Chimeric antigen receptor targeting Ly6G6D and application thereof

Abstract

The invention provides a chimeric antigen receptor targeting to Ly6G6D and application of the chimeric antigen receptor. Specifically, the invention provides a CAR capable of being specifically bound with Ly6G6D, and the CAR comprises a Ly6G6D binding structural domain, a transmembrane structural domain, a co-stimulation structural domain and an intracellular signal transduction structural domain. The invention also provides an application of the CAR in treating a disease or condition related to the expression of Ly6G6D.

Description

Technical Field

[0001] The subject matter disclosed herein provides methods and compositions for treating cancer. Specifically, the present invention provides a chimeric antigen receptor targeting Ly6G6D, immune effector cells comprising the chimeric antigen receptor, and methods of using these immune effector cells for treating cancer. Background Art

[0002] Cell-based immunotherapy is a therapy with therapeutic potential for cancer. T cells and other immune cells can be modified to target tumor antigens by introducing genetic material encoding an artificial or synthetic receptor for an antigen, which receptor is called a chimeric antigen receptor (CAR) and is specific for the selected antigen. Targeted T cell therapy using CAR has achieved clinical success in treating cancer.

[0003] Lymphocyte antigen 6 family member G6D (Ly6G6D) is a leukocyte antigen cluster located in the major histocompatibility complex (MHC) class III region of chromosome 6, encoding a protein of 133 amino acid residues with a molecular weight of approximately 13.7 kDa. Like most family members, Ly6G6D is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Multiple studies have shown that lymphocyte antigen 6 family members are very important for regulating the functions of immunity, the nervous system, and complement. Especially in cancer, numerous lymphocyte antigen 6 family members play key roles in many types of cancer, such as gastric cancer, cervical cancer, breast cancer, ovarian cancer, lung cancer, and bladder cancer. Therefore, the lymphocyte antigen 6 gene family may play an important role in clinical practice, not only as a marker for disease prognosis but also as an important target for developing new drugs. In-depth study of their biological functions will be of great significance for clarifying the functions of the lymphocyte antigen 6 family, analyzing the protein interactions in their structures, exploring the pathogenesis of diseases, and discovering new therapeutic targets.

[0004] Colorectal cancer can be divided into mismatch repair-deficient (dMMR) and mismatch repair-proficient (pMMR), where the dMMR type mainly includes microsatellite-highly unstable (MSI-H) type, and the pMMR type mainly includes microsatellite-low unstable (MSI-L) and microsatellite-stable (MSS) types. For metastatic colorectal cancer of the MSI-H type, the treatment effect of immune checkpoint inhibitors is good; while for the MSI-L and MSS types, which account for about 85% of all metastatic colorectal cancer patients, the response rate of immune checkpoint inhibitors is low and the survival period of patients is short, and there is an urgent need for more effective and safe therapeutic drugs. Research shows that Ly6G6D is highly expressed in advanced colorectal cancer tissues of the pMMR type and is an ideal tumor-specific antigen. Summary of the Invention

[0005] The present invention provides a chimeric antigen receptor targeting Ly6G6D, an immune effector cell comprising the chimeric antigen receptor, and the use of these immune effector cells for treating cancer.

[0006] Specifically, on the one hand, the present invention provides a chimeric antigen receptor targeting Ly6G6D, the chimeric antigen receptor comprising an extracellular antigen-binding domain that binds Ly6G6D, a transmembrane domain, and an intracellular domain, the extracellular antigen-binding domain comprising an antibody or antigen-binding fragment that targets and binds Ly6G6D, wherein the antibody or antigen-binding fragment comprises a heavy-chain variable region, and the heavy-chain variable region comprises complementarity-determining regions: H-CDR1, H-CDR2, and H-CDR3; wherein,

[0007] H-CDR1 has the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:7, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:22, SEQ ID NO:28, SEQ ID NO:34, SEQ ID NO:40, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:127, or SEQ ID NO:138; and

[0008] H-CDR2 has the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:51, or SEQ ID NO:139; and

[0009] H-CDR3 has the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:128, SEQ ID NO:132, or SEQ ID NO:140;

[0010] and the antibody or antigen-binding fragment comprises a light-chain variable region, and the light-chain variable region comprises complementarity-determining regions: L-CDR1, L-CDR2, and L-CDR3; wherein,

[0011] L - CDR1 has the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:53, SEQ ID NO:129, SEQ ID NO:135 or SEQ ID NO:141; and

[0012] L - CDR2 has the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:54, SEQ ID NO:130, SEQ ID NO:133, SEQ ID NO:136 or SEQ ID NO:142; and

[0013] L - CDR3 has the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:131, SEQ ID NO:134, SEQ ID NO:137 or SEQ ID NO:143.

[0014] NYAMH (SEQ ID NO:1).

[0015] VISYDGSKKYYADSVKG (SEQ ID NO:2).

