectively. Including, The antibody or antigen-binding fragment thereof, or antigen-binding domain thereof relates to an isolated monoclonal antibody or antigen-binding fragment thereof, or chimeric antigen receptor (CAR), which specifically binds to EGFRvIII, preferably human EGFRvIII.

[0109] According to another particular aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, or chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the monoclonal antibody or antigen-binding fragment thereof, or the antigen-binding domain is selected from the group consisting of SEQ ID NOs: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 169, 183, 197, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 410, 420, 430, 440, 450, 460, 470, 480, 490, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960 11, 225, 239, 253, 267, 281, 295, 309, 323, 339, 353, 354, 355, 356, 360, 361, 362, 363, 366, 367, 368, 369, 374, 375, 376, 377, 382, ​​383, 384, or 385, and at least 85%, preferably 90%, more preferably 95% or more, e.g., 95%, 96% , 97%, 98%, or 99% identical, or a heavy chain variable region having a polypeptide sequence identical to SEQ ID NOs: 2, 16, 30, 44, 58, 72, 86, 100, 114, 128, 142, 156, 170, 184, 198, 212, 226, 240, 254, 268, 282, 296, 310, 324, 340, 357, 358, 359, 364, 365, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445 73, 378, 379, 380, 381, 386, 387, or 388, or an antigen-binding fragment thereof, or a chimeric antigen receptor (CAR), comprising a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example, 95%, 96%, 97%, 98%, or 99% identical to one ofAccording to one preferred embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof or antigen-binding domain thereof of the present invention has at least 85%, preferably 90%, more preferably 95% or more identical identity to SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 169, 183, 197, 211, 225, 239, 253, 267, 281, 295, 309, 323, 339, 353, 354, 355, 356, 360, 361, 362, 363, 366, 367, 368, 369, 374, 375, 376, 377, 382, ​​383, 384, or 385, e.g., 95% or more identical identity to SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 169, 183, 197, 211, 225, 239, 253, 267, 281, 295, 309, 323, 339, 353, 354, 355, 356, 360, 361, 362, 363, 366, 367, 368, 369, 374, 375, 376, 377, 382, ​​383, 384, or 385. and a heavy chain variable region having a polypeptide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 2, 16, 30, 44, 58, 72, 86, 100, 114, 128, 142, 156, 170, 184, 198, 212, 226, 240, 254, 268, 282, 296, 310, 324, 340, 357, 358, 359, 364, 365, 370, 371, 372, 373, 378, 379, 380, 381, 386, 387, or 388.

[0110] In another particular aspect, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof or an antigen-binding domain thereof of the present invention, comprising: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 29 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 30; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 43 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 44; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 57 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 58; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 71 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 72; (7) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 85 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 86; (8) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 99 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 100; (9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 113 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 114; (10) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 127 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 128; (11) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 141 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 142; (12) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 156; (13) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 170; (14) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 184; (15) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 197 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 198; (16) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 211 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 212; (17) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 225 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 226. (18) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 239 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 240; (19) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 253 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 254. (20) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 267 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 268; (21) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 282; (22) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 295 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 296; (23) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 309 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 310; (24) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 323 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 324. (25) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 339 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 340; (26) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (27) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (28) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359; (29) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (30) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358; (31) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (32) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357; (33) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (34) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (35) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (36) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (37) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (38) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 360 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (39) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 360 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (40) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 361 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (41) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 361 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (42) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 362 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (43) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 362 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (44) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 363 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (45) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 363 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (46) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (47) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (48) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (49) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373. (50) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370; (51) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371; (52) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372; (53) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (54) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (55) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (56) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (57) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (58) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (59) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (60) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (61) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (62) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (63) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (64) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (65) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (66) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (67) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (68) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (69) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (70) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (71) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (72) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (73) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (74) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (75) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (76) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (77) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (78) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (79) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (80) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (81) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (82) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (83) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (84) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (85) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (86) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (87) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (88) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387; or (89) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388.

[0111] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 3, 4, 5, 6, 7, and 8, respectively, or SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1, and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2.

[0112] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively, or SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 15, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 16. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:15 and a light chain variable region having the polypeptide sequence of SEQ ID NO:16.

[0113] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 31, 32, 33, 34, 35, and 36, respectively, or SEQ ID NOs: 37, 38, 39, 40, 41, and 42, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 30. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:29 and a light chain variable region having the polypeptide sequence of SEQ ID NO:30.

[0114] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 45, 46, 47, 48, 49, and 50, respectively, or SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 43, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 44. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:43 and a light chain variable region having the polypeptide sequence of SEQ ID NO:44.

[0115] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 59, 60, 61, 62, 63, and 64, respectively, or SEQ ID NOs: 65, 66, 67, 68, 69, and 70, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 57, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 58. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:57 and a light chain variable region having the polypeptide sequence of SEQ ID NO:58.

[0116] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 73, 74, 75, 76, 77, and 78, respectively, or SEQ ID NOs: 79, 80, 81, 82, 83, and 84, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 71, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 72. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:71 and a light chain variable region having the polypeptide sequence of SEQ ID NO:72.

[0117] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 87, 88, 89, 90, 91, and 92, respectively, or SEQ ID NOs: 93, 94, 95, 96, 97, and 98, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 85, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 86. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:85 and a light chain variable region having the polypeptide sequence of SEQ ID NO:86.

[0118] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 101, 102, 103, 104, 105, and 106, respectively, or SEQ ID NOs: 107, 108, 109, 110, 111, and 112, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 99, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 100. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:99 and a light chain variable region having the polypeptide sequence of SEQ ID NO:100.

[0119] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 115, 116, 117, 118, 119, and 120, respectively, or SEQ ID NOs: 121, 122, 123, 124, 125, and 126, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 113, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 114. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:113 and a light chain variable region having the polypeptide sequence of SEQ ID NO:114.

[0120] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 129, 130, 131, 132, 133, and 134, respectively, or SEQ ID NOs: 135, 136, 137, 138, 139, and 140, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 127, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 128. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:127 and a light chain variable region having the polypeptide sequence of SEQ ID NO:128.

[0121] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 143, 144, 145, 146, 147, and 148, respectively, or SEQ ID NOs: 149, 150, 151, 152, 153, and 154, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 141, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 142. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:141 and a light chain variable region having the polypeptide sequence of SEQ ID NO:142.

[0122] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 157, 158, 159, 160, 161, and 162, respectively, or SEQ ID NOs: 163, 164, 165, 166, 167, and 168, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 155, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 156. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:155 and a light chain variable region having the polypeptide sequence of SEQ ID NO:156.

[0123] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 171, 172, 173, 174, 175, and 176, respectively, or SEQ ID NOs: 177, 178, 179, 180, 181, and 182, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 169, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 170. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:169 and a light chain variable region having the polypeptide sequence of SEQ ID NO:170.

[0124] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 185, 186, 187, 188, 189, and 190, respectively, or SEQ ID NOs: 191, 192, 193, 194, 195, and 196, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 183, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 184. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:183 and a light chain variable region having the polypeptide sequence of SEQ ID NO:184.

[0125] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 199, 200, 201, 202, 203, and 204, respectively, or SEQ ID NOs: 205, 206, 207, 208, 209, and 210, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 197, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 198. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:197 and a light chain variable region having the polypeptide sequence of SEQ ID NO:198.

[0126] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 213, 214, 215, 216, 217, and 218, respectively, or SEQ ID NOs: 219, 220, 221, 222, 223, and 224, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 211, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 212. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:211 and a light chain variable region having the polypeptide sequence of SEQ ID NO:212.

[0127] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 227, 228, 229, 230, 231, and 232, respectively, or SEQ ID NOs: 233, 234, 235, 236, 237, and 238, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 225, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 226. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:225 and a light chain variable region having the polypeptide sequence of SEQ ID NO:226.

[0128] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 241, 242, 243, 244, 245, and 246, respectively, or SEQ ID NOs: 247, 248, 249, 250, 251, and 252, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 239, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 240. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:239 and a light chain variable region having the polypeptide sequence of SEQ ID NO:240.

[0129] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively, or SEQ ID NOs: 261, 262, 263, 264, 265, and 266, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 253, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 254. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:253 and a light chain variable region having the polypeptide sequence of SEQ ID NO:254.

[0130] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 269, 270, 271, 272, 273, and 274, respectively, or SEQ ID NOs: 275, 276, 277, 278, 279, and 280, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 267, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 268. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:267 and a light chain variable region having the polypeptide sequence of SEQ ID NO:268.

[0131] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 283, 284, 285, 286, 287, and 288, respectively, or SEQ ID NOs: 289, 290, 291, 292, 293, and 294, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 281, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 282. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:281 and a light chain variable region having the polypeptide sequence of SEQ ID NO:282.

[0132] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 297, 298, 299, 300, 301, and 302, respectively, or SEQ ID NOs: 303, 304, 305, 306, 307, and 308, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 295, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 296. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:295 and a light chain variable region having the polypeptide sequence of SEQ ID NO:296.

[0133] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 311, 312, 313, 314, 315, and 316, respectively, or SEQ ID NOs: 317, 318, 319, 320, 321, and 322, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 309, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 310. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:309 and a light chain variable region having the polypeptide sequence of SEQ ID NO:310.

[0134] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 325, 326, 327, 328, 329, and 330, respectively, or SEQ ID NOs: 331, 332, 333, 334, 335, and 336, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 323, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 324. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:323 and a light chain variable region having the polypeptide sequence of SEQ ID NO:324.

