Antibodies targeting EGFRvIII and their use in cellular immunotherapy

Abstract

The present invention provides an antibody or antigen-binding fragment thereof that targets EGFRvIII, and further provides a CAR comprising an antigen-binding fragment that targets EGFRvIII, and uses thereof for treatment and detection.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority from Chinese patent application bearing application number CN202210936403.1 and filing date August 5, 2022, which is hereby incorporated by reference in its entirety.

[0002] The present specification relates to an antibody or antigen-binding fragment thereof that targets EGFRvIII. The present specification also relates to a CAR comprising an antigen-binding fragment that targets EGFRvIII and uses thereof. [Background technology]

[0003] Cellular immunotherapy is an emerging technology in tumor treatment, which shows great potential for application in the field of tumor treatment. Engineered T cells have been successfully used in the treatment of hematological tumors, and due to their unprecedented therapeutic effects in clinical trials, up to now, eight chimeric antigen receptor (CAR)-T cell therapies have already been approved for use in the treatment of B-cell malignancies and myeloma worldwide.

[0004] Although cellular immunotherapy has shown remarkable therapeutic effects in hematological malignancies, its effectiveness in the treatment of solid tumors is limited by factors such as the immunosuppressive microenvironment of solid tumors and the limited persistence and infiltration ability of CAR-T cells within tumors, which require novel modifications and breakthroughs in cellular immunotherapy products ( Akhavan et al., 2019 ).

[0005] Another important factor in cellular immunotherapy is target selection, which determines the safety and efficacy of cell therapy products. Tumor antigens are divided into tumor-associated antigens (TAA) and tumor-specific antigens (TSA). Tumor-associated antigens are expressed in normal tissues. When cell therapy products target tumor-associated antigens, they are more likely to cause off-target toxicity, while cell therapy products that target tumor-specific antigens are safer.

[0006] The EGFRvIII antigen is a tumor-specific target antigen and the most common extracellular domain mutation of the EGFR gene (Chistiakov et al., 2017). It is a deletion of 801 bases from exons 2–7 of the EGFR coding region, resulting in a deletion of 267 amino acids in the extracellular domain compared to wild-type EGFR, creating a fusion region between exons 1 and 8 and one new glycine residue (Figure 1; An et al., 2018), making it a tumor-specific deletion mutation. Compared to wild-type EGFR, EGFRvIII is ligand-independent and constitutively activated (Chistiakov et al., 2017). This mutation has been found in many cancers (e.g., breast cancer, lung cancer, head and neck cancer, etc.), but is most commonly found in glioblastoma (Chistiakov et al., 2017; Del Vecchio et al., 2012).

[0007] Glioblastoma accounts for 15% of brain tumors, with nearly 210,000 cases diagnosed worldwide each year. Glioblastoma is a grade 4 tumor characterized by high aggressiveness, a high recurrence rate, and a high mortality and disability rate. Glioblastoma is a rapidly progressing tumor with one of the lowest 5-year survival rates of all human cancers, and no effective treatments are currently available. Scientists are currently working on immunotherapy strategies to address this global challenge (Feldman et al., 2022; Londhe and Date, 2020).

[0008] Research results have shown that EGFRvIII is expressed in glioblastoma but not in normal tissues, and the higher the malignancy in patients, the higher the EGFRvIII expression rate. Therefore, the use of CAR-T cells targeting the EGFRvIII antigen in the treatment of brain gliomas has advantages such as strong specificity, low off-target toxicity, and minimal side effects (Zhu et al., 2021).

[0009] Clinical studies have already shown that anti-tumor drugs targeting EGFRvIII have remarkable safety and initial therapeutic effects (O'Rourke et al., 2017). Antibody drugs have become a relatively mature and effective anti-cancer drug in recent years, targeting specific antigens in tumor cells and utilizing effects such as ADCC and CDC to activate the patient's immune system against cancer cells.

[0010] Currently, there is still a need in the market for antitumor drugs targeting EGFRvIII with better safety and therapeutic efficacy. Summary of the Invention

[0011] In one aspect, the present disclosure provides an antibody or antigen-binding fragment thereof that targets EGFRvIII, wherein the antibody comprises a heavy chain variable region (HCVR), wherein the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and wherein the HCDR1, HCDR2, and HCDR3 are selected from the following combinations: (1) The amino acid sequence of HCDR1 is DFSMH (SEQ ID NO: 1), The amino acid sequence of HCDR2 is WINTETGEPSYADDFKG (SEQ ID NO: 2). The amino acid sequence of HCDR3 is YGYDVRGDY (SEQ ID NO: 3); (2) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 4), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 5). The amino acid sequence of HCDR3 is GWFAY (SEQ ID NO: 6); (3) The amino acid sequence of HCDR1 is DYSIH (SEQ ID NO: 7), The amino acid sequence of HCDR2 is WINTETGEPTYADDFKG (SEQ ID NO: 8). The amino acid sequence of HCDR3 is YGYDVRGDY (SEQ ID NO: 9); (4) The amino acid sequence of HCDR1 is DYYLH (SEQ ID NO: 10), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 11). The amino acid sequence of HCDR3 is GYLTY (SEQ ID NO: 12); (5) The amino acid sequence of HCDR1 is RYWMH (SEQ ID NO: 13), The amino acid sequence of HCDR2 is EINPSNGRANYNEKFMS (SEQ ID NO: 14). The amino acid sequence of HCDR3 is GREITTGFAY (SEQ ID NO: 15); (6) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 16), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 17). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 18); (7) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 19), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 20). The amino acid sequence of HCDR3 is GYLAY (SEQ ID NO: 21); (8) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 22), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 23), The amino acid sequence of HCDR3 is GWFAY (SEQ ID NO: 25); (9) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 25), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 26). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 27); (10) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 28), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 29), The amino acid sequence of HCDR3 is GWFAY (SEQ ID NO: 30); (11) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 31), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 32). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 33); (12) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 34), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 35), The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 36); (13) The amino acid sequence of HCDR1 is NYAMS (SEQ ID NO: 37), The amino acid sequence of HCDR2 is TITSGGSYTYYPDSVKG (SEQ ID NO: 38). The amino acid sequence of HCDR3 is KDYGNYWFAY (SEQ ID NO: 39); (14) The amino acid sequence of HCDR1 is GYAMS (SEQ ID NO: 40), The amino acid sequence of HCDR2 is TITSGGSYTYYPDSVKG (SEQ ID NO: 41). The amino acid sequence of HCDR3 is KDYGNYWFAY (SEQ ID NO: 42); (15) The amino acid sequence of HCDR1 is GYAMS (SEQ ID NO: 43), The amino acid sequence of HCDR2 is TITSGGSYTYYPDSVKG (SEQ ID NO: 44). The amino acid sequence of HCDR3 is KDYGNYWFAY (SEQ ID NO: 45); (16) The amino acid sequence of HCDR1 is SGYSWH (SEQ ID NO: 46), The amino acid sequence of HCDR2 is YIHYSGSTNYNPPLKS (SEQ ID NO: 47). The amino acid sequence of HCDR3 is GVVSNYAMGN (SEQ ID NO: 48); (17) The amino acid sequence of HCDR1 is TYWMH (SEQ ID NO: 49), The amino acid sequence of HCDR2 is YINPNTAYTEYNQNFKD (SEQ ID NO: 50). The amino acid sequence of HCDR3 is GAYYRTYYAMDY (SEQ ID NO: 51); (18) The amino acid sequence of HCDR1 is NYGMN (SEQ ID NO: 52). The amino acid sequence of HCDR2 is WINTYTGEPTYADDFKG (SEQ ID NO: 53). The amino acid sequence of HCDR3 is EEFYSRGAMDY (SEQ ID NO: 54); and (19) The amino acid sequence of HCDR1 is DYYIN (SEQ ID NO: 55), The amino acid sequence of HCDR2 is WIYPGSGNTKYNEKFKG (SEQ ID NO: 56). the amino acid sequence of HCDR3 is SSRCDF (SEQ ID NO:57); or The antibody includes a variant of a combination of any of the HCDR sequences (1) to (19), wherein the variant has at least 90% sequence identity with any of the HCDR sequences (1) to (19), or the HCDR sequences contain a total of at least one and no more than 10, or no more than 5, 4, 3, or 2 amino acid modifications. Antibodies or antigen-binding fragments thereof are provided.

[0012] In some embodiments, the antibody further comprises a light chain variable region (LCVR), wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and wherein the LCDR1, LCDR2, and LCDR3 are one of the following combinations: (1) The amino acid sequence of LCDR1 is SASSSISSNYLH (SEQ ID NO: 58); The amino acid sequence of LCDR2 is GTSNLAS (SEQ ID NO: 59). The amino acid sequence of LCDR3 is HQGSSIPLT (SEQ ID NO: 60); (2) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNHLA (SEQ ID NO: 61); The amino acid sequence of LCDR2 is FASTRAS (SEQ ID NO: 62): The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 63); (3) The amino acid sequence of LCDR1 is SASSGISSNYLH (SEQ ID NO: 64); The amino acid sequence of LCDR2 is STSNLAS (SEQ ID NO: 65). The amino acid sequence of LCDR3 is HQGSDIPLT (SEQ ID NO: 66); (4) The amino acid sequence of LCDR1 is KSSQNLLNSSNQKNYLA (SEQ ID NO: 67); The amino acid sequence of LCDR2 is FASTRY (SEQ ID NO: 68); The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 69); (5) The amino acid sequence of LCDR1 is KASQSVSNDVV (SEQ ID NO: 70); The amino acid sequence of LCDR2 is YASNRYT (SEQ ID NO: 71). The amino acid sequence of LCDR3 is QQDYSSPWT (SEQ ID NO: 72); (6) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 73); The amino acid sequence of LCDR2 is FASTRES (SEQ ID NO: 74), The amino acid sequence of LCDR3 is QQHYSIPLT (SEQ ID NO: 75); (7) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 76); The amino acid sequence of LCDR2 is FASTRKS (SEQ ID NO: 77). The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 78); (8) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNHLA (SEQ ID NO: 79); The amino acid sequence of LCDR2 is FASTRQS (SEQ ID NO: 80). The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 81); (9) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 82); The amino acid sequence of LCDR2 is FASTRQS (SEQ ID NO: 83). The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 84); (10) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNHLA (SEQ ID NO: 85); The amino acid sequence of LCDR2 is FASTRGS (SEQ ID NO: 86). The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 87); (11) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 88); The amino acid sequence of LCDR2 is FASTRDS (SEQ ID NO: 89). The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 90); (12) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 91); The amino acid sequence of LCDR2 is FASTRES (SEQ ID NO: 92), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 93); (13) The amino acid sequence of LCDR1 is RSSQSLVHSDGNTYLH (SEQ ID NO: 94). The amino acid sequence of LCDR2 is KVSNRFS (SEQ ID NO: 95). The amino acid sequence of LCDR3 is SQSIHVPWT (SEQ ID NO: 96); (14) The amino acid sequence of LCDR1 is SASSSVSYMH (SEQ ID NO: 97); The amino acid sequence of LCDR2 is STSNLAS (SEQ ID NO: 98). The amino acid sequence of LCDR3 is QQRSSYPLT (SEQ ID NO: 99); (15) The amino acid sequence of LCDR1 is RSSQSLVHSDGNTYLH (SEQ ID NO: 100). The amino acid sequence of LCDR2 is KVSNRFS (SEQ ID NO: 101). The amino acid sequence of LCDR3 is SQTTQVPWT (SEQ ID NO: 102); (16) The amino acid sequence of LCDR1 is ITNTDIDDDDMN (SEQ ID NO: 103). The amino acid sequence of LCDR2 is EGNTLRP (SEQ ID NO: 104), The amino acid sequence of LCDR3 is LQSDDLPLT (SEQ ID NO: 105); (17) The amino acid sequence of LCDR1 is KASQSVDYDGDSYMN (SEQ ID NO: 106). The amino acid sequence of LCDR2 is AASNLES (SEQ ID NO: 107). The amino acid sequence of LCDR3 is LQSNEDPYT (SEQ ID NO: 108); (18) The amino acid sequence of LCDR1 is RSSQFIVHSNGNTYLE (SEQ ID NO: 109); The amino acid sequence of LCDR2 is KISNRFS (SEQ ID NO: 110). The amino acid sequence of LCDR3 is FQGSHVPFT (SEQ ID NO: 111); and (19) The amino acid sequence of LCDR1 is KASEDIYNRLA (SEQ ID NO: 112). The amino acid sequence of LCDR2 is GATSLET (SEQ ID NO: 113), the amino acid sequence of LCDR3 is QQYWSSPLT (SEQ ID NO: 114); or The antibody includes a variant of a combination of any of the LCDR sequences (1) to (19), wherein the variant has at least 90% sequence identity with any of the LCDR sequences (1) to (19), or contains a total of at least one and no more than 10, or no more than 5, 4, 3, or 2 amino acid modifications in the LCDR sequence.

[0013] In some embodiments, the amino acid sequence of the heavy chain variable region is chosen from any of the following: (1) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 115; (2) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 117; (3) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 119; (4) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 121; (5) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 123; (6) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 125; (7) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 127; (8) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 129; (9) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 131; (10) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 133; (11) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 135; (12) the sequence of a heavy chain variable region having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or the sequence set forth in SEQ ID NO: 137; (13) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 139; (14) The sequence of a heavy chain variable region set forth in SEQ ID NO: 141 or having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (15) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 143; (16) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 145; (17) The sequence of a heavy chain variable region set forth in SEQ ID NO: 147 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (18) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 149; and (19) The sequence of SEQ ID NO: 151 or a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0014] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are selected from any combination of: (1) a heavy chain variable region sequence set forth in SEQ ID NO: 115 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 116 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (2) a heavy chain variable region sequence set forth in SEQ ID NO: 117 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 118 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (3) a heavy chain variable region sequence set forth in SEQ ID NO: 119 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 120 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (4) a heavy chain variable region sequence set forth in SEQ ID NO: 121 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 122 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (5) a heavy chain variable region sequence set forth in SEQ ID NO: 123 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 124 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (6) a heavy chain variable region sequence set forth in SEQ ID NO: 125 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 126 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (7) a heavy chain variable region sequence set forth in SEQ ID NO: 127 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 128 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (8) a heavy chain variable region sequence set forth in SEQ ID NO: 129 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 130 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (9) a heavy chain variable region sequence set forth in SEQ ID NO: 131 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 132 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (10) a heavy chain variable region sequence set forth in SEQ ID NO: 133 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 134 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (11) A heavy chain variable region sequence set forth in SEQ ID NO: 135 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 136 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (12) A heavy chain variable region sequence set forth in SEQ ID NO: 137 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 138 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (13) A heavy chain variable region sequence set forth in SEQ ID NO: 139 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 140 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (14) A heavy chain variable region sequence set forth in SEQ ID NO: 141 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 142 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (15) A heavy chain variable region sequence set forth in SEQ ID NO: 143 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 144 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (16) A heavy chain variable region sequence set forth in SEQ ID NO: 145 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 146 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (17) A heavy chain variable region sequence set forth in SEQ ID NO: 147 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 148 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (18) A heavy chain variable region sequence set forth in SEQ ID NO: 149 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 150 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and (19) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 151, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 152, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0015] In some embodiments, the antibody has a binding KD value for the EGFRvIII antigen of 10 as measured by surface plasmon resonance (SPR). -6 Less than M, preferably 10 -7 It is less than M.

[0016] In some embodiments, the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0017] In some embodiments, the humanized antibody has a binding KD value for the EGFRvIII antigen of 10 as measured by surface plasmon resonance (SPR). -6 Less than M, preferably 10 -7 It is less than M.

[0018] In some embodiments, the antigen-binding fragment comprises a Fab, a Fab', a F(ab')2, a single domain antibody, or a single chain antibody.

[0019] In some embodiments, the humanized antibody is a single chain antibody, the amino acid sequence of which is selected from any of the following: (1) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 167; (2) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 169; (3) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 171; (4) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 173; (5) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 175; (6) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 177; (7) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 179; (8) the sequence of a heavy chain variable region having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or the sequence set forth in SEQ ID NO: 181; (9) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 183; (10) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 185; and (11) The sequence of SEQ ID NO: 187 or a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof further comprises an Fc fragment.

[0021] In some embodiments, the antigen-binding fragment is a single-chain antibody, and preferably, the linker sequence connecting the heavy chain variable region and the light chain variable region in the single-chain antibody comprises the sequence set forth in SEQ ID NO: 166.