[0016] PAGSTSGFDY (SEQ ID NO:3).

[0017] TGTSSDVGGYNYVS (SEQ ID NO:4).

[0018] DVSNRPS (SEQ ID NO:5).

[0019] SSYTSSSTLVV (SEQ ID NO:6).

[0020] DYGMS (SEQ ID NO:7).

[0021] GINWNGGSTGYADSVKG (SEQ ID NO:8).

[0022] SSVKHY (SEQ ID NO:9).

[0023] SGSSSNIGTNTVN (SEQ ID NO:10).

[0024] RNDLRPS (SEQ ID NO:11).

[0025] AAWDDSLNGVV (SEQ ID NO:12).

[0026] NYGIT (SEQ ID NO:13).

[0027] WISPYNGHTIYAQKVQG (SEQ ID NO:14).

[0028] GLNSRLQY (SEQ ID NO:15).

[0029] DVSKRPS (SEQ ID NO:16).

[0030] SSYTSSSTDV (SEQ ID NO:17).

[0031] GYYMH (SEQ ID NO:18).

[0032] WINPVSGGTNYAQKFQG (SEQ ID NO:19).

[0033] DPFDI (SEQ ID NO:20).

[0034] SSYTSRSTPGYV (SEQ ID NO:21).

[0035] NYGFS (SEQ ID NO:22).

[0036] WISSYNGNTKYAQKFRG (SEQ ID NO:23). GGDWPADY (SEQ ID NO:24).

[0037] GLSSGSVSNNYYPS (SEQ ID NO:25). STNTRSS (SEQ ID NO:26).

[0038] VVYMGSGIWV (SEQ ID NO:27).

[0039] SYGIS (SEQ ID NO:28).

[0040] WINPNSGGTNYAQKFQG (SEQ ID NO:29). AGDLRDYYYYGMDV (SEQ ID NO:30). TGSSSNIGAGYDVH (SEQ ID NO:31). GNSNRPS (SEQ ID NO:32).

[0041] QSYDSSLSGWV (SEQ ID NO:33).

[0042] NHFIH (SEQ ID NO:34).

[0043] WISANSGNTDYAQKFQG (SEQ ID NO:35). DLATVDGFDI (SEQ ID NO:36).

[0044] RSSQSLLHSNGYNYLD (SEQ ID NO:37). LGSNRAS (SEQ ID NO:38).

[0045] MQALQTPLT (SEQ ID NO:39).

[0046] SYSVS (SEQ ID NO:40).

[0047] GIIPTFGRTNYAQKFQG (SEQ ID NO:41). VRKPYYGMDV (SEQ ID NO:42).

[0048] RASQSIGRFLN (SEQ ID NO:43).

[0049] GASRLQG (SEQ ID NO:44).

[0050] QQSYSTPPT (SEQ ID NO:45).

[0051] SYGIN (SEQ ID NO:46).

[0052] WINTKTGNPTYVQGFTG (SEQ ID NO:47). DRNPYGMDV (SEQ ID NO:48).

[0053] MQPLQALYT (SEQ ID NO:49).

[0054] SYAIS (SEQ ID NO:50).

[0055] GIIPIFGTANYAQKFQG (SEQ ID NO:51). TGGDYYYYGMDV (SEQ ID NO:52). RASQTISTYLN (SEQ ID NO:53).

[0056] GASSLQT (SEQ ID NO:54).

[0057] DYFLH (SEQ ID NO:127).

[0058] DPFDY (SEQ ID NO:128).

[0059] RSSQSLVHSDGNTYLS (SEQ ID NO:129).

[0060] KISNRFS (SEQ ID NO:130).

[0061] MQATQFPIT (SEQ ID NO:131).

[0062] VGGSYYGGFDY (SEQ ID NO:132).

[0063] LSSNRAS (SEQ ID NO:133).

[0064] MQALQTPPT (SEQ ID NO:134).

[0065] RSSQSLVYSDGNTYLS (SEQ ID NO:135).

[0066] KVSNRFS (SEQ ID NO:136).

[0067] MQATQYPLA (SEQ ID NO:137).

[0068] NYWIG (SEQ ID NO:138).

[0069] IIYPGDSDTRYSPSFQG (SEQ ID NO:139).

[0070] PQGAFDI (SEQ ID NO:140).

[0071] RSSQSLVYSDGNTYLN (SEQ ID NO:141).

[0072] KVSNRDS (SEQ ID NO:142).

[0073] MQGTHWPLT (SEQ ID NO:143).

