Anti-EGFRvIII antibodies and uses thereof

Abstract

Anti-EGFRvIII antibodies, as well as antigen binding fragments thereof, and chimeric antigen receptors (CARs) comprising an 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.

Description

Cross - Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 380,614, filed on October 24, 2022, U.S. Provisional Application No. 63 / 486,261, filed on February 22, 2023, and U.S. Provisional Application No. 63 / 517,461, filed on August 3, 2023. Each disclosure is hereby incorporated by reference in its entirety. Field of the Invention

[0002] The present invention relates to monoclonal anti - EGFRvIII antibodies and chimeric antigen receptors (CARs) comprising anti - EGFRvIII antigen - binding domains, nucleic acids and expression vectors encoding said antibodies and CARs, recombinant cells comprising said vectors and CARs, and compositions comprising said antibodies, CARs, and recombinant cells expressing the CAR. The present invention also provides methods for preparing said antibodies and CARs and methods for treating diseases using said antibodies and CARs, said diseases including cancer, inflammatory diseases, autoimmune diseases, and / or related complications. Reference to a Sequence Listing Submitted in Electronic Form

[0003] This application contains a Sequence Listing submitted in electronic form. The content of the electronic sequence listing (065799.39WO1 SequenceListing.xml; size: 329,805 bytes; created on September 25, 2023) is hereby incorporated by reference in its entirety. Background Art

[0004] The epidermal growth factor receptor, also known as EGFR, HER1 or ERBB1, is a transmembrane receptor tyrosine kinase that upon binding to any number of possible ligands triggers the formation of EGFR homodimers, phosphorylation of the C-terminus, and subsequent downstream signaling (An, Z. et al., Oncogene 2018, (37):1561-1575). A splice variant of EGFR, called EGFRvIII, contains a deletion of exons 2-7 in the extracellular domain and has been identified in a variety of different cancer types, mainly in glioblastoma multiforme (GBM), where the variant is found in up to 40-50% of high-grade gliomas, which also have upregulated EGFR (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, numerous studies have shown that EGFRvIII is not detected in normal tissues, making it an ideal tumor-specific antigen for anti-cancer biologics targeting EGFRvIII-positive tumors (Gan, H. et al., The FEBS Journal. 2013, (280):5350-5370).

[0005] Unlike the parental EGFR, EGFRvIII is constitutively active. Although the signaling activity of EGFRvIII is weaker than that of ligand-bound EGFR, the activation and phosphorylation of EGFRvIII can be induced by wild-type EGFR, and they can act together in a cooperative manner to enhance downstream signaling (Fan, Q. et al., Cancer Cell. 2013, (24):438-439). However, this activity has been reported to be highly oncogenic, and EGFRvIII alone has been reported to be sufficient to confer a growth advantage to tumor cells expressing only the variant (Nishikawa, R. et al., Proceedings of the National Academy of Sciences of the United States of America, 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 have an inhibitory effect on internalization and degradation compared to EGFR, which may play a role in its enhanced oncogenicity (Grandal, M. et al., Carcinogenesis, 2007, (28):1408-1417). Since EGFRvIII has the same cytoplasmic domain as EGFR, it is able to enhance the same signaling pathways. For membrane-bound EGFRvIII, these include the MAPK, ERK, PI3K / AKT, JAK / STAT, and PKC pathways, which together constitute the overall activation of cell survival and proliferation pathways, thereby increasing tumorigenesis (An, Z. et al., Oncogene, 2018, (37):1561-1575; Padfield, E. et al., Frontiers in Oncology, 2015, (5):1-8). Antibodies that block EGFRvIII signaling by preventing dimerization or inducing cell surface turnover have great potential for treating EGFRvIII-related cancers. Alternatively, antibodies that bind to EGFRvIII can promote immune system-mediated killing of cancer cells. SUMMARY OF THE INVENTION

[0006] In general aspects, the present invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind to EGFRvIII.

[0007] The present invention provides an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, and HCDR3, and the light chain variable region comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, having the following polypeptide sequences: (1) SEQ ID NO:3, 4, 5, 6, 7 and 8 respectively, or SEQ ID NO:9, 10, 11, 12, 13 and 14 respectively; (2) SEQ ID NO:17, 18, 19, 20, 21 and 22 respectively, or SEQ ID NO:23, 24, 25, 26, 27 and 28 respectively; (3) SEQ ID NO:31, 32, 33, 34, 35 and 36 respectively, or SEQ ID NO:37, 38, 39, 40, 41 and 42 respectively; (4) SEQ ID NO:45, 46, 47, 48, 49 and 50 respectively, or SEQ ID NO:51, 52, 53, 54, 55 and 56 respectively; (5) SEQ ID NO:59, 60, 61, 62, 63 and 64 respectively, or SEQ ID NO:65, 66, 67, 68, 69 and 70 respectively; (6) SEQ ID NO:73, 74, 75, 76, 77 and 78 respectively, or SEQ ID NO:79, 80, 81, 82, 83 and 84 respectively; (7) SEQ ID NO:87, 88, 89, 90, 91 and 92 respectively, or SEQ ID NO:93, 94, 95, 96, 97 and 98 respectively; (8) SEQ ID NO:101, 102, 103, 104, 105 and 106 respectively, or SEQ ID NO:107, 108, 109, 110, 111 and 112 respectively; (9) SEQ ID NO:115, 116, 117, 118, 119 and 120 respectively, or SEQ ID NO:121, 122, 123, 124, 125 and 126 respectively; (10) SEQ ID NO:129, 130, 131, 132, 133 and 134 respectively, or SEQ ID NO:135, 136, 137, 138, 139 and 140 respectively; (11) SEQ ID NO:143, 144, 145, 146, 147 and 148 respectively, or SEQ ID NO:149, 150, 151, 152, 153 and 154 respectively; (12) are SEQ ID NO:157, 158, 159, 160, 161 and 162, or are SEQ ID NO:163, 164, 165, 166, 167 and 168 respectively; (13) are SEQ ID NO:171, 172, 173, 174, 175 and 176, or are SEQ ID NO:177, 178, 179, 180, 181 and 182 respectively; (14) are SEQ ID NO:185, 186, 187, 188, 189 and 190, or are SEQ ID NO:191, 192, 193, 194, 195 and 196 respectively; (15) are SEQ ID NO:199, 200, 201, 202, 203 and 204, or are SEQ ID NO:205, 206, 207, 208, 209 and 210 respectively; (16) are SEQ ID NO:213, 214, 215, 216, 217 and 218, or are SEQ ID NO:219, 220, 221, 222, 223 and 224 respectively; (17) are SEQ ID NO:227, 228, 229, 230, 231 and 232, or are SEQ ID NO:233, 234, 235, 236, 237 and 238 respectively; (18) are SEQ ID NO:241, 242, 243, 244, 245 and 246, or are SEQ ID NO:247, 248, 249, 250, 251 and 252 respectively; (19) are SEQ ID NO:255, 256, 257, 258, 259 and 260, or are SEQ ID NO:261, 262, 263, 264, 265 and 266; (20) are SEQ ID NO:269, 270, 271, 272, 273 and 274, or are SEQ ID NO:275, 276, 277, 278, 279 and 280; (21) are SEQ ID NO:283, 284, 285, 286, 287 and 288, or are SEQ ID NO:289, 290, 291, 292, 293 and 294; (22) are SEQ ID NO:297, 298, 299, 300, 301 and 302, or are SEQ ID NO:303, 304, 305, 306, 307 and 308; (23) are SEQ ID NO:311, 312, 313, 314, 315 and 316, or are SEQ ID NO:317, 318, 319, 320, 321 and 322; (24) are SEQ ID NO:325, 326, 327, 328, 329 and 330, or are SEQ ID NO:331, 332, 333, 334, 335 and 336; or (25) are SEQ ID NO:341, 342, 343, 344, 345 and 346, or are SEQ ID NO:347, 348, 349, 350, 351 and 352; wherein the antibody or antigen-binding fragment thereof specifically binds to EGFRvIII, preferably human EGFRvIII.

[0008] In certain embodiments, the isolated monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region or a light chain variable region, and the heavy chain variable region has a polypeptide sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the following sequences: 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 the light chain variable region has a polypeptide sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the following sequences: 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.

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

[0010] In certain embodiments, the isolated monoclonal antibody or its antigen-binding fragment binds to EGFRvIII and is capable of inducing effector-mediated tumor cell lysis by antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell 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 or its antigen-binding fragment having cancer-killing effects.

[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, which comprises the monoclonal antibody of the present invention or antigen-binding fragment thereof.

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

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

[0016] Also provided is a host cell comprising the vector, the vector comprising an isolated nucleic acid encoding the monoclonal antibody of the present invention or antigen-binding fragment thereof, or bispecific antibody or antigen-binding fragment thereof.

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

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

[0019] Also provided is a method for treating cancer in a subject in need, the method comprising administering the pharmaceutical composition of the present invention to the subject. The cancer can be any liquid or solid cancer, for example, it can be selected from, 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 lymphoma (NHL), acute lymphoblastic 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 for treating inflammation and / or autoimmune diseases in a subject in need thereof, the method comprising administering to the subject the pharmaceutical composition of the present invention.

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

[0022] Also provided is a method for preparing a pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof, or a bispecific antibody or an antigen-binding fragment thereof, of the present invention, comprising combining the monoclonal antibody or an antigen-binding fragment thereof, or the bispecific antibody or an 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 comprises: (a) obtaining a sample from the subject; (b) contacting the sample with an antibody or an 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 can be, for example, a cancer tissue sample. In certain embodiments, the sample is a blood sample.

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

[0025] The present invention provides an isolated polynucleotide comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR). 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 and a light chain variable region, the heavy chain variable region comprising heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, and the light chain variable region comprising light chain complementarity determining region 1 (LCDR1), LCDR2, and LCDR3, which have the following polypeptide sequences: (1) SEQ ID NO: 3, 4, 5, 6, 7, and 8, respectively, or SEQ ID NO: 9, 10, 11, 12, 13, and 14, respectively; (2) SEQ ID NO: 17, 18, 19, 20, 21, and 22, respectively, or SEQ ID NO: 23, 24, 25, 26, 27, and 28, respectively; (3) SEQ ID NO: 31, 32, 33, 34, 35, and 36, respectively, or SEQ ID NO: 37, 38, 39, 40, 41, and 42, respectively; (4) SEQ ID NO: 45, 46, 47, 48, 49, and 50, respectively, or SEQ ID NO: 51, 52, 53, 54, 55, and 56, respectively; (5) SEQ ID NO: 59, 60, 61, 62, 63, and 64, respectively, or SEQ ID NO: 65, 66, 67, 68, 69, and 70, respectively; (6) SEQ ID NO: 73, 74, 75, 76, 77, and 78, respectively, or SEQ ID NO: 79, 80, 81, 82, 83, and 84, respectively; (7) SEQ ID NO: 87, 88, 89, 90, 91, and 92, respectively, or SEQ ID NO: 93, 94, 95, 96, 97, and 98, respectively; (8) SEQ ID NO: 101, 102, 103, 104, 105, and 106, respectively, or SEQ ID NO: 107, 108, 109, 110, 111, and 112, respectively; (9) SEQ ID NO: 115, 116, 117, 118, 119, and 120, respectively, or SEQ ID NO: 121, 122, 123, 124, 125, and 126, respectively; (10) SEQ ID NO: 129, 130, 131, 132, 133, and 134, respectively, or SEQ ID NO: 135, 136, 137, 138, 139, and 140; (11) SEQ ID NO: 143, 144, 145, 146, 147 and 148 respectively, or SEQ ID NO: 149, 150, 151, 152, 153 and 154 respectively; (12) SEQ ID NO: 157, 158, 159, 160, 161 and 162 respectively, or SEQ ID NO: 163, 164, 165, 166, 167 and 168 respectively; (13) SEQ ID NO: 171, 172, 173, 174, 175 and 176 respectively, or SEQ ID NO: 177, 178, 179, 180, 181 and 182 respectively; (14) SEQ ID NO: 185, 186, 187, 188, 189 and 190 respectively, or SEQ ID NO: 191, 192, 193, 194, 195 and 196 respectively; (15) SEQ ID NO: 199, 200, 201, 202, 203 and 204 respectively, or SEQ ID NO: 205, 206, 207, 208, 209 and 210 respectively; (16) SEQ ID NO: 213, 214, 215, 216, 217 and 218 respectively, or SEQ ID NO: 219, 220, 221, 222, 223 and 224 respectively; (17) SEQ ID NO: 227, 228, 229, 230, 231 and 232 respectively, or SEQ ID NO: 233, 234, 235, 236, 237 and 238 respectively; (18) SEQ ID NO: 241, 242, 243, 244, 245 and 246 respectively, or SEQ ID NO: 247, 248, 249, 250, 251 and 252 respectively; (19) SEQ ID NO: 255, 256, 257, 258, 259 and 260 respectively, or SEQ ID NO: 261, 262, 263, 264, 265 and 266 respectively; (20) SEQ ID NO: 269, 270, 271, 272, 273 and 274 respectively, or SEQ ID NO: 275, 276, 277, 278, 279 and 280 respectively; (21) SEQ ID NO: 283, 284, 285, 286, 287 and 288 respectively, or SEQ ID NO: 289, 290, 291, 292, 293 and 294 respectively; (22) SEQ ID NO: 297, 298, 299, 300, 301 and 302, respectively, or SEQ ID NO: 303, 304, 305, 306, 307 and 308, respectively; (23) SEQ ID NO: 311, 312, 313, 314, 315 and 316, respectively, or SEQ ID NO: 317, 318, 319, 320, 321 and 322, respectively; (24) SEQ ID NO: 325, 326, 327, 328, 329 and 330, respectively, or SEQ ID NO: 331, 332, 333, 334, 335 and 336, respectively; or (25) SEQ ID NO: 341, 342, 343, 344, 345 and 346, respectively, or SEQ ID NO: 347, 348, 349, 350, 351 and 352.

[0027] In certain embodiments, the antigen-binding domain comprises 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 the following sequences: 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 comprises a light 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 the following sequences: 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 shown in SEQ ID NO:1, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:2; (2) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:15, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:16; (3) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:29, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:30; (4) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:43, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:44; (5) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:57, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:58; (6) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:71, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:72; (7) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:85, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:86; (8) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:99, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:100; (9) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:113, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:114; (10) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:127, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:128; (11) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:141, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:142; (12) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:155, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:156; (13) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:169, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:170; (14) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:183, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:184; (15) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 197, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 198; (16) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 211, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 212; (17) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 225, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 226; (18) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 239, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 240; (19) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 253, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 254; (20) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 267, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 268; (21) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 281, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 282; (22) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 295, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 296; (23) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 309, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 310; (24) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 323, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 324; (25) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 339, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 340; (26) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 353, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (27) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 353, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (28) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 353, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (29) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 354, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (30) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 354, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (31) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 354, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (32) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 355, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (33) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 355, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (34) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 355, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (35) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 356, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (36) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 356, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (37) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 356, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (38) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 360, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (39) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 360, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (40) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 361, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (41) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 361, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (42) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 362, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (43) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 362, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (44) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 363, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (45) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 363, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (46) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (47) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (48) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (49) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (50) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (51) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (52) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (53) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (54) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (55) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (56) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (57) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (58) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (59) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (60) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (61) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (62) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (63) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (64) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (65) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (66) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (67) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (68) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (69) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (70) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (71) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (72) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (73) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (74) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (75) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (76) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (77) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (78) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 382, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (79) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 382, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; (80) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 382, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388; (81) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 383, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (82) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 383, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; (83) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 383, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388; (84) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 384, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (85) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 384, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; (86) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 384, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388; (87) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 385, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (88) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 385, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; or (89) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 385, and a light chain variable region having the polypeptide sequence shown in 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, the chimeric antigen receptor (CAR) comprises one or more antigen-binding domains.