[0022] In some embodiments, the present specification provides a chimeric antigen receptor (CAR), the extracellular antigen-binding domain of which comprises one or more antibodies or antigen-binding fragments thereof targeting EGFRvIII, and HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the antibody molecule are selected from one of the following combinations: (1) The amino acid sequence of HCDR1 is DFSMH (SEQ ID NO: 1), The amino acid sequence of HCDR2 is WINTETGEPSYADDFKG (SEQ ID NO: 2). The amino acid sequence of HCDR3 is YGYDVRGDY (SEQ ID NO: 3); (2) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 4), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 5). The amino acid sequence of HCDR3 is GWFAY (SEQ ID NO: 6); (3) The amino acid sequence of HCDR1 is DYSIH (SEQ ID NO: 7), The amino acid sequence of HCDR2 is WINTETGEPTYADDFKG (SEQ ID NO: 8). The amino acid sequence of HCDR3 is YGYDVRGDY (SEQ ID NO: 9); (4) The amino acid sequence of HCDR1 is DYYLH (SEQ ID NO: 10), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 11). The amino acid sequence of HCDR3 is GYLTY (SEQ ID NO: 12); (5) The amino acid sequence of HCDR1 is RYWMH (SEQ ID NO: 13), The amino acid sequence of HCDR2 is EINPSNGRANYNEKFMS (SEQ ID NO: 14). The amino acid sequence of HCDR3 is GREITTGFAY (SEQ ID NO: 15); (6) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 16), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 17). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 18); (7) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 19), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 20). The amino acid sequence of HCDR3 is GYLAY (SEQ ID NO: 21); (8) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 22), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 23), The amino acid sequence of HCDR3 is GWFAY (SEQ ID NO: 25); (9) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 25), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 26). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 27); (10) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 28), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 29), The amino acid sequence of HCDR3 is GWFAY (SEQ ID NO: 30); (11) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 31), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 32). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 33); (12) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 34), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 35), The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 36); (13) The amino acid sequence of HCDR1 is NYAMS (SEQ ID NO: 37), The amino acid sequence of HCDR2 is TITSGGSYTYYPDSVKG (SEQ ID NO: 38). The amino acid sequence of HCDR3 is KDYGNYWFAY (SEQ ID NO: 39); (14) The amino acid sequence of HCDR1 is GYAMS (SEQ ID NO: 40), The amino acid sequence of HCDR2 is TITSGGSYTYYPDSVKG (SEQ ID NO: 41). The amino acid sequence of HCDR3 is KDYGNYWFAY (SEQ ID NO: 42); (15) The amino acid sequence of HCDR1 is GYAMS (SEQ ID NO: 43), The amino acid sequence of HCDR2 is TITSGGSYTYYPDSVKG (SEQ ID NO: 44). The amino acid sequence of HCDR3 is KDYGNYWFAY (SEQ ID NO: 45); (16) The amino acid sequence of HCDR1 is SGYSWH (SEQ ID NO: 46), The amino acid sequence of HCDR2 is YIHYSGSTNYNPPLKS (SEQ ID NO: 47). The amino acid sequence of HCDR3 is GVVSNYAMGN (SEQ ID NO: 48); (17) The amino acid sequence of HCDR1 is TYWMH (SEQ ID NO: 49), The amino acid sequence of HCDR2 is YINPNTAYTEYNQNFKD (SEQ ID NO: 50). The amino acid sequence of HCDR3 is GAYYRTYYAMDY (SEQ ID NO: 51); (18) The amino acid sequence of HCDR1 is NYGMN (SEQ ID NO: 52). The amino acid sequence of HCDR2 is WINTYTGEPTYADDFKG (SEQ ID NO: 53). The amino acid sequence of HCDR3 is EEFYSRGAMDY (SEQ ID NO: 54); and (19) The amino acid sequence of HCDR1 is DYYIN (SEQ ID NO: 55), The amino acid sequence of HCDR2 is WIYPGSGNTKYNEKFKG (SEQ ID NO: 56). the amino acid sequence of HCDR3 is SSRCDF (SEQ ID NO:57); or The antibody includes a variant of a combination of any of the HCDR sequences (1) to (19), wherein the variant has at least 90% sequence identity with any of the HCDR sequences (1) to (19), or the HCDR sequences contain a total of at least one and no more than 10, or no more than 5, 4, 3, or 2 amino acid modifications. Provide CAR.

[0023] In some embodiments, the antibody further comprises a light chain variable region (LCVR), wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and wherein the LCDR1, LCDR2, and LCDR3 are one of the following combinations: (1) The amino acid sequence of LCDR1 is SASSSISSNYLH (SEQ ID NO: 58); The amino acid sequence of LCDR2 is GTSNLAS (SEQ ID NO: 59). The amino acid sequence of LCDR3 is HQGSSIPLT (SEQ ID NO: 60); (2) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNHLA (SEQ ID NO: 61); The amino acid sequence of LCDR2 is FASTRAS (SEQ ID NO: 62): The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 63); (3) The amino acid sequence of LCDR1 is SASSGISSNYLH (SEQ ID NO: 64); The amino acid sequence of LCDR2 is STSNLAS (SEQ ID NO: 65). The amino acid sequence of LCDR3 is HQGSDIPLT (SEQ ID NO: 66); (4) The amino acid sequence of LCDR1 is KSSQNLLNSSNQKNYLA (SEQ ID NO: 67); The amino acid sequence of LCDR2 is FASTRY (SEQ ID NO: 68); The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 69); (5) The amino acid sequence of LCDR1 is KASQSVSNDVV (SEQ ID NO: 70); The amino acid sequence of LCDR2 is YASNRYT (SEQ ID NO: 71). The amino acid sequence of LCDR3 is QQDYSSPWT (SEQ ID NO: 72); (6) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 73); The amino acid sequence of LCDR2 is FASTRES (SEQ ID NO: 74), The amino acid sequence of LCDR3 is QQHYSIPLT (SEQ ID NO: 75); (7) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 76); The amino acid sequence of LCDR2 is FASTRKS (SEQ ID NO: 77). The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 78); (8) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNHLA (SEQ ID NO: 79); The amino acid sequence of LCDR2 is FASTRQS (SEQ ID NO: 80). The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 81); (9) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 82); The amino acid sequence of LCDR2 is FASTRQS (SEQ ID NO: 83). The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 84); (10) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNHLA (SEQ ID NO: 85); The amino acid sequence of LCDR2 is FASTRGS (SEQ ID NO: 86). The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 87); (11) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 88); The amino acid sequence of LCDR2 is FASTRDS (SEQ ID NO: 89). The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 90); (12) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 91); The amino acid sequence of LCDR2 is FASTRES (SEQ ID NO: 92), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 93); (13) The amino acid sequence of LCDR1 is RSSQSLVHSDGNTYLH (SEQ ID NO: 94). The amino acid sequence of LCDR2 is KVSNRFS (SEQ ID NO: 95). The amino acid sequence of LCDR3 is SQSIHVPWT (SEQ ID NO: 96); (14) The amino acid sequence of LCDR1 is SASSSVSYMH (SEQ ID NO: 97); The amino acid sequence of LCDR2 is STSNLAS (SEQ ID NO: 98). The amino acid sequence of LCDR3 is QQRSSYPLT (SEQ ID NO: 99); (15) The amino acid sequence of LCDR1 is RSSQSLVHSDGNTYLH (SEQ ID NO: 100). The amino acid sequence of LCDR2 is KVSNRFS (SEQ ID NO: 101), The amino acid sequence of LCDR3 is SQTTQVPWT (SEQ ID NO: 102); (16) The amino acid sequence of LCDR1 is ITNTDIDDDDMN (SEQ ID NO: 103). The amino acid sequence of LCDR2 is EGNTLRP (SEQ ID NO: 104), The amino acid sequence of LCDR3 is LQSDDLPLT (SEQ ID NO: 105); (17) The amino acid sequence of LCDR1 is KASQSVDYDGDSYMN (SEQ ID NO: 106). The amino acid sequence of LCDR2 is AASNLES (SEQ ID NO: 107). The amino acid sequence of LCDR3 is LQSNEDPYT (SEQ ID NO: 108); (18) The amino acid sequence of LCDR1 is RSSQFIVHSNGNTYLE (SEQ ID NO: 109); The amino acid sequence of LCDR2 is KISNRFS (SEQ ID NO: 110). The amino acid sequence of LCDR3 is FQGSHVPFT (SEQ ID NO: 111); and (19) The amino acid sequence of LCDR1 is KASEDIYNRLA (SEQ ID NO: 112). The amino acid sequence of LCDR2 is GATSLET (SEQ ID NO: 113), the amino acid sequence of LCDR3 is QQYWSSPLT (SEQ ID NO: 114); or The antibody includes a variant of a combination of any of the LCDR sequences (1) to (19), wherein the variant has at least 90% sequence identity with any of the LCDR sequences (1) to (19), or contains a total of at least one and no more than 10, or no more than 5, 4, 3, or 2 amino acid modifications in the LCDR sequence.

[0024] In some embodiments, the amino acid sequence of the heavy chain variable region is chosen from any of the following: (1) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 115; (2) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 117; (3) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 119; (4) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 121; (5) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 123; (6) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 125; (7) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 127; (8) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 129; (9) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 131; (10) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 133; (11) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 135; (12) the sequence of a heavy chain variable region having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or the sequence set forth in SEQ ID NO: 137; (13) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 139; (14) The sequence of a heavy chain variable region set forth in SEQ ID NO: 141 or having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (15) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 143; (16) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 145; (17) The sequence of a heavy chain variable region set forth in SEQ ID NO: 147 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (18) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 149; and (19) The sequence of SEQ ID NO: 151 or a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0025] In some embodiments, the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are selected from any combination of: (1) a heavy chain variable region sequence set forth in SEQ ID NO: 115 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 116 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (2) a heavy chain variable region sequence set forth in SEQ ID NO: 117 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 118 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (3) a heavy chain variable region sequence set forth in SEQ ID NO: 119 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 120 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (4) a heavy chain variable region sequence set forth in SEQ ID NO: 121 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 122 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (5) a heavy chain variable region sequence set forth in SEQ ID NO: 123 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 124 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (6) a heavy chain variable region sequence set forth in SEQ ID NO: 125 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 126 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (7) a heavy chain variable region sequence set forth in SEQ ID NO: 127 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 128 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (8) a heavy chain variable region sequence set forth in SEQ ID NO: 129 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 130 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (9) a heavy chain variable region sequence set forth in SEQ ID NO: 131 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 132 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (10) a heavy chain variable region sequence set forth in SEQ ID NO: 133 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 134 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (11) A heavy chain variable region sequence set forth in SEQ ID NO: 135 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 136 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (12) A heavy chain variable region sequence set forth in SEQ ID NO: 137 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 138 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (13) A heavy chain variable region sequence set forth in SEQ ID NO: 139 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 140 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (14) A heavy chain variable region sequence set forth in SEQ ID NO: 141 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 142 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (15) A heavy chain variable region sequence set forth in SEQ ID NO: 143 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 144 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (16) A heavy chain variable region sequence set forth in SEQ ID NO: 145 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 146 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (17) A heavy chain variable region sequence set forth in SEQ ID NO: 147 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 148 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (18) A heavy chain variable region sequence set forth in SEQ ID NO: 149 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and a light chain variable region sequence set forth in SEQ ID NO: 150 or a sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; and (19) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 151, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 152, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0026] In some embodiments, the antibody has a binding KD value for the EGFRvIII antigen of 10 as measured by surface plasmon resonance (SPR). -6 Less than M, preferably 10 -7 It is less than M.

[0027] In some embodiments, the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

[0028] In some embodiments, the humanized antibody has a binding KD value for the EGFRvIII antigen of 10 as measured by surface plasmon resonance (SPR). -6 Less than M, preferably 10 -7 It is less than M.

[0029] In some embodiments, the antigen-binding fragment is a single-chain antibody, and preferably, the linker sequence connecting the heavy chain variable region and the light chain variable region in the single-chain antibody comprises the sequence set forth in SEQ ID NO: 166.

[0030] In some embodiments, the humanized antibody is a single chain antibody, the amino acid sequence of which is selected from any of the following: (1) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 167; (2) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 169; (3) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 171; (4) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 173; (5) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 175; (6) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 177; (7) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 179; (8) the sequence of a heavy chain variable region having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or the sequence set forth in SEQ ID NO: 181; (9) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 183; (10) a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 185; and (11) The sequence of SEQ ID NO: 187 or a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0031] In some embodiments, the CAR further comprises a hinge region, a transmembrane region, a costimulatory domain, and an activation domain, preferably wherein the hinge region is a CD8 hinge region, the transmembrane region is a CD8α transmembrane region, the costimulatory domain is a 41BB costimulatory domain or a CD28 costimulatory domain, and the activation domain is a CD3ζ activation domain.

[0032] In some embodiments, the hinge region comprises the sequence set forth in SEQ ID NO: 156, the transmembrane region comprises the sequence set forth in SEQ ID NO: 158, the costimulatory domain comprises the sequence set forth in SEQ ID NO: 160 and / or 162, and the activation domain comprises the sequence set forth in SEQ ID NO: 164.

[0033] In some embodiments, the CAR further comprises a signal peptide, preferably the signal peptide is a CD8α signal peptide, more preferably the signal peptide comprises the sequence set forth in SEQ ID NO:154.

[0034] In some embodiments, the signal peptide comprises the sequence set forth in SEQ ID NO:154.

[0035] In another aspect, the description provides 1) a nucleic acid molecule encoding the above-mentioned antibody or antigen-binding fragment thereof, or the heavy chain variable region and / or light chain variable region of the above-mentioned antibody or antigen-binding fragment thereof, or 2) the above-mentioned CAR.

[0036] In some embodiments, the nucleic acid molecule comprises a nucleotide sequence set forth in any of SEQ ID NOs: 153, 155, 157, 159, 161, 163, 165, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, or 188.

[0037] In another aspect, the present disclosure provides an expression vector, preferably a lentiviral vector, comprising the nucleic acid molecule described above.

[0038] In another aspect, the present disclosure provides a cell comprising the expression vector.

[0039] In another aspect, the present description provides a cell expressing the CAR.

[0040] In some embodiments, the cell is an immune cell.

[0041] In some embodiments, the cell is a T cell or an NK cell.

[0042] In some embodiments, the cells are further engineered to express a dominant negative receptor (DNR) or a chimeric switch receptor (CSR), preferably wherein the DNR is dnTGFβRII and / or dnPD1, preferably wherein the intracellular domain of the CSR is derived from IL7Rα, more preferably wherein the dnTGFβRII comprises the sequence set forth in SEQ ID NO: 193 and the intracellular domain of the CSR comprises the sequence set forth in SEQ ID NO: 197.

[0043] In another aspect, the present specification provides a pharmaceutical composition comprising 1) the above-mentioned antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, or cell, and 2) a pharmaceutically acceptable carrier.

[0044] In another aspect, the present description provides the use of the antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector or cell described above in the manufacture of a medicament for treating a tumor.

[0045] In some embodiments, the tumor expresses EGFRvIII.

[0046] In some embodiments, the tumor is a glioblastoma.

[0047] In another aspect, the present specification provides a method for treating a tumor in a subject, the method comprising administering to the subject a therapeutically effective amount of the above-described antibody or antigen-binding fragment thereof, nucleic acid molecule, expression vector, cell, or pharmaceutical composition.

[0048] In some embodiments, the tumor expresses EGFRvIII.

[0049] In some embodiments, the tumor is a glioblastoma.

[0050] In another aspect, the present description provides a multispecific antibody molecule comprising at least a first functional portion and a second functional portion, wherein the first functional portion comprises an antibody or antigen-binding fragment thereof, and the second functional portion has an antigen-binding specificity different from that of the first functional portion.

[0051] In some embodiments, the second functional moiety has binding specificity for a T cell.

[0052] In another aspect, the present disclosure provides an immunoconjugate comprising the above-described antibody or antigen-binding fragment thereof linked to a therapeutic agent.

[0053] In some embodiments, the therapeutic agent is a drug.

[0054] In some embodiments, the therapeutic agent is a cytotoxin.

[0055] In some embodiments, the therapeutic agent is a radioisotope.

[0056] In another aspect, the present specification provides a kit for detecting the presence or amount of EGFRvIII in a sample, the kit comprising the antibody or antigen-binding fragment thereof. [Brief explanation of the drawings]

[0057] [Figure 1] FIG. 1 is a schematic diagram of the structure of EGFRvIII. [Figure 2] FIG. 2 shows the results of flow cytometry detection of the binding ability of the positive subclone supernatants to target cells. [Figure 3] Figure 3 shows the results of detecting the affinity of some purified murine antibodies by the SPR method. [Figure 4] FIG. 4 is a structural schematic of the CAR used herein. [Figure 5] Figure 5 shows the results of the CAR positivity rate of CAR T cells with mouse-derived antibody sequences. [Figure 6] Figure 6 shows the killing efficiency of CAR T cells with murine-derived antibody sequences against U87MG-EGFRvIII target cells. [Figure 7] Figure 7 shows the killing efficiency of CAR T cells with murine-derived antibody sequences against U87MG-EGFR target cells. [Figure 8] Figure 8 shows the in vivo tumor suppression efficiency of CAR T cells with murine-derived antibody sequences. [Figure 9] FIG. 9 shows the results of detecting the affinity of the humanized antibody of the purified #28 clone by the SPR method. [Figure 10] FIG. 10 shows the results of detecting the affinity of the humanized antibody of the #40-2 clone purified by the SPR method. [Figure 11] Figure 11 shows the CAR-positive rate of CAR T cells produced with the humanized antibody of clone #28. [Figure 12] Figure 12 shows the CAR-positive rate of CAR T cells produced with the humanized antibody of clone #40-2. [Figure 13] Figure 13 shows the killing efficiency of CAR T cells of the humanized antibody of clone #28 against U87MG EGFRvIII target cells. [Figure 14] Figure 14 shows the killing efficiency of CAR T cells of the humanized antibody of clone #28 against U87MG EGFR target cells. [Figure 15] Figure 15 shows the killing efficiency of CAR T cells of the humanized antibody of clone #40-2 against U87MG EGFRvIII target cells. [Figure 16] Figure 16 shows the killing efficiency of CAR T cells of the humanized antibody of clone #40-2 against U87MG EGFR target cells. [Figure 17] Figure 17 shows the in vivo tumor-suppressing efficiency of clonal humanized antibody CAR T cells. [Figure 18] Figure 18 shows the in vivo tumor-suppressing efficiency of clonal humanized antibody CAR T cells. [Figure 19]Figure 19 shows the killing efficiency of CAR NK cells produced with the humanized antibody of clone #28 against target cells. (A) U87MG EGFRvIII target cells; (B) U87MG EGFR target cells. [Figure 20] FIG. 20 shows a schematic diagram of CAR constructs carrying DNR (A) or CSR (B). [Figure 21] Figure 21 shows the results of measuring the CAR positivity rate of the fourth-generation CAR T cells constructed in this application. (A) RII cells (B); T7R cells; (C) CAR T cells. [Figure 22] Figure 22 shows the results of detecting the killing efficiency of CAR T cells. (A) Killing rate without TGFβ treatment; (B) Killing rate with TGFβ treatment; (C) Change in killing rate. [Figure 23] Figure 23 shows the results of a study on the in vivo efficacy of CAR T cells. In the figure, CAR-TC represents control CAR T cells, RII represents CAR T cells expressing the dominant negative receptor dnTGF-βRII, and T7R represents CAR T cells expressing the chimeric switch receptor dnTGF-βRII-IL7Rα. DETAILED DESCRIPTION OF THE INVENTION

[0058] Unless otherwise explained, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art.

[0059] Antibodies are immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to neutralize foreign substances (polypeptides, viruses, bacteria, etc.). The basic structure of a typical antibody molecule is a tetramer consisting of two identical heavy chains and two identical light chains. Based on differences in amino acid sequence conservation, the heavy and light chains are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxy terminus. The variable regions of one heavy chain and one light chain interact to form an antigen-binding site (Fv). Within the variable region, some regions are more variable in terms of amino acid residue composition and sequence than other regions (framework regions, or FRs). These regions are called hypervariable regions (HVRs), and are actually the key sites of antibody-antigen binding. Because their sequences are complementary to antigenic determinants, these hypervariable regions are also called complementarity-determining regions (CDRs). Each heavy chain and light chain has three complementarity-determining regions, designated HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3, respectively. As is known in the art, antibody molecules can be further modified in various ways, such as by PEGylation, glycosylation, formation of antibody molecule conjugates (e.g., ADCs), addition of purification tags, or fusion with other proteins, e.g., to form bispecific antibodies. Derivatives of these antibodies obtained by modification based on the antibody molecules provided herein are also within the scope of the present invention.