[0074] In some embodiments, the complementarity determining regions of the heavy chain variable region: H-CDR1, H-CDR2, and H-CDR3 are selected from any combination of the following groups:

[0075] (1) H-CDR1 has the amino acid sequence shown in SEQ ID NO:1, H-CDR2 has the amino acid sequence shown in SEQ ID NO:2, and H-CDR3 has the amino acid sequence shown in SEQ ID NO:3; or

[0076] (2) H-CDR1 has the amino acid sequence shown in SEQ ID NO:7, H-CDR2 has the amino acid sequence shown in SEQ ID NO:8, and H-CDR3 has the amino acid sequence shown in SEQ ID NO:9; or

[0077] (3) H-CDR1 has the amino acid sequence shown in SEQ ID NO:13, H-CDR2 has the amino acid sequence shown in SEQ ID NO:14, and H-CDR3 has the amino acid sequence shown in SEQ ID NO:15; or

[0078] (4) H-CDR1 has the amino acid sequence shown in SEQ ID NO:18, H-CDR2 has the amino acid sequence shown in SEQ ID NO:19, and H-CDR3 has the amino acid sequence shown in SEQ ID NO:20; or

[0079] (5) H-CDR1 has the amino acid sequence shown in SEQ ID NO:22, H-CDR2 has the amino acid sequence shown in SEQ ID NO:23, and H-CDR3 has the amino acid sequence shown in SEQ ID NO:24; or

[0080] (6) H-CDR1 has the amino acid sequence shown in SEQ ID NO:28, H-CDR2 has the amino acid sequence shown in SEQ ID NO:29, and H-CDR3 has the amino acid sequence shown in SEQ ID NO:30; or

[0081] (7) H-CDR1 has the amino acid sequence shown in SEQ ID NO:34, H-CDR2 has the amino acid sequence shown in SEQ ID NO:35, and H-CDR3 has the amino acid sequence shown in SEQ ID NO:36; or

[0082] (8) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 40, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 41, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 42; or

[0083] (9) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 46, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 47, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 48; or

[0084] (10) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 50, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 51, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 52; or

[0085] (11) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 127, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 29, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 128; or

[0086] (12) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 18, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 29, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 132; or

[0087] (13) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 18, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 29, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 128; or

[0088] (14) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 138, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 139, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 140.

[0089] In some embodiments, the complementarity-determining regions of the light chain variable region: L-CDR1, L-CDR2, and L-CDR3 are selected from any combination of the following groups:

[0090] (1) The L-CDR1 has the amino acid sequence shown in SEQ ID NO: 4, the L-CDR2 has the amino acid sequence shown in SEQ ID NO: 5, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO: 6; or

[0091] (2) The L-CDR1 has the amino acid sequence shown in SEQ ID NO: 10, the L-CDR2 has the amino acid sequence shown in SEQ ID NO: 11, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO: 12; or

[0092] (3) The L-CDR1 has the amino acid sequence shown in SEQ ID NO: 4, the L-CDR2 has the amino acid sequence shown in SEQ ID NO: 16, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO: 17; or

[0093] (4) The L-CDR1 has the amino acid sequence shown in SEQ ID NO: 4, the L-CDR2 has the amino acid sequence shown in SEQ ID NO: 16, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO: 21; or

[0094] (5) The L-CDR1 has the amino acid sequence shown in SEQ ID NO: 25, the L-CDR2 has the amino acid sequence shown in SEQ ID NO: 26, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO: 27; or

[0095] (6) The L-CDR1 has the amino acid sequence shown in SEQ ID NO: 31, the L-CDR2 has the amino acid sequence shown in SEQ ID NO: 32, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO: 33; or

[0096] (7) The L-CDR1 has the amino acid sequence shown in SEQ ID NO: 37, the L-CDR2 has the amino acid sequence shown in SEQ ID NO: 38, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO: 39; or

[0097] (8) The L-CDR1 has the amino acid sequence shown in SEQ ID NO: 43, the L-CDR2 has the amino acid sequence shown in SEQ ID NO: 44, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO: 45; or

[0098] (9) The L-CDR1 has the amino acid sequence shown in SEQ ID NO: 37, the L-CDR2 has the amino acid sequence shown in SEQ ID NO: 38, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO: 49; or

[0099] (10) The L-CDR1 has the amino acid sequence shown in SEQ ID NO:53, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:54, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:45; or

[0100] (11) The L-CDR1 has the amino acid sequence shown in SEQ ID NO:129, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:130, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:131; or

[0101] (12) The L-CDR1 has the amino acid sequence shown in SEQ ID NO:37, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:133, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:134; or

[0102] (13) The L-CDR1 has the amino acid sequence shown in SEQ ID NO:135, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:136, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:137; or

[0103] (14) The L-CDR1 has the amino acid sequence shown in SEQ ID NO:141, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:142, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:143.