[0135] In one embodiment, the invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 having the polypeptide sequences of SEQ ID NOs: 341, 342, 343, 344, 345, and 346, respectively, or SEQ ID NOs: 347, 348, 349, 350, 351, and 352, respectively. In another embodiment, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 339, and a light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or more, for example 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 340. Preferably, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence of SEQ ID NO:339 and a light chain variable region having the polypeptide sequence of SEQ ID NO:340.

[0136] In another particular aspect, the present invention relates to the isolated monoclonal antibody, or antigen-binding fragment or antigen-binding domain thereof, of the present invention, which is chimeric.

[0137] In another particular aspect, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment or domain thereof, which is human or humanized.

[0138] According to another particular embodiment, the antigen-binding domain is a single-chain variable fragment (scFv).

[0139] In certain embodiments, the encoded antigen-binding domain is a humanized single-chain variable fragment (scFv).

[0140] According to another particular embodiment, the chimeric antigen receptor comprises one or more antigen-binding domains.

[0141] According to another particular embodiment, the intracellular signaling domain comprises one or more costimulatory domains and one or more activation domains.

[0142] In another general aspect, the present invention relates to an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof, and / or a bispecific antibody or antigen-binding fragment thereof of the present invention. In another general aspect, the present invention relates to an isolated polynucleotide comprising a nucleic acid encoding a chimeric antigen receptor (CAR), wherein the CAR comprises the antigen-binding domain of the present invention. Those skilled in the art will understand that the coding sequence of a protein can be altered (e.g., substituted, deleted, inserted, etc.) without changing the amino acid sequence of the protein. Thus, those skilled in the art will understand that the nucleic acid sequence encoding the monoclonal antibody or antigen-binding fragment thereof of the present invention can be modified without changing the amino acid sequence of the protein.

[0143] In another general aspect, the present invention relates to a vector comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, and / or a CAR of the present invention. In light of the present disclosure, any vector known to those of skill in the art can be used, for example, a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector, for example, a plasmid. The vector can include any elements for establishing the conventional functions of an expression vector, such as a promoter, a ribosome-binding element, a terminator, an enhancer, a selection marker, and an origin of replication. The promoter can be a constitutive, inducible, or repressible promoter. Several expression vectors capable of delivering nucleic acids into cells are known in the art and can be used herein to produce antibodies or antigen-binding fragments thereof in cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to embodiments of the present invention.

[0144] In another general aspect, the present invention relates to a host cell comprising an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment thereof, and / or a bispecific antibody or antigen-binding fragment thereof of the present invention. In light of the present disclosure, any host cell known to those of skill in the art can be used for recombinant expression of an antibody or antigen-binding fragment thereof of the present invention. In some embodiments, the host cell is Escherichia coli (E. coli) TG1 or BL21 cell (e.g., for expression of scFv or Fab antibodies), CHO-DG44 or CHO-K1 cell, or HEK293 cell (e.g., for expression of full-length IgG antibodies). According to certain embodiments, the recombinant expression vector is transformed into the host cell by conventional methods, e.g., chemical transfection, heat shock, or electroporation, where it is stably integrated into the host cell genome for efficient expression of the recombinant nucleic acid.

[0145] In another general aspect, the invention relates to a method for producing a monoclonal antibody or antigen-binding fragment thereof and / or a bispecific antibody or antigen-binding fragment thereof of the invention, comprising culturing cells comprising nucleic acid encoding the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof under conditions for production of the monoclonal antibody or antigen-binding fragment thereof or the bispecific antibody or antigen-binding fragment thereof of the invention, and recovering the antibody or antigen-binding fragment thereof from the cells or cell culture (e.g., from the supernatant). The expressed antibody or antigen-binding fragment thereof can be harvested and purified from the cells according to conventional techniques known in the art and methods described herein.

[0146] In another general aspect, the invention relates to a cell transduced with a vector comprising an isolated nucleic acid encoding a CAR of the invention. The terms "transduced" or "transduction" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transduced" cell is a cell that has been transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny. In certain embodiments, the cell is a CAR-T cell, preferably a human CAR-T cell, where the T cell is engineered to express a CAR of the invention to treat a disease, e.g., cancer. In certain embodiments, the cell is a CAR-NK cell, preferably a human CAR-NK cell, where the NK cell engineered to express a CAR of the invention is used to treat a disease, e.g., cancer.

[0147] In another general aspect, the invention features a method of generating a CAR-T cell by transducing a T cell with a vector comprising an isolated nucleic acid encoding a CAR of the invention.

[0148] In another general aspect, the invention relates to a method of producing a chimeric antigen receptor (CAR) T cell of the invention, comprising culturing T cells comprising a nucleic acid encoding the CAR under CAR-T cell-producing conditions, and recovering the CAR-T cell.

[0149] In another general aspect, the invention features a method of generating CAR-NK cells by transducing NK cells with a vector comprising an isolated nucleic acid encoding a CAR of the invention.

[0150] In another general aspect, the invention features a method of producing a chimeric antigen receptor (CAR)-NK cell of the invention, comprising culturing NK cells comprising a nucleic acid encoding the CAR under CAR-NK cell-producing conditions, and recovering the CAR-NK cell.

[0151] In another general aspect, the invention relates to a method of generating a population of RNA engineered cells comprising a chimeric antigen receptor (CAR) of the invention. The method comprises contacting the population of cells with an isolated polynucleotide comprising a nucleic acid encoding a CAR of the invention, wherein the isolated polynucleotide is an in vitro transcribed RNA or a synthetic RNA.

[0152] Pharmaceutical Composition In another general aspect, the invention relates to a pharmaceutical composition comprising an isolated monoclonal antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, an isolated polynucleotide, an isolated polypeptide, a host cell, and / or an engineered immune cell of the invention, and a pharmaceutically acceptable carrier.

[0153] As used herein, the term "pharmaceutical composition" refers to a product comprising an isolated polynucleotide of the invention, an isolated polypeptide of the invention, a host cell of the invention, an engineered immune cell of the invention, an anti-EGFRvIII monoclonal antibody or antigen-binding fragment thereof, and / or a bispecific antibody of the invention, together with a pharmaceutically acceptable carrier. The polynucleotides, polypeptides, host cells, engineered immune cells, anti-EGFRvIII monoclonal antibodies or antigen-binding fragments thereof, and / or bispecific antibodies of the invention and compositions comprising them are also useful in the manufacture of medicaments for the therapeutic applications described herein.

[0154] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposome encapsulation, or other material known in the art for use in pharmaceutical formulations. It is understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application. As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic material that does not interfere with the efficacy or biological activity of the compositions according to the present invention. According to certain embodiments, any pharmaceutically acceptable carrier suitable for use in antibody pharmaceutical compositions in light of the present disclosure can be used in the present invention.

[0155] Formulation of pharmaceutically active ingredients with pharmaceutically acceptable carriers is known in the art, e.g., Remington: The Science and Practice of Pharmacy (e.g., 21st Edition (2005) and any later editions). Non-limiting examples of additional ingredients include: buffers, diluents, solvents, tonicity adjusters, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used in formulating the pharmaceutical compositions of the present invention.

[0156] In one embodiment of the present invention, the pharmaceutical composition is a liquid formulation. A preferred example of a liquid formulation is an aqueous formulation, i.e., a formulation containing water. Liquid formulations can include solutions, suspensions, emulsions, microemulsions, gels, etc. Aqueous formulations typically contain at least 50% w / w water, or at least 60%, 70%, 75%, 80%, 85%, 90%, or at least 95% w / w water.

[0157] In one embodiment, the pharmaceutical composition can be formulated as an injectable that can be injected, for example, via an injection device (e.g., a syringe or an infusion pump). The injection can be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, intravitreally, or intravenously.

[0158] In another embodiment, the pharmaceutical composition is a solid formulation, such as a freeze-dried or spray-dried composition that can be used as is, or a solid formulation to which the physician or patient can add solvents and / or diluents before use. Solid dosage forms can include tablets, e.g., compressed tablets, and / or coated tablets, and capsules (e.g., hard or soft gelatin capsules). Pharmaceutical compositions can also be in the form of, for example, sachets, dragees, powders, granules, lozenges, or powders for reconstitution.

[0159] Dosage forms may be immediate release, in which case they may include a water soluble or dispersible carrier, or they may be delayed, sustained, or modified release, in which case they may include a water insoluble polymer that modulates the dissolution rate of the dosage form in the gastrointestinal tract or under the skin.

[0160] In other embodiments, the pharmaceutical composition may be delivered intranasally, bucally, or sublingually.

[0161] The pH of the aqueous formulation can be between pH 3 and pH 10. In one embodiment of the invention, the pH of the formulation is from about 7.0 to about 9.5. In another embodiment of the invention, the pH of the formulation is from about 3.0 to about 7.0.

[0162] In another embodiment of the present invention, the pharmaceutical composition comprises a buffering agent. Non-limiting examples of buffering agents include arginine, aspartic acid, bicine, citrate, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinic acid, tartaric acid, tricine, and tris(hydroxymethyl)aminomethane, and mixtures thereof. The buffering agent can be present individually or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, e.g., about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific buffering agents constitute alternative embodiments of the present invention.