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

[0034] Also provided are chimeric antigen receptors (CARs) encoded by the isolated polynucleotides of the present invention.

[0035] Also provided are vectors comprising an isolated polynucleotide comprising a nucleic acid encoding a CAR of the present invention.

[0036] Also provided are host cells comprising the vector of the present invention.

[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. T cells or NK cells can be engineered, for example, to express a CAR of the present invention to treat diseases such as cancer.

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

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

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

[0041] Also provided is a method for treating cancer in a subject in need thereof, comprising administering to the subject the CAR-T cells and / or CAR-NK cells of the present invention. The cancer can be any liquid or solid cancer, for example, it can be selected from, 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 lymphoma (NHL), acute lymphoblastic 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 for treating cancer in a subject in need thereof further comprises administering to the subject an agent that increases the efficacy of cells expressing a CAR molecule.

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

[0044] In certain embodiments, the method for treating cancer in a subject in need thereof further comprises administering to the subject an agent for treating a disease associated with EGFRvIII. Brief Description of the Drawings

[0045] When read in conjunction with the accompanying drawings, the above summary and the following detailed description of the preferred embodiments of the present application will be better understood. However, it should be understood that the present application is not limited to the specific embodiments shown in the drawings.

[0046] Figure 1A-1G Shows the binding of purified chimeric anti-EGFRvIII monoclonal antibody (the VH and VL regions of the rabbit monoclonal antibody are respectively fused with the constant regions of human IgG1 heavy chain and κ light chain) to immobilized recombinant EGFRvIII in an ELISA assay. The antigen is human EGFRvIII (25-378) with a 6xHis tag at the C-terminus (ACROBiosystems, CAT#: EGI-H52H4). Isotype control (IgG1) was used as a negative control.

[0047] Figure 2A-2G Shows the binding of anti-EGFRvIII monoclonal antibody to ExpiCHO-S cells transiently expressing EGFRvIII by FACS analysis. Isotype control (IgG1) was used as a negative control.

[0048] Figure 3A-3DShows the evaluation of the binding activity of anti-EGFRvIII chimeric monoclonal antibody to wild-type EGFR using ELISA.

[0049] Figure 4A-4D Shows the binding of humanized anti-EGFRvIII monoclonal antibody to immobilized recombinant EGFRvIII in an ELISA assay. The antigen was human EGFRvIII (25 - 378) with a 6xHis tag at the C-terminus (ACROBiosystems, CAT#: EGI-H52H4). 16C11-H1L1 refers to the humanized monoclonal antibody constructed from the 16C11-H1 heavy chain and 16C11-L1 light chain shown in Table 7; other humanized clones follow the same naming rule. Isotype control (IgG1) was used as a negative control.

[0050] Figure 5A-5D Shows the binding of humanized anti-EGFRvIII monoclonal antibody to ExpiCHO-S cells transiently expressing EGFRvIII analyzed by FACS. 16C11-H1L1 refers to the humanized monoclonal antibody constructed from the 16C11-H1 heavy chain and 16C11-L1 light chain shown in Table 7; other humanized clones follow the same naming rule. Isotype control (IgG1) was used as a negative control. DETAILED DESCRIPTION OF THE INVENTION

[0051] Various publications, articles, and patents are cited or described in the background and throughout the specification; each of these references is hereby incorporated by reference in its entirety. The discussion of documents, acts, materials, devices, articles, or the like included in the specification of this application is for the purpose of providing the background of the present invention. With respect to any invention disclosed or claimed, such discussion does not admit that any or all of these matters form part of the prior art.

[0052] 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. In addition, certain terms used herein have the meanings set forth in this specification.

[0053] It must be noted that, as used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" include plural referents.

[0054] Unless otherwise indicated, any numerical values (such as concentrations or concentration ranges as described herein) should be understood to be modified by the term "about" in all cases. Thus, numerical values generally include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1 mg / mL. Similarly, a concentration range of 1% - 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges expressly includes all possible sub-ranges, all individual numerical values within that range, including integer and fractional values within that range, unless the context clearly indicates otherwise.

[0055] Unless otherwise indicated, the term "at least" before a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine, using only routine experimentation, many equivalents to the specific embodiments of the invention described herein. The invention is intended to embrace these equivalents.

[0056] As used herein, the terms "comprise / comprising", "include / including", "has / having", "contain / containing" or any other variants thereof will be understood to implicitly include a specified integer or group of integers, but not exclude any other integer or group of integers, and are non-exclusive or open-ended. For example, a composition, mixture, process, method, article or device comprising a series of elements is not necessarily limited to those elements alone, but may include other elements in these compositions, mixtures, processes, methods, articles or devices that are not expressly listed. Further, unless there is a clear contrary statement, "or" refers to an inclusive or, rather than an exclusive or. For example, any of the following satisfies condition A or B: A is true (or present) and B is false (or absent), A is false (or absent) and B is true (or present), A and B are both true (or present).

[0057] As used herein, the conjunctive term "and / or" between multiple recited elements is understood to include both single options and combined options. For example, when two elements are joined by "and / or", the first option refers to the applicability of the first element without the second element. The second option refers to the applicability of the second element without the first element. The third option refers to the applicability of both the first and second elements simultaneously. Any of these options is understood to fall within the meaning, and thus meets the requirements of the term "and / or" as used herein. The simultaneous applicability of multiple options is also understood to be included within this meaning, and thus meets the requirements of the term "and / or".

[0058] As used herein, the term "consisting of" or variations thereof, such as "consisting essentially of" or "consisting solely of", as used throughout the specification and claims, indicates the inclusion of any recited integer or group of integers, but no additional integers or groups of integers can be added to the specified method, structure, and combination.

[0059] As used herein, the term "consisting essentially of" or variations thereof, such as "consisting essentially of", "formed essentially of", as used throughout the specification and claims, indicates the inclusion of any recited integral or group of integrals, and the integral or group of integrals will not materially change the basic or novel properties of the specified method, structure, and composition. See M.P.E.P.§2111.03.

[0060] As used herein, "subject" refers to any animal, preferably a mammal, and most preferably a human. The term "mammal" as used herein includes 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., and more preferably humans.

[0061] The words "right", "left", "lower", and "upper" represent the directions of reference in the drawings.

[0062] It should also be understood that the terms "about", "approximate", "generally", "substantially", and similar terms, as used herein, refer to the more preferred dimensions or characteristics of the components of the present invention, indicating that the stated dimension / characteristic is not a strict boundary or parameter and does not exclude minor variations in function that are the same or similar, as understood by those skilled in the art. At a minimum, these references containing numerical parameters should include the use of mathematical and industrial principles recognized in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.) that do not change the least significant digits.

[0063] In the context of two or more nucleic acid or polypeptide sequences (e.g., an anti-EGFRvIII antibody and the polynucleotide encoding it, a chimeric antigen receptor (CAR) comprising an antigen-binding domain specific for EGFRvIII and the polynucleotide encoding it), the term "identical" or percent "identity" means that two or more sequences or subsequences are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned using one of the following sequence comparison algorithms or by visual inspection to obtain maximum correspondence.

[0064] For sequence alignment, usually one sequence is used as a reference sequence and compared with a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, and if necessary, subsequence coordinates are specified, and the sequence algorithm program parameters are specified. Then, according to the specified program parameters, the sequence comparison algorithm calculates the percentage of sequence identity of the test sequence relative to the reference sequence.

[0065] Optimal alignment of sequences can be performed using the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics and Mechanics, 1981; 2: 482, the homology alignment algorithm of Needleman and Wunsch, Journal of Molecular Biology, 1970, 48: 443, the similarity search method of Pearson and Lipman, Proceedings of the National Academy of Sciences of the United States of America, 1988, 485: 2444, by implementing these algorithms on a computer (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin), or visually (usually see Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., General Techniques, Greene Publishing Associates and John Wiley & Sons, Inc., Suppl. 1995 (Ausubel)).

[0066] Examples of algorithms suitable for determining the percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Journal of Molecular Biology, 1990; 215: 403-410 and Altschul et al., Nucleic Acids Research, 1997; 25: 3389-3402, respectively. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm first identifies high-scoring sequence pairs (HSPs) by identifying short sequences of length W in the query sequence that match or satisfy a positive-valued threshold score T when aligned with sequences of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul et al., supra). Hits to these initial neighboring sequences serve as seeds to initiate a search for longer HSPs that contain them. The hits to the sequences are then extended in each sequence in both directions until the cumulative alignment score increases.

[0067] For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for a mismatched residue, always <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. The extension of a sequence hit in each direction is stopped under the following conditions: the cumulative alignment score drops by the quantity X from its maximum achieved value; the cumulative score is zero or below due to the accumulation of one or more negatively scored residue alignments; 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 nucleic acid sequences) by default uses a word length (W) of 3, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands as defaults. For amino acid sequences, the BLASTP program by default uses a word length (W) of 3, an expectation value (E) of 10, and the BLOSUM62 scoring matrix as defaults (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989; 89:10915).

[0068] In addition to calculating the percent sequence identity, the BLAST algorithm performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA, 1993; 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which gives an indication of the probability that a match between two nucleotide or amino acid sequences occurred by chance. For example, if the minimum sum probability in a comparison of a test nucleic acid to a reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001, the test nucleic acid is considered similar to the reference sequence.

[0069] Further evidence that two nucleic acid sequences or polypeptides are substantially identical is that, as described below, the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid. Thus, for example, when the difference between one polypeptide and a second polypeptide is only in conservative substitutions, the two polypeptides are generally substantially identical. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0070] As used herein, the term "isolated" means that a biological component, such as a nucleic acid, peptide or protein, has been substantially separated, produced or purified from other biological components of the organism in which the component naturally occurs (i.e., 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. An "isolated" nucleic acid, peptide and protein can be part of a composition, and if the composition is not part of the native environment of the nucleic acid, peptide and protein, the composition can be isolated. The term also includes nucleic acids, peptides and proteins prepared by recombinant expression in a host cell and chemically synthesized nucleic acids.

[0071] As used herein, the term "polynucleotide", which is also synonymously referred to as "nucleic acid molecule", "nucleotide" or "nucleic acid", refers to any polyribonucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA, or modified RNA or DNA. "Polynucleotide" includes, but is not limited to, single-stranded and double-stranded DNA, DNA with a mixture of single-stranded and double-stranded regions, single-stranded or double-stranded RNA, and RNA with a mixture of single-stranded and double-stranded regions, and hybrid molecules consisting of single-stranded or more typically double-stranded or a mixture of single-stranded and double-stranded regions. In addition, "polynucleotide" refers to a triple-stranded region containing RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNA or RNA containing one or more modified bases, as well as DNA or RNA with a modified backbone for reasons of stability or otherwise. For example, "modified" bases include, for example, tritylated bases and uncommon bases such as inosine. A variety of modifications can be made to DNA and RNA. Thus, "polynucleotide" includes chemically, enzymatically and metabolically modified forms of polynucleotides commonly found in nature, as well as chemical forms of DNA and RNA unique to viruses and cells. "Polynucleotide" also includes relatively short nucleic acid chains, commonly referred to as oligonucleotides.

[0072] As used herein, the term "vector" is a replicon that is operably inserted into another nucleic acid fragment to effect replication or expression of the fragment.

[0073] As used herein, the term "host cell" refers to a cell that contains a nucleic acid molecule of the present invention. The "host cell" can be any type of cell, for example, a primary cell, a cell in culture or a cell in a cell line. In one embodiment, the "host cell" is a cell transfected with a nucleic acid molecule of the present invention. In another embodiment, the "host cell" is a progeny or potential progeny of such a transfected cell. The progeny of the cell can be the same as or different from the parental cell. For example, due to mutations that may occur in the offspring or environmental influences, or due to integration of the nucleic acid molecule into the host cell genome.

[0074] As used herein, the term "expression" refers to the biosynthesis of a gene product. The term encompasses the process of transcription of a gene into RNA. The term also encompasses the translation of RNA into one or more polypeptides, and further encompasses all naturally occurring post-transcriptional and post-translational modifications. The expressed antibody can be within the cytoplasm of the host cell, secreted into the extracellular environment such as the growth medium of cell culture, or anchored to the cell membrane.

[0075] As used herein, the terms "peptide", "polypeptide" or "protein" can refer to a molecule composed of amino acids and can be identified as a protein by those skilled in the art. Conventional single-letter or three-letter codes for amino acid residues are used herein. The terms "peptide", "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer can be linear or branched, it can contain modified amino acids, and can be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified by natural or intervening modifications, such as formation of disulfide bonds, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeling component. This definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), and other modifications known in the art.

[0076] The peptide sequences described herein are written according to the usual convention, i.e., 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 otherwise specifically stated, the L-form of the amino acid is represented. Chimeric antigen receptor (CAR)

[0077] As used herein, the term "chimeric antibody receptor" (CAR) refers to a recombinant polypeptide containing 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. The extracellular domain of the CAR binds to the target antigen on the surface of the target cell, resulting in aggregation of the CAR and transmission of an activation stimulus to the cell containing the CAR. The CAR redirects the specificity of immune effector cells and triggers proliferation, cytokine production, phagocytosis, and / or the production of molecules that mediate the death of cells expressing the target antigen in a major histocompatibility (MHC)-independent manner.

[0078] In one aspect, the CAR comprises an antigen-binding domain, a hinge region, a co-stimulatory domain, an activation domain, and a transmembrane region. In one aspect, the CAR comprises an antigen-binding domain, a hinge region, two co-stimulatory domains, an activation domain, and a transmembrane region. In one aspect, the CAR comprises two antigen-binding domains, a hinge region, a co-stimulatory domain, an activation domain, and a transmembrane region. In one aspect, the CAR comprises two antigen-binding domains, a hinge region, two co-stimulatory domains, an activation domain, and a transmembrane region.

[0079] As used herein, the term "signal peptide" refers to the leading sequence at the amino terminus (N-terminus) of the nascent CAR protein that co-translationally or post-translationally directs the nascent CAR protein into the endoplasmic reticulum and subsequent surface expression.

[0080] As used herein, the terms "extracellular antigen-binding domain", "extracellular domain", or "extracellular ligand-binding domain" refer to the portion of the CAR that is located external to the cell membrane and is capable of binding an antigen, target, or ligand.

[0081] As used herein, the term "hinge region" refers to the portion of the CAR that connects two adjacent domains of the CAR protein, e.g., the extracellular domain and the transmembrane domain.