[0104] In some embodiments, the complementarity determining regions of the heavy chain variable region: H-CDR1, H-CDR2, and H-CDR3, and the complementarity determining regions of the light chain variable region: L-CDR1, L-CDR2, and L-CDR3 are selected from any combination of the following groups:

[0105] (1) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:1, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:2, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:3; and

[0106] The L-CDR1 has the amino acid sequence shown in SEQ ID NO:4, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:5, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:6; or

[0107] (2) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:7, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:8, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:9; and

[0108] The L-CDR1 has the amino acid sequence shown in SEQ ID NO:10, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:11, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:12; or

[0109] (3) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:13, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:14, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:15; and

[0110] The L-CDR1 has the amino acid sequence shown in SEQ ID NO:4, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:16, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:17; or

[0111] (4) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:18, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:19, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:20; and

[0112] The L-CDR1 has the amino acid sequence shown in SEQ ID NO:4, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:16, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:21; or

[0113] (5) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:22, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:23, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:24; and

[0114] The L-CDR1 has the amino acid sequence shown in SEQ ID NO:25, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:26, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:27; or

[0115] (6) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:28, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:29, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:30; and

[0116] The L-CDR1 has the amino acid sequence shown in SEQ ID NO:31, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:32, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:33; or

[0117] (7) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:34, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:35, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:36; and

[0118] The L-CDR1 has the amino acid sequence shown in SEQ ID NO:37, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:38, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:39; or

[0119] (8) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:40, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:41, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:42; and

[0120] The L-CDR1 has the amino acid sequence shown in SEQ ID NO:43, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:44, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:45; or

[0121] (9) The H-CDR1 has the amino acid sequence shown in SEQ ID NO:46, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:47, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:48; and

[0122] The L-CDR1 has the amino acid sequence shown in SEQ ID NO:37, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:38, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:49; or

[0123] The H-CDR1 has the amino acid sequence shown in SEQ ID NO:50, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:51, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:52; and

[0124] The L-CDR1 has the amino acid sequence shown in SEQ ID NO:53, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:54, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:45.

[0125] The H-CDR1 has the amino acid sequence shown in SEQ ID NO:127, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:29, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:128; and

[0126] The L-CDR1 has the amino acid sequence shown in SEQ ID NO:129, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:130, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:131.

[0127] The H-CDR1 has the amino acid sequence shown in SEQ ID NO:18, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:29, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:132; and

[0128] The L-CDR1 has the amino acid sequence shown in SEQ ID NO:37, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:133, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:134.

[0129] The H-CDR1 has the amino acid sequence shown in SEQ ID NO:18, the H-CDR2 has the amino acid sequence shown in SEQ ID NO:29, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO:128; and

[0130] The L-CDR1 has the amino acid sequence shown in SEQ ID NO:135, the L-CDR2 has the amino acid sequence shown in SEQ ID NO:136, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO:137.

[0131] (14) The H-CDR1 has the amino acid sequence shown in SEQ ID NO: 138, the H-CDR2 has the amino acid sequence shown in SEQ ID NO: 139, and the H-CDR3 has the amino acid sequence shown in SEQ ID NO: 140; and

[0132] The L-CDR1 has the amino acid sequence shown in SEQ ID NO: 141, the L-CDR2 has the amino acid sequence shown in SEQ ID NO: 142, and the L-CDR3 has the amino acid sequence shown in SEQ ID NO: 143.

[0133] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable region framework sequence and a light chain variable region framework sequence, and the framework sequences are derived from a murine antibody, a human antibody, a primate antibody, or a mutant thereof.

[0134] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 55-64 or SEQ ID NO: 144-147.

[0135] In some embodiments, the light chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO: 65-76 or SEQ ID NO: 148-151.

[0136] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 55; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 65.

[0137] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 55; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 65.

[0138] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:56; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:66.

[0139] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:56; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:66.

[0140] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:57; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:67.

[0141] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:57; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:67.

[0142] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:58; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with SEQ ID NO:68.

[0143] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 58; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68.

[0144] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 59; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 69.

[0145] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 59; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 69.

[0146] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 60; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 70.

[0147] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 60; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 70.

[0148] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:61; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:71.

[0149] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:61; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:71.

[0150] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:62; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:72 or 73.

[0151] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO:62; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO:72 or 73.

[0152] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:63; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO:74.

[0153] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 63; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 74.

[0154] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 64; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 75 or 76.

[0155] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 64; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 75 or 76.

[0156] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 144; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 148.

[0157] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence shown in SEQ ID NO: 144; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 148.

[0158] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 145; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 149.

[0159] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 145; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 149.

[0160] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 146; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 150.

[0161] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 146; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 150.

[0162] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 147; and the light chain variable region comprises an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to SEQ ID NO: 151.

[0163] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises the amino acid sequence set forth in SEQ ID NO: 147; and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 151.

[0164] EVHLVESGGGVVQPGRSLRLSCAASGFTFSNYAMHWVRQAPGKGLEWVAVISYDGSK

[0165] KYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCAKPAGSTSGFDYWGQGTLVTV SS(SEQID NO:55).

[0166] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSG VSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVVFGGGTKLTVL(SEQ ID NO:65).