[0060] If the sequences of the complementarity-determining regions of an antibody are known, one skilled in the art can easily use, for example, DNA recombinant technology, to replace a portion of the antibody molecule (e.g., the constant region or framework region) with a sequence derived from another species to form a chimeric antibody, which essentially retains the binding specificity of the original antibody. For example, if the original antibody is a murine antibody, its constant region can be replaced with that of a human antibody to reduce the immunogenicity of the antibody when used in a different species or to utilize a specific function of the constant region, such as ADCC-related activity. Conversely, if the original antibody is a human antibody, its constant region can be replaced with that of a murine antibody. Furthermore, to reduce the immunogenicity of the antibody or for other purposes, all sequences other than the CDR sequences in the antibody molecule can be replaced with the corresponding sequences (which may optionally contain one or more amino acid mutations) of another antibody molecule (derived from the same or a different species), while essentially retaining the binding specificity of the original antibody. In one specific example, those skilled in the art often humanize a murine-derived antibody by CDR grafting.

[0061] An "antigen-binding fragment" of an antibody molecule refers to a fragment of amino acids in the antibody molecule that is involved in antigen-specific binding, such as Fab, Fab', or (Fab')2. Methods for obtaining these antigen-binding fragments are known to those skilled in the art. For example, a typical antibody molecule is digested with papain to obtain Fab fragments, with pepsin to obtain F(ab')2, and treated with a reducing agent to cleave the disulfide bond between the hinge regions of F(ab')2, thereby forming Fab' fragments. Single-chain antibodies and single-domain antibodies that have antigen-binding ability are single-peptide chain antibody molecules and can be considered "antigen-binding fragments" of typical antibody molecules.

[0062] The term "Fc fragment" refers to the Y-shaped stalk region of an antibody molecule; i.e., the crystallizable fragment (Fc) contains the second and third constant domains (CH2 and CH3 domains) of the heavy chain. The Fc region of an antibody can be obtained by hydrolysis of an antibody molecule with a protease (e.g., papain). In some instances, the Fc region may contain a hinge, CH2, and CH3. When the Fc region contains a hinge, it can mediate dimerization between two Fc-containing polypeptides. The Fc fragment may be derived from IgG, IgM, IgD, IgE, or IgA. In some instances, the Fc region is derived from IgG1, IgG2, IgG3, or IgG4. The term "Fc fragment" also includes variant Fc fragments that are derived from a native Fc fragment and have been modified but retain their effective functions. A "variant Fc fragment" contains an amino acid sequence with at least one amino acid modification in the amino acid sequence of a native Fc fragment. In some embodiments, the variant Fc fragment has at least one amino acid substitution compared to the parent Fc fragment (native Fc fragment), for example, about 1 to about 10 amino acid substitutions in the parent Fc fragment, and preferably about 1 to about 5 amino acid substitutions. In some embodiments, the Fc region of the variant Fc fragment has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the parent Fc fragment. Effect functions of the "Fc fragment" may include binding to an Fc receptor, Clq binding, and mediating complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, etc.

[0063] A single-chain antibody (single chain fragment variable, scFv) consists of the heavy and light chain variable regions of an antibody linked together into a single peptide chain via a single peptide. By correctly folding, the variable regions from the heavy and light chains interact non-covalently to form an Fv fragment, allowing the scFv to retain its affinity activity for its antigen.

[0064] A "single domain antibody (sdAb)," also known as a "VHH antibody," refers to an antibody molecule that has antigen-binding ability and contains a heavy chain variable region but lacks a light chain. From a structural perspective, a single domain antibody can also be considered a type of antigen-binding fragment of an antibody molecule. It was first discovered in camelids, and researchers subsequently discovered more single domain antibodies with antigen-binding ability through screening of antibody libraries (e.g., phage display libraries). Single domain antibodies have several advantages over conventional antibody molecules (e.g., typical tetrameric antibody molecules) or antigen-binding fragments thereof, including, but not limited to, their smaller molecular weight, which allows them to more easily reach tissues or sites in the human body that are difficult for conventional antibody molecules to reach, or to access antigen epitopes in proteins or polypeptides that are difficult for conventional antibody molecules to access, and their greater stability, such as resistance to temperature and pH changes and the action of denaturants and proteases.

[0065] A "bispecific antibody" is an antibody molecule that has two distinct binding sites and can recognize and bind to two different antigens. For example, one binding site of a bispecific antibody can be used to bind to immune cells (e.g., T cells) and the other to bind to tumor cells, thereby enhancing the immune cell's ability to kill tumor cells while reducing side effects such as off-target toxicity. Such dual-function antibodies have greater therapeutic efficacy as tumor treatment drugs than conventional monoclonal antibody drugs. Similarly, antibody molecules can be engineered to contain multiple distinct binding sites, generating "trispecific antibodies," "tetraspecific antibodies," and the like. As used herein, "multispecific antibody" includes these bispecific antibodies, trispecific antibodies, tetraspecific antibodies, and the like. Preferably, the multispecific antibody used is a bispecific antibody.

[0066] As used herein, the term "humanized antibody" refers to an antibody that retains the reactivity of a non-human antibody while having reduced immunogenicity in humans. For example, this can be achieved by retaining the non-human CDR regions and replacing the remainder of the antibody with its human counterparts (i.e., the constant and variable region framework regions).

[0067] "Immunoconjugate" refers to an EGFRvIII-targeting antibody or antigen-binding fragment thereof disclosed herein conjugated to a therapeutic agent, such as a cytotoxin, a drug (e.g., an immunosuppressant), or a radiotoxin.

[0068] A "fusion protein" refers to a protein molecule that is artificially created (e.g., by genetic engineering) and is composed of at least two distinct peptide fragments that do not occur in nature or in a single protein molecule. Common examples of fusion proteins containing antibody fragments include antibody-cytokine fusion proteins, antibody-cytotoxin fusion proteins (also called immunotoxins), enzyme-labeled antibodies for immunodetection, chimeric antigen receptors (CARs), etc.

[0069] "Targeting" or "specifically binding" refers to a molecule (e.g., an antibody or antigen-binding fragment thereof) having a higher binding affinity for another molecule (e.g., an antigen on the surface of a tumor cell) than for other molecules present in the environment at the same time. "Targeting" or "specifically binding" does not exclude that the molecule may have binding affinity for more than one molecule; for example, a bispecific antibody may have high affinity for two different antigens. The modifier "anti" used before an antigen indicates that the following antibody "targets" or "specifically binds" to that antigen; for example, an anti-EGFRvIII antibody means that the antibody "targets" or "specifically binds" to EGFRvIII. Preferably, the anti-EGFRvIII antibody does not "target" or "specifically bind" to normal EGFR molecules. An antibody's "specific binding to an antigen" or the "antigen-binding specificity" of an antibody refers to the antibody's ability to bind to a target antigen with higher or relatively higher binding affinity than other antigens. The ability of an antibody to bind to a target antigen can be qualitatively or quantitatively determined by various methods, for example, by surface plasmon resonance (SPR) to determine the K D The K value can be measured, or the ability of an antibody to compete with other antibodies for binding to a target antigen. D is the equilibrium dissociation constant, which can be used to estimate the strength of the binding affinity between an antibody and its antigen. K D A smaller value indicates a stronger affinity. The fact that an antibody specifically binds to a target antigen does not mean that it does not bind to other antigens, for example, immune cross-reactivity is also known in the art.

[0070] A "binding complex" is a complex formed between a molecule and its binding target. Examples of binding complexes include antigen-antibody complexes, ligand-receptor complexes, and protein dimers. The forces that form binding complexes primarily involve non-covalent forces such as hydrogen bonds, van der Waals forces, and ionic bonds. Detecting the complex formed between an antigen and an antibody can detect the presence or absence of the target antigen or antibody in a sample, and determine the content or concentration of the target antigen or antibody.

[0071] "Peptide fragment" refers to a polypeptide fragment, which is a short amino acid sequence, eg, about 2-20 amino acids in length, that is typically part of a polypeptide or protein.

[0072] EC50 (concentration for 50% of maximal effect) is the concentration that produces 50% of the maximum effect. For example, when used to express the binding ability of an antibody molecule to its corresponding antigen in cells in FACS measurement, it refers to the antibody molecule concentration at which half of the maximum fluorescence intensity occurs. The lower the EC50 value, the greater the binding affinity to the antigen in cells.

[0073] "Purification tag" refers to an amino acid sequence of a protein or polypeptide used for purification purposes, which is co-expressed with a protein or polypeptide of interest in the form of a fusion protein, and includes, but is not limited to, His6 tag, Flag tag, MBP (maltose-binding protein) tag, GST (glutathione S-transferase) tag, SUMO (small ubiquitin-related modifier), etc. These tags may be removed by enzymatic cleavage after purification, or may remain attached to the protein or polypeptide of interest without affecting its normal function (e.g., His6 tag).

[0074] A "detectable tag" refers to an amino acid sequence or other chemical group linked to a protein or polypeptide to indicate the presence or amount of that protein or polypeptide in a sample or to track the location of that protein or polypeptide within a subject's body or cells. Examples of detectable tags include various enzymes that can be used in immunodetection, e.g., horseradish peroxidase (HRP), alkaline phosphatase (ALP), fluorescent groups (e.g., FAM, FITC) or fluorescent proteins (e.g., GFP), radioisotopes (e.g., 3 H, 14 C. 35 When the detectable tag is an enzyme, the presence or amount of a protein or polypeptide linked to the enzyme can be determined by the enzymatic activity of the enzyme.

[0075] The terms "polypeptide" and "protein" refer to polymers of amino acid residues and can be used interchangeably. Such polymers of amino acid residues can contain naturally occurring or unnatural amino acid residues, and include, but are not limited to, peptides, oligopeptides, dimers, trimers, and multimers composed of amino acid residues. The term also encompasses post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and similar modifications. For purposes of the present invention, "polypeptide" also refers to proteins containing modifications to the native sequence, such as deletions, additions, and substitutions (usually conservative in nature), so long as the protein retains the desired activity. These modifications may be purposeful, e.g., due to site-directed mutagenesis, or may be accidental, resulting from, for example, mutations in hosts producing the protein or errors due to PCR amplification.

[0076] When referring to an antibody or antigen-binding fragment thereof, the term "variant" or "functional variant" refers to a variant or functional variant that contains one or more (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more) amino acid residue insertions, deletions, or substitutions compared to its parent sequence (antibody or antigen-binding fragment), while retaining at least 50%, 60%, 70%, 80%, 90%, or even higher binding affinity for the corresponding antigen (e.g., EGFRvIII) than the parent sequence. As known to those skilled in the art, the sequences of the VH or VL framework regions can be varied considerably, as long as the spatial positions of the corresponding CDR sequences are maintained. For example, functional variants that essentially retain antigen-binding ability can be obtained by modifying the framework region sequence in the amino acid sequence of VH, for example, by swapping framework regions of antibody molecules from different species.Furthermore, functional variants that retain antigen-binding ability can be obtained by modifying a small number of residues in the CDR sequence, for example, one, two, or three amino acid residues, and then detecting the antigen-binding ability of the modified antibody molecule.

[0077] As used herein, the terms "nucleic acid molecule," "nucleic acid," and "polynucleotide" refer to a polymer of nucleotides and can be used interchangeably. Such nucleotide polymers may contain natural and / or non-natural nucleotides and include, but are not limited to, DNA, RNA, and PNA. A "nucleic acid sequence" refers to the linear sequence of nucleotides contained in a nucleic acid molecule or polynucleotide.

[0078] The term "vector" refers to a nucleic acid molecule (e.g., a nucleic acid, a plasmid, or a virus) that has been engineered to contain a polynucleotide of interest (e.g., a coding sequence for a polypeptide of interest) or that is capable of replicating in a host cell. A vector may contain one or more of the following elements: an origin of replication, regulatory sequences (e.g., a promoter and / or enhancer) that control the expression of one or more polynucleotides of interest, and / or one or more selectable marker genes (e.g., antibiotic resistance genes and genes that can be used in colorimetric assays, e.g., β-galactose). The term "expression vector" refers to a vector for expressing a polypeptide of interest in a host cell.

[0079] A "host cell" refers to a cell that can be or has been the recipient of a vector or an isolated polynucleotide. A host cell can be a prokaryotic or eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, e.g., primate or non-primate cells, fungal cells, e.g., yeast, plant cells, and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, 293 and CHO cells, and cells derived therefrom, e.g., 293-6E, CHO-DG44, CHO-K1, CHO-S, and CHO-DS cells. A host cell includes the progeny of a single host cell, and progeny may not necessarily be completely identical (in terms of morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell also includes cells transfected in vivo with a nucleic acid molecule or expression vector provided herein.

[0080] "Treatment" refers to treatment of a subject to achieve a beneficial or desired clinical result. As used herein, "treatment" encompasses a variety of treatment modalities, including administration of any available medication to a subject, surgery, radiation, etc. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviation of one or more symptoms, reduction in the severity of the disease, prevention or delay of disease spread (e.g., metastasis, e.g., to the lungs or lymph nodes), prevention or delay of disease recurrence, delay or alleviation of disease progression, amelioration of disease symptoms, inhibition of the disease or its progression, arrest and alleviation (partial or complete alleviation) of its progression. The methods provided herein include one or more of these treatments. In accordance with the foregoing, "treatment" does not necessarily mean complete elimination or complete alleviation of the disease or all symptoms of the disease.

[0081] The term "therapeutically effective amount" refers to an amount of an active compound sufficient to elicit the biological or medical response desired by a clinician in a subject. The "therapeutically effective amount" of the fusion protein of the present invention can be determined by one skilled in the art based on factors such as the route of administration, the subject's weight, age, and medical condition. For example, a typical daily dosage range may be 0.01 mg to 100 mg / kg body weight or more of the active ingredient.

[0082] When referring to a pharmaceutical composition, the term "pharmaceutically acceptable carrier" refers to a solid or liquid diluent, filler, antioxidant, stabilizer, or other substance that can be safely used, is suitable for administration to humans and / or animals, does not cause excessive adverse side effects, and is suitable for maintaining the activity of the drug or active agent contained therein. Depending on the route of administration, various carriers known in the art can be used, including, but not limited to, sugars, starch, cellulose and its derivatives, maltose, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyhydric alcohols, alginic acid, phosphate buffer, emulsifiers, isotonic saline, and / or pyrogen-free distilled water. The pharmaceutical compositions provided herein may be in a clinically acceptable dosage form, such as a powder or an injectable solution. The pharmaceutical compositions of the present invention can be administered to a subject by any suitable route, such as oral administration, intravenous infusion, intramuscular injection, subcutaneous injection, subperitoneal administration, rectal administration, sublingual administration, inhalation administration, transdermal administration, etc.

[0083] "Subject" refers to an animal, e.g., a mammal, including, but not limited to, humans, rodents, simians, felines, canines, equines, bovines, porcines, sheep, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and pet mammals. A subject may be male or female and of any age, including infants, children, adolescents, adults, and geriatric subjects. In some instances, a subject refers to an individual in need of treatment for a disease or condition. In some instances, a subject receiving treatment may be a patient suffering from or at risk for a condition related to the treatment. In certain instances, the subject is a human, e.g., a human patient. The term is generally used interchangeably with "patient," "subject to be tested," "subject to be treated," etc.

[0084] "Biological sample" means a quantity of a substance from a living or cadaveric organism, including, but not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0085] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also referred to as "sequence identity") refers to the amount of match between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Typically, before calculating the percentage of identity between two amino acid or nucleotide sequences, a sequence alignment is first performed to introduce gaps, if any. If the amino acid residues or bases in the two sequences are similar at a certain alignment position, the two sequences are considered to match at that position. If the amino acid residues or bases in the two sequences are different, the position is considered to be mismatched. Some algorithms calculate the sequence identity by dividing the number of matching positions by the total number of positions in the alignment window. Other algorithms further consider the number of gaps and / or gap length. For purposes of the present invention, the open alignment software BLAST (accessible at the homepage ncbi.nlm.nih.gov) can be used to obtain optimal sequence alignment with default settings and calculate the sequence identity between two amino acid or nucleotide sequences.

[0086] Chimeric antigen receptors (CARs), also known as chimeric T cell receptors or chimeric immune receptors, are engineered protein receptor molecules that can confer desired specificity, such as the ability to bind to specific tumor antigens, to immune effector cells. Chimeric antigen receptors typically consist of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain. The antigen-binding domain is often a single scFv sequence, responsible for recognizing and binding to a specific antigen. The intracellular signaling domain typically contains an immunoreceptor tyrosine-based activation motif (ITAM), such as a signaling domain derived from the CD3ζ molecule, and is responsible for activating immune effector cells and resulting in their killing. Chimeric antigen receptors can also contain a signal peptide at the amino terminus responsible for intracellular localization of the nascent protein, and a hinge region between the antigen-binding domain and the transmembrane domain. In addition to the signaling domain, the intracellular signaling domain may also contain a costimulatory domain, such as that derived from the 4-1BB molecule.

[0087] "CAR-T cells" refer to T cells that express a CAR, and are typically obtained by transducing T cells with an expression vector encoding the CAR. Commonly used expression vectors are viral vectors, such as lentiviral expression vectors. Chimeric antigen receptor-modified T cells (CAR-T) are not restricted by the major histocompatibility complex and have specific target-killing activity and the ability to proliferate continuously. In addition to T cells, other lymphocytes, such as NK cells, can also be transformed with an expression vector encoding a CAR to obtain target-killing cells expressing the CAR.