[0082] As used herein, the term "transmembrane domain" refers to the portion of the CAR that extends across the cell membrane and anchors the CAR to the cell membrane. It is sometimes referred to as the "transmembrane region". Co-stimulatory domain

[0083] As used herein, chimeric antibody receptors can incorporate co-stimulatory (signaling) domains to increase their potency. The co-stimulatory (signaling) domains can be derived from co-stimulatory molecules. Co-stimulatory molecules are cell surface molecules that are not antigen receptors or their ligands required for an effective immune response. The co-stimulatory domain can be derived from a co-stimulatory molecule, including but not limited to CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3 (α, β, δ, ε, γ, ζ), 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 co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1; CD11a and CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a), DAP10, Fcγ receptor, MHC class I molecule, TNFR, integrin, signaling lymphocytic activation molecule, BTLA, Toll ligand receptor, ICAM-1, CDS, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGAL, CD1a, CD1b, CD1c, CD1d, ITGAM, ITGAX, ITGB1, CD29, ITGB2 (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 fragments thereof, or any combination thereof. Activation domain

[0084] As used herein, a chimeric antibody receptor can comprise an activation domain. The activation domain can include, but is not limited to, CD3. CD3 is an element of the T cell receptor on naive T cells and has been shown to be an important intracellular activation element in CARs. In a preferred embodiment, the CD3 is CD3ζ. Hinge region

[0085] As used herein, a chimeric antibody receptor can comprise a hinge region. This is part of the extracellular domain and is sometimes referred to as the "spacer" region. According to the present invention, a variety of hinge regions can be used, including co-stimulatory molecules, immunoglobulin (Ig) sequences or other suitable molecules as described above, to achieve a desired special distance from the target cell. In certain embodiments, the entire extracellular region contains the hinge region. Transmembrane region

[0086] As used herein, a chimeric antibody receptor can include a transmembrane region / domain. The CAR can be designed to include a transmembrane domain that is fused to the extracellular domain of the CAR. Similarly, it 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 domains to the transmembrane domains of the same or different surface membrane proteins, thereby reducing interactions with other members of the receptor complex. The transmembrane domain can be from natural or synthetic sources. When the source is natural, the domain can be from any membrane-bound or transmembrane protein.The transmembrane regions specifically used in the present invention can be derived from (i.e., comprise or be engineered from), but are not limited to, CD28, CD28T, OX40, 4-1BB / CD137, CD2, CD3 (α, β, δ, ε, γ, ζ), 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 co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1; CD11a and CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Igα (CD79a), DAP10, Fcγ receptor, MHC class I molecule, TNFR, integrin, signaling lymphocytic activation molecule, BTLA, Toll ligand receptor, ICAM-1, CDS, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, ITGAE, CD103, ITGAL, CD1a, CD1b, CD1c, CD1d, ITGAM, ITGAX, ITGB1, CD29, ITGB2 (CD18), ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (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 fragments thereof, or any combination thereof. immune cell

[0087] According to specific aspects, the cells provided by the present invention are immune cells, which contain an isolated polynucleotide or vector, and the isolated polynucleotide or vector contains a nucleotide sequence encoding a CAR. The immune cells containing the isolated polynucleotide and / or vector of the present invention can be referred to as "engineered immune cells". Preferably, the engineered immune cells are derived from humans (human-derived before recombination).

[0088] For example, the engineered immune cells can be lymphoid cells. Non-limiting examples of lymphoid cells include T cells and natural killer (NK) cells. T cells express T cell receptors (TCRs), and most cells express α and β chains, while a small fraction of cells express γ and δ chains. The T cells that can be used as the engineered immune cells of the present invention can be CD4 + or CD8 + , and can include, but are not limited to, T helper cells (CD4 + ), cytotoxic T cells (also known as cytotoxic T lymphocytes, CTLs; CD8 + cells), and memory T cells, including central memory T cells, stem cell-like memory T cells, effector T cells, natural killer T cells, mucosa-associated invariant T cells, and γδ T cells. Other exemplary immune cells include, but are not limited to, phagocytes, antigen-presenting cells (APCs), or any immune cells expressing inhibitors of cell-mediated immune responses, such as immune checkpoint inhibitor pathway receptors (e.g., PD-1). The precursor cells of the immune cells that can be used according to the present invention include hematopoietic stem cells and / or progenitor cells. Hematopoietic stem cells and / or progenitor cells can be obtained from bone marrow, umbilical cord blood, and peripheral blood of adults after cytokine mobilization by methods known in the art. The immune cells are engineered to recombinantly express the CAR of the present invention.

[0089] The immune cells and their precursor cells can be isolated by methods known in the art, including commercially available methods (see, for example, Rowland Jones et al., Lymphocytes: A Practical Approach, Oxford University Press, New York, 2009). The sources of immune cells or their precursor cells 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 isolate cells to isolate or enrich the desired immune cells. For example, negative selection methods can be used to remove unwanted immune cells. In addition, positive selection methods can be used to isolate or enrich the desired immune cells or their precursors, or a combination of positive and negative selection methods can be used. If a specific type of cell, such as a specific T cell, is to be isolated, various cell surface markers or combinations of markers (such as CD3, CD4, CD8, CD34) can be used to isolate the cells.

[0090] The immune cells or their precursor cells can be autologous or non - autologous to the subject administered in the treatment methods of the present invention. Autologous cells are isolated from the subject and recombinant engineered immune cells expressing a CAR are administered thereto. Optionally, the cells can be obtained by leukapheresis, i.e., selectively removing white blood cells from the drawn blood, engineering them, and then transfusing them back into the donor. Alternatively, allogeneic cells from a non - subject non - autologous donor can be used. In the case of a non - autologous donor, the cells are typed and matched for human leukocyte antigen (HLA) to determine an appropriate level of compatibility. For both autologous and non - autologous cells, the cells are optionally cryopreserved until ready for use.

[0091] A variety of methods for isolating immune cells that can be used for recombinant expression of a CAR of the present invention have been previously described and can be used, including but not limited to, using peripheral donor lymphocytes (Sadelain et al., Nature Reviews Cancer, 2003; 3:35 - 45; Morgan et al., Nature, 2006; 314:126 - 9), using lymphocyte cultures from tumor - infiltrating lymphocytes (TILs) in tumor biopsies (Panelli et al., Journal of Immunology, 2000; 164:495 - 504; Panelli et al., Journal of Immunology, 2000; 164:4382 - 92) and using artificial antigen - presenting cells (AAPCs) or dendritic cells, selectively using in vitro - expanded antigen - specific peripheral blood leukocytes (Dupont et al., Cancer Research, 2005; 65:5417 - 427; Papanicolaou et al., Blood, 2003; 102:2498 - 505). In the case of using stem cells, the cells can be isolated by methods known in the art (see, e.g., Klug et al., Methods of Hematopoietic Stem Cells, Humana Press, New Jersey, 2002; Freshney et al., Culture of Human Stem Cells, John Wiley & Sons, 2007).

[0092] According to a specific embodiment, the method for preparing engineered immune cells includes transfecting or transducing immune effector cells isolated from an individual to make the immune effector cells express one or more CARs according to an embodiment of the present invention. For example, methods for preparing immune cells for immunotherapy are described in WO2014 / 130635, WO2013 / 176916, and WO2013 / 176915, which are hereby incorporated by reference in their entireties. The individual steps for preparing engineered immune cells are disclosed, for example, in WO2014 / 039523, WO2014 / 184741, WO2014 / 191128, WO2014 / 184744, and WO2014 / 184143, which are hereby incorporated by reference in their entireties.

[0093] In a specific embodiment, immune effector cells, such as T cells, are genetically modified with a CAR of the present invention (such as being transduced with a viral vector containing a nucleic acid encoding the CAR), and then activated and expanded in vitro. In various embodiments, T cells can be activated or expanded before or after genetic modification to express the CAR, using methods described, for example, in US6352694, US6534055, US6905680, US6692964, US5858358, US6887466, US6905681, US7144575, US7067318, US7172869, US7232566, US7175843, US5883223, US6905874, US6797514, US6867041, US2006 / 121005, which are incorporated herein by reference in their entireties. T cells can be expanded in vivo or in vitro. Generally, the T cells of the present invention can be expanded by contacting a reagent that stimulates signals associated with the CD3 / TCR complex attached to its surface and a ligand that stimulates co-stimulatory molecules on the surface of the T cells. As non-limiting examples, a population of T cells can be stimulated as described herein, for example, by contacting with an anti-CD3 antibody or an antigen-binding fragment thereof, or an anti-CD antibody immobilized on a surface, or by contacting with a calcium ionophore protein kinase C activator (such as bryostatin) conjugated thereto, or by activating the CAR itself. For co-stimulation of co-stimulatory molecules on the surface of T cells, ligands that bind to the co-stimulatory molecules are used. For example, under conditions suitable for stimulating T cell proliferation, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD38 antibody. Conditions suitable for T cell culture include, for example, a suitable culture medium (such as minimum essential medium or RPMI medium or X-vivo 5 (Lonza), which can contain factors necessary for proliferation and survival (such as serum (such as fetal bovine or human serum), cytokines (such as IL-2, IL-7, IL-15, and / or IL-17), insulin, IFN-γ, GM-CSF, TGF-β, and / or any other additives known to those skilled in the art for cell growth). In other embodiments, T cells can be activated and stimulated to proliferate together with feeder layer cells and appropriate antibodies and cytokines, using the methods described in US6040177, US5827642, and WO2012129514, which are incorporated herein by reference. Antibodies and antigen-binding domains

[0094] 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 of making the antibodies and CARs, and methods of using the antibodies and CARs to treat diseases including cancer, inflammatory diseases, and autoimmune diseases. The antigen-binding domains of the antibodies and CARs of the present invention have one or more desired 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 cell phagocytosis (ADCP), and / or antibody-dependent cell-mediated cytotoxicity (ADCC) against cells expressing EGFRvIII, and the ability to inhibit tumor growth in a subject and in animal models when administered alone or in combination with other anti-cancer therapies.

[0095] In general aspects, the present invention relates to isolated monoclonal antibodies or antigen-binding fragments thereof that bind EGFRvIII.

[0096] As used herein, the term "antibody" is used in a broad sense and includes monoclonal or polyclonal human, humanized, composite, and chimeric antibodies and immunoglobulin or antibody molecules of antigen fragments. Generally, an antibody is a protein or peptide chain that exhibits binding specificity to a particular antigen. The structure of an antibody is well known. Immunoglobulins can be classified into five major classes (i.e., IgA, IgD, IgE, IgG, and IgM) according to the amino acid sequence of the heavy chain constant domain. IgA and IgG are further subdivided into IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4 isotypes. Thus, the antibodies of the present invention can be any of the five major classes or the corresponding subclasses. Preferably, the antibodies of the present invention are IgG1, IgG2, IgG3, or IgG4. The light chains of antibodies of vertebrates can be divided into one of two distinct types according to the amino acid sequence of their constant domains, i.e., κ and λ. Thus, the antibodies of the present invention can comprise κ and λ light chain constant domains. According to specific embodiments, the antibodies of the present invention include heavy and / or light chain constant regions from rat or human antibodies. In addition to the heavy and light chain constant domains, an antibody also comprises an antigen-binding region, which is composed of a light chain variable region and a heavy chain variable region, and each heavy chain variable region contains three domains (i.e., complementarity-determining regions 1-3: CDR1, CDR2, and CDR3). The light chain variable region domains can alternatively be referred to as LCDR1, LCDR2, and LCDR3, and the heavy chain variable region domains can alternatively be referred to as HCDR1, HCDR2, and HCDR3.

[0097] "Complementary determining regions" (CDRs) are the antibody regions that bind antigen. CDRs can be defined using various descriptions, such as Kabat (Wu et al., Journal of Experimental Medicine 132:211-50, 1970) (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland, 1991), Chothia (Chothia et al., Journal of Molecular Biology, 196:901-17, 1987), IMGT (Lefranc et al., Developmental and Comparative Immunology, 27:55-77, 2003) and AbM (Martin and Thornton J, Journal of Molecular Biology 263:800-15, 1996). The correspondence between the various depictions and variable region numbering is described (see, e.g., Lefranc et al., Developmental and Comparative Immunology, 27:55-77, 2003; Honegger and Pluckthun, Journal of Molecular Biology 309:657-70, 2001; International ImMunoGeneTics (IMGT) database; web resource, http: / / www_imgt_org). Available programs such as abYsis from UCL Business PLC can be used to describe CDRs. Unless otherwise expressly stated in the specification, the terms "CDR", "HCDR1", "HCVR2", "HDDR3", "LCDR1", "LCDR2" and "LCDR3" as used herein include the CDRs defined by any of the above methods, Kabat, Chothia, IMGT or AbM. The correspondence between numbering systems, including, for example, Kabat numbering and the IMGT unique numbering system, is well known to those of skill in the art (see, e.g., Kabat; Chothia; Martin; Lefranc et al.).

[0098] Table A. CDRs determined by different methods. IMGT Kabat AbM Chothia <![CDATA[V H CDR1]]> 27-38 31-35 26-35 26-32 <![CDATA[V H CDR2]]> 56-65 50-65 50-58 53-55 <![CDATA[V H CDR3]]> 105-117 95-102 95-102 96-101 <![CDATA[V L CDR1]]> 27-38 24-34 24-34 26-32 <![CDATA[V L CDR2]]> 56-65 50-56 50-56 50-52 <![CDATA[V L CDR3]]> 105-117 89-97 89-97 91-96

[0099] As used herein, the term "isolated antibody" means an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds EGFRvIII is substantially free of antibodies that do not bind the same EGFRvIII). In addition, an isolated antibody is substantially free of other cellular materials and / or chemicals.

[0100] As used herein, the term "monoclonal antibody" refers to an antibody obtained from antibodies that are substantially homogeneous, i.e., the same antibody of the population is identical except for minor mutations that may occur naturally. The monoclonal antibodies described in the present invention can be prepared by hybridoma methods, phage display techniques, single lymphocyte gene cloning techniques, or DNA recombinant methods. For example, the monoclonal antibody can be produced by a hybridoma that includes B cells obtained from a transgenic non-human animal (such as a transgenic mouse or rat) whose genome includes a human heavy chain transgenic and a light chain transgenic.

[0101] As used herein, the terms "antigen-binding fragment" and / or "antigen-binding domain" refer to antibody fragments, e.g., diabodies, Fab, Fab', F(ab')2, 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 a portion of an antibody containing one or more CDRs, camelid single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or other antibody fragments that bind to an antigen but do not contain the complete antibody structure. The antigen-binding fragment is capable of binding to the same antigen to which the parental antibody or parental antibody fragment binds. According to specific embodiments, the antigen-binding fragment includes a light chain variable region, a light chain constant region, and a heavy chain Fd segment. According to other specific embodiments, the antigen-binding fragment includes Fab and F(ab'). The antigen-binding domain is capable of binding to the same antigen to which the parental antibody binds. According to specific embodiments, the antigen-binding domain comprises a single-chain antibody molecule (scFv).

[0102] As used herein, the term "single-chain antibody" refers to a conventional single-chain antibody in the art, which includes a heavy chain variable region and a light chain variable region linked by a short peptide consisting 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 contains a heavy chain variable region and a heavy chain constant region or only contains a heavy chain variable region.

[0103] 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 that of an antibody produced by a human using any technique known in the art. The definition of a human antibody includes a complete or full-length antibody, fragments thereof, and / or an antibody containing at least one human heavy chain and / or light chain polypeptide.

[0104] As used herein, the terms "humanized antibody" and / or "humanized antigen-binding domain" refer to non-human antibodies and / or non-human antigen-binding domains that have been modified to increase sequence homology with human antibodies and / or antigen-binding domains, thereby retaining the antigen-binding properties of the antibody and / or antigen-binding domain, but having reduced immunogenicity in the human body.

[0105] 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 molecules are derived from two or more species. The variable regions of the light and heavy chains typically correspond to the antibodies and / or antigen-binding domains from one mammalian species (such as mouse, rat, rabbit, etc.) and have variable regions with the desired specificity, affinity, and performance, while the constant regions correspond to the sequences of antibodies and / or antigen-binding domains from another mammalian species (such as human) to avoid eliciting an immune response in that species.

[0106] As used herein, the term "multispecific antibody" refers to an antibody that contains multiple immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence of the multiple has binding specificity for a first epitope and the second immunoglobulin variable domain sequence of the multiple has binding specificity for a second epitope. In one embodiment, the first and second epitopes are located on the same antigen, for example, on 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 located on different antigens, for example, on different proteins (or different subunits of a multimeric protein). In one embodiment, the multispecific antibody includes a third, fourth, or fifth immunoglobulin variable domain. In one embodiment, the multispecific antibody is a bispecific antibody molecule, a trispecific antibody molecule, or a tetra-specific antibody molecule.