[0167] QVQLVQSGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGG

[0168] STGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATSSVKHYWGQGTLVTVSS(SEQ IDNO:56).

[0169] QPVLTQPPSASATPGQRVTISCSGSSSNIGTNTVNWYQQVPGTAPKVLIYRNDLRPSGVP DRFSGSKSGTSASLAISGLQSDDEADYYCAAWDDSLNGVVFGGGTKVTVL(SEQ ID NO:66).

[0170] QVQLVQSGPEMKKPGASVKVACKTSGYIFINYGITWVRQAPGQGLEWMGWISPYNGH

[0171] TIYAQKVQGRGTMTTETPTSTAYLDLTNLTPDDTAIYYCATGLNSRLQYWGQGALVTVSS(SEQ IDNO:57).

[0172] QPVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSG VSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTDVFGTGTKVTVL(SEQ ID NO:67).

[0173] EVQLVQSGAEVKKPGASMKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPVS

[0174] GGTNYAQKFQGRVTMTRDTSIGTAYMELTGLRSDDTAVYYCAADPFDIWGQGTMVTVSS(SEQ IDNO:58).

[0175] QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSRSTPGYVFGTGTKLTVL(SEQ ID NO:68).

[0176] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGFSWVRQAPGQGLEWMGWISSYNG

[0177] NTKYAQKFRGRVTMTTDTSTSTGYMELRSLRSDDTAVYYCARGGDWPADYWGQGTLVTVSS(SEQ IDNO:59).

[0178] QAVVTQEPSFSVSPGGTVTLTCGLSSGSVSNNYYPSWYQQIPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGAQADDEADYYCVVYMGSGIWVFGGGTKLTVL(SEQ ID NO:69).

[0179] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINPNSGG

[0180] TNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARAGDLRDYYYYGMDVWGQGTTVTVSS(SEQ ID NO:60).

[0181] QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTQLTAL(SEQ ID NO:70).

[0182] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNHFIHWVRQAPGQGLEWMGWISANSG

[0183] NTDYAQKFQGRVTMTTDTSTSTAYMELRSLRYDDTAVYYCARDLATVDGFDIWGQGTMVTVSS(SEQID NO:61).

[0184] DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGPGTKVDIK(SEQ ID NO:71).

[0185] EVQLVQSGSEVKKPGSSVKVSCKAPGGTFSSYSVSWVRQAPGQGLEWMGGIIPTFGRTNYAQKFQGRVTISAHESTGTAYMELSSLRSEDTAVYYCASVRKPYYGMDVWGQGTMVTVSS(SEQ ID NO:62).

[0186] DIQMTQSPSSLSASVGDRVTITCRASQSIGRFLNWYQQKPGTAPKLLISGASRLQGGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK(SEQ ID NO:72).

[0187] AIWMTQSPSSLSASVGDRVTITCRASQSIGRFLNWYQQKPGTAPKLLISGASRLQGGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK(SEQ ID NO:73).

[0188] QVQLVQSGSELKKPGASVKISCKASGYTLTSYGINWVRQAPGKGLELMGWINTKTGNP

[0189] TYVQGFTGQVVFSFDTSVNTAYLQINNLKSDDTAVYFCARDRNPYGMDVWGQGTTVTVSS(SEQ IDNO:63)。

[0190] EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQPLQALYTFGQGTKVEIK(SEQ ID NO:74)。

[0191] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARTGGDYYYYGMDVWGQGTTVTVSS(SEQ ID NO:64)。

[0192] DIQMTQSPSSLSASVGDRVTITCRASQTISTYLNWYQQKPGKAPKLLIYGASSLQTGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKLEIK(SEQ ID NO:75)。

[0193] AIWMTQSPSSLSASVGDRVTITCRASQTISTYLNWYQQKPGKAPKLLIYGASSLQTGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVDIK(SEQ ID NO:76)。

[0194] EVQLVESGAEVKKPGASVKVSCKASGYTFTDYFLHWVRQAPGQGLEWMGWINPNSG GTNYAQKFQGWVTMTRDTSISTAYLELSSLTSDDTAVYYCAVDPFDYWGQGTLVTVSS(SEQ ID NO:144)。

[0195] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNS GGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARVGGSYYGGFDYWGQGTL VTVSS(SEQ ID NO:145).

[0196] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNS GGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASDPFDYWGQGTLVTVSS(SEQ ID NO:146).

[0197] QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSD TRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARPQGAFDIWGQGTMVTVSS(SEQ ID NO:147).

[0198] DVVMTQSPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLHQRPGQPPRLLIHKISNRF SGVPDRFSGSGAGTDFTLKISRVEAEDVGVFYCMQATQFPITFGQGTRLEIK(SEQ ID NO:148).

[0199] EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLMYLSSNR ASGVPDRFSGSGSGRDFTLKISRVEAEDVGLYYCMQALQTPPTFGQGTRLEIK(SEQ ID NO:149).