[0163] In another embodiment of the present invention, the pharmaceutical composition comprises a preservative. Non-limiting examples of preservatives include benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl 4-hydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl 4-hydroxybenzoate, imidurea, methyl 4-hydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl 4-hydroxybenzoate, sodium dehydroacetate, thiomerosal, and mixtures thereof. The preservatives can be present individually or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, e.g., about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific preservatives constitute alternative embodiments of the present invention.

[0164] In another embodiment of the present invention, the pharmaceutical composition comprises an isotonicity agent. Non-limiting examples of isotonicity agents include salts (e.g., sodium chloride), amino acids (e.g., glycine, histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, and threonine), alditols (e.g., glycerol, 1,2-propanediol, propylene glycol), 1,3-propanediol, and 1,3-butanediol), polyethylene glycols (e.g., PEG 400), and mixtures thereof. Another example of an isotonicity agent includes a sugar. Non-limiting examples of sugars may be monosaccharides, disaccharides, or polysaccharides, or water-soluble glucans, such as fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, alpha- and beta-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethylcellulose. Another example of an isotonicity agent is a sugar alcohol, where the term "sugar alcohol" is defined as a C(4-8) hydrocarbon having at least one -OH group. Non-limiting examples of sugar alcohols include mannitol, sorbitol, inositol, galactitol, dulcitol, xylitol, and arabitol. The isotonicity agent can be present individually or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, e.g., about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific isotonicity agents constitute alternative embodiments of the invention.

[0165] In another embodiment of the invention, the pharmaceutical composition comprises a chelating agent. Non-limiting examples of chelating agents include citric acid, aspartic acid, salts of ethylenediaminetetraacetic acid (EDTA), and mixtures thereof. The chelating agent can be present individually or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, e.g., about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific chelating agents constitute alternative embodiments of the invention.

[0166] In another embodiment of the invention, the pharmaceutical composition comprises a stabilizer, non-limiting examples of which include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and / or one or more protease inhibitors.

[0167] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer, which is carboxy- / hydroxycellulose and its derivatives (e.g., HPC, HPC-SL, HPC-L, and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (e.g., PEG3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (e.g., sodium chloride), sulfur-containing substances (e.g., monothioglycerol), or thioglycolic acid. The stabilizer can be present individually or in aggregates at a concentration of about 0.01 mg / ml to about 50 mg / ml, e.g., about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific stabilizers constitute alternative embodiments of the present invention.

[0168] In a further embodiment of the present invention, the pharmaceutical composition comprises one or more surfactants, preferably one surfactant, at least one surfactant, or two different surfactants. The term "surfactant" refers to any molecule or ion composed of a water-soluble (hydrophilic) portion and a fat-soluble (lipophilic) portion. The surfactant may be selected, for example, from the group consisting of anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants. The surfactant may be present individually or in aggregates at a concentration of about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific surfactants constitute alternative embodiments of the present invention.

[0169] In a further embodiment of the invention, the pharmaceutical composition comprises one or more protease inhibitors, such as EDTA and / or benzamidine hydrochloride (HCl). The protease inhibitors can be present individually or in aggregates at a concentration of about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions comprising each one of these specific protease inhibitors constitute alternative embodiments of the invention.

[0170] In another general aspect, the invention relates to a method for producing a pharmaceutical composition comprising a monoclonal antibody or antigen-binding fragment thereof, and / or a bispecific antibody or antigen-binding fragment thereof of the invention, the method comprising combining the monoclonal antibody or antigen-binding fragment thereof, and / or the bispecific antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0171] How to use In another general aspect, the present invention relates to a method for targeting EGFRvIII on the surface of cancer cells in a subject to achieve cell killing, the method comprising administering to the subject an isolated monoclonal antibody or antigen-binding fragment thereof, and / or a bispecific antibody or antigen-binding fragment thereof, that specifically binds to EGFRvIII, or a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof, and / or the bispecific antibody or antigen-binding fragment thereof of the present invention. Binding of the EGFRvIII monoclonal or bispecific antibody or antigen-binding fragment to EGFRvIII can mediate complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and / or antibody-dependent cellular cytotoxicity (ADCC), or other effects that result in the death of the targeted cancer cells. The monoclonal or bispecific antibody or antigen-binding fragment thereof can, for example, serve to recruit a conjugated drug and / or can form a bispecific antibody with another monoclonal antibody to mediate the death of the targeted cancer cells.

[0172] The functional activity of antibodies and antigen-binding fragments thereof that bind to EGFRvIII can be characterized by methods known in the art and by methods described herein. Methods for characterizing antibodies and antigen-binding fragments thereof that bind to EGFRvIII include, but are not limited to, affinity and specificity assays, including Biacore, ELISA, OctetRed, and GatorBio analysis, and FACS to detect binding of antibodies and antigen-binding fragments to EGFRvIII on cells (either cells transfected with EGFRvIII or cells that naturally express EGFRvIII). According to certain embodiments, methods for characterizing antibodies and antigen-binding fragments thereof that bind to EGFRvIII include those described below.

[0173] In another general aspect, the present invention relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject an isolated monoclonal antibody or antigen-binding fragment thereof, and / or bispecific antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, or a pharmaceutical composition of the invention. The cancer can be selected from, for example, but not limited to, lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma, and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0174] In another general aspect, the present invention relates to a method of treating an inflammatory disease and / or an autoimmune disease in a subject in need thereof, comprising administering to the subject an isolated monoclonal antibody or antigen-binding fragment thereof, and / or bispecific antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, or a pharmaceutical composition of the invention.

[0175] In another general aspect, the invention relates to a method of treating cancer in a subject in need thereof, comprising administering to the subject CAR-T cells and / or CAR-NK cells of the invention. The cancer can be selected from, for example, but not limited to, lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma, and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0176] According to embodiments of the present invention, the CAR-T cells or CAR-NK cells comprise a therapeutically effective amount of the expressed CAR of the present invention, and the pharmaceutical composition comprises a therapeutically effective amount of an anti-EGFRvIII antibody or an antigen-binding fragment thereof (e.g., an anti-EGFRvIII antibody). As used herein, the term "therapeutically effective amount" refers to the amount of active ingredients or components that elicits a desired biological or pharmaceutical response in a subject. A therapeutically effective amount can be determined empirically and routinely for the stated purpose.

[0177] As used herein with respect to a CAR, a therapeutically effective amount refers to the amount of a CAR molecule expressed in a transduced T cell or NK cell that modulates an immune response in a subject in need thereof. Further, as used herein with respect to a CAR, a therapeutically effective amount refers to the amount of a CAR molecule expressed in a transduced T cell or NK cell that provides treatment for a disease, disorder, or condition, prevents or slows the progression of the disease, disorder, or condition, or reduces or completely alleviates symptoms associated with the disease, disorder, or condition.

[0178] As used herein with respect to CAR-T cells or CAR-NK cells, a therapeutically effective amount refers to the amount of CAR-T cells or CAR-NK cells that modulates an immune response in a subject in need thereof. Further, as used herein with respect to CAR-T cells or CAR-NK cells, a therapeutically effective amount refers to the amount of CAR-T cells or CAR-NK cells that results in treatment of a disease, disorder, or condition, prevents or slows the progression of the disease, disorder, or condition, or reduces or completely alleviates symptoms associated with the disease, disorder, or condition.

[0179] As used herein with respect to an anti-EGFRvIII antibody or antigen-binding fragment thereof, a therapeutically effective amount refers to the amount of an anti-EGFRvIII antibody or antigen-binding fragment thereof that modulates an immune response in a subject in need thereof, and also refers to the amount of an anti-EGFRvIII antibody or antigen-binding fragment thereof that provides treatment for a disease, disorder, or condition, prevents or slows the progression of the disease, disorder, or condition, or reduces or completely alleviates symptoms associated with the disease, disorder, or condition.

[0180] According to certain embodiments, the disease, disorder, or condition to be treated is cancer, preferably a cancer selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, bile duct cancer, cholangiocarcinoma, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma, and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors. According to other certain embodiments, the disease, disorder, or condition to be treated is an inflammatory disease and / or an autoimmune disease.

[0181] According to certain embodiments, a therapeutically effective amount refers to the amount of therapy that is sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or ameliorating the severity of the disease, disorder, or condition being treated or the symptoms associated therewith; (ii) reducing the duration of the disease, disorder, or condition being treated or the symptoms associated therewith; (iii) preventing the progression of the disease, disorder, or condition being treated or the symptoms associated therewith; (iv) causing regression of the disease, disorder, or condition being treated or the symptoms associated therewith; or (v) preventing the development or onset of the disease, disorder, or condition being treated or the symptoms associated therewith. (vi) preventing the recurrence of the disease, disorder or condition to be treated or symptoms associated therewith; (vii) reducing hospitalization of a subject having the disease, disorder or condition to be treated or symptoms associated therewith; (viii) reducing the length of hospitalization of a subject having the disease, disorder or condition to be treated or symptoms associated therewith; (ix) increasing survival of a subject having the disease, disorder or condition to be treated or symptoms associated therewith; (xi) inhibiting or reducing the disease, disorder or condition to be treated or symptoms associated therewith in a subject; and / or (xii) enhancing or improving the prophylactic or therapeutic efficacy of another therapy.

[0182] The therapeutically effective amount or dosage can vary depending on a variety of factors, such as the disease, disorder, or condition to be treated, the means of administration, the target site, the physiological state of the subject (including, for example, age, weight, and health), whether the subject is human or animal, other pharmaceutical agents administered, and whether the treatment is prophylactic or therapeutic. Treatment dosages are optimally titrated to optimize safety and efficacy.