[0107] As used herein, the term "bispecific antibody" refers to a multispecific antibody that binds no more than two epitopes or two antigens. Bispecific antibodies are characterized by having a first immunoglobulin variable domain sequence with binding specificity for a first epitope and a second immunoglobulin variable domain sequence with binding specificity for a second epitope. In one embodiment, the first and second epitopes are located on the same antigen, e.g., on the same protein (or multimeric protein subunit). In one embodiment, the first and second epitopes overlap or substantially overlap. In one embodiment, the first and second epitopes are located on different antigens, e.g., on different proteins (or different multimeric protein subunits). In one embodiment, a bispecific antibody comprises a heavy chain variable domain sequence and a light chain variable domain sequence with binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence with binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises a half antibody or fragment thereof with binding specificity for a first epitope, and a half antibody or fragment thereof with binding specificity for a second epitope. In one embodiment, a bispecific antibody comprises a scFv or fragment thereof with binding specificity for a first epitope, and a scFv or fragment thereof with binding specificity for a second epitope. In one embodiment, the first epitope is located on the 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, Claudin 18.2, MUC16, mesothelin, IL13Ra2, PSCA, p95HER2, ROR1, ROR2, NKp46, and / or other tumor-associated immunosuppressors or surface antigens. In one embodiment, the first and second epitopes are located on the same EGFRvIII of the present invention.

[0108] As used herein, an antibody and / or antigen-binding domain that "specifically binds to EGFRvIII" refers to an antibody and / or antigen-binding domain that binds to EGFRvIII (preferably human EGFRvIII) with a KD of 1×10 -7 M or lower, preferably 1×10 -8 M or lower, more preferably 5×10 -9 M or lower, 1×10 -9 M or lower, 5×10 -10 M or lower, or 1×10 -10M or lower. The term "KD" refers to the dissociation constant, which is obtained as the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed in molar concentration (M). Given the present disclosure, methods in the art can be used to determine the KD value of an antibody and / or antigen-binding domain. For example, the KD of an antibody and / or antigen-binding domain can be determined by using surface plasmon resonance, such as by using a biosensor system (e.g., system), or by using biolayer interferometry techniques (e.g., Octet RED96 system).

[0109] The smaller the KD value of an antibody and / or antigen-binding domain, the higher the affinity of the antibody and / or antigen-binding domain for binding to the target antigen.

[0110] As used herein, the term "IC 50 " refers to the half-maximal inhibitory concentration of the monoclonal or bispecific antibody or antigen-binding fragment thereof of the present invention. IC 50 is a measure of the potency of the monoclonal or bispecific antibody or antigen-binding fragment thereof of the present invention to inhibit the binding of a target antigen (i.e., a 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 KD of a monoclonal antibody or antigen-binding fragment thereof or a bispecific antibody or antigen-binding fragment thereof is less than about 10 -7 M, less than about 10 -8 M, less than about 10 -9 M, less than about 10 -10 M, less than about 10 -11 M, less than about 10 -12 M or less than about 10 -13 M.

[0111] As used herein, the term "EC 50 " refers to the half-maximal effective concentration of the monoclonal or bispecific antibody or antigen-binding fragment thereof of the present invention or a CAR comprising an antigen-binding domain. EC 50 refers to the half concentration at which a monoclonal or bispecific antibody or antigen-binding fragment thereof or a CAR comprising an antigen-binding domain induces a biological response (i.e., cell death) between baseline and maximum within a specific exposure time. 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 50 of less than about 1 μM, from about 1000 nM to about 100 nM, from about 100 nM to about 10 nM, from about 10 nM to about 1 nM, from about 1000 pM to about 500 pM, from about 500 pM to about 200 pM, less than about 200 pM, from about 200 pM to about 150 pM, from about 200 pM to about 100 pM, from about 100 pM to about 10 pM, or from about 10 pM to about 1 pM.

[0112] According to certain aspects, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof or a chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the monoclonal antibody or the antigen-binding fragment thereof or the antigen-binding domain comprises a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region comprising heavy-chain complementarity-determining region 1 (HCDR1), HCDR2, HCDR3, and the light-chain variable region comprising light-chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, having the following polypeptide sequences: (1) SEQ ID NO:3, 4, 5, 6, 7, and 8, respectively, or SEQ ID NO:9, 10, 11, 12, 13, and 14, respectively; (2) SEQ ID NO:17, 18, 19, 20, 21, and 22, respectively, or SEQ ID NO:23, 24, 25, 26, 27, and 28, respectively; (3) SEQ ID NO:31, 32, 33, 34, 35, and 36, respectively, or SEQ ID NO:37, 38, 39, 40, 41, and 42, respectively; (4) SEQ ID NO:45, 46, 47, 48, 49, and 50, respectively, or SEQ ID NO:51, 52, 53, 54, 55, and 56, respectively; (5) SEQ ID NO:59, 60, 61, 62, 63, and 64, respectively, or SEQ ID NO:65, 66, 67, 68, 69, and 70, respectively; (6) SEQ ID NO:73, 74, 75, 76, 77, and 78, respectively, or SEQ ID NO:79, 80, 81, 82, 83, and 84, respectively; (7) SEQ ID NO:87, 88, 89, 90, 91, and 92, respectively, or SEQ ID NO:93, 94, 95, 96, 97, and 98, respectively; (8) SEQ ID NO:101, 102, 103, 104, 105, and 106, respectively, or SEQ ID NO: 107, 108, 109, 110, 111, and 112, respectively; (9) are SEQ ID NO:115, 116, 117, 118, 119 and 120 respectively, or are SEQ ID NO: 121, 122, 123, 124, 125 and 126 respectively; (10) are SEQ ID NO:129, 130, 131, 132, 133 and 134 respectively, or are SEQ ID NO: 135, 136, 137, 138, 139 and 140 respectively; (11) are SEQ ID NO:143, 144, 145, 146, 147 and 148 respectively, or are SEQ ID NO: 149, 150, 151, 152, 153 and 154 respectively; (12) are SEQ ID NO:157, 158, 159, 160, 161 and 162 respectively, or are SEQ ID NO: 163, 164, 165, 166, 167 and 168 respectively; (13) are SEQ ID NO:171, 172, 173, 174, 175 and 176 respectively, or are SEQ ID NO: 177, 178, 179, 180, 181 and 182 respectively; (14) are SEQ ID NO:185, 186, 187, 188, 189 and 190 respectively, or are SEQ ID NO: 191, 192, 193, 194, 195 and 196 respectively; (15) are SEQ ID NO:199, 200, 201, 202, 203 and 204 respectively, or are SEQ ID NO: 205, 206, 207, 208, 209 and 210 respectively; (16) are SEQ ID NO:213, 214, 215, 216, 217 and 218 respectively, or are SEQ ID NO: 219, 220, 221, 222, 223 and 224 respectively; (17) are SEQ ID NO:227, 228, 229, 230, 231 and 232 respectively, or are SEQ ID NO: 233, 234, 235, 236, 237 and 238 respectively; (18) are SEQ ID NO:241, 242, 243, 244, 245 and 246 respectively, or are SEQ ID NO: 247, 248, 249, 250, 251 and 252; (19) are SEQ ID NO:255, 256, 257, 258, 259 and 260, respectively, or are SEQ ID NO: 261, 262, 263, 264, 265 and 266; (20) are SEQ ID NO:269, 270, 271, 272, 273 and 274, respectively, or are SEQ ID NO: 275, 276, 277, 278, 279 and 280; (21) are SEQ ID NO:283, 284, 285, 286, 287 and 288, respectively, or are SEQ ID NO: 289, 290, 291, 292, 293 and 294; (22) are SEQ ID NO:297, 298, 299, 300, 301 and 302, respectively, or are SEQ ID NO: 303, 304, 305, 306, 307 and 308; (23) are SEQ ID NO:311, 312, 313, 314, 315 and 316, respectively, or are SEQ ID NO: 317, 318, 319, 320, 321 and 322; (24) are SEQ ID NO:325, 326, 327, 328, 329 and 330, respectively, or are SEQ ID NO: 331, 332, 333, 334, 335 and 336; or (25) are SEQ ID NO:341, 342, 343, 344, 345 and 346, respectively, or are SEQ ID NO:347, 348, 349, 350, 351 and 352; wherein the antibody or its antigen-binding fragment or its antigen-binding domain specifically binds to EGFRvIII, preferably human EGFRvIII.

[0113] According to another specific aspect, the present invention relates to a separated monoclonal antibody or an antigen-binding fragment thereof or a chimeric antigen receptor (CAR) comprising an antigen-binding domain, wherein the monoclonal antibody or an antigen-binding fragment thereof or the antigen-binding domain comprises a heavy-chain variable region or a light-chain variable region, and the heavy-chain variable region has a polypeptide sequence having at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98% or 99% identity with one of the following sequences: 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 the light-chain variable region has a polypeptide sequence having at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98% or 99% identity with the following sequence: 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.According to a preferred embodiment, the isolated monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof of the present invention comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence having at least 85%, preferably 90%, more preferably 95% or higher, such as 95%, 96%, 97%, 98% or 99% identity with the following sequences: 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 the light chain variable region has a polypeptide sequence having at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the following sequences: 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.

[0114] According to another specific aspect, the present invention relates to the isolated monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof of the present invention, which comprises: (1) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 1, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 2; (2) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 15, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 16; (3) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 29, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 30; (4) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 43, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 44; (5) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 57, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 58; (6) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 71, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 72; (7) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 85, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 86; (8) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 99, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 100; (9) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 113, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 114; (10) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 127, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 128; (11) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 141, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 142; (12) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 156; (13) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 170; (14) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 183, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 184; (15) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 197, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 198; (16) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 211, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 212; (17) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 225, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 226; (18) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 239, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 240 shown in the polypeptide sequence; (19) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 253, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 254; (20) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 267, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 268; (21) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 281, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 282; (22) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 295, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 296; (23) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 309, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 310; (24) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 323, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 324; (25) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 339, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 340; (26) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 353, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (27) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 353, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (28) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 353, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (29) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 354, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (30) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 354, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (31) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 354, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (32) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 355, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (33) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 355, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358 shown; (34) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 355, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (35) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 356, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (36) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 356, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (37) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 356, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (38) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 360, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (39) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 360, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (40) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 361, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (41) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 361, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (42) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 362, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (43) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 362, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (44) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 363, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (45) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 363, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (46) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (47) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (48) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372 shown; (49) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (50) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (51) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (52) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (53) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (54) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (55) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (56) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (57) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (58) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (59) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (60) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (61) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (62) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (63) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379 shown; (64) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (65) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (66) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (67) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (68) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (69) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (70) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (71) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (72) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (73) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (74) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (75) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (76) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (77) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (78) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 382, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386 shown; (79) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 382, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; (80) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 382, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388; (81) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 383, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (82) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 383, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; (83) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 383, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388; (84) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 384, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (85) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 384, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; (86) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 384, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388; (87) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 385, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (88) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 385, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; or (89) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 385, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388.

[0115] In one embodiment, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 3, 4, 5, 6, 7 and 8 respectively, or have the polypeptide sequences shown in SEQ ID NO: 9, 10, 11, 12, 13 and 14 respectively. In another embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 1, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 2. Preferably, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 1; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 2.

[0116] In one embodiment, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 17, 18, 19, 20, 21 and 22, respectively, or have the polypeptide sequences shown in SEQ ID NO: 23, 24, 25, 26, 27 and 28, respectively. In another embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 15, and the light-chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 16. Preferably, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 15; and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 16.

[0117] In one embodiment, the present invention relates to an isolated monoclonal antibody or an antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 31, 32, 33, 34, 35 and 36, respectively, or have the polypeptide sequences shown in SEQ ID NO: 37, 38, 39, 40, 41 and 42, respectively. In another embodiment, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 29, and the light-chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 30. Preferably, the isolated monoclonal antibody or an antigen-binding fragment thereof comprises a heavy-chain variable region having the polypeptide sequence shown in SEQ ID NO: 29; and a light-chain variable region having the polypeptide sequence shown in SEQ ID NO: 30.

[0118] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 45, 46, 47, 48, 49 and 50 respectively, or the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO: 43, and the light chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity 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 shown in SEQ ID NO: 43; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 44.

[0119] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 59, 60, 61, 62, 63 and 64 respectively, or the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO: 57, and the light chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity 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 shown in SEQ ID NO: 57; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 58.

[0120] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 73, 74, 75, 76, 77 and 78 respectively, or have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO: 71, and the light chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity 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 shown in SEQ ID NO: 71; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 72.

[0121] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 87, 88, 89, 90, 91 and 92 respectively, or have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO: 85, and the light chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity 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 shown in SEQ ID NO: 85; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 86.

[0122] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 101, 102, 103, 104, 105 and 106, respectively, or have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 99, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 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 shown in SEQ ID NO: 99; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 100.

[0123] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 115, 116, 117, 118, 119 and 120, respectively, or have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 113, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 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 shown in SEQ ID NO: 113; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 114.

[0124] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 129, 130, 131, 132, 133 and 134 respectively, or have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 127, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 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 shown in SEQ ID NO: 127; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 128.

[0125] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 143, 144, 145, 146, 147 and 148 respectively, or have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 141, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 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 shown in SEQ ID NO: 141; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 142.

[0126] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 157, 158, 159, 160, 161 and 162 respectively, or which have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 155, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 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 shown in SEQ ID NO: 155; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 156.

[0127] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 171, 172, 173, 174, 175 and 176 respectively, or which have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 169, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (such as 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 shown in SEQ ID NO: 169; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 170.

[0128] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 185, 186, 187, 188, 189 and 190, respectively, or have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 183, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 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 shown in SEQ ID NO: 183; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 184.

[0129] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 199, 200, 201, 202, 203 and 204, respectively, or have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 197, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 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 shown in SEQ ID NO: 197; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 198.

[0130] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 213, 214, 215, 216, 217 and 218 respectively, or have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO: 211, and the light chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity 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 shown in SEQ ID NO: 211; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 212.

[0131] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 227, 228, 229, 230, 231 and 232 respectively, or have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO: 225, and the light chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity 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 shown in SEQ ID NO: 225; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 226.

[0132] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 241, 242, 243, 244, 245 and 246 respectively, or which have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO: 239, and the light chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity 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 shown in SEQ ID NO: 239; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 240.

[0133] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 255, 256, 257, 258, 259 and 260 respectively, or which have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO: 253, and the light chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity 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 shown in SEQ ID NO: 253; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 254.

[0134] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 269, 270, 271, 272, 273 and 274 respectively, or have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO: 267, and the light chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity 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 shown in SEQ ID NO: 267; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 268.

[0135] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 283, 284, 285, 286, 287 and 288 respectively, or have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO: 281, and the light chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity 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 shown in SEQ ID NO: 281; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 282.

[0136] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have polypeptide sequences shown in SEQ ID NO: 297, 298, 299, 300, 301 and 302 respectively, or have polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO: 295, and the light chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity 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 shown in SEQ ID NO: 295; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 296.

[0137] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have polypeptide sequences shown in SEQ ID NO: 311, 312, 313, 314, 315 and 316 respectively, or have polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity to SEQ ID NO: 309, and the light chain variable region having a polypeptide sequence with at least 85%, preferably 90%, more preferably 95% or higher (such as 95%, 96%, 97%, 98% or 99%) identity 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 shown in SEQ ID NO: 309; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 310.

[0138] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 325, 326, 327, 328, 329 and 330, respectively, or which have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 323, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 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 shown in SEQ ID NO: 323; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 324.