[0088] A "dominant negative receptor (DNR)" refers to a receptor that can bind to its ligand but does not induce a signal cascade within the cell. DNRs typically have an intact ligand-binding domain but lack the domain of intracellular enzymatic activity (e.g., kinase activity). They may be a mutant form of the full-length receptor or a truncated form of the receptor. After CAR T cell immunotherapy, some cancers, particularly solid tumors, may upregulate inhibitory ligands that bind to inhibitory receptors in CAR T cells. Such adaptive resistance impairs the efficacy of CAR T cell therapy. Some cancers, particularly solid tumors, are known to generate an immunosuppressive environment by secreting transforming growth factor β (TGF-β). TGF-β can induce or promote metastasis and effectively suppress the immune system. Therefore, in some embodiments, a truncated form of the TGF-β receptor TGFBRII is used as a TGF-β DNR to improve the antitumor function of the CAR T cells disclosed herein. In some embodiments, the CAR and TGF-β DNR are co-expressed on the surface of T cells with a 2A self-cleaving peptide. In some embodiments, the CAR and TGF-β DNR are expressed on the surface of T cells using two expression vectors, respectively. We have found that introducing TGF-β DNR into the CAR T cells disclosed herein can enhance the cytotoxicity of the CAR T cells against some EGFRvIII-positive cancer cells. Similarly, in some embodiments, a truncated form of the PD-1 receptor is used as the PD-1 DNR to improve the anti-tumor function of the CAR T cells disclosed herein.

[0089] A "chimeric switch receptor (CSR)" refers to a receptor engineered to reverse the outcome of its original signaling pathway, thereby conferring a desired activity to immune cells (e.g., CAR T cells), such as the ability to overcome an immunosuppressive tumor microenvironment and achieve greater in vivo persistence. In some embodiments, the CSR can further stimulate CAR T cells with inhibitory molecules expressed by cancer cells. In one non-limiting example, CAR T cells may be engineered with a CSR that expresses the PD-1 extracellular ligand domain fused to the transmembrane and intracellular costimulatory signaling domains of CD28. When the CAR T cells are administered to a subject with a cancer that expresses EGFRvIII and PD-L1, the expressed CAR can bind to EGFRvIII, and the simultaneously expressed CSR can bind to PD-L1. The properties of the PD-1 / CD28 chimeric switch receptor fusion protein stimulate CAR T cells by promoting signaling through the CD28 domain while blocking normal PD1 / PD-L1-mediated T cell suppression. Therefore, by switching the transmembrane and intracellular domains of PD-1 with those of CD28, PD-L1 is converted into a costimulatory ligand for CAR T cells, which induces enhanced toxicity against PD-L1-expressing cancer cells. In another non-limiting example, by switching the transmembrane and intracellular domains of TGFBRII with those of the IL7Rα receptor, the inhibitory signal of TGF-β can be converted into a stimulatory signal for CAR T cells in the tumor microenvironment, promoting their proliferation or activity.

[0090] Anti-EGFRvIII antibodies and antigen-binding fragments thereof The present specification provides anti-EGFRvIII antibodies that specifically bind to EGFRvIII. The EGFRvIII antibodies bind to the EGFRvIII antigen with relatively high binding affinity. As described in the Examples below, the binding ability of anti-EGFRvIII antibodies to EGFRvIII can be measured by a measurement method such as surface plasmon resonance (SPR) technology. It can also be measured by other protein interaction measurement methods known in the art, such as biolayer interference (BLI) technology, enzyme-linked immunosorbent assay (ELISA), and fluorescence-activated cell sorting (FACS).

[0091] The present specification provides CDR sequences of multiple types of anti-EGFRvIII antibodies, for example, the heavy chain CDR sequences of which are represented by SEQ ID NOs: 1-3, 4-6, 7-9, 10-12, 13-15, 16-18, 19-21, 22-24, 25-27, 28-30, 31-33, 34-36, 37-39, 40-42, 43-45, 46-48, 49-51, 52-54, or 55-57, respectively. Preferably, the corresponding light chain CDR sequences are those set forth in SEQ ID NOs: 58-60, 61-63, 64-66, 67-69, 70-72, 73-75, 76-78, 79-81, 82-84, 85-87, 88-90, 91-93, 94-96, 97-99, 100-102, 103-105, 106-108, 109-111 or 112-114, respectively.

[0092] In some embodiments, the heavy chain variable region of an anti-EGFRvIII antibody provided by the present invention comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99% or even 100% sequence identity) to the sequence set forth in SEQ ID NO: 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149 or 151. Preferably, the corresponding light chain variable region comprises an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99% or even 100% sequence identity) to the sequence set forth in SEQ ID NO: 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150 or 152.

[0093] Based on the CDR sequences of the anti-EGFRvIII antibodies provided herein, those skilled in the art can construct polypeptide constructs with various EGFRvIII-binding abilities, including combining these CDR sequences with framework regions (FRs) from different antibody molecules. These framework regions may include natural framework region sequences from human antibodies or animal (e.g., mouse, rat, sheep, camel, etc.) antibodies. These framework regions may also include variants of framework region sequences generated by modifying the natural framework region sequences. Polypeptide constructs that specifically bind to EGFRvIII can be easily obtained by combining the CDR sequences provided herein with different framework region sequences to form heavy chain variable regions and then detecting their binding ability to EGFRvIII. Preferably, humanized antibodies, such as humanized single-chain fragments (scFVs), may be constructed based on these CDR sequences.

[0094] In some embodiments, the humanized single chain antibodies (scFVs) provided by the present invention comprise an amino acid sequence having at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99% or even 100% sequence identity) to the sequence set forth in SEQ ID NO: 169, 171, 173, 175, 177, 179, 181, 183, 185 or 187.

[0095] It is anticipated that the anti-EGFRvIII antibodies or antigen-binding fragments thereof described herein may contain conservative amino acid substitutions. Conservative amino acid substitutions are generally described as substitutions of one amino acid residue with another amino acid residue of similar chemical structure, while having little or no effect on the function, activity, or other biological properties of the polypeptide. Conservative amino acid substitutions are well known in the art. Conservative substitutions include, for example, substitutions of one amino acid from the following sets (a) to (e) for another amino acid from the same set: (a) small aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Pro, and Gly; (b) polar negatively charged residues and their (uncharged) amides: Asp, Asn, Glu, and Gln; (c) polar positively charged residues: His, Arg, and Lys; (d) large aliphatic nonpolar residues: Met, Leu, Ile, Val, and Cys; and (e) aromatic residues: Phe, Tyr, and Trp. Therefore, those skilled in the art will understand that by substituting, deleting, or adding a small number of amino acids based on the specific sequences provided herein and verifying or screening the binding ability or biological activity of the resulting products with the corresponding antigen EGFRvIII, corresponding variants of the antibody molecules targeting EGFRvIII provided herein can be obtained, and these variants are also included within the scope of the present invention. For example, the antibody molecules or single-chain antibodies provided herein may have at least one and not more than 10, for example, not more than 5, 4, 3, 2, or 1 amino acid alterations in their full-length or variable region sequences or CDR sequences.

[0096] In some embodiments related to the application of the anti-EGFRvIII antibody, an antigen-binding fragment of the anti-EGFRvIII antibody binds to the corresponding antigen (EGFRvIII). Preferably, the antigen-binding fragment is a single-chain antibody (scFv). For example, when the antibody is used in a CAR, the single-chain antibody (scFv)(s) formed therefrom can serve as the extracellular antigen-binding domain of the CAR.

[0097] Fusion proteins comprising anti-EGFRvIII antibodies or antigen-binding fragments thereof In addition, the anti-EGFRvIII antibody or its antigen-binding fragment may form a fusion protein with another polypeptide.

[0098] In some embodiments, an antigen-binding fragment capable of binding to EGFRvIII can be linked to an Fc fragment (modified or derived from a different species) to form a fusion protein, i.e., a typical antibody molecule. The Fc fragment may be located at the C-terminus or N-terminus of the antigen-binding fragment of an EGFRvIII antibody. Preferably, the Fc fragment is located at the C-terminus of the antigen-binding fragment of an EGFRvIII antibody. A fusion protein consisting of an antigen-binding fragment of an EGFRvIII antibody and an Fc fragment retains the ability to specifically bind to EGFRvIII and simultaneously retains the functional properties of the Fc fragment, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), and phagocytosis. Furthermore, fusion with an Fc fragment increases the half-life of the antigen-binding fragment of an anti-EGFRvIII antibody in vivo, thereby allowing for shorter administration intervals when the antigen-binding fragment of an anti-EGFRvIII antibody is used as a therapeutic drug.

[0099] In some embodiments, the anti-EGFRvIII antibody or antigen-binding fragment thereof may be linked to a protein tag to form a fusion protein. The protein tag may include a purification tag and a detectable tag. Purification tags include, but are not limited to, His6 tag, Flag tag, MBP tag, GST tag, SUMO tag, etc. The detectable tag indicates the presence or amount of the anti-EGFRvIII antibody or antigen-binding fragment thereof in a sample or tracks the location of the anti-EGFRvIII antibody or antigen-binding fragment thereof within a subject's body or cells. Examples of detectable tags include various enzymes that can be used in immunodetection, such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP), and fluorescent proteins, such as GFP. Due to the specific binding ability of the anti-EGFRvIII antibody or antigen-binding fragment thereof to EGFRvIII, the amount of the anti-EGFRvIII antibody or antigen-binding fragment thereof can be determined by the amount of the detectable tag linked to the anti-EGFRvIII antibody or antigen-binding fragment thereof, and further the presence or amount of EGFRvIII in a sample can be determined.

[0100] In another embodiment, the anti-EGFRvIII antibody or antigen-binding fragment thereof may be linked to a cytokine or therapeutic protein to form a fusion protein. In such a case, the specific binding ability of the anti-EGFRvIII antibody or antigen-binding fragment thereof to EGFRvIII allows the cytokine or therapeutic protein to be delivered to a specific tissue or cell (e.g., tumor tissue expressing EGFRvIII) for a specific purpose, thereby achieving the therapeutic effect of the cytokine or therapeutic protein.

[0101] Multispecific antibodies comprising anti-EGFRvIII antibodies or antigen-binding fragments thereof In some embodiments, provided herein are multispecific antibody molecules comprising at least one EGFRvIII-binding domain (or functional unit) and one or more additional binding domains that bind to a second antigen or protein other than EGFRvIII.

[0102] In some embodiments, the present disclosure provides a multispecific antibody molecule comprising at least two EGFRvIII-binding domains that differ in amino acid sequence and preferably each bind to a different antigenic epitope on EGFRvIII.

[0103] In some embodiments, the domain that binds to EGFRvIII is in the form of a single chain antibody, and the one or more additional binding domains may be single domain antibodies, single chain antibodies, or other antigen-binding fragments.

[0104] In some embodiments, the second antigen is a tumor-associated antigen (TAA) or a tumor microenvironment-associated antigen (TMEAA). In some embodiments, the second antigen is an immunomodulatory antigen, wherein the antigen is associated with enhancing or suppressing a signaling pathway in an immune cell. In some embodiments, the second antigen is a surface molecule of a T cell, e.g., a component of the T cell receptor complex, e.g., CD3 (including the gamma, delta, epsilon, zeta, and eta chains).

[0105] Preferably, said multispecific binding peptide is a bispecific antibody molecule.

[0106] In some embodiments, the multispecific binding peptide further comprises an Fc fragment, the presence of which can facilitate the formation of multimers of the binding domains and provide the associated effector function.

[0107] Immune complexes (or antibody complexes) containing anti-EGFRvIII antibodies or antigen-binding fragments thereof The present disclosure provides a conjugate comprising at least one anti-EGFRvIII antibody or antigen-binding fragment thereof that specifically binds to EGFRvIII provided herein and one or more other functional moieties. The other moieties may be chemical groups, such as therapeutic agents, e.g., cytotoxic agents, or tracers. In some instances, the moieties may be targeting moieties, small molecule drugs (e.g., non-polypeptide drugs less than 500 Da), toxins, cell growth inhibitors, cytotoxic agents, immunosuppressants, radioactive reagents suitable for diagnostic purposes, radioactive metal ions for therapeutic purposes, etc.

[0108] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) comprising one or more anti-EGFRvIII antibodies or antigen-binding fragments thereof provided herein and a therapeutic agent that is cytotoxic, inhibits cell growth, or provides some therapeutic benefit. In some examples, the cytotoxic agent is a chemotherapeutic agent, a drug, a growth inhibitor, a toxin (e.g., an enzymatically active toxin or fragment thereof of bacterial, fungal, plant, or animal origin), or a radioisotope (i.e., a radioconjugate). In some instances, the antibody-drug conjugates provided by the present invention are designed to deliver the drug moiety to tumors in a targeted manner. In some cases, this results in targeted killing of tumor cells.

[0109] In some instances, the therapeutic agent includes, for example, daunorubicin, doxorubicin, methotrexate, vindesine, maytansinoids, etc. In some instances, the therapeutic agent has intracellular activity. In some instances, an immune complex conjugated with an anti-EGFRvIII antibody or antigen-binding fragment thereof is taken up by a cell, and the therapeutic agent has activity that blocks cellular protein synthesis or nucleic acid synthesis, leading to stunted cell growth or death.

[0110] In some embodiments, an immunoconjugate comprises one or more anti-EGFRvIII antibodies or antigen-binding fragments thereof provided herein and a tracer. The immunoconjugate may be for research or diagnostic purposes, e.g., for the detection of cancer in vivo. The tracer can generate a detectable signal directly or indirectly. For example, the tracer can be a radioisotope, e.g., 3 H, 14 C. 32 P, 35 S, 123 I, fluorescent (fluorophore) or chemiluminescent (chromophore) compounds, such as fluorescein isothiocyanate, rhodamine, or fluorescein, contrast agents, or metal ions. In some embodiments, the tracer is a radioactive atom for scintigraphic studies, e.g., 99 Tc and 123 I, or spin labels for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), e.g. 89 Zr, 123 I, 19 F, 13 C. 15 N, 17 In O, 89 Zr can be coordinated with many metal chelators and attached to antibodies, for example, for use in PET imaging.

[0111] The anti-EGFRvIII antibody or its antigen-binding fragment may be linked to other functional moieties via covalent or non-covalent bonds. Examples of non-covalent bonds include the biotin-avidin system. Examples of covalent bonds include various chemical linkers, including peptide linkers, cleavable linkers, or non-cleavable linkers. Exemplary linker moieties include 6-maleimidocaproyl ("MC"), maleimidopropionyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carbonate ("SMCC"), and N-succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB").

[0112] In some embodiments, the linker may comprise an amino acid residue. Exemplary amino acid linker moieties include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues contained in the amino acid linker moiety include naturally occurring residues and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker moieties can be designed and optimized for selectivity to be enzymatically catalyzed by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, and plasmin.

[0113] Conjugates of anti-EGFRvIII antibodies or antigen-binding fragments thereof and cytotoxic agents can be prepared with a number of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate dihydrochloride), activated esters (e.g., disuccinimidyl sublate), aldehydes (e.g., glutaraldehyde), bisazide compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bisdiazonium derivatives (e.g., bis(p-azidobenzoyl)ethylenediamine), diisocyanates (e.g., toluene-2,6-diisocyanate), and diactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene).

[0114] Chimeric antigen receptor (CAR) comprising an anti-EGFRvIII antibody or its antigen-binding fragment The chimeric antigen receptors (CARs) provided herein are artificially constructed chimeric proteins that contain an extracellular antigen-binding domain that specifically binds to EGFRvIII, a transmembrane domain, and one or more intracellular signaling domains. Characteristics of these CARs include their ability to specifically and reactively redirect T cells to cells expressing EGFRvIII in a non-MHC-restricted manner. Non-MHC-restricted recognition of EGFRvIII allows T cells or NK cells expressing the disclosed CARs to recognize antigens independently of antigen processing and kill cells expressing EGFRvIII (e.g., tumor cells).

[0115] The intracellular signaling domain may comprise a T cell receptor signaling domain, a T cell costimulatory signaling domain, or both. The T cell receptor signaling domain may comprise the intracellular domain of a T cell receptor, e.g., the intracellular portion of the CD3 zeta protein. The costimulatory signaling domain is the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or its ligand, that is required for an effective response of lymphocytes to antigens.

[0116] In some embodiments, the CARs provided herein comprise an extracellular antigen-binding domain that specifically binds to EGFRvIII. For example, the antigen-binding domain may be an scFv, or the heavy and light chain variable regions of any antibody or antigen-binding fragment thereof linked by a peptide linker that specifically binds to EGFRvIII. Also, for example, in a CAR constructed from a single-domain antibody that specifically binds to EGFRvIII, the extracellular antigen-binding domain may comprise a VHH derived from the single-domain antibody, but may not include any light chain variable region.

[0117] In some embodiments, the extracellular antigen-binding domain of a CAR provided herein comprises one or more antigen-binding fragments from the anti-EGFRvIII antibodies provided herein, e.g., non-humanized or humanized single chain antibodies (scFv).

[0118] When the extracellular antigen-binding domain comprises two or more antigen-binding fragments, they are directly linked in tandem or linked in tandem via a peptide linker. The use of two or more tandemly linked antigen-binding fragments as the extracellular antigen-binding domain or a portion thereof is advantageous in promoting the recognition and binding of the constructed CAR to a target molecule (e.g., EGFRvIII). Preferably, each of these antigen-binding fragments binds to a different epitope on EGFRvIII.

[0119] The CAR may contain a signal peptide sequence, for example, at the N-terminus of the antigen-binding domain. Any suitable signal peptide sequence may be used. In one embodiment, the signal peptide sequence is a CD8α signal peptide. Although a signal peptide sequence can promote the expression of the CAR on the cell surface, the presence of the signal peptide sequence in the expressed CAR is not essential for the CAR to function. When the CAR is expressed on the cell surface, the signal peptide sequence may be cleaved from the CAR. Therefore, in some embodiments, the CAR lacks a signal peptide sequence.

[0120] Between the antigen-binding domain and the transmembrane domain of the CAR, there may be a hinge region (or spacer domain), which comprises a polypeptide sequence. The hinge region may comprise up to 300 amino acids, preferably 10-100 amino acids. In one embodiment, the CAR provided herein comprises the hinge region of the CD8α protein.

[0121] The CAR may comprise a transmembrane domain fused to the extracellular domain of the CAR, hi one embodiment, a transmembrane domain that is naturally associated with other elements in the CAR (e.g., a binding region or an intracellular signaling domain) is used.

[0122] The transmembrane domain may be of natural or synthetic origin. If naturally derived, the domain may be derived from any membrane-bound or transmembrane protein. Exemplary transmembrane domains used in the disclosed CARs may include at least the transmembrane domains of the α, β, or ζ chain of the T cell receptor, CD28, CD3 ε, CD45, CD4, CD5, CDS, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. Alternatively, the transmembrane domain may be synthetic, in which case it contains primarily hydrophobic residues, such as leucine and valine. In some embodiments, a trimer of phenylalanine, tryptophan, and valine appears at each end of the synthetic transmembrane domain.