[0200] DVVMTQSPLSSPVTLGQPASISCRSSQSLVYSDGNTYLSWLQQRPGQPPRLLIYKVSNR FSGVPDRFSGSGAGTDFTLTISRVEAEDVGVYYCMQATQYPLAFGGGTKLEIK(SEQ ID NO:150).

[0201] EIVMTQTPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPLTFGQGTKVEIK(SEQ ID NO:151).

[0202] In some embodiments, the heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain are linked by a linker.

[0203] The linker is a connecting peptide for connecting the heavy chain variable region and the light chain variable region of the antibody, and can also be a linker peptide modified by researchers. Examples of the amino acid sequence of the linker peptide are: (GnS)m, where n is a natural number from 3 to 5, preferably 4, and m is a natural number from 1 to 5, preferably 3.

[0204] GGGS(SEQ ID NO:77).

[0205] GGGGS(SEQ ID NO:78).

[0206] GGGGSGGGGS(SEQ ID NO:79).

[0207] GGGGSGGGGSGGGGS(SEQ ID NO:80).

[0208] In some embodiments, the extracellular antigen-binding domain is a single-chain antibody (scFv).

[0209] The extracellular antigen-binding domain comprises a single-chain antibody (scFv) shown by an amino acid sequence having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with any one of SEQ ID NOs: 81-90 or SEQ ID NOs: 152-155.

[0210] EVHLVESGGGVVQPGRSLRLSCAASGFTFSNYAMHWVRQAPGKGLEWVAVISYDGSKKYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCAKPAGSTSGFDYWGQGTLVTVSS GGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVVFGGGTKLTVL(SEQ ID NO:81)。

[0211] QVQLVQSGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATSSVKHYWGQGTLVTVSS GGGGSGGGGSGGGGS QPVLTQPPSASATPGQRVTISCSGSSSNIGTNTVNWYQQVPGTAPKVLIYRNDLRPSGVPDRFSGSKSGTSASLAISGLQSDDEADYYCAAWDDSLNGVVFGGGTKVTVL(SEQ ID NO:82)。

[0212] QVQLVQSGPEMKKPGASVKVACKTSGYIFINYGITWVRQAPGQGLEWMGWISPYNGHTIYAQKVQGRGTMTTETPTSTAYLDLTNLTPDDTAIYYCATGLNSRLQYWGQGALVTVSS GGGGSGGGGSGGGGS QPVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTDVFGTGTKVTVL(SEQ ID NO:83)。

[0213] EVQLVQSGAEVKKPGASMKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPVSGGTNYAQKFQGRVTMTRDTSIGTAYMELTGLRSDDTAVYYCAADPFDIWGQGTMVTVSS GGGGSGGGGSGGGGS QSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSRSTPGYVFGTGTKLTVL(SEQ ID NO:84)。

[0214] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGFSWVRQAPGQGLEWMGWISSYNGNTKYAQKFRGRVTMTTDTSTSTGYMELRSLRSDDTAVYYCARGGDWPADYWGQGTLVTVSS GGGGSGGGGSGGGGS QAVVTQEPSFSVSPGGTVTLTCGLSSGSVSNNYYPSWYQQIPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGAQADDEADYYCVVYMGSGIWVFGGGTKLTVL(SEQ ID NO:85)。

[0215] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARAGDLRDYYYYGMDVWGQGTTVTVSS GGGGSGGGGSGGGGS QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTQLTAL(SEQ ID NO:86)。

[0216] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNHFIHWVRQAPGQGLEWMGWISANSGNTDYAQKFQGRVTMTTDTSTSTAYMELRSLRYDDTAVYYCARDLATVDGFDIWGQGTMVTVSS GGGGSGGGGSGGGGS DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGPGTKVDIK(SEQ ID NO:87)。

[0217] EVQLVQSGSEVKKPGSSVKVSCKAPGGTFSSYSVSWVRQAPGQGLEWMGGIIPTFGRTNYAQKFQGRVTISAHESTGTAYMELSSLRSEDTAVYYCASVRKPYYGMDVWGQGTMVTVSS GGGGSGGGGSGGGGSAIWMTQSPSSLSASVGDRVTITCRASQSIGRFLNWYQQKPGTAPKLLISGASRLQGGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIK(SEQ ID NO:88).

[0218] QVQLVQSGSELKKPGASVKISCKASGYTLTSYGINWVRQAPGKGLELMGWINTKTGNPTYVQGFTGQVVFSFDTSVNTAYLQINNLKSDDTAVYFCARDRNPYGMDVWGQGTTVTVSS GGGGSGGGGSGGGGS EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQPLQALYTFGQGTKVEIK(SEQ ID NO:89).