[0139] In one embodiment, the present invention relates to an isolated monoclonal antibody or antigen-binding fragment thereof, which comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, which have the polypeptide sequences shown in SEQ ID NO: 341, 342, 343, 344, 345 and 346, respectively, or which have the polypeptide sequences shown in SEQ ID NO: 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 and a light chain variable region, the heavy chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 95%, 96%, 97%, 98% or 99%) identical to SEQ ID NO: 339, and the light chain variable region having a polypeptide sequence that is at least 85%, preferably 90%, more preferably 95% or higher (e.g., 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 shown in SEQ ID NO: 339; and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 340.

[0140] According to another specific aspect, the present invention relates to an isolated monoclonal antibody of the present invention or an antigen-binding fragment or antigen-binding domain thereof, wherein the antibody or its antigen-binding fragment or antigen-binding domain is chimeric.

[0141] According to another specific aspect, the present invention relates to an isolated monoclonal antibody of the present invention or an antigen-binding fragment or antigen-binding domain thereof, wherein the isolated monoclonal antibody or its antigen-binding fragment or antigen-binding domain is human or humanized.

[0142] According to another specific aspect, the antigen-binding domain is a single-chain variable fragment (scFv).

[0143] In certain embodiments, the encoded antigen-binding domain is a humanized single-chain variable fragment (scFv).

[0144] According to another specific aspect, the chimeric antigen receptor comprises one or more antigen-binding domains.

[0145] According to another specific aspect, the intracellular signaling domain comprises one or more co-stimulatory domains and one or more activation domains.

[0146] In another general aspect, the present invention relates to an isolated nucleic acid encoding the monoclonal antibody of the present invention or an antigen-binding fragment thereof and / or a bispecific antibody or an antigen-binding fragment thereof. In another general aspect, the present invention relates to an isolated polynucleotide comprising a nucleic acid encoding the chimeric antigen receptor (CAR) of the present invention, 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 of the present invention or an antigen-binding fragment thereof can be altered without changing the amino acid sequence of the protein.

[0147] In another general aspect, the present invention relates to a vector comprising a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof, a bispecific antibody or an antigen-binding fragment thereof, and / or a CAR of the present invention. According to the present disclosure, any vector known to those skilled in the art can be used, such as a plasmid, cosmid, phage vector or viral vector. In some embodiments, the vector is a recombinant expression vector, such as a plasmid. The vector may include any element that performs the conventional functions of an expression vector, such as a promoter, ribosome-binding element, terminator, enhancer, selection marker, and origin of replication. The promoter may be a constitutive, inducible or repressible promoter. Many expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to produce an antibody or an antigen-binding fragment thereof in a cell. According to an embodiment of the present invention, recombinant expression vectors can be generated using conventional cloning techniques or artificial gene synthesis.

[0148] In another general aspect, the present invention relates to a host cell comprising a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof and / or a bispecific antibody or an antigen-binding fragment thereof of the present invention. According to the present disclosure, any host cell known to those skilled in the art can be used for recombinant expression of the antibody or an antigen-binding fragment thereof of the present invention. In some embodiments, the host cell is an Escherichia coli TG1 or BL21 cell (for expressing, for example, scFv or Fab antibodies), a CHO-DG44 or CHO-K1 cell or a HEK293 cell (for expressing, for example, full-length IgG antibodies). According to a specific embodiment, the recombinant expression vector is transformed into the host cell by conventional methods (such as chemical transfection, heat shock or electroporation), wherein it is stably integrated into the host cell genome such that the recombinant nucleic acid is effectively expressed.

[0149] In another general aspect, the present invention relates to a method for producing a monoclonal antibody or an antigen-binding fragment thereof and / or a bispecific antibody or an antigen-binding fragment thereof of the present invention, comprising culturing a cell comprising a nucleic acid encoding a monoclonal antibody or an antigen-binding fragment thereof or a bispecific antibody or an antigen-binding fragment thereof under conditions for producing the monoclonal antibody or an antigen-binding fragment thereof or the bispecific antibody or an antigen-binding fragment thereof of the present invention, and recovering the antibody or an antigen-binding fragment thereof from the cell or cell culture (for example, from the supernatant). The expressed antibody or an antigen-binding fragment thereof can be harvested from the cell and purified according to conventional techniques known in the art and the techniques described herein.

[0150] In another general aspect, the present invention relates to cells transduced with a vector comprising a nucleic acid encoding the CAR of the present invention. The term "transduced" or "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transduced" cell is a cell that has been transduced with exogenous nucleic acid. The cells include primary subject cells and their progeny. In certain embodiments, the cells are CAR-T cells, preferably human CAR-T cells, wherein the T cells are engineered to express the CAR of the present invention for treating diseases such as cancer. In certain embodiments, the cells are CAR-NK cells, preferably human CAR-NK cells, wherein the NK cells engineered to express the CAR of the present invention are used for treating diseases such as cancer.

[0151] In another general aspect, the present invention relates to a method for preparing CAR-T cells by transducing T cells with a vector comprising a nucleic acid encoding the CAR of the present invention.

[0152] In another general aspect, the present invention relates to a method for producing the CAR-T cells of the present invention, the method comprising culturing T cells comprising a nucleic acid encoding a chimeric antigen receptor (CAR) of the present invention under conditions for producing CAR-T cells and recovering the CAR-T cells.

[0153] In another general aspect, the present invention relates to a method for preparing CAR-NK cells by transducing NK cells with a vector comprising a nucleic acid encoding the CAR of the present invention.

[0154] In another general aspect, the present invention relates to a method for producing the CAR-NK cells of the present invention, the method comprising: culturing NK cells containing a nucleic acid of the chimeric antigen receptor (CAR) of the present invention under conditions for producing CAR-NK cells and recovering the CAR-NK cells.

[0155] In another general aspect, the present invention relates to a method for producing a population of RNA-engineered cells containing the chimeric antigen receptor (CAR) of the present invention. The method comprises contacting the cell population with a polynucleotide comprising a nucleic acid encoding the CAR of the present invention, wherein the polynucleotide is in vitro transcribed RNA or synthetic RNA. Pharmaceutical composition

[0156] In another general aspect, the present invention relates to a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof, bispecific antibody or antigen-binding fragment thereof, isolated polynucleotide, isolated polypeptide, host cell and / or engineered immune cell of the present invention, and a pharmaceutically acceptable carrier.

[0157] 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 an antigen-binding fragment thereof of the invention, and / or a bispecific antibody and 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, as well as compositions containing them, can also be used to prepare pharmaceuticals for the therapeutic uses mentioned herein.

[0158] As used herein, the term "carrier" refers to any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid-containing vesicle, microsphere, liposomal encapsulant, or other material known in the art for pharmaceutical formulations. It is understood that the properties 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 effectiveness or biological activity of the compositions of the invention. In accordance with specific embodiments, and given the present disclosure, any pharmaceutically acceptable carrier suitable for antibody pharmaceutical compositions can be used in the present invention.

[0159] Formulations of the pharmaceutically active ingredient with pharmaceutically acceptable carriers are known in the art, for example, in Remington: The Science and Practice of Pharmacy (e.g., 21st Edition (2005) and any subsequent editions). Non-limiting examples of additional ingredients include buffers, diluents, solvents, tonicity modifiers, preservatives, stabilizers, and chelating agents. One or more pharmaceutically acceptable carriers can be used in formulating the pharmaceutical compositions of the invention.

[0160] In one embodiment of the invention, the pharmaceutical composition is a liquid formulation. Preferred examples of liquid formulations are aqueous formulations, i.e., formulations that contain water. Liquid formulations can include solutions, suspensions, emulsions, microemulsions, gels, and the like. 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.

[0161] In one embodiment, the pharmaceutical composition can be formulated as an injectable, which can be injected, for example, by an injection device (such as a syringe or an infusion pump). The injectable can be delivered, for example, subcutaneously, intramuscularly, intraperitoneally, intravitreally, or intravenously.

[0162] In another embodiment, the pharmaceutical composition is a solid preparation, such as a freeze-dried or spray-dried composition, which can be used as such or with a solvent and / or diluent added by a doctor or a patient before use. The solid dosage forms can include tablets, such as compressed tablets and / or coated tablets, and capsules (such as hard or soft gelatin capsules). The pharmaceutical composition can also be in the form of, for example, sachets, dragees, powders, granules, lozenges or powders for reconstitution.

[0163] The dosage forms can be immediate-release, in which case they can contain a water-soluble or dispersible carrier, or can be delayed-release, sustained-release or modified-release, in which case they can contain a water-insoluble polymer that regulates the dissolution rate of the dosage form in the gastrointestinal tract or subcutaneously.

[0164] In other embodiments, the pharmaceutical composition can be delivered intranasally, buccally or sublingually.

[0165] The pH value of the aqueous preparation can be between pH 3 and pH 10. In one embodiment of the present invention, the pH value of the preparation is from about 7.0 to about 9.5. In another embodiment of the present invention, the pH value of the preparation is from about 3.0 to about 7.0.

[0166] In another embodiment of the present invention, the pharmaceutical composition contains a buffer. Non-limiting examples of buffers include: arginine, aspartic acid, diglycine, citric acid, disodium hydrogen phosphate, fumaric acid, glycine, glycylglycine, histidine, lysine, maleic acid, malic acid, sodium acetate, sodium carbonate, sodium dihydrogen phosphate, sodium phosphate, succinate, tartaric acid, tricine and tris(hydroxymethyl)-aminomethane and mixtures thereof. The buffer can be present alone or in aggregates, at a concentration of from about 0.01 mg / ml to about 50 mg / ml, such as from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions containing each of these specific buffers constitute alternative embodiments of the present invention.

[0167] In another embodiment of the present invention, the pharmaceutical composition contains a preservative. Non-limiting examples of preservatives include: benzethonium chloride, benzoic acid, benzyl alcohol, bronopol, butyl parahydroxybenzoate, chlorobutanol, chlorocresol, chlorhexidine, chlorphenesin, o-cresol, m-cresol, p-cresol, ethyl parahydroxybenzoate, imidurea, methyl parahydroxybenzoate, phenol, 2-phenoxyethanol, 2-phenylethanol, propyl parahydroxybenzoate, sodium dehydroacetate, thimerosal and mixtures thereof. The preservative can be present alone or in aggregates, at a concentration of from about 0.01 mg / ml to about 50 mg / ml, such as from about 0.1 mg / ml to about 20 mg / ml. Pharmaceutical compositions containing each of these specific preservatives constitute alternative embodiments of the present invention.

[0168] In another embodiment of the present invention, the pharmaceutical composition comprises an isotonic agent. Non-limiting examples of isotonic 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, 1,3-propanediol, and 1,3-butanediol), polyethylene glycols (e.g., PEG400), and mixtures thereof. Another example of an isotonic agent includes sugars. Non-limiting examples of sugars can be monosaccharides, disaccharides, or polysaccharides, or water-soluble dextrans, including, for example, fructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose, trehalose, dextran, pullulan, dextrin, cyclodextrin, α- and β-HPCD, soluble starch, hydroxyethyl starch, and sodium carboxymethyl cellulose. Another example of an isotonic agent is 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 arabinitol. The isotonic agent can be present alone or in aggregates, at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. The pharmaceutical compositions containing each of these specific isotonic agents constitute alternative embodiments of the present invention.

[0169] In another embodiment of the present 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 alone or in aggregates, at a concentration of about 0.01 mg / ml to about 50 mg / ml, for example, about 0.1 mg / ml to about 20 mg / ml. The pharmaceutical compositions containing each of these specific chelating agents constitute alternative embodiments of the present invention.

[0170] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer. Non-limiting examples of stabilizers include one or more aggregation inhibitors, one or more oxidation inhibitors, one or more surfactants, and / or one or more protease inhibitors.

[0171] In another embodiment of the present invention, the pharmaceutical composition comprises a stabilizer, wherein the stabilizer is carboxy / hydroxy cellulose and its derivatives (such as HPC, HPC-SL, HPC-L, and HPMC), cyclodextrin, 2-methylthioethanol, polyethylene glycol (such as PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, salts (such as sodium chloride), sulfur-containing substances (such as monothioglycerol), or mercaptoacetic acid. The stabilizer can exist alone or in the form of aggregates, at a concentration of about 0.01 mg / ml to about 50 mg / ml, such as about 0.1 mg / ml to about 20 mg / ml. The pharmaceutical composition containing each of these specific stabilizers constitutes an alternative embodiment of the present invention.

[0172] 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) part and a lipid-soluble (lipophilic) part. Surfactants can be selected, for example, from the group consisting of: anionic surfactants, cationic surfactants, nonionic surfactants, and / or zwitterionic surfactants. The surfactant can exist alone or in the form of aggregates, at a concentration of about 0.1 mg / ml to about 20 mg / ml. The pharmaceutical composition containing each of these specific surfactants constitutes an alternative embodiment of the present invention.

[0173] In another embodiment of the present invention, the pharmaceutical composition comprises one or more protease inhibitors, such as EDTA and / or benzamidine hydrochloride (HCl). The protease inhibitor can exist alone or in the form of aggregates, at a concentration of about 0.1 mg / ml to about 20 mg / ml. The pharmaceutical composition containing each of these specific protease inhibitors constitutes an alternative embodiment of the present invention.

[0174] In another general aspect, the present invention relates to a method for preparing a pharmaceutical composition comprising the monoclonal antibody or its antigen-binding fragment and / or bispecific antibody or its antigen-binding fragment of the present invention, comprising combining the monoclonal antibody or its antigen-binding fragment, and / or bispecific antibody or its antigen-binding fragment with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition. Method of use

[0175] In another general aspect, the present invention relates to a method for targeting EGFRvIII on the surface of cancer cells of a subject to effect 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 EGFRvIII, or a pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof and / or bispecific antibody or antigen-binding fragment thereof of the present invention. The monoclonal or bispecific antibody or antigen-binding fragment of EGFRvIII that binds EGFRvIII can mediate complement-dependent cytotoxicity (CDC), antibody-dependent cell phagocytosis (ADCP), and / or antibody-dependent cell cytotoxicity (ADCC) or other effects that result in the death of target cancer cells. The monoclonal or bispecific antibody or antigen-binding fragment thereof can, for example, be used to recruit conjugated drugs, and / or can form a bispecific antibody with another monoclonal antibody to mediate the death of target cancer cells.

[0176] The functional activities of antibodies and antigen-binding fragments thereof that bind EGFRvIII can be characterized by methods known in the art and described herein. Methods for characterizing antibodies and antigen-binding fragments thereof that bind EGFRvIII include, but are not limited to, affinity and specificity assays, including Biacore, ELISA, OctetRed, and GatorBio assays, and detection of the binding of the antibody and antigen-binding fragment thereof to EGFRvIII on cells (cells transfected with EGFRvIII or cells that naturally express EGFRvIII) by FACS. According to specific embodiments, the methods for characterizing antibodies and antigen-binding fragments thereof that bind EGFRvIII include the methods described below.

[0177] In another general aspect, the present invention relates to a method for treating cancer in a subject in need thereof, comprising administering to the subject an isolated monoclonal antibody or antigen-binding fragment thereof and / or a bispecific antibody or antigen-binding fragment thereof or a pharmaceutical composition that specifically binds EGFRvIII of the present invention. The cancer can, for example, be selected from, but not limited to, lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocellular carcinoma, 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, and non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors.

[0178] In another general aspect, the present invention relates to a method for treating inflammation and / or autoimmune diseases in a subject in need thereof, comprising administering to the subject an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to EGFRvIII of the present invention and / or a specific antibody or an antigen-binding fragment thereof or a pharmaceutical composition.

[0179] In another general aspect, the present invention relates to a method for treating cancer in a subject in need thereof, comprising administering to the subject the CAR-T cells and / or CAR-NK cells of the present invention. The cancer can be selected from, but not limited to, lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocellular carcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, 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 lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

[0180] According to an embodiment of the present invention, the CAR-T cells or CAR-NK cells comprise a therapeutically effective amount of the CAR expressed by 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 an active ingredient or component that elicits a desired biological or medical response in a subject. The therapeutically effective amount can be determined empirically and routinely according to the purpose.