[0183] According to certain embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to a subject. For example, the compositions described herein can be formulated to be suitable for intravenous, subcutaneous, or intramuscular administration.

[0184] The cells of the present invention can be administered in any convenient manner known to those skilled in the art. For example, the cells of the present invention can be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation, and / or transplantation. Compositions comprising the cells of the present invention can be administered intraarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intrapleurally, by intravenous (iv) injection, or intraperitoneally. In certain embodiments, the cells of the present invention can be administered with or without lymphodepletion of the subject.

[0185] Pharmaceutical compositions comprising the cells of the invention expressing a CAR of the invention can be provided in sterile liquid formulations, generally isotonic aqueous solutions with cell suspensions, or optionally as emulsions, dispersions, etc., which are generally buffered to a selected pH. The compositions can include carriers suitable for cell integrity and viability and for administration of the cell composition, such as water, saline, phosphate-buffered saline, etc.

[0186] Sterile injectable solutions can be prepared by incorporating the cells of the present invention in a suitable solvent in a suitable amount, along with various other ingredients, as desired. Such compositions can contain pharmaceutically acceptable carriers, diluents, or excipients, such as sterile water, physiological saline, glucose, dextrose, etc., that are suitable for use in cell compositions and for administration to subjects, e.g., humans. Buffers suitable for providing cell compositions are well known in the art. Any vehicle, diluent, or additive used will be compatible with preserving the integrity and viability of the cells of the present invention.

[0187] The cells of the present invention can be administered in any physiologically acceptable vehicle. A population of cells containing the cells of the present invention can include a purified cell population. Those skilled in the art can easily determine the purity of cells in a population of cells using various well-known methods. The purity range of a population of cells containing genetically modified cells of the present invention can be about 50% to about 55%, about 55% to about 60%, about 60% to about 65%, about 65% to about 70%, about 70% to about 75%, about 75% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 100%. The dosage can be easily adjusted by those skilled in the art; for example, a decrease in purity may require an increase in dosage.

[0188] The cells of the invention are generally administered as a dose based on the number of cells per kilogram of body weight (cells / kg) of the subject to whom the cells are administered. Generally, cell doses range from about 10 cells per kg of body weight depending on the mode and location of administration. 4 ~about 10 10 Cell numbers, e.g., about 10 5 ~about 10 9 , about 10 5 ~about 10 8 , about 10 5 ~about 10 7 or about 10 5 ~about 10 6 Generally, for systemic administration, higher doses are used than for local administration, where the immune cells of the invention are administered within the region of the tumor and / or cancer. Exemplary dose ranges include, but are not limited to, 1 x 10 4 ~1×10 8 , 2 × 10 4 ~1×10 8 , 3×10 4 ~1×10 8 , 4×10 4 ~1×10 8 , 5×10 4 ~6×10 8 , 7×10 4 ~1×10 8 , 8×10 4 ~1×10 8 , 9×10 4 ~1×10 8 , 1×105 ~1×10 8 、1×10 5 ~9×10 7 、1×10 5 ~8×10 7 、1×10 5 ~7×10 7 、1×10 5 ~6×10 7 、1×10 5 ~5×10 7 、1×10 5 ~4×10 7 、1×10 5 ~4×10 7 、1×10 5 ~3×10 7 、1×10 5 ~2×10 7 、1×10 5 ~1×10 7 、1×10 5 ~9×10 6 、1×10 5 ~8×10 6 、1×10 5 ~7×10 6 、1×10 5 ~6×10 6 、1×10 5 ~5×10 6 、1×10 5 ~4×10 6 、1×10 5 ~4×10 6 、1×10 5 ~3×10 6 、1×10 5 ~2×10 6 、1×10 5 ~1×10 6 、2×10 5 ~9×10 7 、2×10 5 ~8×10 7 、2×10 5 ~7×10 7 、2×10 5 ~6×10 7 、2×10 5 ~5×10 7 、2×10 5 ~4×10 7 、2×10 5 ~4×10 7, 2 × 10 5 ~3×10 7 , 2 × 10 5 ~2×10 7 , 2 × 10 5 ~1×10 7 , 2 × 10 5 ~9×10 6 , 2 × 10 5 ~8×10 6 , 2 × 10 5 ~7×10 6 , 2 × 10 5 ~6×10 6 , 2 × 10 5 ~5×10 6 , 2 × 10 5 ~4×10 6 , 2 × 10 5 ~4×10 6 , 2 × 10 5 ~3×10 6 , 2 × 10 5 ~2×10 6 , 2 × 10 5 ~1×10 6 , 3×10 5 ~3×10 6 The dosage may include the number of cells / kg, etc. Additionally, the dosage may be adjusted to account for whether a single dose or multiple doses are administered. The precise determination of what is considered an effective dose may be based on individual factors for each subject.

[0189] As used herein, the terms "treat," "treating," and "treatment" all refer to an improvement or reversal of at least one measurable physical parameter associated with cancer and / or an inflammatory or autoimmune disease, disorder, or condition, which may be, but is not necessarily, discernible in the subject. The terms "treat," "treating," and "treatment" can also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In a particular embodiment, "treat," "treating," and "treatment" refer to alleviating, preventing the development or onset, or reducing the duration of one or more symptoms associated with a disease, disorder, or condition, such as a tumor, more preferably a cancer. In a particular embodiment, "treat," "treating," and "treatment" refer to preventing the recurrence of a disease, disorder, or condition. In a particular embodiment, "treat," "treating," and "treatment" refer to increasing the survival of a subject having a disease, disorder, or condition. In a particular embodiment, "treat," "treating," and "treatment" refer to eliminating a disease, disorder, or condition in a subject.

[0190] According to certain embodiments, compositions are provided for use in the treatment of cancer and / or inflammatory or autoimmune diseases, disorders, or conditions. For cancer therapy, the provided compositions can be used in combination with another treatment, including, but not limited to, chemotherapy, anti-CD20 mAb, anti-TIM-3 mAb, anti-LAG-3 mAb, anti-EGFR mAb, anti-HER-2 mAb, anti-CD19 mAb, anti-CD33 mAb, anti-CD47 mAb, anti-CD73 mAb, anti-DLL-3 mAb, anti-Apelin mAb, anti-FOLR1 mAb, anti-CTLA-4 mAb, anti-PD-L1 mAb, anti-PD-1 mAb, anti-Claudin18.2 mAb, other immuno-oncology agents, anti-angiogenic agents, radiation therapy, antibody-drug conjugates (ADCs), targeted therapies, or other anti-cancer agents. Antibodies against EGFRvIII can be used to construct bispecific antibodies with partner mAbs against PD-1, PD-L1, LAG3, TIM-3, CTLA-4, EGFR, HER-2, CD19, CD20, CD33, CD73, CD47, CD3, apelin, DLL-3, TIP-1, GPC3, Claudin 18.2, folate receptor alpha (FOLR1), MUC16, mesothelin, PSCA, IL13Ra2, p95HER2, and / or any other tumor-associated antigen (TAA) to treat cancers / tumors expressing both antigens. Two antibodies recognizing two different epitopes on EGFRvIII can also be used to construct bispecific antibodies to treat cancers / tumors expressing EGFRvIII.

[0191] According to certain embodiments, a method of treating cancer in a subject in need thereof comprises administering to the subject the CAR-T cells and / or CAR-NK cells of the invention in combination with an agent that increases the efficacy of cells expressing a CAR molecule, including, but not limited to, an antibody fragment that binds to CD73, CD39, PD1, PD-L1, PD-L2, CTLA4, TIM3, or LAG3, or an adenosine A2a receptor antagonist.

[0192] According to certain embodiments, a method of treating cancer in a subject in need thereof comprises administering to the subject the CAR-T cells and / or CAR-NK cells of the invention in combination with an agent that ameliorates one or more side effects associated with the administration of cells expressing a CAR molecule. Such agents include, but are not limited to, steroids, inhibitors of TNFα, or inhibitors of IL-6.

[0193] According to certain embodiments, a method of treating cancer in a subject in need thereof comprises administering to the subject the CAR-T cells and / or CAR-NK cells of the invention in combination with an agent that treats an EGFRvIII-associated disease, including, but not limited to, an anti-EGFRvIII monoclonal antibody or bispecific antibody.

[0194] As used herein, in the context of administering two or more therapies to a subject, the term "in combination" refers to the use of more than one therapy.The use of the term "in combination" does not limit the order in which therapies are administered to a subject.For example, a first therapy (such as a composition described herein) can be administered before (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks before), simultaneously with, or after (for example, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks or 12 weeks after) the administration of a second therapy to a subject.

[0195] In another general aspect, the invention relates to a method for determining the level of EGFRvIII in a subject, the method comprising: (a) obtaining a sample from the subject; (b) contacting the sample with a monoclonal antibody or antigen-binding fragment thereof of the invention; and (c) determining the level of EGFRvIII in the subject.

[0196] As used herein, "sample" refers to a biological sample isolated from a subject, and can include, but is not limited to, whole blood, serum, plasma, blood cells, endothelial cells, tissue biopsy (e.g., cancer tissue), lymph, ascites, interstitial fluid, bone marrow, cerebrospinal fluid, saliva, mucus, sputum, sweat, urine, or any other secretion, excretion, or other bodily fluid. A "blood sample" refers to whole blood or any fraction thereof, including blood cells, serum, and plasma.