[0219] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARTGGDYYYYGMDVWGQGTTVTVSS GGGGSGGGGSGGGGS AIWMTQSPSSLSASVGDRVTITCRASQTISTYLNWYQQKPGKAPKLLIYGASSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVDIK(SEQ ID NO:90).

[0220] EVQLVESGAEVKKPGASVKVSCKASGYTFTDYFLHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGWVTMTRDTSISTAYLELSSLTSDDTAVYYCAVDPFDYWGQGTLVTVSS GGGGSGGGGSGGGGS DVVMTQSPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLHQRPGQPPRLLIHKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVFYCMQATQFPITFGQGTRLEIK(SEQ ID NO:152).

[0221] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARVGGSYYGGFDYWGQGTLVTVSS GGGGSGGGGSGGGGS EIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLMYLSSNRASGVPDRFSGSGSGRDFTLKISRVEAEDVGLYYCMQALQTPPTFGQGTRLEIK(SEQ ID NO:153)。

[0222] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASDPFDYWGQGTLVTVSS GGGGSGGGGSGGGGS DVVMTQSPLSSPVTLGQPASISCRSSQSLVYSDGNTYLSWLQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLTISRVEAEDVGVYYCMQATQYPLAFGGGTKLEIK(SEQ ID NO:154)。

[0223] QVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARPQGAFDIWGQGTMVTVSS GGGGSGGGGSGGGGS EIVMTQTPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPLTFGQGTKVEIK(SEQ ID NO:155)。

[0224] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from a protein selected from the group consisting of the α, β, or ζ chain of a T cell receptor, CD28, CD3ζ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, 4-1BB, CD154, or a combination thereof.

[0225] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from the CD8α protein.

[0226] In some embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:92.

[0227] IWAPLAGTCGVLLLSLVITLYC (SEQ ID NO:92).

[0228] In some embodiments, the chimeric antigen receptor further comprises a hinge region.

[0229] The hinge region generally refers to the connecting region between the antigen-binding region and the immunocyte Fc receptor (FcR)-binding region.

[0230] In some embodiments, the hinge region connects the extracellular antigen-binding structure and the transmembrane domain.

[0231] In some embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO:93.

[0232] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY (SEQ ID NO:93).

[0233] In some embodiments, the intracellular domain of the chimeric antigen receptor comprises a signaling domain derived from CD3ζ.

[0234] In some embodiments, the intracellular domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:94.

[0235] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:94).

[0236] In some embodiments, the intracellular domain further comprises at least one co-stimulatory domain.

[0237] In some embodiments, the co-stimulatory domain comprises a co-stimulatory domain derived from the following proteins: CD28, 4-1BB, OX-40, ICOS, or a combination thereof.

[0238] In some embodiments, the co-stimulatory domain comprises a domain derived from 4-1BB.

[0239] In some embodiments, the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 95.

[0240] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 95).

[0241] In some embodiments, the costimulatory domain comprises a domain derived from ICOS.

[0242] In some embodiments, the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 96.

[0243] CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 96).

[0244] In some embodiments, the costimulatory domain comprises a domain derived from a combination of 4-1BB and ICOS.

[0245] In some embodiments, the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO: 97.

[0246] CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 97).

[0247] In some embodiments, the intracellular domain comprises the amino acid sequence shown in SEQ ID NO: 98.

[0248] CWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 98).

[0249] In some embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NOs: 99-108 or SEQ ID NOs: 156-159.

[0250] Specifically, the chimeric antigen receptor D9-CAR comprises the amino acid sequence shown in SEQ ID NO:99.

[0251] EVHLVESGGGVVQPGRSLRLSCAASGFTFSNYAMHWVRQAPGKGLEWVAVISYDGSKKYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCAKPAGSTSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVVFGGGTKLTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:99).

[0252] Specifically, the chimeric antigen receptor D10-CAR comprises the amino acid sequence shown in SEQ ID NO:100.

[0253] QVQLVQSGGGVVRPGGSLRLSCAASGFTFDDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATSSVKHYWGQGTLVTVSSGGGGSGGGGSGGGGSQPVLTQPPSASATPGQRVTISCSGSSSNIGTNTVNWYQQVPGTAPKVLIYRNDLRPSGVPDRFSGSKSGTSASLAISGLQSDDEADYYCAAWDDSLNGVVFGGGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:100).

[0254] Specifically, the chimeric antigen receptor D28-CAR comprises the amino acid sequence shown in SEQ ID NO: 101.

[0255] QVQLVQSGPEMKKPGASVKVACKTSGYIFINYGITWVRQAPGQGLEWMGWISPYNGHTIYAQKVQGRGTMTTETPTSTAYLDLTNLTPDDTAIYYCATGLNSRLQYWGQGALVTVSSGGGGSGGGGSGGGGSQPVLTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTDVFGTGTKVTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:101).

[0256] Specifically, the chimeric antigen receptor D30-CAR comprises the amino acid sequence shown in SEQ ID NO:102.