[0123] Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, may form the link between the transmembrane domain and the intracellular T cell signaling domain and / or T cell costimulatory domain of the CAR. Exemplary linker sequences include one or more glycine-serine dimers.

[0124] In some embodiments, the transmembrane domain comprises a transmembrane domain of a T cell receptor, for example, a CD8α transmembrane domain.

[0125] The intracellular region of the CAR comprises one or more intracellular T cell signaling domains and is responsible for activating at least one normal effector function of the T cell expressing the CAR. Exemplary T cell signaling domains are provided herein and known to those of skill in the art.

[0126] While the complete intracellular T cell signaling domain may be used in a CAR, in many cases the complete chain need not be used. For use of truncated portions of the intracellular T cell signaling domain, the truncated portions can be used in place of the complete chain, provided that they transmit the relevant T cell effector function signals.

[0127] Examples of intracellular T cell signaling domains for use in CARs include the intracellular sequences of the T cell receptor (TCR) and costimulatory molecules that cooperate to initiate signaling upon antigen binding to the receptor, as well as any derivatives or variants of these sequences and any synthetic sequences capable of similar function.

[0128] T cell receptor signaling domains regulate the activation of the T cell receptor complex in a stimulatory or inhibitory manner. The CARs disclosed herein may comprise an intracellular signaling sequence that acts in a stimulatory manner, which may include a signaling motif called an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of primary intracellular signaling sequences containing ITAMs that may be included in the disclosed CARs include intracellular domains derived from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, and CD66d proteins. In some embodiments, the intracellular signaling molecule in the CAR comprises an intracellular T cell signaling domain derived from CD3 zeta.

[0129] The intracellular domain of the CAR provided herein may comprise a CD3 zeta chain portion and an intracellular costimulatory signaling domain. The costimulatory signaling domain may comprise the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an effective response of lymphocytes to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0130] In some embodiments, a CAR may comprise a CD3ζ signaling domain, a CD8 signaling domain, a CD28 signaling domain, a 4-1BB signaling domain, or a combination of two or more thereof. In one embodiment, the intracellular domain comprises a CD3ζ signaling domain and a CD28 signaling domain. In another embodiment, the intracellular domain comprises a CD3ζ signaling domain and a 4-1BB signaling domain. In yet another embodiment, the intracellular domain comprises a CD3ζ signaling domain and CD28 and CD137 signaling domains. One skilled in the art can change the order of one or more T cell signaling domains in a CAR as needed. The intracellular signaling sequences in the intracellular signaling portion of a CAR of the present invention may be linked to each other randomly or in a specified order. Optionally, a short polypeptide linker, preferably between 2 and 10 amino acids in length, may form the linkage. A glycine-serine dimer provides a particularly suitable linker. A spacer domain, including a polypeptide sequence, may also be present between the signaling domain and the transmembrane domain of the CAR. The spacer domain may contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0131] Based on the CAR provided herein (as a parent CAR), one skilled in the art can modify it, for example, fuse it with other polypeptides, or leave only fragments of the CAR provided herein, and these fusion molecules or fragments have at least some of the biological activity of the parent CAR, for example, the activity of recognizing or detecting target cells (e.g., tumor cells expressing EGFRvIII), treating or preventing disease.

[0132] The CARs provided herein may further comprise additional amino acids at the amino or carboxy terminus, or at both termini, which additional amino acids are not present in the amino acid sequence of the parent CAR. Desirably, the additional amino acids do not interfere with the biological function of the CAR or functional portion, such as target cell recognition, cancer detection, cancer treatment or prevention, etc. More desirably, the additional amino acids enhance the biological activity compared to the biological activity of the parent CAR.

[0133] Also provided herein are functional variants of the CARs described herein that have substantial or significant sequence identity or similarity with the parent CAR, and the functional variants retain the biological activity of the CAR as the variant. Functional variants include, for example, variants of the CARs (parent CARs) described herein that retain the ability to recognize target cells to a similar, equal, or greater extent than the parent CAR. With reference to the parent CAR, functional variants have, for example, at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more amino acid sequence identity with the parent CAR.

[0134] A functional variant may comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. Alternatively, a functional variant may comprise the amino acid sequence of a parent CAR with at least one non-conservative amino acid substitution. In such cases, preferably, the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution can enhance the biological activity of the functional variant, making the biological activity of the functional variant higher than that of the parent CAR.

[0135] The CARs provided herein may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, n-leucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, dihydrochloride, cyclohexylglycerin ... These include indole-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and α-t-butylglycine.

[0136] The CARs provided herein may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized, e.g., by disulfides, or converted to acid addition salts, and / or optionally dimerized or multimerized, or conjugated.

[0137] Methods for generating chimeric antigen receptors, T cells containing such receptors, and their uses (e.g., for treating cancer) are known in the art. For example, a nucleic acid molecule encoding a disclosed chimeric antigen-binding receptor may be included in an expression vector (e.g., a lentiviral vector) such that it is expressed in a host cell, e.g., a T cell, to produce the disclosed CAR. In some embodiments, a method for using a chimeric antigen receptor includes isolating T cells from a subject, transforming the T cells with an expression vector (e.g., a lentiviral vector) encoding the chimeric antigen receptor, and administering the engineered T cells expressing the chimeric antigen receptor to the subject for use in therapy, e.g., treating a tumor in the subject.

[0138] The CARs provided herein can also be used in combination with safety mechanisms to control the activity of the CAR or cells expressing the CAR. In one embodiment, the CAR is coexpressed with "EGFRt." "EGFRt" refers to a truncated human epidermal growth factor receptor polypeptide that lacks the membrane-distal EGF-binding domain and intracellular signaling tail but retains an extracellular epitope recognized by anti-EGFR antibodies. EGFRt is useful as a non-immunogenic selection tool and tracking marker for genetically modified cellular functions. While useful as a marker molecule for CAR-T cells, it can also be used as a safety switch during clinical transformation, for example, by eliminating CAR-T cells in vivo using the ADCC pathway mediated by EGFR antibodies (e.g., cetuximab) (see US8802374B2). In addition to using tEGFR, the CAR-T cells provided herein can also be used in combination with safety switches based on other mechanisms. For example, the CARs provided herein can be engineered to control CAR activity using a chemical inducer of dimerization (CID). In one example, the complete CAR provided herein is expressed as a separate fusion protein (e.g., the CD3ζ intracellular domain is expressed separately from other portions), and the complete CAR is reconstituted in cells using the dimerization mechanism of an FKBP / FRB-based binding domain and rapamycin (or a rapamycin analog), allowing the integrity and activity of the CAR to be controlled by the presence or absence of rapamycin (see, for example, U.S. Patent 11,084,880). Furthermore, when a self-cleaving peptide is used to express a fusion protein with suicide capabilities, the activity of CAR-T cells can be controlled by inducing degradation of the CAR molecule or death of the CAR-T cells. Examples of such an example include the use of proteins capable of inducing apoptosis, such as caspase-9, and degradation of the CAR molecule using PROTAC technology.

[0139] Pharmaceutical compositions and treatment methods comprising anti-EGFRvIII antibodies or antigen-binding fragments thereof The anti-EGFRvIII antibodies or antigen-binding fragments thereof disclosed herein, as well as fusion proteins, multispecific antibody molecules, and immunoconjugates containing the anti-EGFRvIII antibodies or antigen-binding fragments thereof (which may be formulated into pharmaceutical compositions together with a pharmaceutically acceptable carrier) can be administered to subjects to prevent or treat tumors. The effective dosage for such use depends on the severity of the disease and the overall state of the patient's immune system. Dosage regimens vary depending on the state of the disease and the subject's condition, but typically range from a single bolus or continuous infusion to multiple doses per day (e.g., every 4-6 hours). A clinician skilled in the art can easily determine whether a subject is a candidate for such treatment, for example, based on clinical tests, physical examination, and the subject's family history.

[0140] In some embodiments, the anti-EGFRvIII antibodies or antigen-binding fragments thereof disclosed herein, and fusion proteins, multispecific antibody molecules, and immunoconjugates comprising the anti-EGFRvIII antibodies or antigen-binding fragments thereof, can be administered in combination with one or more other drugs (e.g., anti-tumor agents).

[0141] In some embodiments, the disease or condition being treated is a tumor, preferably glioblastoma. The anti-EGFRvIII antibodies or antigen-binding fragments thereof disclosed herein (and fusion proteins, multispecific antibody molecules, and immunoconjugates comprising the anti-EGFRvIII antibodies or antigen-binding fragments thereof) can be administered by any suitable method or means, optionally in combination with other anti-tumor agents. Routes of administration include, for example, oral, intravenous, intraperitoneal, subcutaneous, or intramuscular administration.

[0142] The nucleic acid molecules encoding the anti-EGFRvIII antibodies or antigen-binding fragments thereof provided herein, expression vectors containing the nucleic acid molecules, and host cells transfected with the nucleic acid molecules alone or with the expression vectors can also be used for the above-mentioned therapeutic purposes by various means. For example, the expression vector can be introduced into the body of a subject by gene therapy methods known in the art to express the desired protein or polypeptide (anti-EGFRvIII antibody or antigen-binding fragment thereof) for therapeutic purposes.

[0143] Detection, diagnostic or therapeutic kits comprising anti-EGFRvIII antibodies or antigen-binding fragments thereof The anti-EGFRvIII antibodies or antigen-binding fragments thereof provided herein, as well as other forms of molecules (e.g., fusion proteins or bispecific antibody molecules) containing the anti-EGFRvIII antibodies or antigen-binding fragments thereof, can specifically bind to EGFRvIII in a sample. The content (or presence or absence) of EGFRvIII in a sample can be conveniently determined by detecting the amount of the anti-EGFRvIII antibody or antigen-binding fragment-EGFRvIII complex formed, or the amount of the anti-EGFRvIII antibody or antigen-binding fragment thereof in the anti-EGFRvIII antibody or antigen-binding fragment-EGFRvIII complex formed.

[0144] As described above, for this purpose, the anti-EGFRvIII antibodies or antigen-binding fragments thereof provided herein can be linked to various detection tags so that they can be detected by various means, including, but not limited to, bioluminescence, fluorescence, radioactive labeling, and the amount of product produced by an enzyme-catalyzed reaction. After detecting the EGFRvIII content in a sample, the content can be compared with the normal EGFRvIII content in a group of healthy individuals and used to determine the condition or severity of the disease in the subject who provided the sample. Changes in the EGFRvIII content can be periodically detected during the course of treatment of the subject and used to determine the effectiveness of the treatment, providing a basis for modifying the treatment plan.

[0145] The anti-EGFRvIII antibodies or antigen-binding fragments thereof provided herein, other forms of molecules containing the anti-EGFRvIII antibodies or antigen-binding fragments thereof (e.g., fusion proteins or bispecific antibody molecules), and cells expressing CARs may be placed in containers to form detection, diagnostic, or therapeutic kits. These containers may be in the form of boxes, ampoules, vials, tubes, bags, or other suitable containers known in the art. These containers may be made of plastic, glass, laminated paper, metal foil, or other materials suitable for storing drugs. If necessary, the containers may further include instructions for use. The instructions may typically include information about how to use the anti-EGFRvIII antibody or antigen-binding fragment thereof, composition comprising the anti-EGFRvIII antibody or antigen-binding fragment thereof, or CAR-T cells to treat or prevent tumors (e.g., glioblastoma), e.g., a description of the therapeutic agent (e.g., anti-EGFRvIII antibody or antigen-binding fragment thereof), dosage regimens for treating or preventing tumor (e.g., glioblastoma) formation, precautions, warnings, indications, contraindications, adverse reactions, animal pharmacology, clinical studies, and / or reference materials. The instructions can be printed directly on the container (if present), or can be a label affixed to the container, or a separate paper, booklet, card, or foldout provided in or with the container.

[0146] In some embodiments, the anti-EGFRvIII antibodies or antigen-binding fragments thereof provided herein have binding affinity to EGFRvIII that is similar to or better than that of an existing control antibody. The anti-EGFRvIII antibodies or antigen-binding fragments thereof provided herein are humanized and have binding affinity to EGFRvIII that is similar to or better than that of an existing control antibody.

[0147] This study immunized mice using different immunization methods, compared the specificity and affinity of the antibodies obtained by different immunization methods, and screened for the optimal immunization conditions. A series of novel murine monoclonal antibodies against the target antigen of EGFRvIII were screened using government screening methods. The affinity of the antibodies was detected using methods such as ELISA, FACS, and SPR. High-affinity antibodies were selected, and then the antibodies were sequenced. CAR T cells targeting EGFRvIII were designed and prepared using the antibody sequences. The optimal antibody sequences were screened through in vitro and in vivo efficacy studies. The optimal murine antibody sequences were selected for further humanization. The affinity of the humanized antibodies was detected using SPR technology, and the humanized antibodies with the optimal affinity were screened. The antibodies were sequenced, and the high-affinity humanized antibodies were selected to prepare a series of CAR T cells targeting the EGFRvIII antigen. The optimal humanized antibody sequences were selected through in vitro and in vivo efficacy studies. CAR NK cells were prepared with the selected optimal antibody sequences, and the efficacy of CAR NK in treating solid tumors was determined by in vitro efficacy studies.

[0148] To overcome the inhibitory effects of cellular immunotherapy for solid tumors, this study simultaneously performed genetic modification on second-generation CAR T cells to improve the resistance of CAR T cells to the inhibitory effects of the tumor immune microenvironment and increase their persistence and ability to infiltrate into solid tumors. This study simultaneously expressed dnTGFβRII in the independently developed EGFRvIII CAR T cells, or converted the inhibitory signal of dnTGFβRII into an IL7-activating signal. The increased expression significantly improved the tumor-suppressing efficiency of CAR T cells and allowed them to resist the inhibitory effects of TGFβ.

[0149] Example 1. Experimental Procedure 1.1 Development of murine antibodies 1.1.1 Immunization of mice Before immunization, 100 μl of blood was collected from each mouse and incubated at 37°C for 5 minutes before centrifugation. Serum was collected, with negative serum used as a control. Mice were immunized in three different ways: Group 1 was immunized with a protein / polypeptide conjugate, Group 2 with overexpressing EGFRvIII target cells, and Group 3 with only the polypeptide. Adjuvant and antigen were mixed at a 1:1 ratio and emulsified. The emulsified immunogen was injected subcutaneously into mice. One week after the second immunization, blood was collected from the mice, serum was separated, and antibody titers were measured. Depending on the antibody titer, booster immunizations were administered until the serum titer reached the desired level, followed by a final immunization.

[0150] 1.1.2 Titer detection The serum titer was detected by flow cytometry. Positive and negative cells were identified with F98npEGFRvIII and F98npEGFR (ATCC), respectively. The cells were digested and collected in a centrifuge tube. The cells were centrifuged at 300g for 5 minutes. The supernatant was discarded, and the cells were washed twice with an appropriate amount of 1x PBS. The supernatant was discarded, and the cells were resuspended in 1x PBS. 50 μl of the cell suspension was placed in a 96-well plate. Approximately 2 x 10^5 cells were added to each well. Each well contained 1 / 50 and 1 / 500 diluted serum, positive control antibody C097VEJ300, isotype control mouse IgG, and negative control PBS. Each well contained 100 μl of diluted fluorescent secondary antibody Alexa Fluor 647 AffiniPure Goat Anti-Mouse IgG, Fcγ fragment specific (min X Hu Bov Hrs). After incubation at 4°C for 30 minutes with shaking, the 96-well plate was removed, 150 μl of 1×PBS was added to each well, and the plate was washed twice. The supernatant was discarded, and 150 μl of 1×PBS was added to each well to resuspend the cells. The cells were then detected by flow cytometry and the values were read using a flow cytometer.

[0151] 1.1.3 Preparation of hybridoma cells The two mice with the best titer detection results were sacrificed, and their spleens were removed using sterilized forceps under high pressure in a clean bench and then immersed in sterile saline. The spleens were thoroughly minced using sterile scissors, transferred to a sterile 70 μm cell sieve mesh, and thoroughly ground using a disposable sterile syringe. The ground spleen cells on the cell sieve mesh were washed three times with RPMI-1640 medium, and the washings were collected to obtain the corresponding cell suspension. The spleen cells were collected by centrifugation at 1000 rpm for 5 minutes. Well-growing SP2 / 0 cells were resuspended and collected, centrifuged at 1000 rpm for 5 minutes, washed, and counted. Mouse splenocytes and SP2 / 0 cells were mixed at a 5:1 ratio and centrifuged. The plate was gently flicked to disperse the cell pellet and placed in a 37°C water bath for 1 minute. PEG-1450 solution (37°C) was slowly added dropwise. After addition, the sample was centrifuged and the supernatant discarded. The cell suspension prepared after spleen fusion from each mouse was diluted in HAT screening medium and seeded into a 96-well plate at 150 μl per well. The plate was then cultured in a CO2 cell incubator. The medium was replenished every 3-5 days, and once the appropriate cell density was reached at the bottom of the plate, the supernatant was collected and detected.

[0152] 1.1.4 Detection of antibody titers in hybridoma supernatants The supernatants were screened for positive results using the mirrorball method. The positive and negative cells were F98npEGFRvIII and F98npEGFR, respectively. The supernatant samples and controls were gradient diluted into 384-well plates, each containing approximately 1 x 10^3 cells. An appropriate amount of CFSE was added to stain the cells, and after thorough homogeneity, the corresponding secondary antibody was added. The cells were incubated at room temperature and then detected and read using an instrument. Positive clones were further screened using FACS.

[0153] 1.1.5 Preparation and detection of subclones Positive clones were subcloned by finite dilution, and the subclones were cultured in 1×HT medium in a 5% carbon dioxide incubator at 37°C. Clones were screened by flow cytometry.

[0154] 1.1.6 Expression, Purification, and Detection of Subcloned Antibodies The supernatant of the subclone was taken and subjected to affinity purification of the protein. The concentration of the purified monoclonal antibody was detected, and it was diluted to a series of gradient concentrations and subjected to SPR detection, and the EC50 was calculated (Figure 3).

[0155] 1.1.7 Antibody sequencing of candidate clones Total RNA from the monoclonal hybridoma cells was extracted using a total RNA extraction kit, and the total RNA extracted using a reverse transcription kit was used as a template for reverse transcription. The antibody sequence was then amplified by PCR, and the antibody was sequenced.