[0181] As used for the CAR mentioned herein, the therapeutically effective amount refers to the amount of the CAR molecule expressed in the transduced T cells or NK cells, which regulates the immune response of a subject in need thereof. In addition, as used for the CAR herein, the therapeutically effective amount refers to the amount of the CAR molecule expressed in the transduced T cells or NK cells, which results in the treatment of a disease, disorder or condition; prevention or slowing of the progression of a disease, disorder or condition; or alleviation or complete remission of symptoms associated with a disease, disorder or condition.

[0182] As used for the CAR-T cells or CAR-NK cells mentioned herein, the therapeutically effective amount refers to the amount of CAR-T cells or CAR-NK cells that regulate the immune response of a subject in need thereof. In addition, as used for the CAR-T cells or CAR-NK cells herein, the therapeutically effective amount refers to the amount of CAR-T cells or CAR-NK cells, which results in the treatment of a disease, disorder or condition; prevention or slowing of the progression of a disease, disorder or condition; or alleviation or complete remission of symptoms associated with a disease, disorder or condition.

[0183] As used herein with respect to the anti-EGFRvIII antibody or antigen-binding fragment thereof, a therapeutically effective amount refers to the amount of the anti-EGFRvIII antibody or antigen-binding fragment thereof that modulates the immune response of a subject in need thereof. Additionally, as used herein with respect to the anti-EGFRvIII antibody or antigen-binding fragment thereof, a therapeutically effective amount refers to the amount of the anti-EGFRvIII antibody or antigen-binding fragment thereof that results in the treatment of a disease, disorder, or condition; the prevention or slowing of the progression of a disease, disorder, or condition; or the reduction or complete alleviation of symptoms associated with a disease, disorder, or condition.

[0184] According to specific embodiments, the disease, disorder, or condition to be treated is cancer, preferably, the cancer is selected from the group consisting of: lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocellular carcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, 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 lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML), and other liquid tumors. According to other specific embodiments, the disease, disorder, or condition to be treated is an inflammatory and / or autoimmune disease.

[0185] According to specific embodiments, a therapeutically effective amount refers to a therapeutic amount sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or improving the severity of the disease, disorder, or condition to be treated or symptoms associated therewith; (ii) reducing the duration of the disease, disorder, or condition to be treated or symptoms associated therewith; (iii) preventing the progression of the disease, disorder, or condition to be treated or symptoms associated therewith; (iv) causing the regression of the disease, disorder, or condition to be treated or symptoms associated therewith; (v) preventing the development or onset of the disease, disorder, or condition to be treated or symptoms associated therewith; (vi) preventing the recurrence of the disease, disorder, or condition to be treated or symptoms associated therewith; (vii) reducing the 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, or symptoms associated therewith; (ix) increasing the survival rate of a subject having the disease, disorder, or condition, or symptoms associated therewith; (xi) inhibiting or alleviating 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 effect of another therapy.

[0186] The therapeutically effective amount or dose can vary depending on various factors such as the disease, disorder or condition to be treated, the mode of administration, the target, the physiological state of the subject (including, for example, age, weight, health status), whether the subject is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. The therapeutic dose is optimally titrated to optimize safety and efficacy.

[0187] According to specific embodiments, the compositions described herein are formulated to be suitable for the intended route of administration to the subject. For example, the compositions described herein can be formulated for intravenous, subcutaneous or intramuscular administration.

[0188] 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 by arterial, subcutaneous, intradermal, intratumoral, intranodular, intramedullary, intramuscular, intrapleural, intravenous (iv) injection or intraperitoneal administration. In certain embodiments, the cells of the present invention can be administered with or without lymphatic failure in the subject.

[0189] A pharmaceutical composition comprising the cells of the present invention expressing the CAR of the present invention can be provided in the form of a sterile liquid preparation, typically an isotonic aqueous solution with a cell suspension, or optionally as an emulsion, dispersion, etc., usually buffered to a selected pH. The composition can comprise a carrier such as water, saline, phosphate buffered saline, etc., suitable for the integrity and viability of the cells, and suitable for the administration of the cell composition.

[0190] If desired, a sterile injectable solution can be prepared by adding the cells of the present invention together with various other ingredients to a suitable solvent in an appropriate amount. Such compositions can include pharmaceutically acceptable carriers, diluents or excipients such as sterile water, physiological saline, glucose, dextrose, etc., which are suitable for use with the cell composition and for administration to a subject (such as a human). Suitable buffers for providing the cell composition are well known in the art. Any carrier, diluent or additive used is compatible with maintaining the integrity and viability of the cells of the present invention.

[0191] The cells of the present invention can be administered in any physiologically acceptable carrier. A cell population comprising the cells of the present invention can comprise a purified cell population. A person skilled in the art can readily determine the cells in the cell population using various well-known methods. The purity range of a cell population comprising the genetically modified cells of the present invention can be from 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%. A person skilled in the art can readily adjust the dose, for example, a decrease in purity may require an increase in dose.

[0192] The cells of the present invention are typically administered at a dose based on the number of cells per kilogram body weight of the subject to whom the cells are administered (cells / kg). Generally, the cell dose is in the range of about 10 4 to about 10 10 cells / kg body weight, for example, depending on the mode and location of administration, about 10 5 to about 10 9 , about 10 5 to about 10 8 , about 10 5 to about 10 7 or about 10 5 to about 10 6 . Generally, in the case of systemic administration, a higher dose is used than in local administration, in which the immune cells of the present invention are administered in the area of a tumor and / or cancer. Exemplary dose ranges include, but are not limited to, 1x10 4 to 1x10 8 , 2x10 4 to 1x10 8 , 3x10 4 to 1x10 8 , 4x10 4 to 1x10 8 , 5x10 4 to 6x10 8 , 7x10 4 to 1x10 8 , 8x10 4 to 1x10 8 , 9x10 4 to 1x10 8 , 1x10 5 to 1x10 8 , 1x10 5 to 9x10 7 , 1x10 5 to 8x10 7 , 1x10 5 to 7x107 and 10 5 to 6x10 7 and 1x10 5 to 5x10 7 and 1x10 5 to 4x10 7 and 1x10 5 to 4x10 7 and 1x10 5 to 3x10 7 and 1x10 5 to 2x10 7 and 1x10 5 to 1x10 7 and 1x10 5 to 9x10 6 and 1x10 5 to 8x10 6 and 1x10 5 to 7x10 6 and 1x10 5 to 6x10 6 and 1x10 5 to 5x10 6 and 1x10 5 to 4x10 6 and 1x10 5 to 4x10 6 and 1x10 5 to 3x10 6 and 1x10 5 to 2x10 6 and 1x10 5 to 1x10 6 and 2x10 5 to 9x10 7 and 2x10 5 to 8x10 7 and 2x10 5 to 7x10 7 and 2x10 5 to 6x10 7 and 2x10 5 to 5x10 7 and 2x10 5 to 4x10 7 and 2x10 5 to 4x10 7 and 2x10 5 to 3x10 7 and 2x10 5 to 2x10 7 and 2x10 5 to 1x10 7 and 2x10 5to 9x10 6 、2x10 5 to 8x10 6 、2x10 5 to 7x10 6 、2x10 5 to 6x10 6 、2x10 5 to 5x10 6 、2x10 5 to 4x10 6 、2x10 5 to 4x10 6 、2x10 5 to 3x10 6 、2x10 5 to 2x10 6 、2x10 5 to 1x10 6 、3x10 5 to 3x10 6 cells / kg, etc. In addition, the dose can be adjusted according to whether it is a single dose or multiple doses. The effective dose can be precisely determined based on the individual factors of each subject.

[0193] As used herein, the terms "treat / treating / treatment" are all intended to mean improving or reversing at least one measurable physical parameter associated with cancer, inflammation, and / or autoimmune diseases, disorders, or conditions, which do not have to be distinguishable in a subject but can be distinguishable in a subject. The term "treatment" can also mean causing the regression of a disease, disorder, or condition, preventing its progression, or at least slowing its progression. In a specific embodiment, "treatment" means alleviating, preventing the development or onset of, or shortening the duration of one or more symptoms associated with a disease, disorder, or condition (such as a tumor or more preferably cancer). In a specific embodiment, "treatment" means preventing the recurrence of a disease, disorder, or condition. In a specific embodiment, "treatment" means increasing the survival rate of a subject suffering from a disease, disorder, or condition. In a specific embodiment, "treatment" means eliminating the disease, disorder, or condition of a subject.

[0194] According to specific embodiments, compositions are provided for treating cancer, inflammation, and / or autoimmune diseases, disorders, or conditions. For cancer treatment, the provided compositions can be used in combination with another treatment method, including but not limited to chemotherapy, anti-CD20 monoclonal antibody, anti-TIM-3 monoclonal antibody, anti-LAG-3 monoclonal antibody, anti-EGFR monoclonal antibody, anti-HER-2 monoclonal antibody, anti-CD19 monoclonal antibody, anti-CD33 monoclonal antibody, anti-CD47 monoclonal antibody, anti-CD73 monoclonal antibody, anti-DLL-3 monoclonal antibody, anti-apelin monoclonal antibody, anti-FOLR1 monoclonal antibody, anti-CTLA-4 monoclonal antibody, anti-PD-L1 monoclonal antibody, anti-PD-1 monoclonal antibody, anti-Claudin18.2 monoclonal antibody, other immuno-oncology drugs, anti-angiogenic agents, radiotherapy, antibody-drug conjugates (ADCs), targeted therapies, or other anti-cancer drugs. Antibodies against EGFRvIII can be used to construct bispecific antibodies together with paired monoclonal antibodies 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, Claudin18.2, folate receptor alpha (FOLR1), MUC16, mesothelin, PSCA, IL13Ra2, p95HER2, and / or any other tumor-associated antigen (TAA) to treat cancers / tumors expressing two antigens. Two antibodies recognizing two different epitopes on EGFRvIII can also be used to construct bispecific antibodies to treat cancers / tumors expressing EGFRvIII.

[0195] According to specific embodiments, a method of treating cancer in a subject in need thereof includes co-administering the CAR-T cells and / or CAR-NK cells of the present invention to the subject in combination with an agent that increases the efficacy of cells expressing the CAR molecule. Such agents include but are not limited to antibody fragments that bind to CD73, CD39, PD1, PD-L1, PD-L2, CTLA4, TIM3, or LAG3, or adenosine A2a receptor antagonists.

[0196] According to specific embodiments, a method of treating cancer in a subject in need thereof includes co-administering the CAR-T cells and / or CAR-NK cells of the present invention to the subject in combination with an agent that ameliorates one or more side effects associated with cells expressing the CAR molecule. Such agents include but are not limited to steroids, TNFα inhibitors, or IL-6 inhibitors.

[0197] According to specific embodiments, a method of treating cancer in a subject in need thereof includes co-administering to the subject the CAR-T cells and / or CAR-NK cells of the present invention and an agent for treating a disease associated with EGFRvIII. Such agents include, but are not limited to, anti-EGFRvIII monoclonal antibodies or bispecific antibodies.

[0198] As used herein, in the context of co-administering two or more therapies to a subject, the term "co-administer" refers to the use of more than one therapy. The use of the term "co-administer" does not limit the order in which the therapies are administered to the subject. For example, a first therapy (e.g., a composition described herein) can be administered before (e.g., 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 (e.g., 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) a second therapy is administered to the subject.

[0199] In another general aspect, the present invention relates to a method of determining the level of EGFRvIII in a subject. The method includes (a) obtaining a sample from the subject; (b) contacting the sample with a monoclonal antibody of the present invention or an antigen-binding fragment thereof; and (c) determining the level of EGFRvIII in the subject.

[0200] 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 fluid, ascites, interstitial fluid, bone marrow, cerebrospinal fluid, saliva, mucus, sputum, sweat, urine, or any other secretion, excretory product, or other body fluid. "Blood sample" refers to whole blood or any portion thereof, including blood cells, serum, and plasma.

[0201] In certain embodiments, assays selected from, but not limited to, Western blot analysis, immunohistochemistry (IHC), and ELISA assays can be utilized to determine the EGFRvIII level in a subject. Western blot analysis and IHC can be used to determine relative protein levels, and ELISA assays can be used to determine absolute protein levels. When determining the relative level of EGFRvIII, the EGFRvIII level can be determined between at least two samples (e.g., between samples from different time points of the same subject, between samples from different tissues of the same subject, and / or between samples from different subjects). Alternatively, when determining the absolute level of EGFRvIII, e.g., by ELISA assay, the absolute level of EGFRvIII in a sample can be determined by creating a standard for the ELISA assay prior to testing the sample. Those skilled in the art will understand which assay techniques to use to determine the EGFRvIII level in a subject sample using the antibodies or antigen-binding fragments thereof of the present invention.

[0202] Methods for determining the EGFRvIII level in a subject sample can be used to diagnose abnormal (elevated, decreased, or deficient) EGFRvIII levels in a disease and make appropriate treatment decisions. Such diseases can be selected from, but not limited to, cancer, inflammatory diseases, or autoimmune diseases. In addition, by monitoring the EGFRvIII level in a subject, the risk of occurrence of the above diseases can be determined based on the understanding of the EGFRvIII level in a specific disease and / or during the progression of a specific disease. Embodiment

[0203] The present invention also provides the following non-limiting embodiments.