[0197] In certain embodiments, the level of EGFRvIII in a subject can be determined using an assay selected from, but not limited to, Western blot assay, immunohistochemistry (IHC), and ELISA assay. Relative protein levels can be determined using Western blot analysis and IHC, and absolute protein levels can be determined using ELISA assay. When determining the relative level of EGFRvIII, the level of EGFRvIII can be determined between at least two samples, for example, between samples from the same subject at different time points, between samples from different tissues within the same subject, and / or between samples from different subjects. Alternatively, when determining the absolute level of EGFRvIII, for example, by ELISA assay, the absolute level of EGFRvIII in a sample can be determined by producing a standard for the ELISA assay before testing the sample. Those skilled in the art will understand the analytical techniques to be used to determine the level of EGFRvIII in a sample from a subject using the antibody or antigen-binding fragment thereof of the present invention.

[0198] Utilizing a method for determining the level of EGFRvIII in a sample from a subject can lead to the diagnosis of abnormal (elevated, decreased, or insufficient) EGFRvIII levels in diseases, allowing appropriate treatment decisions to be made. Such diseases can be selected from, but are not limited to, cancer, inflammatory diseases, and autoimmune diseases. Furthermore, by monitoring the level of EGFRvIII in a subject, the risk of developing such diseases can be determined based on knowledge of the level of EGFRvIII in a particular disease and / or during the progression of a particular disease.

[0199] Embodiment The present invention also provides the following non-limiting embodiments.

[0200] Embodiment 1 is an isolated monoclonal antibody or antigen-binding fragment thereof, comprising a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, and a light chain variable region comprising light chain complementarity determining region 1 (LCDR1), LCDR2, LCDR3, having the following polypeptide sequences: (1) SEQ ID NOs: 3, 4, 5, 6, 7, and 8, respectively, or SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively; (2) SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively, or SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively; (3) SEQ ID NOs: 31, 32, 33, 34, 35, and 36, respectively, or SEQ ID NOs: 37, 38, 39, 40, 41, and 42, respectively; (4) SEQ ID NOs: 45, 46, 47, 48, 49, and 50, respectively, or SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively; (5) SEQ ID NOs: 59, 60, 61, 62, 63, and 64, respectively, or SEQ ID NOs: 65, 66, 67, 68, 69, and 70, respectively; (6) SEQ ID NOs: 73, 74, 75, 76, 77, and 78, or SEQ ID NOs: 79, 80, 81, 82, 83, and 84, respectively; (7) SEQ ID NOs: 87, 88, 89, 90, 91, and 92, respectively, or SEQ ID NOs: 93, 94, 95, 96, 97, and 98, respectively; (8) SEQ ID NOs: 101, 102, 103, 104, 105, and 106, respectively, or SEQ ID NOs: 107, 108, 109, 110, 111, and 112, respectively; (9) SEQ ID NOs: 115, 116, 117, 118, 119, and 120, respectively, or SEQ ID NOs: 121, 122, 123, 124, 125, and 126, respectively; (10) SEQ ID NOs: 129, 130, 131, 132, 133, and 134, respectively, or SEQ ID NOs: 135, 136, 137, 138, 139, and 140, respectively; (11) SEQ ID NOs: 143, 144, 145, 146, 147, and 148, respectively, or SEQ ID NOs: 149, 150, 151, 152, 153, and 154, respectively; (12) SEQ ID NOs: 157, 158, 159, 160, 161, and 162, respectively, or SEQ ID NOs: 163, 164, 165, 166, 167, and 168, respectively; (13) SEQ ID NOs: 171, 172, 173, 174, 175, and 176, respectively, or SEQ ID NOs: 177, 178, 179, 180, 181, and 182, respectively; (14) SEQ ID NOs: 185, 186, 187, 188, 189, and 190, respectively, or SEQ ID NOs: 191, 192, 193, 194, 195, and 196, respectively; (15) SEQ ID NOs: 199, 200, 201, 202, 203, and 204, respectively, or SEQ ID NOs: 205, 206, 207, 208, 209, and 210, respectively; (16) SEQ ID NOs: 213, 214, 215, 216, 217, and 218, respectively, or SEQ ID NOs: 219, 220, 221, 222, 223, and 224, respectively; (17) SEQ ID NOs: 227, 228, 229, 230, 231, and 232, respectively, or SEQ ID NOs: 233, 234, 235, 236, 237, and 238, respectively; (18) SEQ ID NOs: 241, 242, 243, 244, 245, and 246, respectively, or SEQ ID NOs: 247, 248, 249, 250, 251, and 252, respectively; (19) SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively, or SEQ ID NOs: 261, 262, 263, 264, 265, and 266, respectively; (20) SEQ ID NOs: 269, 270, 271, 272, 273, and 274, respectively, or SEQ ID NOs: 275, 276, 277, 278, 279, and 280, respectively; (21) SEQ ID NOs: 283, 284, 285, 286, 287, and 288, or SEQ ID NOs: 289, 290, 291, 292, 293, and 294, respectively; (22) SEQ ID NOs: 297, 298, 299, 300, 301, and 302, respectively, or SEQ ID NOs: 303, 304, 305, 306, 307, and 308, respectively; (23) SEQ ID NOs: 311, 312, 313, 314, 315, and 316, respectively, or SEQ ID NOs: 317, 318, 319, 320, 321, and 322, respectively; (24) SEQ ID NOs: 325, 326, 327, 328, 329, and 330, respectively, or SEQ ID NOs: 331, 332, 333, 334, 335, and 336, respectively; or (25) SEQ ID NOs: 341, 342, 343, 344, 345, and 346, respectively, or SEQ ID NOs: 347, 348, 349, 350, 351, and 352, respectively. wherein the antibody or antigen-binding fragment thereof is an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, preferably human EGFRvIII.

[0201] Embodiment 2 is directed to a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 169, 183, 197, 211, 225, 239, 253, 267, 281, 295, 309, 323, 339, 353, 354, 355, 356, 360, 361, 362, 363, 366, 367, 368, 369, 374, 375, 376, 377, 382, ​​383, 384, or 385, or a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 2, 1 6, 30, 44, 58, 72, 86, 100, 114, 128, 142, 156, 170, 184, 198, 212, 226, 240, 254, 268, 282, 296, 310, 324, 340, 357, 358, 359, 364, 365, 370, 371, 372, 373, 378, 379, 380, 381, 386, 387, or 388.

[0202] Embodiment 3 is the isolated monoclonal antibody or antigen-binding fragment thereof of embodiment 1 or 2, comprising: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 29 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 30; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 43 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 44; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 57 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 58; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 71 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 72; (7) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 85 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 86; (8) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 99 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 100; (9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 113 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 114; (10) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 127 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 128; (11) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 141 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 142; (12) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 156; (13) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 170; (14) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 184; (15) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 197 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 198; (16) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 211 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 212; (17) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 225 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 226. (18) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 239 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 240; (19) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 253 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 254. (20) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 267 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 268; (21) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 282; (22) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 295 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 296; (23) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 309 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 310; (24) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 323 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 324. (25) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 339 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 340; (26) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (27) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (28) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359; (29) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (30) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358; (31) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (32) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357; (33) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (34) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (35) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (36) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (37) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (38) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 360 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (39) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 360 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (40) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 361 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (41) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 361 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (42) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 362 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (43) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 362 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (44) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 363 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (45) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 363 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (46) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (47) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (48) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (49) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373. (50) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370; (51) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371; (52) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372; (53) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (54) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (55) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (56) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (57) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373. (58) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (59) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (60) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (61) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (62) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (63) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (64) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (65) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (66) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (67) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (68) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (69) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (70) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (71) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (72) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (73) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (74) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (75) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (76) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (77) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (78) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (79) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (80) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (81) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (82) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (83) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (84) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (85) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (86) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (87) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (88) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387; or (89) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388.

[0203] Embodiment 4 is the isolated monoclonal antibody or antigen-binding fragment of any one of Embodiments 1 to 3, which is chimeric and / or human or humanized.

[0204] Embodiment 5 is an isolated monoclonal antibody or antigen-binding fragment of any one of embodiments 1 to 4, which is capable of inducing effector-mediated tumor cell lysis via antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC), and / or mediating the recruitment of a conjugated drug, and / or forming a bispecific antibody with another monoclonal antibody (mAb) or antigen-binding fragment thereof that has cancericidal activity.

[0205] Embodiment 6 is an isolated bispecific antibody or antigen-binding fragment thereof comprising the monoclonal antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 5.

[0206] Embodiment 7 is an isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment of any one of Embodiments 1 to 5, or the bispecific antibody or antigen-binding fragment of Embodiment 6.

[0207] Embodiment 8 is a vector comprising the isolated nucleic acid of embodiment 7.

[0208] Embodiment 9 is a host cell comprising the vector of embodiment 8.

[0209] Embodiment 10 is a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment of any one of Embodiments 1 to 5, or the bispecific antibody or antigen-binding fragment thereof of Embodiment 6, and a pharmaceutically acceptable carrier.

[0210] Embodiment 11 is a method of targeting EGFRvIII on the surface of cancer cells and / or treating cancer, treating an inflammatory disease, and / or treating an autoimmune disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of Embodiment 10, optionally wherein the cancer is selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma, and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0211] Embodiment 12 is a method for producing the monoclonal antibody or antigen-binding fragment of any one of Embodiments 1 to 5, or the bispecific antibody or antigen-binding fragment thereof of Embodiment 6, comprising culturing cells containing nucleic acids encoding the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof, under conditions for production of the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof, and recovering the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof from the cells or culture.