[0156] 1.1.8 Detection of antibody affinity by flow cytometry (1) 1x10^5 F98npEGFRvIII cells were taken per well, washed twice with 2mL of 2% BAS BSA, and resuspended in 100μL of PBS. (2) The maximum antigen concentration was 16 μg, diluted in a gradient of 8 steps, and the dilution series is shown in the table below. [Table A] (3) The diluted antibody was added and allowed to react at 4°C for 30 minutes. (4) After washing the cells three times with 2 mL of 2% BSA, a BV421-labeled anti-human IgG Fc secondary antibody was added and allowed to react at 4°C for 30 minutes. BV421 anti-mouse IgG was used as the secondary antibody for the mouse antibody. (5) The cells were washed twice with PBS, resuspended in 500 μL of PBS, loaded into the device, and detected and analyzed. (6) The mean fluorescence intensity (MFI) was calculated, a graph was created based on the MFI and antibody concentration, and the EC50 value was calculated to compare the affinity of different antibodies.

[0157] 1.2 Preparation and functional study of murine antibody CAR T cells 1.2.1 CAR structural design The CAR structure comprises an ScFv structure of the mouse-derived antibody sequence of the anti-EGFRvIII antigen to be developed, and further comprises, but is not limited to, the CD8 hinge region and transmembrane region, the 41-BB activation signal, and the CD3ζ activation domain (Figure 4). At the same time, other modifications may be made based on this, including cytokine secretion, chemokine secretion, immune checkpoint inhibitory gene modification, blocking of tumor microenvironment inhibitory signals, and conversion to a fourth-generation CAR T, such as activation type.

[0158] 1.2.2 Construction of expression plasmids The synthesized CAR gene sequence was linked to a lentivirus expression plasmid by molecular cloning, and the synthesized plasmid was transfected into susceptible Stbl3 cells. The culture solution was shaken to perform a plasmid maxiprep, which was then used for subsequent lentivirus packaging.

[0159] 1.2.3 Packaging and titering lentivirus Each CAR plasmid was co-transfected with a packaging plasmid into 293T cells. 48 h post-transfection, the cell supernatant was collected and centrifuged at 4,000 rpm for 10 minutes. The supernatant was then filtered through a 0.45 μm filter and the lentivirus pellet was collected in an ultracentrifuge. The centrifugation conditions were 4°C, 100,000 × g, no brake, and 90 minutes. The concentrated lentivirus was aliquoted into 0.5 mL cryopreservation tubes at 200 μl per tube and frozen at -80°C. The lentivirus titer was determined by flow cytometry.

[0160] 1.2.4 Preparation of CAR T cells Twenty milliliters of peripheral blood from a healthy donor was placed in a 50-ml centrifuge tube. 1 ml of RosetteSep™ Cocktail was added to the blood and mixed uniformly to achieve an antibody concentration of 50 μL / ml. 20 ml of GE Ficoll solution was then placed in a new 50-ml centrifuge tube. 20 ml of the diluted blood sample was carefully pipetted onto the top layer of the Ficoll solution in a 1:1 ratio. The tube containing the blood sample was then placed in a centrifuge and centrifuged at 400 g for 30 minutes. The upper layer of plasma and platelets was then aspirated using a sterile pipette. The second layer of milky white, circular lymphocytes was carefully aspirated into a new tube without touching the mononuclear cell layer. Each tube of lymphocyte suspension was washed with three volumes of PBS and centrifuged at 400 g for 10 minutes. The supernatant was discarded, and the resulting cells were resuspended in 5 ml of PBS and centrifuged at 1200 rpm for 10 minutes. Finally, the cells were suspended in complete culture medium supplemented with X-VIVO + 100U IL-2 and counted. 1 mL of isolated T cells at a density of 1 x 10^6 was seeded into one well of a 24-well plate, and 25 μL of CD3 / CD28 magnetic beads were added. The cells were cultured in a 5% CO2, 37°C incubator for 48 hours before being used for lentiviral infection. The corresponding lentivirus was added at an MOI of 0.5. The following day, the cells were replenished, and each well was filled to 30 mL. The cells were then transferred to a T75 vial and cultured horizontally.

[0161] 1.2.5 CAR T cell CAR positive rate expression detection After 3 days, CAR-T cells were transferred to sterile flow cytometry tubes, and the cells from each tube were placed on a Biolegend magnet for 2 minutes to remove the magnetic beads. Afterwards, a portion of the cells were stained with a flow cytometry antibody (AF647-protein L) to detect CAR expression.

[0162] 1.2.6 Detection of in vitro killing efficiency of CAR T cells In the detection plate, U87MG-EGFRvIII target cells were seeded at 1 x 10^4 cells / well in six duplicate wells, along with six control wells, at 100 μl / well. After overnight incubation, the supernatant was removed and CAR-T cells were added at an effector-target ratio of 2.5:1 in 100 μl / well. After 6 hours, the plates were centrifuged at 300 g for 5 minutes, and the supernatant was discarded as much as possible from all wells. Each well was filled with 100 μl of digestion solution, digested at room temperature for 10 minutes, and then loaded into the device for detection. Each well was filled with 100 μL of firefly luciferase detection reagent. The formula for killing efficiency was: killing efficiency (%) = (fluorescence intensity of control wells - killing fluorescence intensity) / (fluorescence intensity of control wells - fluorescence intensity of blank wells) x 100%.

[0163] 1.2.7 Research into the in vivo efficacy of CAR T cells Male NOG mice aged 6-8 weeks were used as experimental animals. After digestion, U87MG-EGFRvIII cells were resuspended in sterile 1x PBS and washed twice. Viable cells were counted and adjusted to a cell density of 5x10^5 / 50μl per mouse. At the time of cell injection, 50μl of Matrigel was mixed with each mouse at a 1:1 volume, i.e., a total volume of 100μl of cell suspension was injected per mouse. The implantation site was subcutaneous on the lower right side of the mouse's back. After tumor implantation, tumor volume was measured three times a week with a vernier caliper. The formula for tumor volume calculation was TV = 0.5 a x b 2 The tumor size is 100 mm 3 When tumor size reached 100%, CAR-T cells or saline were infused at 5x10^6 / mouse, and the tumor size and survival status of the mice were subsequently observed.

[0164] 1.3 Humanization of murine antibody sequences The two clones with the best in vivo efficacy were selected for antibody sequence humanization. (1) The hybridoma V-genes were codon-optimized for mammalian expression and synthesized by overlap extension PCR. (2) The mouse monoclonal antibody V-genes were humanized by grafting them into the optimal framework using CDR grafting. 12–16 humanized variants were designed to minimize differences in the framework from the human V-genes. (3) Up to three variants were designed for each site to eliminate critical PTM sites in the murine antibody sequence. (4) After codon optimization for mammalian expression, humanized V-genes and PTM-free V-genes were synthesized. The mouse and all variant genes were cloned into a human IgG expression vector to generate human IgG1 constructs of the desired isotype. The antibody genes were synthesized, expressed, and purified. After purification, the antibody affinities were identified using SPR, and the humanized antibody sequences with the optimal affinities were screened.

[0165] 1.4 Preparation and functional study of humanized antibody CAR T cells 1.4.1 CAR structural design The CAR structure comprises an ScFv structure of the mouse-derived antibody sequence of the anti-EGFRvIII antigen to be developed, and further comprises, but is not limited to, the CD8 hinge region and transmembrane region, the 41-BB activation signal, and the CD3ζ activation domain. At the same time, other modifications may be made based on this, including cytokine secretion, chemokine secretion, immune checkpoint inhibitory gene modification, blocking of tumor microenvironment inhibitory signals, and conversion to a fourth-generation CAR T, such as activation type. 1.4.2 Synthesis of CAR gene The synthesized CAR gene sequence was linked to a lentivirus expression plasmid by molecular cloning, and the synthesized plasmid was transfected into susceptible Stbl3 cells. The culture solution was shaken to perform a plasmid maxiprep, which was then used for subsequent lentivirus packaging.

[0166] 1.4.3 Packaging and titering lentivirus Each CAR plasmid was co-transfected with a packaging plasmid into 293T cells. 48 h post-transfection, the cell supernatant was collected and centrifuged at 4,000 rpm for 10 minutes. The supernatant was then filtered through a 0.45 μm filter and the lentivirus pellet was collected in an ultracentrifuge. The centrifugation conditions were 4°C, 100,000 × g, no brake, and 90 minutes. The concentrated lentivirus was aliquoted into 0.5 mL cryopreservation tubes at 200 μl per tube and frozen at -80°C. The lentivirus titer was determined by flow cytometry.

[0167] 1.4.4 Preparation of CAR T cells Twenty milliliters of peripheral blood from a healthy donor was placed in a 50-ml centrifuge tube. 1 ml of RosetteSep™ Cocktail was added to the blood and mixed uniformly to achieve an antibody concentration of 50 μL / ml. 20 ml of GE Ficoll solution was then placed in a new 50-ml centrifuge tube. 20 ml of the diluted blood sample was carefully pipetted onto the top layer of the Ficoll solution in a 1:1 ratio. The tube containing the blood sample was then placed in a centrifuge and centrifuged at 400 g for 30 minutes. The upper layer of plasma and platelets was then aspirated using a sterile pipette. The second layer of milky white, circular lymphocytes was carefully aspirated into a new tube without touching the mononuclear cell layer. Each tube of lymphocyte suspension was washed with three volumes of PBS and centrifuged at 400 g for 10 minutes. The supernatant was discarded, and the resulting cells were resuspended in 5 ml of PBS and centrifuged at 1200 rpm for 10 minutes. Finally, the cells were suspended in complete culture medium supplemented with X-VIVO + 100U IL-2 and counted. 1 mL of isolated T cells at a density of 1 x 10^6 was seeded into one well of a 24-well plate, and 25 μL of CD3 / CD28 magnetic beads were added. The cells were cultured in a 5% CO2, 37°C incubator for 48 hours before being used for lentiviral infection. The corresponding lentivirus was added at an MOI of 0.5. The following day, the cells were replenished, and each well was filled to 30 mL. The cells were then transferred to a T75 vial and cultured horizontally.

[0168] 1.4.5 CAR T cell CAR positive rate expression detection After 3 days, CAR-T cells were transferred to sterile flow cytometry tubes, and the cells from each tube were placed on a Biolegend magnet for 2 minutes to remove the magnetic beads. Afterwards, a portion of the cells were stained with a flow cytometry antibody (AF647-protein L) to detect CAR expression.

[0169] 1.4.6 Detection of in vitro killing efficiency of CAR T cells In the detection plate, U87MG-EGFRvIII target cells were seeded at 1 x 10^4 cells / well in six duplicate wells, along with six control wells, at 100 μl / well. After overnight incubation, the supernatant was removed and CAR-T cells were added at an effector-target ratio of 2.5:1 in 100 μl / well. After 6 hours, the plates were centrifuged at 300 g for 5 minutes, and the supernatant was discarded as much as possible from all wells. Each well was filled with 100 μl of digestion solution, digested at room temperature for 10 minutes, and then loaded into the device for detection. Each well was filled with 100 μL of firefly luciferase detection reagent. The formula for killing efficiency was: killing efficiency (%) = (fluorescence intensity of control wells - killing fluorescence intensity) / (fluorescence intensity of control wells - fluorescence intensity of blank wells) x 100%.

[0170] 1.4.7 Research into the in vivo efficacy of CAR T cells Male NOG mice aged 6-8 weeks were used as experimental animals. After digestion, U87MG-EGFRvIII cells were resuspended in sterile 1x PBS and washed twice. Viable cells were counted and adjusted to a cell density of 5x10^5 / 50μl per mouse. For cell injection, 50μl of Matrigel was mixed with each mouse at a 1:1 volume, i.e., a total volume of 100μl of cell suspension was injected per mouse. The implantation site was subcutaneous on the lower right side of the mouse's back. After tumor implantation, tumor volume was measured three times a week with a vernier caliper. The formula for tumor volume calculation was TV = 0.5 a x b 2 The tumor size is 100 mm 3 When tumor size reached 100%, CAR-T cells or saline were infused at 5x10^6 / mouse, and the tumor size and survival status of the mice were subsequently observed.

[0171] 1.5 Preparation and functional study of humanized antibody CAR NK cells 1.5.1 Preparation of CAR NK cells (1) NK cells were cultured and placed in a retronectin-coated 24-well plate. Lentivirus was then added to the cultured NK cells at an MOI of 20. The cells were then centrifuged at 32°C for 30 minutes and transferred to a 37°C, 5% CO2 incubator for culture. (2) 48 hours after infection, the cells were washed with complete medium and cultured in complete medium containing 300 IU / ml of IL2.

[0172] 1.5.2 Expression detection of CAR positivity of CAR NK cells CAR-NK cells were transferred to a sterile flow cytometry tube, and the cells were stained with a flow cytometry antibody (AF647-protein L) to detect the CAR expression rate.

[0173] 1.5.3 Detection of in vitro killing efficiency of CAR NK cells In the detection plate, U87MG-EGFRvIII target cells were seeded at 1 x 10^4 cells / well in six duplicate wells, and six control wells were simultaneously seeded, with 100 μl / well. After overnight incubation, the supernatant was aspirated, and CAR-NK cells were added at an effector-target ratio of 1:1 in 100 μl / well. After 6 hours, the plates were centrifuged at 300 g for 5 minutes, and the supernatant was discarded as much as possible from all wells. Each well was filled with 100 μl of digestion solution, digested at room temperature for 10 minutes, and then loaded into the device for detection. Each well was filled with 100 μL of firefly luciferase detection reagent. The formula for killing efficiency was: killing efficiency (%) = (fluorescence intensity of control wells - killing fluorescence intensity) / (fluorescence intensity of control wells - fluorescence intensity of blank wells) x 100%.

[0174] 1.6 Preparation and functional study of CAR T cells resistant to TGFβ inhibition 1.6.1 CAR structural design Two types of CAR structures were designed that resist TGFβ inhibition. The first type ligated the dominant-negative receptor dnTGFβRII with a T2A self-cleaving peptide (Figure 20A). dnTGFβRII has the ability to bind to TGFβ as a receptor, but lacks the ability to convert TGFβ binding into an intracellular signal (e.g., it lacks the intracellular domain or kinase activity required for signal transduction). The second type ligated a chimeric switch receptor (dnTGFβRII-IL7RA TM-IL7RA intracellular domain) with a T2A self-cleaving peptide (Figure 20B). The presence of this chimeric switch receptor ligated the CAR T cells to resist TGFβ inhibition and convert TGFβ binding into an intracellular IL7RA receptor-stimulating signal, promoting proliferation and / or activity of CAR T cells.

[0175] 1.6.2 Preparation of CAR T cells The specific method is the same as the procedure for preparing humanized antibody CAR T cells. 1.6.3 Studying the Function of CAR T Cells In the detection plate, U87MG-EGFRvIII target cells were seeded at 1 x 10^4 cells / well in six duplicate wells, along with six control wells, at 100 μl / well. After overnight incubation, the supernatant was removed and CAR-T cells were added at an effector-target ratio of 2.5:1 in 100 μl / well. After 6 hours, the plates were centrifuged at 300 g for 5 minutes, and the supernatant was discarded as much as possible from all wells. Each well was filled with 100 μl of digestion solution, digested at room temperature for 10 minutes, and then loaded into the device for detection. Each well was filled with 100 μL of firefly luciferase detection reagent. The formula for killing efficiency was: killing efficiency (%) = (fluorescence intensity of control wells - killing fluorescence intensity) / (fluorescence intensity of control wells - fluorescence intensity of blank wells) x 100%. To compare the resistance of modified fourth-generation CAR T cells to TGFβ inhibition, a group was set up to simultaneously suppress TGFβ and kill the cells. Before conducting the killing experiment with CAR T cells, some cells were taken and the CAR T cells were suppressed with 30 ng / ml of TGFβ, and the killing efficiency of the suppressed and unsuppressed CAR T cells was then monitored.

[0176] 1.6.4 Research into the in vivo function of CAR T cells Male NOG mice aged 6-8 weeks were used as experimental animals. After digestion, U87MG-EGFRvIII cells were resuspended in sterile 1x PBS and washed twice. Viable cells were counted and adjusted to a cell density of 5x10^5 / 50μl per mouse. At the time of cell injection, 50μl of Matrigel was mixed with each mouse at a 1:1 volume, i.e., a total volume of 100μl of cell suspension was injected per mouse. The implantation site was subcutaneous on the lower right side of the mouse's back. After tumor implantation, tumor volume was measured three times a week with a vernier caliper. The formula for tumor volume calculation was TV = 0.5 a x b 2 The tumor size is 100 mm 3 When tumor size reached 100%, CAR-T cells or saline were infused at 3x10^6 / mouse, and the tumor size and survival status of the mice were subsequently observed.

[0177] The partial sequence information for this example is as follows: [Table B] TIFF2025525941000003.tif41170

[0178] Experimental results 1. Measurement results of the affinity of murine antibodies 1.1 Results of antibody affinity detection for mouse-derived subclone supernatants The binding ability of the subclones to F98npEGFR and F98npEGFRvIII was detected by flow cytometry. The results showed that the screened positive subclones specifically bound to the target cells F98npEGFRvIII overexpressing EGFRvIII, but not to the target cells F98npEGFR overexpressing EGFR. This indicates that the screening yielded specific antibodies capable of specifically binding to the mutated EGFRvIII target antigen (Figure 2).

[0179] 1.2 Results of affinity detection of purified murine antibodies SPR results showed that all murine-derived antibodies with good in vitro efficacy had high affinity for the EGFRvIII target antigen (Table 1 and Figure 3). [Table 1]

[0180] 2. Murine antibody sequences (using the IMGT coding system) [Table C] TIFF2025525941000006.tif245170TIFF2025525941000007.tif172170Note: When a single clone of 20-2-40 was sequenced, different antibody sequences were obtained and designated 20-2-40-1 and 20-2-40-2, respectively.

[0181] 3. CAR structure sequence [Table D]

[0182] 4. Research results on the in vitro efficacy of CAR T cells produced with mouse-derived antibodies 4.1 CAR positive rate of CAR T cells produced with murine antibodies The CAR positive rate of CAR T cells was detected by flow cytometry, and the results showed that the CAR positive rate of CAR T cells manufactured with murine-derived antibody sequences was between 50 and 90% ( Figure 5 ).