[0204] Embodiment 1 is an isolated monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy-chain variable region and a light-chain variable region, the heavy-chain variable region comprising heavy-chain complementarity-determining region 1 (HCDR1), HCDR2, HCDR3, and the light-chain variable region comprising light-chain complementarity-determining region 1 (LCDR1), LCDR2, and LCDR3, having the following polypeptide sequences: (1) SEQ ID NO: 3, 4, 5, 6, 7, and 8, respectively, or SEQ ID NO: 9, 10, 11, 12, 13, and 14, respectively; (2) SEQ ID NO: 17, 18, 19, 20, 21, and 22, respectively, or SEQ ID NO: 23, 24, 25, 26, 27, and 28, respectively; (3) SEQ ID NO: 31, 32, 33, 34, 35, and 36, respectively, or SEQ ID NO: 37, 38, 39, 40, 41, and 42; (4) SEQ ID NO:45, 46, 47, 48, 49, and 50, respectively, or SEQ ID NO:51, 52, 53, 54, 55, and 56, respectively; (5) SEQ ID NO:59, 60, 61, 62, 63, and 64, respectively, or SEQ ID NO:65, 66, 67, 68, 69, and 70, respectively; (6) SEQ ID NO:73, 74, 75, 76, 77, and 78, respectively, or SEQ ID NO:79, 80, 81, 82, 83, and 84, respectively; (7) SEQ ID NO:87, 88, 89, 90, 91, and 92, respectively, or SEQ ID NO:93, 94, 95, 96, 97, and 98, respectively; (8) SEQ ID NO:101, 102, 103, 104, 105, and 106, respectively, or SEQ ID NO:107, 108, 109, 110, 111, and 112, respectively; (9) SEQ ID NO:115, 116, 117, 118, 119, and 120, respectively, or SEQ ID NO:121, 122, 123, 124, 125, and 126, respectively; (10) SEQ ID NO:129, 130, 131, 132, 133, and 134, respectively, or SEQ ID NO:135, 136, 137, 138, 139, and 140, respectively; (11) SEQ ID NO:143, 144, 145, 146, 147, and 148, respectively, or SEQ ID NO:149, 150, 151, 152, 153, and 154, respectively; (12) SEQ ID NO:157, 158, 159, 160, 161, and 162, respectively, or SEQ ID NO:163, 164, 165, 166, 167, and 168, respectively; (13) SEQ ID NO:171, 172, 173, 174, 175, and 176, respectively, or SEQ ID NO:177, 178, 179, 180, 181, and 182, respectively; (14) SEQ ID NO:185, 186, 187, 188, 189, and 190, respectively, or SEQ ID NO:191, 192, 193, 194, 195, and 196, respectively; (15) are SEQ ID NO:199, 200, 201, 202, 203 and 204 respectively, or are SEQ ID NO:205, 206, 207, 208, 209 and 210 respectively; (16) are SEQ ID NO:213, 214, 215, 216, 217 and 218 respectively, or are SEQ ID NO:219, 220, 221, 222, 223 and 224 respectively; (17) are SEQ ID NO:227, 228, 229, 230, 231 and 232 respectively, or are SEQ ID NO:233, 234, 235, 236, 237 and 238 respectively; (18) are SEQ ID NO:241, 242, 243, 244, 245 and 246 respectively, or are SEQ ID NO:247, 248, 249, 250, 251 and 252 respectively; (19) are SEQ ID NO:255, 256, 257, 258, 259 and 260 respectively, or are SEQ ID NO: 261, 262, 263, 264, 265 and 266; (20) are SEQ ID NO:269, 270, 271, 272, 273 and 274 respectively, or are SEQ ID NO: 275, 276, 277, 278, 279 and 280; (21) are SEQ ID NO:283, 284, 285, 286, 287 and 288 respectively, or are SEQ ID NO: 289, 290, 291, 292, 293 and 294; (22) are SEQ ID NO:297, 298, 299, 300, 301 and 302 respectively, or are SEQ ID NO: 303, 304, 305, 306, 307 and 308; (23) are SEQ ID NO:311, 312, 313, 314, 315 and 316 respectively, or are SEQ ID NO: 317, 318, 319, 320, 321 and 322; (24) are SEQ ID NO:325, 326, 327, 328, 329 and 330 respectively, or are SEQ ID NO: 331, 332, 333, 334, 335 and 336; or (25) are SEQ ID NO:341, 342, 343, 344, 345 and 346, or are SEQ ID NO: 347, 348, 349, 350, 351 and 352; wherein the antibody or antigen-binding fragment thereof specifically binds to EGFRvIII, preferably specifically binds to human EGFRvIII.

[0205] Embodiment 2 is an isolated monoclonal antibody or antigen-binding fragment as described in Embodiment 1, comprising a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence having at least 95% identity with the following sequences: 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 the light chain variable region has a polypeptide sequence having at least 95% identity with the following sequences: 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.

[0206] Embodiment 3 is an isolated monoclonal antibody or antigen-binding fragment as described in Embodiment 1 or 2, comprising: (1) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:1, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:2; (2) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:15, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:16; (3) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:29, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:30; (4) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO:43, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO:44; (5) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 57, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 58; (6) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 71, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 72; (7) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 85, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 86; (8) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 99, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 100; (9) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 113, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 114; (10) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 127, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 128; (11) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 141, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 142; (12) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 156; (13) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 170; (14) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 183, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 184; (15) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 197, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 198; (16) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 211, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 212; (17) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 225, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 226; (18) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 239, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 240; (19) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 253, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 254; (20) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 267, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 268; (21) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 281, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 282; (22) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 295, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 296; (23) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 309, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 310; (24) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 323, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 324; (25) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 339, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 340; (26) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 353, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (27) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 353, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (28) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 353, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (29) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 354, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (30) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 354, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 354, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (32) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 355, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (33) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 355, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (34) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 355, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (35) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 356, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (36) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 356, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (37) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 356, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (38) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 360, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (39) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 360, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (40) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 361, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (41) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 361, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (42) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 362, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (43) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 362, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (44) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 363, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (45) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 363, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (46) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (47) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (48) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (49) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373 polypeptide sequence; (50) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (51) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (52) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (53) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (54) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (55) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (56) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (57) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (58) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (59) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (60) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (61) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (62) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (63) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (64) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380 ; (65) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (66) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (67) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (68) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (69) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (70) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (71) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (72) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (73) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (74) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (75) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (76) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (77) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (78) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 382, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (79) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 382, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; (80) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 382, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388; (81) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 383, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (82) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 383, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; (83) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 383, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388; (84) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 384, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (85) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 384, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; (86) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 384, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388; (87) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 385, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (88) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 385, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; or (89) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 385, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388.

[0207] Embodiment 4 is an isolated monoclonal antibody or antigen-binding fragment as described in any one of Embodiments 1-3, wherein the antibody or its antigen-binding fragment is chimeric and / or human or humanized.

[0208] Embodiment 5 is an isolated monoclonal antibody or antigen-binding fragment as described in any one of Embodiments 1-4, wherein the isolated antibody or its antigen-binding fragment is capable of inducing effector-mediated tumor cell lysis by antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell 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 its antigen-binding fragment having cancer-killing effects.

[0209] Embodiment 6 is an isolated bispecific antibody or its antigen-binding fragment, comprising a monoclonal antibody or its antigen-binding fragment as described in any one of Embodiments 1-5.

[0210] Embodiment 7 is an isolated nucleic acid encoding a monoclonal antibody or antigen-binding fragment as described in any one of Embodiments 1-5 or a bispecific antibody or antigen-binding fragment as described in Embodiment 6.

[0211] Embodiment 8 is a vector that comprises the isolated nucleic acid as described in Embodiment 7.

[0212] Embodiment 9 is a host cell that comprises the vector as described in Embodiment 8.

[0213] Embodiment 10 is a pharmaceutical composition that comprises the isolated monoclonal antibody or antigen-binding fragment as described in any one of Embodiments 1-5, or the bispecific antibody or its antigen-binding fragment as described in Embodiment 6, and a pharmaceutically acceptable carrier.

[0214] 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 as described in Embodiment 10. Optionally, wherein the cancer is selected from the group consisting of: lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocellular carcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, 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 lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

[0215] Embodiment 12 is a method of producing the monoclonal antibody or antigen-binding fragment as described in any one of Embodiments 1-5, or the bispecific antibody or its antigen-binding fragment of Embodiment 6, comprising culturing a cell that comprises a nucleic acid encoding the monoclonal antibody or its antigen-binding fragment or the bispecific antibody or its antigen-binding fragment under conditions for producing the monoclonal antibody or its antigen-binding fragment or the bispecific antibody or its antigen-binding fragment, and recovering the monoclonal antibody or its antigen-binding fragment or the bispecific antibody or its antigen-binding fragment from the cell or the culture.

[0216] Embodiment 13 is a method of preparing a pharmaceutical composition that comprises the monoclonal antibody or its antigen-binding fragment as described in any one of Embodiments 1-5, or the bispecific antibody or its antigen-binding fragment as described in Embodiment 6, comprising combining the monoclonal antibody or its antigen-binding fragment or the bispecific antibody or its antigen-binding fragment with a pharmaceutically acceptable carrier to obtain the pharmaceutical composition.

[0217] Embodiment 14 is a method of determining the level of EGFRvIII in a subject, the method comprising: (a) obtaining a sample from the subject; (b) contacting the sample with the isolated monoclonal antibody or antigen-binding fragment thereof as described in any one of embodiments 1-5; and (c) determining the level of EGFRvIII in the subject.

[0218] Embodiment 15 is the method as described in Embodiment 14, wherein the sample is a tissue sample or a blood sample, optionally, wherein the tissue sample is a cancer tissue sample.

[0219] 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) a hinge region; (c) a transmembrane region; and (d) an intracellular signaling domain.

[0220] Embodiment 17 is the isolated polynucleotide as described in Embodiment 16, wherein the antigen-binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises heavy chain complementarity determining region 1 (HCDR1), HCDR2, HCDR3, the light chain variable region comprises light chain complementarity determining region 1 (LCDR1), LCDR2 and LCDR3, having the following polypeptide sequences: (1) SEQ ID NO:3, 4, 5, 6, 7 and 8 respectively, or SEQ ID NO:9, 10, 11, 12, 13 and 14 respectively; (2) SEQ ID NO:17, 18, 19, 20, 21 and 22 respectively, or SEQ ID NO:23, 24, 25, 26, 27 and 28 respectively; (3) SEQ ID NO:31, 32, 33, 34, 35 and 36 respectively or SEQ ID NO:37, 38, 39, 40, 41 and 42 respectively; (4) SEQ ID NO:45, 46, 47, 48, 49 and 50 respectively, or SEQ ID NO:51, 52, 53, 54, 55 and 56 respectively; (5) SEQ ID NO:59, 60, 61, 62, 63 and 64 respectively, or SEQ ID NO:65, 66, 67, 68, 69 and 70 respectively; (6) are SEQ ID NO:73, 74, 75, 76, 77 and 78 respectively, or are SEQ ID NO:79, 80, 81, 82, 83 and 84 respectively; (7) are SEQ ID NO:87, 88, 89, 90, 91 and 92 respectively, or are SEQ ID NO:93, 94, 95, 96, 97 and 98 respectively; (8) are SEQ ID NO:101, 102, 103, 104, 105 and 106 respectively, or are SEQ ID NO:107, 108, 109, 110, 111 and 112 respectively; (9) are SEQ ID NO:115, 116, 117, 118, 119 and 120 respectively, or are SEQ ID NO:121, 122, 123, 124, 125 and 126 respectively; (10) are SEQ ID NO:129, 130, 131, 132, 133 and 134 respectively, or are SEQ ID NO:135, 136, 137, 138, 139 and 140 respectively; (11) are SEQ ID NO:143, 144, 145, 146, 147 and 148 respectively, or are SEQ ID NO:149, 150, 151, 152, 153 and 154 respectively; (12) are SEQ ID NO:157, 158, 159, 160, 161 and 162 respectively, or are SEQ ID NO:163, 164, 165, 166, 167 and 168 respectively; (13) are SEQ ID NO:171, 172, 173, 174, 175 and 176 respectively, or are SEQ ID NO:177, 178, 179, 180, 181 and 182 respectively; (14) are SEQ ID NO:185, 186, 187, 188, 189 and 190 respectively, or are SEQ ID NO:191, 192, 193, 194, 195 and 196 respectively; (15) are SEQ ID NO:199, 200, 201, 202, 203 and 204 respectively, or are SEQ ID NO:205, 206, 207, 208, 209 and 210 respectively; (16) are SEQ ID NO:213, 214, 215, 216, 217 and 218 respectively, or are SEQ ID NO:219, 220, 221, 222, 223 and 224 respectively; (17) are SEQ ID NO:227, 228, 229, 230, 231 and 232 respectively, or are SEQ ID NO:233, 234, 235, 236, 237 and 238 respectively; (18) are SEQ ID NO:241, 242, 243, 244, 245 and 246 respectively, or are SEQ ID NO:247, 248, 249, 250, 251 and 252 respectively; (19) are SEQ ID NO:255, 256, 257, 258, 259 and 260 respectively, or are SEQ ID NO:261, 262, 263, 264, 265 and 266 respectively; (20) are SEQ ID NO:269, 270, 271, 272, 273 and 274 respectively, or are SEQ ID NO:275, 276, 277, 278, 279 and 280 respectively; (21) are SEQ ID NO:283, 284, 285, 286, 287 and 288 respectively, or are SEQ ID NO:289, 290, 291, 292, 293 and 294 respectively; (22) are SEQ ID NO:297, 298, 299, 300, 301 and 302 respectively, or are SEQ ID NO:303, 304, 305, 306, 307 and 308 respectively; (23) are SEQ ID NO:311, 312, 313, 314, 315 and 316 respectively, or are SEQ ID NO:317, 318, 319, 320, 321 and 322 respectively; (24) are SEQ ID NO:325, 326, 327, 328, 329 and 330 respectively, or are SEQ ID NO: 331, 332, 333, 334, 335 and 336; or (25) are SEQ ID NO:341, 342, 343, 344, 345 and 346 respectively, or are SEQ ID NO: 347, 348, 349, 350, 351 and 352.

[0221] Embodiment 18 is an isolated polynucleotide as described in Embodiment 16 or 17, wherein the antigen-binding domain comprises a heavy chain variable region or a light chain variable region, and the heavy chain variable region has a polypeptide sequence having at least 95% identity with the following sequences: 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 the light chain variable region has a polypeptide sequence having at least 95% identity with the following sequences: 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.

[0222] Embodiment 19 is an isolated polynucleotide as described in Embodiment 18, wherein the antigen-binding domain comprises: (1) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 1, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 2; (2) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 15, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 16; (3) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 29, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 30; (4) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 43, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 44; (5) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 57, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 58; (6) a heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 71, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 72; (7) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 85, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 86; (8) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 99, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 100; (9) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 113, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 114; (10) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 127, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 128; (11) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 141, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 142; (12) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 155, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 156; (13) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 169, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 170; (14) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 183, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 184; (15) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 197, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 198; (16) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 211, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 212; (17) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 225, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 226; (18) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 239, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 240; (19) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 253, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 254; (20) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 267, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 268; (21) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 281, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 282; (22) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 295, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 296; (23) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 309, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 310; (24) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 323, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 324; (25) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 339, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 340; (26) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 353, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (27) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 353, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (28) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 353, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (29) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 354, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (30) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 354, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (31) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 354, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (32) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 355, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (33) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 355, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (34) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 355, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (35) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 356, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 357; (36) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 356, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 358; (37) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 356, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 359; (38) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 360, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (39) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 360, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (40) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 361, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (41) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 361, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (42) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 362, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (43) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 362, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (44) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 363, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 364; (45) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 363, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 365; (46) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (47) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (48) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (49) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 366, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (50) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (51) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (52) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (53) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 367, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (54) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; (55) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (56) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (57) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 368, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (58) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 370; A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 371; (60) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 372; (61) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 369, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 373; (62) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (63) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (64) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (65) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 374, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (66) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (67) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (68) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (69) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 375, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (70) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (71) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (72) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (73) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 376, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (74) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 378; (75) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 379; (76) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 380; (77) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 377, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 381; (78) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 382, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (79) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 382, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; (80) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 382, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388; (81) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 383, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (82) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 383, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; (83) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 383, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388; (84) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 384, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (85) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 384, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; (86) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 384, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388; (87) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 385, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 386; (88) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 385, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 387; or (89) A heavy chain variable region having the polypeptide sequence shown in SEQ ID NO: 385, and a light chain variable region having the polypeptide sequence shown in SEQ ID NO: 388.

[0223] Embodiment 20 is the isolated polynucleotide according to any one of Embodiments 16 - 19, wherein the antigen - binding domain is a single - chain variable fragment (scFv), optionally, wherein the scFv is humanized.

[0224] Embodiment 21 is the isolated polynucleotide according to any one of Embodiments 16 - 20, wherein the chimeric antigen receptor (CAR) comprises one or more antigen - binding domains, and / or wherein the intracellular signaling domain comprises one or more co - stimulatory domains and one or more activation domains.

[0225] Embodiment 22 is a chimeric antigen receptor (CAR) encoded by the isolated polynucleotide according to any one of Embodiments 16 - 21.

[0226] Embodiment 23 is a vector comprising the isolated polynucleotide according to any one of Embodiments 16 - 21.

[0227] Embodiment 24 is a host cell comprising the vector as shown in Embodiment 23, optionally, wherein the cell is a T cell or an NK cell, preferably a human T cell or a human NK cell.

[0228] Embodiment 25 is a method for expressing a chimeric antigen receptor (CAR) in a host cell, the method comprising transducing a T cell or an NK cell with the vector as described in Embodiment 23.