[0212] Embodiment 13 is a method for producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 5, or the bispecific antibody or antigen-binding fragment thereof of Embodiment 6, comprising combining the monoclonal antibody or antigen-binding fragment thereof, or the bispecific antibody or antigen-binding fragment thereof with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0213] Embodiment 14 is a method for determining the level of EGFRvIII in a subject, comprising: (a) obtaining a sample from a subject; (b) contacting the sample with the isolated monoclonal antibody or antigen-binding fragment thereof of any one of embodiments 1 to 5; and (c) determining the level of EGFRvIII in the subject; The method includes:

[0214] Embodiment 15 is the method of embodiment 14, wherein the sample is a tissue sample or a blood sample, and optionally the tissue sample is a cancer tissue sample.

[0215] Embodiment 16 is an isolated polynucleotide comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) an extracellular domain comprising at least one antigen-binding domain that specifically binds to EGFRvIII, preferably human EGFRvIII; (b) hinge region; (c) a transmembrane domain, and (d) Intracellular signaling domain It is an isolated polynucleotide comprising:

[0216] Embodiment 17 is the isolated polynucleotide of embodiment 16, wherein the antigen-binding domain comprises a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, and a light chain variable region comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having the following polypeptide sequences: (1) SEQ ID NOs: 3, 4, 5, 6, 7, and 8, respectively, or SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively; (2) SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively, or SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively; (3) SEQ ID NOs: 31, 32, 33, 34, 35, and 36, respectively, or SEQ ID NOs: 37, 38, 39, 40, 41, and 42, respectively; (4) SEQ ID NOs: 45, 46, 47, 48, 49, and 50, respectively, or SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively; (5) SEQ ID NOs: 59, 60, 61, 62, 63, and 64, respectively, or SEQ ID NOs: 65, 66, 67, 68, 69, and 70, respectively; (6) SEQ ID NOs: 73, 74, 75, 76, 77, and 78, or SEQ ID NOs: 79, 80, 81, 82, 83, and 84, respectively; (7) SEQ ID NOs: 87, 88, 89, 90, 91, and 92, respectively, or SEQ ID NOs: 93, 94, 95, 96, 97, and 98, respectively; (8) SEQ ID NOs: 101, 102, 103, 104, 105, and 106, respectively, or SEQ ID NOs: 107, 108, 109, 110, 111, and 112, respectively; (9) SEQ ID NOs: 115, 116, 117, 118, 119, and 120, respectively, or SEQ ID NOs: 121, 122, 123, 124, 125, and 126, respectively; (10) SEQ ID NOs: 129, 130, 131, 132, 133, and 134, respectively, or SEQ ID NOs: 135, 136, 137, 138, 139, and 140, respectively; (11) SEQ ID NOs: 143, 144, 145, 146, 147, and 148, respectively, or SEQ ID NOs: 149, 150, 151, 152, 153, and 154, respectively; (12) SEQ ID NOs: 157, 158, 159, 160, 161, and 162, respectively, or SEQ ID NOs: 163, 164, 165, 166, 167, and 168, respectively; (13) SEQ ID NOs: 171, 172, 173, 174, 175, and 176, respectively, or SEQ ID NOs: 177, 178, 179, 180, 181, and 182, respectively; (14) SEQ ID NOs: 185, 186, 187, 188, 189, and 190, respectively, or SEQ ID NOs: 191, 192, 193, 194, 195, and 196, respectively; (15) SEQ ID NOs: 199, 200, 201, 202, 203, and 204, respectively, or SEQ ID NOs: 205, 206, 207, 208, 209, and 210, respectively; (16) SEQ ID NOs: 213, 214, 215, 216, 217, and 218, respectively, or SEQ ID NOs: 219, 220, 221, 222, 223, and 224, respectively; (17) SEQ ID NOs: 227, 228, 229, 230, 231, and 232, respectively, or SEQ ID NOs: 233, 234, 235, 236, 237, and 238, respectively; (18) SEQ ID NOs: 241, 242, 243, 244, 245, and 246, respectively, or SEQ ID NOs: 247, 248, 249, 250, 251, and 252, respectively; (19) SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively, or SEQ ID NOs: 261, 262, 263, 264, 265, and 266, respectively; (20) SEQ ID NOs: 269, 270, 271, 272, 273, and 274, respectively, or SEQ ID NOs: 275, 276, 277, 278, 279, and 280, respectively; (21) SEQ ID NOs: 283, 284, 285, 286, 287, and 288, or SEQ ID NOs: 289, 290, 291, 292, 293, and 294, respectively; (22) SEQ ID NOs: 297, 298, 299, 300, 301, and 302, respectively, or SEQ ID NOs: 303, 304, 305, 306, 307, and 308, respectively; (23) SEQ ID NOs: 311, 312, 313, 314, 315, and 316, respectively, or SEQ ID NOs: 317, 318, 319, 320, 321, and 322, respectively; (24) SEQ ID NOs: 325, 326, 327, 328, 329, and 330, respectively, or SEQ ID NOs: 331, 332, 333, 334, 335, and 336, respectively; or (25) SEQ ID NOs: 341, 342, 343, 344, 345, and 346, respectively, or SEQ ID NOs: 347, 348, 349, 350, 351, and 352, respectively.

[0217] Embodiment 18 is a method for producing a human immunodeficiency virus (HIV)-associated HIV-1 antibody comprising a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 169, 183, 197, 211, 225, 239, 253, 267, 281, 295, 309, 323, 339, 353, 354, 355, 356, 360, 361, 362, 363, 366, 367, 368, 369, 374, 375, 376, 377, 382, ​​383, 384, or 385. 18. The isolated polynucleotide of embodiment 16 or 17, comprising a light chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 2, 16, 30, 44, 58, 72, 86, 100, 114, 128, 142, 156, 170, 184, 198, 212, 226, 240, 254, 268, 282, 296, 310, 324, 340, 357, 358, 359, 364, 365, 370, 371, 372, 373, 378, 379, 380, 381, 386, 387, or 388.

[0218] Embodiment 19 is the isolated polynucleotide of embodiment 18, wherein the antigen-binding domain comprises: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 29 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 30; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 43 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 44; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 57 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 58; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 71 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 72; (7) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 85 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 86; (8) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 99 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 100; (9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 113 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 114; (10) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 127 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 128; (11) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 141 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 142; (12) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 156; (13) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 170; (14) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 184; (15) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 197 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 198; (16) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 211 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 212; (17) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 225 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 226. (18) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 239 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 240; (19) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 253 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 254. (20) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 267 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 268; (21) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 282; (22) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 295 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 296; (23) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 309 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 310; (24) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 323 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 324. (25) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 339 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 340; (26) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (27) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (28) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359; (29) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (30) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358; (31) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (32) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357; (33) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (34) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (35) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357. (36) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358. (37) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359. (38) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 360 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (39) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 360 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (40) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 361 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (41) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 361 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (42) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 362 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (43) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 362 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (44) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 363 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 364. (45) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 363 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 365. (46) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (47) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (48) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (49) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373. (50) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370; (51) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371; (52) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372; (53) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (54) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (55) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (56) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (57) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (58) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370. (59) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371. (60) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372. (61) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (62) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (63) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (64) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (65) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (66) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (67) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (68) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (69) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (70) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (71) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (72) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (73) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (74) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 378. (75) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 379. (76) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 380. (77) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 381. (78) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (79) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (80) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (81) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (82) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (83) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (84) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (85) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387. (86) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388. (87) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 386. (88) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387; or (89) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 388.

[0219] Embodiment 20 is the isolated polynucleotide of any one of embodiments 16 to 19, wherein the antigen-binding domain is a single-chain variable fragment (scFv), and optionally the scFv is humanized.

[0220] Embodiment 21 is the isolated polynucleotide of any one of Embodiments 16 to 20, wherein the chimeric antigen receptor (CAR) comprises one or more antigen-binding domains, and / or the intracellular signaling domain comprises one or more costimulatory domains and one or more activation domains.

[0221] Embodiment 22 is a chimeric antigen receptor (CAR) encoded by the isolated polynucleotide of any one of Embodiments 16 to 21.

[0222] Embodiment 23 is a vector comprising the isolated polynucleotide of any one of embodiments 16 to 21.

[0223] Embodiment 24 is a host cell comprising the vector of embodiment 23, which is optionally a T cell or an NK cell, preferably a human T cell or a human NK cell.

[0224] Embodiment 25 is a method of producing a host cell expressing a chimeric antigen receptor (CAR), comprising transducing a T cell or an NK cell with the vector of embodiment 23.

[0225] Embodiment 26 is a method of producing chimeric antigen receptor (CAR)-T cells or chimeric antigen receptor (CAR)-NK cells, comprising culturing T cells or NK cells comprising an isolated polynucleotide comprising a nucleic acid encoding the chimeric antigen receptor (CAR) of any one of Embodiments 16 to 21 under CAR-T cell or CAR-NK cell-producing conditions, and recovering the CAR-T cells or CAR-NK cells.

[0226] Embodiment 27 is a method of generating a cell comprising a chimeric antigen receptor (CAR), comprising contacting a cell with an isolated polynucleotide comprising a nucleic acid encoding the chimeric antigen receptor (CAR) of any one of embodiments 16 to 21, wherein the isolated polynucleotide is an in vitro transcribed RNA or a synthetic RNA.