[0183] 4.2 Killing efficiency of CAR T cells produced with murine antibodies The killing efficiency of CAR T cells engineered with the mouse-derived antibody sequences against EGFRvIII-overexpressing target cells U87MG-EGFRvIII and EGFR-expressing target cells U87MG-EGFR was measured. Results showed that all engineered CAR T cells specifically killed EGFRvIII-overexpressing target cells, but had little killing efficiency against EGFR-expressing target cells. This demonstrates the specific killing efficiency of CAR T cells engineered with the developed antibodies (Figures 6 and 7). Among these, 20-2-1, 20-2-4, 20-2-25, 20-2-28, 20-2-34, and 20-2-40-2 had relatively high killing efficiency.

[0184] 5. In vivo tumor suppression efficiency of CAR T cells engineered with murine antibodies Subcutaneous tumor formation was performed in mice using U87MG-EGFRvIII, and tumor volumes were 100 mm 3 After the tumors had grown to a certain size, they were infused with CAR T cells. The tumor volumes of the mice were measured every week after the CAR T cell infusion. The results showed that CAR T cells produced with the antibody sequences of clones 4, 28, and 40-2 had significant tumor-suppressing effects in vivo (Figure 8).

[0185] 6. Measurement results of affinity of humanized antibodies Murine antibodies were humanized by CDR grafting and post-translational modification site removal. As a result, the sequence of the humanized antibody #28 was obtained, which maintained the same affinity as the murine antibody after humanization. However, the affinity of the murine antibody #40-2 clone was shown to decrease approximately two-fold after humanization (Tables 2 and 3, Figures 9 and 10).

[0186] [Table 2]

[0187] [Table 3]

[0188] 7. Humanized antibody sequence [Table E] TIFF2025525941000012.tif237170TIFF2025525941000013.tif235170TIFF20255259410 00014.tif235170TIFF2025525941000015.tif235170TIFF2025525941000016.tif239170

[0189] 8. Research results on the in vitro efficacy of CAR T cells manufactured with humanized antibodies 8.1 Detection of CAR-positive rate of humanized antibody CAR T cells The CAR positive rate of CAR T cells was detected by flow cytometry, and the results showed that the CAR positive rate of CAR T cells manufactured with mouse-derived antibody sequences was between 50 and 90% (Figures 11 and 12).

[0190] 8.2 Results of in vitro killing efficiency detection of humanized antibody CAR T and CAR NK cells The killing efficiency of CAR T cells produced with the humanized antibody sequences against target cells U87MG-EGFRvIII overexpressing EGFRvIII and target cells U87MG-EGFR expressing EGFR was measured. The results showed that both CAR T cells specifically killed target cells overexpressing EGFRvIII, but had little killing efficiency against target cells expressing EGFR, demonstrating that CAR T cells produced with the developed humanized antibodies have specific killing efficiency (Figures 13, 14, 15, and 16). Similarly, CAR NK cells produced with the humanized antibody sequences specifically killed target cells overexpressing EGFRvIII, but had little killing efficiency against target cells expressing EGFR (Figures 19A and 19B).

[0191] 9. Study of the in vivo tumor suppression efficiency of CAR T cells manufactured with humanized antibodies Subcutaneous tumor formation was performed in mice using U87MG-EGFRvIII, and tumor volumes were 100 mm 3 After the tumors had grown to about 100% tumor size, CAR T cells were infused. The tumor volumes of the mice were measured every week after CAR T cell infusion. The results showed that CAR T cells produced with the antibody sequences of clones 28-H9, 40-2-H10, 40-2-H12, 40-2-H13, and 40-2-H15 had significant tumor-suppressing effects in vivo (Figures 17 and 18).

[0192] 10. Research results on the in vitro efficacy of CAR T cells resistant to TGFβ 10.1 CAR positive rate detection results The results of detecting the CAR positivity rate are shown in Table 4 and Figures 21A-C. The CAR positivity rate was detected using the PE-EGFRvIII antigen protein in RII (CAR T cells expressing dnTGFβRII), T7R (CAR T cells expressing the chimeric switch receptor dnTGFβRII-IL7Rα), or CAR T cells (conventional second-generation CAR T cells that do not express DNR or CSR).

[0193] [Table 4] The results showed that the produced CAR T cells had a CAR positivity rate of over 50%.

[0194] 10.2 Results of in vitro killing efficiency detection of CAR T cells The results showed that CAR T cells overexpressing TGFβRII and TGFβRII-IL7R did not show significant differences in killing efficiency in the absence of TGFβ suppression (Figure 22A). After TGFβ suppression was added, the killing efficiency of CAR T cells overall was significantly reduced (Figure 22B), but the difference in the rate of reduction in killing efficiency was significantly lower for CAR T cells overexpressing TGFβRII and TGFβRII-IL21R (Figure 22C).

[0195] 10.3 Results of detection of tumor-suppressing activity of CAR T cells in vivo The results showed that CAR T cells overexpressing TGFβRII and TGFβRII-IL21R had significantly higher tumor suppression efficiency in vivo than control CAR T cells (Figure 23).

[0196] Consider EGFRvIII is a mutation of EGFR in which exons 2-7 of EGFR are deleted. A single glycine residue is added at the junction of the deleted sequence, creating a new antigenic epitope. The high homology between EGFRvIII and EGFR poses significant challenges in antibody sequence screening. While EGFR is expressed in all epithelial cells, EGFRvIII is a tumor-specific antigen expressed only in tumors. Developing drugs targeting EGFRvIII requires screening for antibodies that specifically recognize the EGFRvIII target antigen but not the wild-type EGFR antigen. This requires high antibody specificity, and concurrent recognition of the EGFR antigen poses the risk of off-target activity. This study optimized multiple mouse immunization strategies and antibody screening strategies. Through screening, we obtained murine antibody sequences with high specificity and affinity, and simultaneously generated immune cells with murine sequences that exhibited significant tumor-suppressing activity. In order to reduce the immunogenicity of drugs, this study humanized the sequence of a murine antibody based on that of a murine antibody. Through screening, a humanized antibody with high affinity and specificity was obtained. This led to the research and development of immune cells made from humanized antibodies with significant tumor-suppressing activity, which laid the foundation for the subsequent development of anti-tumor drugs.

[0197] Antibody drugs and cellular immunotherapy have made significant progress in the field of tumor treatment in recent years. Glioma is a highly malignant tumor with no effective treatment available at present, so there is a strong need for research and development of drugs to effectively treat brain glioma. Research has shown that 30-60% of brain glioma patients express EGFRvIII antigen. Therefore, the developed antibodies targeting EGFRvIII and immune cells produced with this antibody sequence can be applied to brain gliomas expressing EGFRvIII and other tumors expressing EGFRvIII.

[0198] Antibodies allow drugs to target, and currently antibody drugs come in many forms, including monospecific antibodies, bispecific antibodies, multispecific antibodies, ADCs, and immune cells, so the antibodies developed in this study can be applied in a variety of fields. Furthermore, since current targeted therapies all require corresponding targeted diagnostics, the antibodies developed in this study are also useful for the development of diagnostic kits and diagnostic-related applications.

[0199] Currently, CAR T cells have made significant progress in hematological tumors, and CAR T cells are already commercially available. The CAR structure consists of an antigen recognition domain, hinge region, transmembrane region, costimulatory domain, and activation domain. The antibodies developed in this study, when combined with different structures, can produce different types of immune cells, including but not limited to T cells, NK cells, macrophages, monocytes, B cells, and erythrocytes. Treatment of solid tumors is limited by the immunosuppressive microenvironment, infiltration, and persistence. Therefore, immune cells developed in this study must be modified with different techniques, including but not limited to gene editing, gene knockout, and RNAi, to confer in vivo persistence, infiltration, and the ability to overcome the suppressive immune microenvironment. Therefore, immune cells produced with the antibodies developed in this study can be modified to improve their tumor-suppressing ability.

[0200] References: 1. Akhavan, D., Alizadeh, D., Wang, D., Weist, M.R., Shepphird, J.K., and Brown, C.E. (2019). CAR T cells for brain tumors: Lessons learned and road ahead. Immunological reviews 290, 60-84. An, Z., Aksoy, O., Zheng, T., Fan, Q., and Weiss, W.J.O. (2018). Epidermal growth factor receptor and EGFRvIII in glioblastoma: signaling pathways and targeted therapies. 37, 1561-1575. Chistiakov, D., Chekhonin, I., and Chekhonin, V.J.E.j.o.p. (2017). The EGFR variant III mutant as a target for immunotherapy of glioblastoma multiforme. 810, 70-82. Del Vecchio, C., Li, G., and Wong, A.J.E.r.o.v. (2012). Targeting EGF receptor variant III: tumor-specific peptide vaccination for malignant gliomas. 11, 133-144. Feldman, L., Brown, C., and Badie, B. (2022). Chimeric Antigen Receptor (CAR) T Cell Therapy for Glioblastoma. Neuromolecular Med 24, 35-40. Londhe, V., and Date, V.J.E.r.o.v. (2020). Personalized neoantigen vaccines: a glimmer of hope for glioblastoma. 19, 407-417. O’Rourke, D.M., Nasrallah, M.P., Desai, A., Melenhorst, J.J., Mansfield, K., Morrissette, J.J.D., Martinez-Lage, M., Brem, S., Maloney, E., Shen, A., et al. (2017). A single dose of peripherally infused EGFRvIII-directed CAR T cells mediates antigen loss and induces adaptive resistance in patients with recurrent glioblastoma. Science translational medicine 9. Zhu, G., Zhang, Q., Zhang, J., and Liu, F. (2021). Targeting Tumor-Associated Antigen: A Promising CAR-T Therapeutic Strategy for Glioblastoma Treatment. Frontiers in pharmacology 12, 661606.

Claims

1. An antibody or antigen-binding fragment thereof targeting EGFRvIII, wherein the antibody comprises a heavy chain variable region (HCVR), the heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the HCDR1, HCDR2, and HCDR3 are one of the following combinations: (1) The amino acid sequence of HCDR1 is DFSMH (SEQ ID NO: 1), The amino acid sequence of HCDR2 is WINTETGEPSYADDFKG (SEQ ID NO: 2). The amino acid sequence of HCDR3 is YGYDVRGDY (SEQ ID NO: 3); (2) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 4), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 5). The amino acid sequence of HCDR3 is GWFAY (SEQ ID NO: 6); (3) The amino acid sequence of HCDR1 is DYSIH (SEQ ID NO: 7), The amino acid sequence of HCDR2 is WINTETGEPTYADDFKG (SEQ ID NO: 8). The amino acid sequence of HCDR3 is YGYDVRGDY (SEQ ID NO: 9); (4) The amino acid sequence of HCDR1 is DYYLH (SEQ ID NO: 10), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 11). The amino acid sequence of HCDR3 is GYLTY (SEQ ID NO: 12); (5) The amino acid sequence of HCDR1 is RYWMH (SEQ ID NO: 13), The amino acid sequence of HCDR2 is EINPSNGRANYNEKFMS (SEQ ID NO: 14). The amino acid sequence of HCDR3 is GREITTGFAY (SEQ ID NO: 15); (6) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 16), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 17). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 18); (7) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 19), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 20). The amino acid sequence of HCDR3 is GYLAY (SEQ ID NO: 21); (8) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 22), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 23). The amino acid sequence of HCDR3 is GWFAY (SEQ ID NO: 25); (9) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 25), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 26). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 27); (10) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 28), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 29). The amino acid sequence of HCDR3 is GWFAY (SEQ ID NO: 30); (11) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 31), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 32). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 33); (12) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 34), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 35). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 36); (13) The amino acid sequence of HCDR1 is NYAMS (SEQ ID NO: 37); The amino acid sequence of HCDR2 is TITSGGSYTYYPDSVKG (SEQ ID NO: 38). The amino acid sequence of HCDR3 is KDYGNYWFAY (SEQ ID NO: 39); (14) The amino acid sequence of HCDR1 is GYAMS (SEQ ID NO: 40); The amino acid sequence of HCDR2 is TITSGGSYTYYPDSVKG (SEQ ID NO: 41). The amino acid sequence of HCDR3 is KDYGNYWFAY (SEQ ID NO: 42); (15) The amino acid sequence of HCDR1 is GYAMS (SEQ ID NO: 43); The amino acid sequence of HCDR2 is TITSGGSYTYYPDSVKG (SEQ ID NO: 44). The amino acid sequence of HCDR3 is KDYGNYWFAY (SEQ ID NO: 45); (16) The amino acid sequence of HCDR1 is SGYSWH (SEQ ID NO: 46); The amino acid sequence of HCDR2 is YIHYSGSTNYNPPLKS (SEQ ID NO: 47). The amino acid sequence of HCDR3 is GVVSNYAMGN (SEQ ID NO: 48); (17) The amino acid sequence of HCDR1 is TYWMH (SEQ ID NO: 49); The amino acid sequence of HCDR2 is YINPNTAYTEYNQNFKD (SEQ ID NO: 50). The amino acid sequence of HCDR3 is GAYYRTYYAMDY (SEQ ID NO: 51); (18) The amino acid sequence of HCDR1 is NYGMN (SEQ ID NO: 52); The amino acid sequence of HCDR2 is WINTYTGEPTYADDFKG (SEQ ID NO: 53). The amino acid sequence of HCDR3 is EEFYSRGAMDY (SEQ ID NO:54); and (19) The amino acid sequence of HCDR1 is DYYIN (SEQ ID NO: 55); The amino acid sequence of HCDR2 is WIYPGSGNTKYNEKFKG (SEQ ID NO: 56). the amino acid sequence of HCDR3 is SSRCDF (SEQ ID NO:57); or The antibody comprises a variant of a combination of any of the HCDR sequences (1) to (19), wherein the variant has at least 90% sequence identity with any of the HCDR sequences (1) to (19), or contains a total of at least one and no more than 10, or no more than 5, 4, 3, or 2 amino acid alterations in the HCDR sequence. An antibody or antigen-binding fragment thereof.

2. The antibody further comprises a light chain variable region (LCVR), wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and wherein the LCDR1, LCDR2, and LCDR3 are one of the following combinations: (1) The amino acid sequence of LCDR1 is SASSISSNYLH (SEQ ID NO: 58); The amino acid sequence of LCDR2 is GTSNLAS (SEQ ID NO: 59), The amino acid sequence of LCDR3 is HQGSSIPLT (SEQ ID NO: 60); (2) the amino acid sequence of LCDR1 is KSSQSLLNSSNQKNHLA (SEQ ID NO: 61); The amino acid sequence of LCDR2 is FASTRAS (SEQ ID NO: 62), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 63); (3) the amino acid sequence of LCDR1 is SASSGISSNYLH (SEQ ID NO: 64); The amino acid sequence of LCDR2 is STSNLAS (SEQ ID NO: 65), The amino acid sequence of LCDR3 is HQGSDIPLT (SEQ ID NO: 66); (4) The amino acid sequence of LCDR1 is KSSQNLLNSSNQKNYLA (SEQ ID NO: 67); The amino acid sequence of LCDR2 is FASTRY (SEQ ID NO: 68), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 69); (5) the amino acid sequence of LCDR1 is KASQSVSNDVV (SEQ ID NO: 70); The amino acid sequence of LCDR2 is YASNRYT (SEQ ID NO: 71), The amino acid sequence of LCDR3 is QQDYSSPWT (SEQ ID NO: 72); (6) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 73); The amino acid sequence of LCDR2 is FASTRES (SEQ ID NO: 74), The amino acid sequence of LCDR3 is QQHYSIPLT (SEQ ID NO: 75); (7) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 76); The amino acid sequence of LCDR2 is FASTRKS (SEQ ID NO: 77), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 78); (8) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNHLA (SEQ ID NO: 79); The amino acid sequence of LCDR2 is FASTRQS (SEQ ID NO: 80), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 81); (9) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 82); The amino acid sequence of LCDR2 is FASTRQS (SEQ ID NO: 83), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 84); (10) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNHLA (SEQ ID NO: 85); The amino acid sequence of LCDR2 is FASTRGS (SEQ ID NO: 86), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 87); (11) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 88); The amino acid sequence of LCDR2 is FASTRDS (SEQ ID NO: 89): The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 90); (12) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 91); The amino acid sequence of LCDR2 is FASTRES (SEQ ID NO: 92), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 93); (13) The amino acid sequence of LCDR1 is RSSQSLVHSDGNTYLH (SEQ ID NO: 94); The amino acid sequence of LCDR2 is KVSNRFS (SEQ ID NO: 95), The amino acid sequence of LCDR3 is SQSIHVPWT (SEQ ID NO: 96); (14) The amino acid sequence of LCDR1 is SASSSVSYMH (SEQ ID NO: 97); The amino acid sequence of LCDR2 is STSNLAS (SEQ ID NO: 98), The amino acid sequence of LCDR3 is QQRSSYPLT (SEQ ID NO: 99); (15) The amino acid sequence of LCDR1 is RSSQSLVHSDGNTYLH (SEQ ID NO: 100); The amino acid sequence of LCDR2 is KVSNRFS (SEQ ID NO: 101), The amino acid sequence of LCDR3 is SQTTQVPWT (SEQ ID NO: 102); (16) The amino acid sequence of LCDR1 is ITNTDIDDDMN (SEQ ID NO: 103). The amino acid sequence of LCDR2 is EGNTLRP (SEQ ID NO: 104), The amino acid sequence of LCDR3 is LQSDDLPLT (SEQ ID NO: 105); (17) The amino acid sequence of LCDR1 is KASQSVDYDGDSYMN (SEQ ID NO: 106); The amino acid sequence of LCDR2 is AASNLES (SEQ ID NO: 107), The amino acid sequence of LCDR3 is LQSNEDPYT (SEQ ID NO: 108); (18) The amino acid sequence of LCDR1 is RSSQFIVHSNGNTYLE (SEQ ID NO: 109); The amino acid sequence of LCDR2 is KISNRFS (SEQ ID NO: 110), The amino acid sequence of LCDR3 is FQGSHVPFT (SEQ ID NO: 111); and (19) The amino acid sequence of LCDR1 is KASEDIYNRLA (SEQ ID NO: 112); The amino acid sequence of LCDR2 is GATSLET (SEQ ID NO: 113), the amino acid sequence of LCDR3 is QQYWSSPLT (SEQ ID NO: 114); or The antibody comprises a variant of a combination of any of the LCDR sequences (1) to (19), wherein the variant has at least 90% sequence identity with any of the LCDR sequences (1) to (19), or the LCDR sequence contains a total of at least one and no more than 10, or no more than 5, 4, 3, or 2 amino acid alterations. The antibody or antigen-binding fragment thereof according to claim 1.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the amino acid sequence of the heavy chain variable region is selected from the following: (1) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 115; (2) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 117; (3) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 119; (4) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 121; (5) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 123; (6) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 125; (7) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 127; (8) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 129; (9) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 131; (10) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 133; (11) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 135; (12) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 137; (13) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 139; (14) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 141; (15) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 143; (16) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 145; (17) The sequence of SEQ ID NO: 147 or a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (18) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 149; and (19) The sequence of SEQ ID NO: 151 or a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are selected from any combination of the following: (1) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 115, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 116; (2) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 117, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 118; (3) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 119, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 120; (4) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 121, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 122; (5) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 123, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 124; (6) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 125, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 126; (7) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 127, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 128; (8) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 129, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 130; (9) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 131, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 132; (10) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 133, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 134; (11) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 135, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 136; (12) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 137, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 138; (13) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 139, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 140; (14) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 141, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 142; (15) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 143, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 144; (16) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 145, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 146; (17) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 147, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 148; (18) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 149, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 150; and (19) The sequence set forth in SEQ ID NO: 151 or a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and the sequence set forth in SEQ ID NO: 152 or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

5. The binding KD value of the antibody to the EGFRvIII antigen as measured by surface plasmon resonance (SPR) is 10 －６ Less than M, preferably 10 －７ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof has a molecular weight of less than M.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is selected from a murine antibody, a chimeric antibody, a humanized antibody, or a human antibody.