[0229] Embodiment 26 is a method for producing chimeric antigen receptor (CAR)-T cells or chimeric antigen receptor (CAR)-NK cells, the method comprising: culturing T cells or NK cells comprising an isolated polynucleotide encoding a chimeric antigen receptor (CAR) as described in any one of Embodiments 16-21 under conditions for generating CAR-T cells or CAR-NK cells, and recovering the CAR-T cells or CAR-NK cells.

[0230] Embodiment 27 is a method for generating cells comprising a chimeric antigen receptor (CAR), the method comprising contacting cells with an isolated polynucleotide comprising a nucleic acid encoding a chimeric antigen receptor (CAR) as described in any one of Embodiments 16-21, wherein the isolated polynucleotide is in vitro transcribed RNA or synthetic RNA.

[0231] Embodiment 28 is a method for treating cancer in a subject in need thereof, comprising administering to the subject in need thereof a host cell as described in Embodiment 24, optionally, wherein the cancer is selected from lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocellular carcinoma, 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 lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

[0232] Embodiment 29 is the method as shown in Embodiment 28, which further comprises administering to a subject in need thereof an agent that increases the efficacy of cells expressing CAR, or an agent that ameliorates one or more side effects associated with the administration of cells expressing CAR, or an agent for treating a disease associated with EGFRvIII. Examples Example 1: Identification of anti-EGFRvIII monoclonal antibody

[0233] Rabbits were immunized with an antigen (a mixture of Aplysia hemocyanin (KLH)-CLEEKKGNYVVTDHGS (SEQ ID NO: 337) and LEEKKGNYVVTDHGSC-KLH (SEQ ID NO: 338)), B cell clones from the immunized rabbits were cultured, the supernatants were screened by ELISA, and the cDNA of the IgG mRNA of the positive B cell clones was sequenced.

[0234] The sequences of the variable regions of the heavy and light chains (VH region and VL region, respectively) of the anti-EGFRvIII monoclonal antibody are shown in Tables 1 and 2, and the CDR regions of the anti-EGFRvIII monoclonal antibody are shown in Tables 3 - 6. Table 1: Variable region sequence of the heavy chain of the anti-EGFRvIII monoclonal antibody VH: Variable region of the heavy chain Table 2: Variable region sequence of the light chain of the anti-EGFRvIII monoclonal antibody VL: Variable region of the light chain Table 3: Heavy chain CDR regions 1 - 3 of the anti-EGFRvIII monoclonal antibody HC: Heavy chain; CDR: Complementary determining region; NO: SEQ ID NO The HC CDR of the anti-EGFRvIII monoclonal antibody was determined using the IMGT method (Lefranc, M.-P. et al., Nucleic Acids Research, 1999; 27:209 - 212). Table 4: Light chain CDR regions 1 - 3 of the anti-EGFRvIII monoclonal antibody LC: Light chain; CDR: Complementary determining region; NO: SEQ ID NO The LC CDR of the anti-EGFRvIII monoclonal antibody was determined using the IMGT method (Lefranc, M.-P. et al., Nucleic Acids Research, 1999; 27:209 - 212). Table 5: Heavy chain CDR regions 1 - 3 of the anti-EGFRvIII monoclonal antibody HC: Heavy chain; CDR: Complementary determining region; NO: SEQ ID NO The HC CDR of the anti-EGFRvIII monoclonal antibody was determined using the Kabat method (Elvin A. Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991). Table 6: Light chain CDR regions 1 - 3 of the anti-EGFRvIII monoclonal antibody LC: Light chain; CDR: Complementary determining region; NO: SEQ ID NO The LC CDRs of the anti-EGFRvIII monoclonal antibody were determined using the Kabat method (Elvin A. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, 1991). Example 2: Production and purification of monoclonal antibodies from the culture medium of transfected cells

[0235] To obtain recombinant anti-EGFRvIII chimeric monoclonal antibodies, expression vectors containing rabbit variable regions (VH and VL) fused to human IgG1 heavy chain and κ light chain constant regions, respectively, were transiently transfected into ExpiCHO-S cells. The recombinant antibodies produced in the ExpiCHO-S cell suspension were then purified using Protein A affinity chromatography. Example 3: ELISA binding assay of purified chimeric monoclonal antibody to EGFRvIII

[0236] The ability of the purified chimeric monoclonal antibody to bind to immobilized EGFRvIII was tested in an ELISA assay. Recombinant human EGFRvIII (ACROBiosystems, CAT#: EGI-H52H4) in DPBS buffer was coated onto a 96-well plate at room temperature for 1 hour. The plate was blocked with 5% BSA in TBST at room temperature for 1 hour. In each well of a single plate, different concentrations of the monoclonal antibody were incubated at room temperature for 1 hour. The plate was washed, and binding to EGFRvIII was detected by incubating with a mouse anti-human IgG Fc antibody (50B4A9)[HRP], monoclonal antibody (Genescript, CAT#: A01854) at room temperature for 1 hour. After washing, ELISA color development was performed using a one-step detection solution (ThermoFisher Scientific, CAT#: 34028), and the absorbance was measured at 450 nm. The results are as Figure 1A-1G shown. Example 4: FACS binding assay of purified monoclonal antibody to cell surface EGFRvIII

[0237] Approximately 50,000 ExpiCHO-S cells expressing the EGFRvIII antigen were transferred to a 96-well plate and incubated with different concentrations of purified chimeric anti-EGFRvIII monoclonal antibody (the variable regions of the rabbit monoclonal antibody were fused to the constant regions of human IgG1 heavy chain and κ light chain respectively) for 15 minutes at 4°C. The cells were then centrifuged for 5 minutes 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 / Cyanine7 anti-human IgG Fc recombinant antibody (clone QA19A42) (Biolegend, CAT#: 366908) and incubated for another 15 minutes on ice. The cells were subsequently washed twice with FACS buffer and resuspended in FACS buffer. The cells were then placed in AttuneNxT and the data was analyzed using AttuneNxT software. The results are as Figure 2A-2G shown. Example 5: ELISA Binding Analysis of Purified Monoclonal Antibody to Wild-Type EGFR

[0238] To test the binding activity of the monoclonal antibody to wild-type EGFR, the ability of the purified chimeric monoclonal antibody to bind to immobilized wild-type EGFR was tested in an ELISA assay. Recombinant human EGFR (ACROBiosystems, CAT#: EGR-H5222) in DPBS buffer was coated on a 96-well plate at room temperature for 1 hour. The plate was blocked with 5% BSA in TBST at room temperature for 1 hour. Solutions containing different concentrations of the monoclonal antibody were added to each well of the plate and the plate was incubated at room temperature for 1 hour. 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 at room temperature for 1 hour. After washing the plate, ELISA color development was performed using a one-step detection solution (ThermoFisher Scientific, CAT#: 34028) and the absorbance was measured at 450 nm. The anti-EGFR antibody cetuximab (Selleckchem, Catalog: A2000) was added as a positive control. The results are as Figure 3A-3D shown. Example 6: Construction of Chimeric Antigen Receptor Constructs Containing Anti-EGFRvIII Antigen-Binding Domains

[0239] To construct the CAR construct, the monoclonal antibody was converted to scFv using VH, VL, and (G4S)n linker, and the scFv was fused to the N-terminus of the hinge and transmembrane domains derived from human CD8a (aa 114 - 118, Boursier JP et al., The Journal of Biological Chemistry, 268(3):2013 - 20(1993)). The C-terminal intracellular signaling domain of the CAR was constructed by fusing the intracellular co-stimulatory domain of CD28 (aa 162 - 202, Aruffo A and Seed B, Proceedings of the National Academy of Sciences of the United States of America, 84(23):8573 - 7(1987)) followed by the activation domain from the CD3ζ chain (aa 52 - 162, Letourneur F and Klausner RD, Proceedings of the National Academy of Sciences of the United States of America, 88(20):8905 - 9(1991)). Using standard molecular biology cloning techniques, the DNA sequence encoding the CAR was assembled and cloned into an expression vector (retroviral, lentiviral, episomal, or integrated) to generate the CAR construct. Example 7: Humanization of Anti - EGFRvIII Monoclonal Antibody

[0240] The rabbit anti - EGFRvIII monoclonal antibody was humanized to reduce the likelihood of generating immunogenicity when used in human patients. The variable region sequences of the heavy and light chains (VH and VL) were aligned with human antibody sequences in the Protein Data Bank (PDB) database, and a homology model was established. The CDRs in the heavy and light chains of the rabbit monoclonal antibody were transplanted into the human framework most likely to maintain the appropriate structure required for antigen binding. Reverse mutations or other mutations from human residues to rabbit residues were designed when necessary. 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 chain and κ light chain, respectively. The binding of the humanized monoclonal antibody to EGFRvIII was evaluated using ELISA assay ( Figure 4A-4D ) and FACS assay ( Figure 5A-5D ). 16C11 - H1L1 refers to the humanized monoclonal antibody constructed from the 16C11 - H1 heavy chain and 16C11 - L1 light chain shown in Table 7; other humanized clones follow the same naming rule.

[0241] Table 7: Variable Region Sequences of Heavy and Light Chains of Humanized Anti - EGFRvIII Monoclonal Antibody NO:SEQ ID NO

[0242] Those skilled in the art will recognize that modifications may be made to the above-described embodiments without departing from their broad inventive concept. Accordingly, it is to be understood that the invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined by this specification.

Claims

1. An isolated monoclonal antibody or an antigen-binding fragment or antigen-binding domain thereof, comprising a heavy-chain variable region and a light-chain variable region, wherein the heavy-chain variable region comprises heavy-chain complementarity-determining region 1 (HCDR1), HCDR2 and HCDR3, and the light-chain variable region comprises light-chain complementarity-determining region 1 (LCDR1), LCDR2 and LCDR3, having the following polypeptide sequences: (1) SEQ ID NO:3, 4, 5, 6, 7 and 8 respectively, or SEQ ID NO:9, 10, 11, 12, 13 and 14 respectively; (2) SEQ ID NO:17, 18, 19, 20, 21 and 22 respectively, or SEQ ID NO:23, 24, 25, 26, 27 and 28 respectively; (3) SEQ ID NO:31, 32, 33, 34, 35 and 36 respectively or SEQ ID NO:37, 38, 39, 40, 41 and 42 respectively; (4) SEQ ID NO:45, 46, 47, 48, 49 and 50 respectively, or SEQ ID NO:51, 52, 53, 54, 55 and 56 respectively; (5) SEQ ID NO:59, 60, 61, 62, 63 and 64 respectively, or SEQ ID NO:65, 66, 67, 68, 69 and 70 respectively; (6) SEQ ID NO:73, 74, 75, 76, 77 and 78 respectively, or SEQ ID NO:79, 80, 81, 82, 83 and 84 respectively; (7) SEQ ID NO:87, 88, 89, 90, 91 and 92 respectively, or SEQ ID NO:93, 94, 95, 96, 97 and 98 respectively; (8) SEQ ID NO:101, 102, 103, 104, 105 and 106 respectively, or SEQ ID NO:107, 108, 109, 110, 111 and 112 respectively; (9) SEQ ID NO:115, 116, 117, 118, 119 and 120 respectively, or SEQ ID NO:121, 122, 123, 124, 125 and 126 respectively; (10) SEQ ID NO:129, 130, 131, 132, 133 and 134 respectively, or SEQ ID NO: 135, 136, 137, 138, 139 and 140; (11) SEQ ID NO: 143, 144, 145, 146, 147, and 148, respectively, or SEQ ID NO: 149, 150, 151, 152, 153, and 154; (12) SEQ ID NO: 157, 158, 159, 160, 161, and 162, respectively, or SEQ ID NO: 163, 164, 165, 166, 167, and 168; (13) SEQ ID NO: 171, 172, 173, 174, 175, and 176, respectively, or SEQ ID NO: 177, 178, 179, 180, 181, and 182; (14) SEQ ID NO: 185, 186, 187, 188, 189, and 190, respectively, or SEQ ID NO: 191, 192, 193, 194, 195, and 196; (15) SEQ ID NO: 199, 200, 201, 202, 203, and 204, respectively, or SEQ ID NO: 205, 206, 207, 208, 209, and 210; (16) SEQ ID NO: 213, 214, 215, 216, 217, and 218, respectively, or SEQ ID NO: 219, 220, 221, 222, 223, and 224; (17) SEQ ID NO: 227, 228, 229, 230, 231, and 232, respectively, or SEQ ID NO: 233, 234, 235, 236, 237, and 238; (18) SEQ ID NO: 241, 242, 243, 244, 245, and 246, respectively, or SEQ ID NO: 247, 248, 249, 250, 251, and 252; (19) SEQ ID NO: 255, 256, 257, 258, 259, and 260, respectively, or SEQ ID NO: 261, 262, 263, 264, 265, and 266; (20) SEQ ID NO: 269, 270, 271, 272, 273, and 274, respectively, or SEQ ID NO: 275, 276, 277, 278, 279, and 280; (21) are SEQ ID NO:283, 284, 285, 286, 287, and 288, respectively, or are SEQ ID NO: 289, 290, 291, 292, 293, and 294; (22) are SEQ ID NO:297, 298, 299, 300, 301, and 302, respectively, or are SEQ ID NO: 303, 304, 305, 306, 307, and 308; (23) are SEQ ID NO:311, 312, 313, 314, 315, and 316, respectively, or are SEQ ID NO: 317, 318, 319, 320, 321, and 322; (24) are SEQ ID NO:325, 326, 327, 328, 329, and 330, respectively, or are SEQ ID NO: 331, 332, 333, 334, 335, and 336; or (25) are SEQ ID NO:341, 342, 343, 344, 345, and 346, respectively, or are SEQ ID NO: 347, 348, 349, 350, 351, and 352; wherein the antibody or its antigen-binding fragment or antigen-binding domain specifically binds to EGFRvIII, preferably human EGFRvIII.

2. The isolated monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof according to claim 1, comprising a heavy chain variable region or a light chain variable region, wherein the heavy chain variable region has a polypeptide sequence having at least 95% identity with the following sequences: 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 the light chain variable region has a polypeptide sequence having at least 95% identity with the following sequences: 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.

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

388.

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

5. The isolated monoclonal antibody or antigen-binding fragment or antigen-binding domain thereof according to any one of claims 1-4, wherein the antibody or antigen-binding fragment or antigen-binding domain thereof is capable of inducing effector-mediated tumor cell lysis by antibody-dependent cell cytotoxicity (ADCC), antibody-dependent cell 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 having cancer-killing effects.

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 1-5.

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

8. A vector comprising the isolated nucleic acid according to claim 7.

9. A host cell comprising the vector according to claim 8.

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

11. A method of targeting EGFRvIII on the surface of cancer cells, and / or treating cancer in a subject in need thereof, and / or treating an inflammatory disease in a subject in need thereof, and / or treating an autoimmune disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition according to claim 10. Optionally, wherein the cancer is selected from the group consisting of: lung cancer, gastric cancer, esophageal cancer, cholangiocarcinoma, cholangiocellular carcinoma, colon cancer, hepatocellular carcinoma, renal cell carcinoma, urothelial carcinoma of the bladder, 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 lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), multiple myeloma (MM), acute myeloid leukemia (AML) and other liquid tumors.

12. A method of producing the monoclonal antibody or antigen-binding fragment or antigen-binding domain according to any one of claims 1-5, or the bispecific antibody or antigen-binding fragment or bispecific antigen-binding domain according to claim 6, comprising culturing a cell comprising a nucleic acid encoding the monoclonal antibody or antigen-binding fragment or antigen-binding domain or 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 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 bispecific antibody or antigen-binding fragment or bispecific antigen-binding domain from the cell or culture.

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

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

15. 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 thereof according to any one of claims 1-5 or a bispecific antigen-binding domain thereof according to claim 6; (b) a hinge region; (c) a transmembrane region; and (d) an intracellular signaling domain.

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

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

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