[0227] Embodiment 28 is a method of treating cancer in a subject in need thereof, comprising administering to a subject in need thereof the host cell of Embodiment 24, optionally wherein the cancer is selected from lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma, and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0228] Embodiment 29 is the method of embodiment 28, further comprising administering to a subject in need thereof an agent that increases the efficacy of the CAR-expressing cells, or an agent that ameliorates one or more side effects associated with administration of the CAR-expressing cells, or an agent that treats an EGFRvIII-associated disease. [Example]

[0229] Example 1: Identification of anti-EGFRvIII monoclonal antibodies Rabbits were immunized with antigen (a mixture of keyhole limpet hemocyanin (KLH)-CLEEKKGNYVVTDHGS (SEQ ID NO: 337) and LEEKKGNYVVTDHGSC-KLH (SEQ ID NO: 338)). B cell clones from the immunized rabbits were cultured and the supernatants were screened by ELISA. IgG mRNA cDNA from positive B cell clones was sequenced.

[0230] The sequences of the heavy and light chain variable regions (VH and VL regions, respectively) of anti-EGFRvIII monoclonal antibodies are provided in Tables 1 and 2, and the CDR regions of anti-EGFRvIII monoclonal antibodies are provided in Tables 3-6.

[0231] [Table 1-2] TIFF2025535476000003.tif108150

[0232] [Table 2] TIFF2025535476000005.tif110152

[0233] [Table 3] TIFF2025535476000007.tif28154

[0234] [Table 4]

[0235] [Table 5] TIFF2025535476000010.tif83154

[0236] [Table 6]

[0237] Example 2: Production and purification of mAb from the culture medium of transfected cells To obtain the recombinant anti-EGFRvIII chimeric mAb, an expression vector containing rabbit variable regions (VH and VL) fused to the constant regions of human IgG1 heavy and kappa light chains, respectively, was transiently transfected into ExpiCHO-S cells. The recombinant antibody produced in suspension in ExpiCHO-S cells was purified using Protein A affinity chromatography.

[0238] Example 3 ELISA binding analysis of purified chimeric mAb to EGFRvIII The purified chimeric mAb was tested for its ability to bind to immobilized EGFRvIII in an ELISA assay. Recombinant human EGFRvIII (ACROBiosystems, CAT#: EGI-H52H4) in DPBS buffer was coated onto a 96-well plate for 1 hour at room temperature. The plate was blocked with 5% BSA in TBST for 1 hour at room temperature. Various concentrations of mAb were incubated in each well of each plate for 1 hour at room temperature. The plate was washed, and binding to EGFRvIII was detected by incubating with mouse anti-human IgG Fc antibody (50B4A9) [HRP], mAb (Genescript, CAT#: A01854) for 1 hour at room temperature. After washing, the ELISA was then developed using One-Step Detection Solution (ThermoFisher Scientific, CAT#: 34028), and absorbance at 450 nm was measured. The results are shown in Figures 1A-1G.

[0239] Example 4: FACS binding analysis of purified mAb to cell surface EGFRvIII Approximately 50,000 ExpiCHO-S cells expressing the EGFRvIII antigen were transferred to a 96-well plate and incubated with various concentrations of purified chimeric anti-EGFRvIII mAb (rabbit mAb variable regions fused to human IgG1 heavy and kappa light chain constant regions, respectively) for 15 min at 4°C. The cells were then centrifuged for 5 min and washed three times with FACS buffer (HBSS supplemented with 0.1% BSA and 0.05% sodium azide). The cells were then incubated with PE / Cyanine 7 anti-human IgG Fc recombinant antibody (clone QA19A42) (Biolegend, CAT#: 366908) and incubated on ice for an additional 15 min. The cells were then washed twice in FACS buffer and resuspended in FACS buffer. The cells were then run on an Attune NxT, and the data were analyzed using Attune NxT software. The results are shown in Figures 2A-2G.

[0240] Example 5 ELISA binding analysis of purified mAbs to wild-type EGFR To test the binding activity of the mAb to wild-type EGFR, the purified chimeric mAb was tested for its ability to bind to immobilized wild-type EGFR in an ELISA assay. Recombinant human EGFR (ACROBiosystems, CAT#: EGR-H5222) in DPBS buffer was coated onto a 96-well plate for 1 hour at room temperature. The plate was blocked with 5% BSA in TBST for 1 hour at room temperature. Solutions containing various concentrations of mAb were added to each well of the plate, and the plate was incubated for 1 hour at room temperature. After washing the plate, mouse anti-human IgG Fc antibody (50B4A9) [HRP] (Genescript, CAT#: A01854) was added to each well, and the plate was incubated for 1 hour at room temperature. After washing the plate, the ELISA was developed using One-Step Detection Solution (ThermoFisher Scientific, CAT#: 34028), and the absorbance was measured at 450 nm. As a positive control, the anti-EGFR antibody cetuximab (Selleckchem, catalog number: A2000) was added. The results are shown in Figures 3A to 3D.

[0241] Example 6: Construction of a chimeric antigen receptor construct containing an anti-EGFRvIII antigen-binding domain To construct the CAR construct, the mAb is converted to an scFv using a VH, VL, and (G4S)n linker, and the scFv is fused to the N-terminus of the hinge and transmembrane domains derived from human CD8a (aa 114-118, Boursier JP et al., J. Biol. Chem. 268(3):2013-20 (1993)). The C-terminal intracellular signaling domain of the CAR is constructed by fusing the intracellular costimulatory domain of CD28 (aa 162-202, Aruffo A and Seed B, Proc. Natl. Acad. Sci. USA 84(23):8573-7 (1987)) followed by the activation domain from the CD3 zeta chain (aa 52-162, Letourneur F and Klausner RD, Proc. Natl. Acad. Sci. USA 88(20):8905-9 (1991)). The DNA sequence encoding the CAR is assembled and cloned into an expression vector (either retroviral, lentiviral, extrachromosomal, or integrative) to generate the CAR construct using standard molecular biology cloning techniques.

[0242] Example 7: Humanization of anti-EGFRvIII mAb The rabbit anti-EGFRvIII mAb was humanized to reduce the potential for immunogenicity when used in human patients. The sequences of the variable regions of the heavy and light chains (VH and VL) were compared with human antibody sequences in the Protein Data Bank (PDB) database, and a homology model was constructed. The CDRs of both the heavy and light chains of the rabbit mAb were grafted onto human frameworks most likely to maintain the proper structure required for antigen binding. Backmutations of human residues to rabbit residues or other mutations were designed as needed. The sequences of the humanized VH and VL regions are shown in Table 7. The humanized VH and VL regions were fused to the constant regions of human IgG1 heavy and kappa light chains, respectively. Binding of the humanized mAb to EGFRvIII was assessed using ELISA assays (Figures 4A-4D) and FACS assays (Figures 5A-5D). 16C11-H1L1 refers to a humanized mAb constructed using the 16C11-H1 heavy chain and 16C11-L1 light chain shown in Table 7, and other humanized clones follow the same naming convention.

[0243] [Table 7] TIFF2025535476000013.tif141156

[0244] Those skilled in the art will appreciate that changes could be made to the above-described embodiments without departing from the broad inventive concept thereof. It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but that it is intended to cover modifications within the scope of the invention as defined herein.