7. The binding KD value of the humanized antibody to the EGFRvIII antigen as measured by surface plasmon resonance (SPR) is 10 －６ Less than M, preferably 10 －７ The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, having a molecular weight of less than M.

8. The antigen-binding fragments include Fab, Fab', F(ab') ２ 8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 7, comprising a single domain antibody or a single chain antibody.

9. The humanized antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the humanized antibody is a single-chain antibody and the amino acid sequence thereof is selected from the following: (1) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 167; (2) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 169; (3) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 171; (4) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 173; (5) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 175; (6) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 177; (7) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 179; (8) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 181; (9) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 183; (10) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 185; and (11) The sequence set forth in SEQ ID NO: 187 or a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

10. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, further comprising an Fc fragment.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antigen-binding fragment is a single-chain antibody, and preferably, a linker sequence for linking the heavy chain variable region and the light chain variable region in the single-chain antibody comprises the sequence shown in SEQ ID NO:

166.

12. A chimeric antigen receptor (CAR), the extracellular antigen-binding domain of which comprises one or more antibodies or antigen-binding fragments thereof targeting EGFRvIII, wherein HCDR1, HCDR2, and HCDR3 of the heavy chain variable region of the antibody molecule are one selected from the following combinations: (1) The amino acid sequence of HCDR1 is DFSMH (SEQ ID NO: 1), The amino acid sequence of HCDR2 is WINTETGEPSYADDFKG (SEQ ID NO: 2). The amino acid sequence of HCDR3 is YGYDVRGDY (SEQ ID NO: 3); (2) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 4), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 5). The amino acid sequence of HCDR3 is GWFAY (SEQ ID NO: 6); (3) The amino acid sequence of HCDR1 is DYSIH (SEQ ID NO: 7), The amino acid sequence of HCDR2 is WINTETGEPTYADDFKG (SEQ ID NO: 8). The amino acid sequence of HCDR3 is YGYDVRGDY (SEQ ID NO: 9); (4) The amino acid sequence of HCDR1 is DYYLH (SEQ ID NO: 10), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 11). The amino acid sequence of HCDR3 is GYLTY (SEQ ID NO: 12); (5) The amino acid sequence of HCDR1 is RYWMH (SEQ ID NO: 13), The amino acid sequence of HCDR2 is EINPSNGRANYNEKFMS (SEQ ID NO: 14). The amino acid sequence of HCDR3 is GREITTGFAY (SEQ ID NO: 15); (6) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 16), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 17). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 18); (7) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 19), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 20). The amino acid sequence of HCDR3 is GYLAY (SEQ ID NO: 21); (8) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 22), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 23). The amino acid sequence of HCDR3 is GWFAY (SEQ ID NO: 25); (9) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 25), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 26). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 27); (10) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 28), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 29). The amino acid sequence of HCDR3 is GWFAY (SEQ ID NO: 30); (11) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 31), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 32). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 33); (12) The amino acid sequence of HCDR1 is DYYMH (SEQ ID NO: 34), The amino acid sequence of HCDR2 is WIDPENGNTIYDPKFQG (SEQ ID NO: 35). The amino acid sequence of HCDR3 is GYLVY (SEQ ID NO: 36); (13) The amino acid sequence of HCDR1 is NYAMS (SEQ ID NO: 37); The amino acid sequence of HCDR2 is TITSGGSYTYYPDSVKG (SEQ ID NO: 38). The amino acid sequence of HCDR3 is KDYGNYWFAY (SEQ ID NO: 39); (14) The amino acid sequence of HCDR1 is GYAMS (SEQ ID NO: 40); The amino acid sequence of HCDR2 is TITSGGSYTYYPDSVKG (SEQ ID NO: 41). The amino acid sequence of HCDR3 is KDYGNYWFAY (SEQ ID NO: 42); (15) The amino acid sequence of HCDR1 is GYAMS (SEQ ID NO: 43); The amino acid sequence of HCDR2 is TITSGGSYTYYPDSVKG (SEQ ID NO: 44). The amino acid sequence of HCDR3 is KDYGNYWFAY (SEQ ID NO: 45); (16) The amino acid sequence of HCDR1 is SGYSWH (SEQ ID NO: 46); The amino acid sequence of HCDR2 is YIHYSGSTNYNPPLKS (SEQ ID NO: 47). The amino acid sequence of HCDR3 is GVVSNYAMGN (SEQ ID NO: 48); (17) The amino acid sequence of HCDR1 is TYWMH (SEQ ID NO: 49); The amino acid sequence of HCDR2 is YINPNTAYTEYNQNFKD (SEQ ID NO: 50). The amino acid sequence of HCDR3 is GAYYRTYYAMDY (SEQ ID NO: 51); (18) The amino acid sequence of HCDR1 is NYGMN (SEQ ID NO: 52); The amino acid sequence of HCDR2 is WINTYTGEPTYADDFKG (SEQ ID NO: 53). The amino acid sequence of HCDR3 is EEFYSRGAMDY (SEQ ID NO:54); and (19) The amino acid sequence of HCDR1 is DYYIN (SEQ ID NO: 55); The amino acid sequence of HCDR2 is WIYPGSGNTKYNEKFKG (SEQ ID NO: 56). the amino acid sequence of HCDR3 is SSRCDF (SEQ ID NO:57); or The antibody comprises a variant of a combination of any of the HCDR sequences (1) to (19), wherein the variant has at least 90% sequence identity with any of the HCDR sequences (1) to (19), or contains a total of at least one and no more than 10, or no more than 5, 4, 3, or 2 amino acid alterations in the HCDR sequence. CAR.

13. The antibody further comprises a light chain variable region (LCVR), wherein the light chain variable region comprises LCDR1, LCDR2, and LCDR3, and wherein the LCDR1, LCDR2, and LCDR3 are one of the following combinations: (1) The amino acid sequence of LCDR1 is SASSISSNYLH (SEQ ID NO: 58); The amino acid sequence of LCDR2 is GTSNLAS (SEQ ID NO: 59), The amino acid sequence of LCDR3 is HQGSSIPLT (SEQ ID NO: 60); (2) the amino acid sequence of LCDR1 is KSSQSLLNSSNQKNHLA (SEQ ID NO: 61); The amino acid sequence of LCDR2 is FASTRAS (SEQ ID NO: 62), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 63); (3) the amino acid sequence of LCDR1 is SASSGISSNYLH (SEQ ID NO: 64); The amino acid sequence of LCDR2 is STSNLAS (SEQ ID NO: 65), The amino acid sequence of LCDR3 is HQGSDIPLT (SEQ ID NO: 66); (4) The amino acid sequence of LCDR1 is KSSQNLLNSSNQKNYLA (SEQ ID NO: 67); The amino acid sequence of LCDR2 is FASTRY (SEQ ID NO: 68), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 69); (5) the amino acid sequence of LCDR1 is KASQSVSNDVV (SEQ ID NO: 70); The amino acid sequence of LCDR2 is YASNRYT (SEQ ID NO: 71), The amino acid sequence of LCDR3 is QQDYSSPWT (SEQ ID NO: 72); (6) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 73); The amino acid sequence of LCDR2 is FASTRES (SEQ ID NO: 74), The amino acid sequence of LCDR3 is QQHYSIPLT (SEQ ID NO: 75); (7) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 76); The amino acid sequence of LCDR2 is FASTRKS (SEQ ID NO: 77), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 78); (8) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNHLA (SEQ ID NO: 79); The amino acid sequence of LCDR2 is FASTRQS (SEQ ID NO: 80), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 81); (9) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 82); The amino acid sequence of LCDR2 is FASTRQS (SEQ ID NO: 83), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 84); (10) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNHLA (SEQ ID NO: 85); The amino acid sequence of LCDR2 is FASTRGS (SEQ ID NO: 86), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 87); (11) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 88); The amino acid sequence of LCDR2 is FASTRDS (SEQ ID NO: 89): The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO: 90); (12) The amino acid sequence of LCDR1 is KSSQSLLNSSNQKNYLA (SEQ ID NO: 91); The amino acid sequence of LCDR2 is FASTRES (SEQ ID NO: 92), The amino acid sequence of LCDR3 is QQHYSTPLT (SEQ ID NO:93); (13) The amino acid sequence of LCDR1 is RSSQSLVHSDGNTYLH (SEQ ID NO: 94); The amino acid sequence of LCDR2 is KVSNRFS (SEQ ID NO: 95), The amino acid sequence of LCDR3 is SQSIHVPWT (SEQ ID NO: 96); (14) The amino acid sequence of LCDR1 is SASSSVSYMH (SEQ ID NO: 97); The amino acid sequence of LCDR2 is STSNLAS (SEQ ID NO: 98), The amino acid sequence of LCDR3 is QQRSSYPLT (SEQ ID NO: 99); (15) The amino acid sequence of LCDR1 is RSSQSLVHSDGNTYLH (SEQ ID NO: 100); The amino acid sequence of LCDR2 is KVSNRFS (SEQ ID NO: 101), The amino acid sequence of LCDR3 is SQTTQVPWT (SEQ ID NO: 102); (16) The amino acid sequence of LCDR1 is ITNTDIDDDMN (SEQ ID NO: 103). The amino acid sequence of LCDR2 is EGNTLRP (SEQ ID NO: 104), The amino acid sequence of LCDR3 is LQSDDLPLT (SEQ ID NO: 105); (17) The amino acid sequence of LCDR1 is KASQSVDYDGDSYMN (SEQ ID NO: 106); The amino acid sequence of LCDR2 is AASNLES (SEQ ID NO: 107), The amino acid sequence of LCDR3 is LQSNEDPYT (SEQ ID NO: 108); (18) The amino acid sequence of LCDR1 is RSSQFIVHSNGNTYLE (SEQ ID NO: 109); The amino acid sequence of LCDR2 is KISNRFS (SEQ ID NO: 110), The amino acid sequence of LCDR3 is FQGSHVPFT (SEQ ID NO: 111); and (19) The amino acid sequence of LCDR1 is KASEDIYNRLA (SEQ ID NO: 112); The amino acid sequence of LCDR2 is GATSLET (SEQ ID NO: 113), the amino acid sequence of LCDR3 is QQYWSSPLT (SEQ ID NO: 114); or The antibody comprises a variant of a combination of any of the LCDR sequences (1) to (19), wherein the variant has at least 90% sequence identity with any of the LCDR sequences (1) to (19), or contains a total of at least one and no more than 10, or no more than 5, 4, 3, or 2 amino acid alterations in the LCDR sequence. The CAR according to claim 12.

14. The CAR of claim 12 or 13, wherein the amino acid sequence of the heavy chain variable region is selected from the following: (1) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 115; (2) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 117; (3) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 119; (4) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 121; (5) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 123; (6) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 125; (7) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 127; (8) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 129; (9) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 131; (10) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 133; (11) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 135; (12) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 137; (13) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 139; (14) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 141; (15) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 143; (16) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 145; (17) The sequence of SEQ ID NO: 147 or a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto; (18) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 149; and (19) The sequence of SEQ ID NO: 151 or a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

15. The CAR according to any one of claims 12 to 14, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the antibody are selected from any combination of the following: (1) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 115, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 116; (2) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 117, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 118; (3) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 119, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 120; (4) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 121, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 122; (5) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 123, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 124; (6) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 125, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 126; (7) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 127, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 128; (8) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 129, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 130; (9) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 131, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 132; (10) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 133, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 134; (11) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 135, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 136; (12) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 137, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 138; (13) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 139, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 140; (14) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 141, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 142; (15) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 143, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 144; (16) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 145, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 146; (17) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 147, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 148; (18) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 149, or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, as set forth in SEQ ID NO: 150; and (19) The sequence set forth in SEQ ID NO: 151 or a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, and the sequence set forth in SEQ ID NO: 152 or a light chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

16. The binding KD value of the antibody to the EGFRvIII antigen as measured by surface plasmon resonance (SPR) is 10 －６ Less than M, preferably 10 －７ The CAR according to any one of claims 12 to 15, wherein the molecular weight is less than M.

17. The CAR according to any one of claims 12 to 16, wherein the antibody is selected from a mouse-derived antibody, a chimeric antibody, a humanized antibody, or a human antibody.

18. The binding KD value of the humanized antibody to the EGFRvIII antigen as measured by surface plasmon resonance (SPR) is 10 －６ Less than M, preferably 10 －７ The CAR according to any one of claims 12 to 17, wherein the molecular weight is less than M.

19. The CAR according to any of claims 12 to 18, wherein the antigen-binding fragment is a single-chain antibody, and preferably, a linker sequence for linking a heavy chain variable region and a light chain variable region in the single-chain antibody comprises the sequence shown in SEQ ID NO:

166.

20. The CAR according to any one of claims 12 to 19, wherein the humanized antibody is a single-chain antibody, and the amino acid sequence thereof is selected from the following: (1) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 167; (2) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 169; (3) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 171; (4) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 173; (5) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 175; (6) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 177; (7) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 179; (8) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 181; (9) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 183; (10) A heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto, or a sequence set forth in SEQ ID NO: 185; and (11) The sequence set forth in SEQ ID NO: 187 or a heavy chain variable region sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

21. The CAR according to any of claims 12 to 20, further comprising a hinge region, a transmembrane region, a costimulatory domain, and an activation domain, wherein preferably the hinge region is a CD8 hinge region, the transmembrane region is a CD8α transmembrane region, the costimulatory domain is a 41BB costimulatory domain or a CD28 costimulatory domain, and the activation domain is a CD3ζ activation domain.

22. The CAR according to any of claims 12 to 21, wherein the hinge region comprises the sequence shown in SEQ ID NO: 156, the transmembrane region comprises the sequence shown in SEQ ID NO: 158, the costimulatory domain comprises the sequence shown in SEQ ID NO: 160 and / or 162, and the activation domain comprises the sequence shown in SEQ ID NO:

164.

23. The CAR according to any one of claims 12 to 22, further comprising a signal peptide, preferably the signal peptide is a CD8α signal peptide, and more preferably the signal peptide comprises the sequence shown in SEQ ID NO:

154.

24. The CAR according to any one of claims 12 to 23, wherein the signal peptide comprises the sequence shown in SEQ ID NO:

154.

25. A nucleic acid molecule encoding: 1) An antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or a heavy chain variable region and / or a light chain variable region of said antibody or antigen-binding fragment thereof; or 2) The CAR according to any one of claims 12 to 24.

26. 26. The nucleic acid molecule of claim 25, comprising a nucleotide sequence set forth in any of SEQ ID NOs: 153, 155, 157, 159, 161, 163, 165, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186 or 188.

27. 27. An expression vector comprising a nucleic acid molecule according to claim 25 or 26, preferably a lentiviral vector.

28. A cell comprising the expression vector of claim 27.

29. A cell expressing the CAR according to any one of claims 12 to 24.

30. 30. The cell of claim 28 or 29, which is an immune cell.

31. The cell according to any one of claims 28 to 30, which is a T cell or an NK cell.

32. 32. The cell of any of claims 29 to 31, further modified to express a dominant negative receptor (DNR) or a chimeric switch receptor (CSR), preferably wherein the DNR is dnTGFβRII and / or dnPD1, preferably wherein the intracellular domain of the CSR is derived from IL7Rα, more preferably wherein the dnTGFβRII comprises the sequence set forth in SEQ ID NO: 193 and wherein the intracellular domain of the CSR comprises the sequence set forth in SEQ ID NO:

197.

33. A pharmaceutical composition comprising: 1) an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, a nucleic acid molecule according to claim 25 or 26, an expression vector according to claim 27, or a cell according to any one of claims 29 to 32; and 2) A pharmaceutically acceptable carrier.

34. Use of an antibody or antigen-binding fragment thereof of any of claims 1 to 11, a nucleic acid molecule of claim 25 or 26, an expression vector of claim 27, or a cell of any of claims 29 to 32 in the manufacture of a medicament for treating a tumor.

35. 35. The use of claim 34, wherein the tumor expresses EGFRvIII.

36. 36. The use of claim 34 or 35, wherein the tumor is a glioblastoma.

37. A method for treating a tumor in a subject, comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment thereof of any one of claims 1 to 11, a nucleic acid molecule of claim 25 or 26, an expression vector of claim 27, a cell of any one of claims 29 to 32, or a pharmaceutical composition of claim 33.

38. 38. The method of claim 37, wherein the tumor expresses EGFRvIII.

39. 39. The method of claim 37 or 38, wherein the tumor is a glioblastoma.

40. A multispecific antibody molecule comprising at least a first functional portion and a second functional portion, wherein the first functional portion comprises an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, and the second functional portion has an antigen-binding specificity different from that of the first functional portion.

41. The multispecific antibody molecule of claim 40, wherein the second functional portion has binding specificity for a T cell.

42. An immunoconjugate comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11 linked to a therapeutic agent.

43. 43. The immunoconjugate of claim 42, wherein the therapeutic agent is a drug.

44. 44. The immunoconjugate of claim 42 or 43, wherein the therapeutic agent is a cytotoxin.

45. The immunoconjugate of any one of claims 42 to 44, wherein the therapeutic agent is a radioisotope.

46. A kit for detecting the presence or absence or content of EGFRvIII in a sample, comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

Images