Claims

1. An isolated monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof, comprising a heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and a light chain variable region comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having the following polypeptide sequences: (1) SEQ ID NOs: 3, 4, 5, 6, 7, and 8, respectively, or SEQ ID NOs: 9, 10, 11, 12, 13, and 14, respectively; (2) SEQ ID NOs: 17, 18, 19, 20, 21, and 22, respectively, or SEQ ID NOs: 23, 24, 25, 26, 27, and 28, respectively; (3) SEQ ID NOs: 31, 32, 33, 34, 35, and 36, respectively, or SEQ ID NOs: 37, 38, 39, 40, 41, and 42, respectively; (4) SEQ ID NOs: 45, 46, 47, 48, 49, and 50, respectively, or SEQ ID NOs: 51, 52, 53, 54, 55, and 56, respectively; (5) SEQ ID NOs: 59, 60, 61, 62, 63, and 64, respectively, or SEQ ID NOs: 65, 66, 67, 68, 69, and 70, respectively; (6) SEQ ID NOs: 73, 74, 75, 76, 77, and 78, or SEQ ID NOs: 79, 80, 81, 82, 83, and 84, respectively; (7) SEQ ID NOs: 87, 88, 89, 90, 91, and 92, respectively, or SEQ ID NOs: 93, 94, 95, 96, 97, and 98, respectively; (8) SEQ ID NOs: 101, 102, 103, 104, 105, and 106, respectively, or SEQ ID NOs: 107, 108, 109, 110, 111, and 112, respectively; (9) SEQ ID NOs: 115, 116, 117, 118, 119, and 120, respectively, or SEQ ID NOs: 121, 122, 123, 124, 125, and 126, respectively; (10) SEQ ID NOs: 129, 130, 131, 132, 133, and 134, respectively, or SEQ ID NOs: 135, 136, 137, 138, 139, and 140, respectively; (11) SEQ ID NOs: 143, 144, 145, 146, 147, and 148, respectively, or SEQ ID NOs: 149, 150, 151, 152, 153, and 154, respectively; (12) SEQ ID NOs: 157, 158, 159, 160, 161, and 162, respectively, or SEQ ID NOs: 163, 164, 165, 166, 167, and 168, respectively; (13) SEQ ID NOs: 171, 172, 173, 174, 175, and 176, respectively, or SEQ ID NOs: 177, 178, 179, 180, 181, and 182, respectively; (14) SEQ ID NOs: 185, 186, 187, 188, 189, and 190, respectively, or SEQ ID NOs: 191, 192, 193, 194, 195, and 196, respectively; (15) SEQ ID NOs: 199, 200, 201, 202, 203, and 204, respectively, or SEQ ID NOs: 205, 206, 207, 208, 209, and 210, respectively; (16) SEQ ID NOs: 213, 214, 215, 216, 217, and 218, respectively, or SEQ ID NOs: 219, 220, 221, 222, 223, and 224, respectively; (17) SEQ ID NOs: 227, 228, 229, 230, 231, and 232, respectively, or SEQ ID NOs: 233, 234, 235, 236, 237, and 238, respectively; (18) SEQ ID NOs: 241, 242, 243, 244, 245, and 246, respectively, or SEQ ID NOs: 247, 248, 249, 250, 251, and 252, respectively; (19) SEQ ID NOs: 255, 256, 257, 258, 259, and 260, respectively, or SEQ ID NOs: 261, 262, 263, 264, 265, and 266, respectively; (20) SEQ ID NOs: 269, 270, 271, 272, 273, and 274, respectively, or SEQ ID NOs: 275, 276, 277, 278, 279, and 280, respectively; (21) SEQ ID NOs: 283, 284, 285, 286, 287, and 288, or SEQ ID NOs: 289, 290, 291, 292, 293, and 294, respectively; (22) SEQ ID NOs: 297, 298, 299, 300, 301, and 302, respectively, or SEQ ID NOs: 303, 304, 305, 306, 307, and 308, respectively; (23) SEQ ID NOs: 311, 312, 313, 314, 315, and 316, respectively, or SEQ ID NOs: 317, 318, 319, 320, 321, and 322, respectively; (24) SEQ ID NOs: 325, 326, 327, 328, 329, and 330, respectively, or SEQ ID NOs: 331, 332, 333, 334, 335, and 336, respectively; or (25) SEQ ID NOs: 341, 342, 343, 344, 345, and 346, respectively, or SEQ ID NOs: 347, 348, 349, 350, 351, and 352, respectively. Including, The antibody or antigen-binding fragment thereof or antigen-binding domain thereof is an isolated monoclonal antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII, preferably human EGFRvIII.

2. a heavy chain variable region having a polypeptide sequence at least 95% identical to SEQ ID NO: 1, 15, 29, 43, 57, 71, 85, 99, 113, 127, 141, 155, 169, 183, 197, 211, 225, 239, 253, 267, 281, 295, 309, 323, 339, 353, 354, 355, 356, 360, 361, 362, 363, 366, 367, 368, 369, 374, 375, 376, 377, 382, ​​383, 384, or 385; 8, 72, 86, 100, 114, 128, 142, 156, 170, 184, 198, 212, 226, 240, 254, 268, 282, 296, 310, 324, 340, 357, 358, 359, 364, 365, 370, 371, 372, 373, 378, 379, 380, 381, 386, 387, or 388. The isolated monoclonal antibody or antigen-binding fragment thereof or antigen-binding domain thereof of claim 1, comprising a light chain variable region having a polypeptide sequence at least 95% identical to

3. 3. The isolated monoclonal antibody or antigen-binding fragment thereof or antigen-binding domain thereof of claim 1 or 2, comprising: (1) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 1 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 2; (2) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 15 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 16; (3) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 29 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 30; (4) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 43 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 44; (5) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 57 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 58; (6) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 71 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 72; (7) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 85 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 86; (8) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 99 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 100; (9) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 113 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 114; (10) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 127 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 128; (11) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 141 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 142; (12) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 155 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 156; (13) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 169 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 170; (14) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 183 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 184; (15) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 197 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 198; (16) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 211 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 212; (17) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 225 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

226. (18) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 239 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 240; (19) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 253 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

254. (20) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 267 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 268; (21) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 281 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 282; (22) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 295 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 296; (23) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 309 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 310; (24) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 323 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

324. (25) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 339 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 340; (26) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

357. (27) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

358. (28) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 353 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 359; (29) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

357. (30) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 358; (31) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 354 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

359. (32) a heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 357; (33) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

358. (34) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 355 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

359. (35) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

357. (36) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

358. (37) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 356 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

359. (38) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 360 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

364. (39) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 360 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

365. (40) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 361 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

364. (41) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 361 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

365. (42) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 362 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

364. (43) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 362 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

365. (44) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 363 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

364. (45) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 363 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

365. (46) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

370. (47) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

371. (48) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

372. (49) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 366 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

373. (50) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 370; (51) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 371; (52) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 372; (53) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 367 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (54) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

370. (55) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

371. (56) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

372. (57) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 368 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

373. (58) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

370. (59) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

371. (60) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

372. (61) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 369 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 373; (62) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

378. (63) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

379. (64) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

380. (65) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 374 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

381. (66) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

378. (67) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

379. (68) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

380. (69) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 375 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

381. (70) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

378. (71) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

379. (72) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

380. (73) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 376 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

381. (74) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

378. (75) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

379. (76) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

380. (77) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 377 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

381. (78) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

386. (79) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

387. (80) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 382 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

388. (81) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

386. (82) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

387. (83) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 383 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

388. (84) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

386. (85) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

387. (86) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 384 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

388. (87) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

386. (88) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO: 387; or (89) A heavy chain variable region having the polypeptide sequence of SEQ ID NO: 385 and a light chain variable region having the polypeptide sequence of SEQ ID NO:

388.

4. 4. An isolated monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof according to any one of claims 1 to 3, which is a chimeric, human or humanized antibody or antigen-binding fragment or antigen-binding domain thereof.

5. 5. The isolated monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof according to any one of claims 1 to 4, which is capable of inducing effector-mediated tumor cell lysis via antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and / or complement-dependent cytotoxicity (CDC), and / or mediating the recruitment of conjugated drugs, and / or forming a bispecific antibody with another monoclonal antibody (mAb) or antigen-binding fragment thereof that has cancer-killing activity.

6. A bispecific antibody or antigen-binding fragment, or bispecific antigen-binding domain thereof, comprising the monoclonal antibody or antigen-binding fragment, or antigen-binding domain thereof, according to any one of claims 1 to 5.

7. An isolated nucleic acid encoding the monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof of any one of claims 1 to 5, or the bispecific antibody or antigen-binding fragment or bispecific antigen-binding domain thereof of claim 6.

8. A vector comprising the isolated nucleic acid of claim 7.

9. A host cell comprising the vector of claim 8.

10. 10. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof of any one of claims 1 to 5, or the bispecific antibody or antigen-binding fragment or bispecific antigen-binding domain thereof of claim 6, and a pharmaceutically acceptable carrier.

11. 11. A method of targeting EGFRvIII on the surface of cancer cells and / or treating cancer and / or treating an inflammatory disease and / or treating an autoimmune disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 10, wherein optionally the cancer is selected from the group consisting of lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocarcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, bladder urothelial carcinoma, metastatic melanoma, breast cancer, ovarian cancer, cervical cancer, head and neck cancer, pancreatic cancer, glioma, glioblastoma, mesothelioma and other solid tumors, as well as non-Hodgkin's lymphoma (NHL), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

12. A method for producing the monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof according to any one of claims 1 to 7, or the bispecific antibody or antigen-binding fragment or bispecific antigen-binding domain thereof according to claim 8, the method comprising culturing cells containing a nucleic acid encoding the monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof, or the bispecific antibody or antigen-binding fragment or bispecific antigen-binding domain, under conditions for producing the monoclonal antibody or antigen-binding fragment or antigen-binding domain, or the bispecific antibody or antigen-binding fragment or bispecific antigen-binding domain, and recovering the monoclonal antibody or antigen-binding fragment or antigen-binding domain, or the bispecific antibody or antigen-binding fragment or bispecific antigen-binding domain from the cells or culture.

13. 10. A method for producing a pharmaceutical composition comprising the monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof according to any one of claims 1 to 5, or the bispecific antibody or antigen-binding fragment or bispecific antigen-binding domain thereof according to claim 6, the method comprising combining the monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof, or the bispecific antibody or antigen-binding fragment or bispecific antigen-binding domain thereof with a pharmaceutically acceptable carrier to obtain a pharmaceutical composition.

14. 1. A method for determining the level of EGFRvIII in a subject, comprising: (a) obtaining a sample from a subject; (b) contacting the sample with the isolated monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof of any one of claims 1 to 5; and (c) determining the level of EGFRvIII in the subject; Including, Optionally, the sample is a tissue sample or a blood sample, and optionally, the tissue sample is a cancer tissue sample.

15. 1. An isolated polynucleotide comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) an extracellular domain comprising at least one antigen-binding domain of any one of claims 1 to 5 or a bispecific antigen-binding domain of claim 6; (b) hinge region; (c) a transmembrane domain, and (d) Intracellular signaling domain An isolated polynucleotide comprising:

16. 16. A chimeric antigen receptor (CAR) encoded by the isolated polynucleotide of claim 15.

17. 16. A host cell comprising a vector comprising the isolated polynucleotide of claim 15, optionally wherein the host cell is a T cell or a NK cell, preferably a human T cell or a human NK cell.