[0257] EVQLVQSGAEVKKPGASMKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPVSGGTNYAQKFQGRVTMTRDTSIGTAYMELTGLRSDDTAVYYCAADPFDIWGQGTMVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSRSTPGYVFGTGTKLTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:102).

[0258] Specifically, the chimeric antigen receptor D32-CAR comprises the amino acid sequence shown in SEQ ID NO: 103.

[0259] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYGFSWVRQAPGQGLEWMGWISSYNGNTKYAQKFRGRVTMTTDTSTSTGYMELRSLRSDDTAVYYCARGGDWPADYWGQGTLVTVSSGGGGSGGGGSGGGGSQAVVTQEPSFSVSPGGTVTLTCGLSSGSVSNNYYPSWYQQIPGQAPRTLIYSTNTRSSGVPDRFSGSILGNKAALTITGAQADDEADYYCVVYMGSGIWVFGGGTKLTVLTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:103).

[0260] Specifically, the chimeric antigen receptor D46-CAR comprises the amino acid sequence shown in SEQ ID NO:104.

[0261] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINPNSGGTN

[0262] YAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARAGDLRDYYYYGMDVWGQGTT

[0263] VTVSSGGGGSGGGGSGGGGSQSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQL

[0264] PGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFG

[0265] GGTQLTALTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTC

[0266] GVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYI

[0267] FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRR

[0268] EEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:104).

[0269] Specifically, the chimeric antigen receptor D59-CAR comprises the amino acid sequence shown in SEQ ID NO:105.

[0270] QVQLVQSGAEVKKPGASVKVSCKASGYTFTNHFIHWVRQAPGQGLEWMGWISANSGNTDYAQKFQGRVTMTTDTSTSTAYMELRSLRYDDTAVYYCARDLATVDGFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPLTFGPGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:105).

[0271] Specifically, the chimeric antigen receptor D61-CAR comprises the amino acid sequence shown in SEQ ID NO: 106.

[0272] EVQLVQSGSEVKKPGSSVKVSCKAPGGTFSSYSVSWVRQAPGQGLEWMGGIIPTFGRTNYAQKFQGRVTISAHESTGTAYMELSSLRSEDTAVYYCASVRKPYYGMDVWGQGTMVTVSSGGGGSGGGGSGGGGSAIWMTQSPSSLSASVGDRVTITCRASQSIGRFLNWYQQKPGTAPKLLISGASRLQGGIPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 106).

[0273] Specifically, the chimeric antigen receptor D70-CAR comprises the amino acid sequence shown in SEQ ID NO: 107.

[0274] QVQLVQSGSELKKPGASVKISCKASGYTLTSYGINWVRQAPGKGLELMGWINTKTGNPTYVQGFTGQVVFSFDTSVNTAYLQINNLKSDDTAVYFCARDRNPYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSEIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQPLQALYTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:107).

[0275] Specifically, the chimeric antigen receptor D72-CAR comprises the amino acid sequence shown in SEQ ID NO: 108.

[0276] QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARTGGDYYYYGMDVWGQGTTVTVSSGGGGSGGGGSGGGGSAIWMTQSPSSLSASVGDRVTITCRASQTISTYLNWYQQKPGKAPKLLIYGASSLQTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKVDIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:108).

[0277] Specifically, the chimeric antigen receptor D76-CAR comprises the amino acid sequence shown in SEQ ID NO: 156.

[0278] MGVKVLFALICIAVAEAEVQLVESGAEVKKPGASVKVSCKASGYTFTDYFLHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGWVTMTRDTSISTAYLELSSLTSDDTAVYYCAVDPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSSPVTLGQPASISCRSSQSLVHSDGNTYLSWLHQRPGQPPRLLIHKISNRFSGVPDRFSGSGAGTDFTLKISRVEAEDVGVFYCMQATQFPITFGQGTRLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:156).

[0279] Specifically, the chimeric antigen receptor D79-CAR comprises the amino acid sequence shown in SEQ ID NO:157.

[0280] MGVKVLFALICIAVAEAQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCARVGGSYYGGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSEIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLMYLSSNRASGVPDRFSGSGSGRDFTLKISRVEAEDVGLYYCMQALQTPPTFGQGTRLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:157).

[0281] Specifically, the chimeric antigen receptor D85-CAR comprises the amino acid sequence shown in SEQ ID NO:158.

[0282] MGVKVLFALICIAVAEAQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCASDPFDYWGQGTLVTVSSGGGGSGGGGSGGGGSDVVMTQSPLSSPVTLGQPASISCRSSQSLVYSDGNTYLSWLQQRPGQPPRLLIYKVSNRFSGVPDRFSGSGAGTDFTLTISRVEAEDVGVYYCMQATQYPLAFGGGTKLEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:158).

[0283] Specifically, the chimeric antigen receptor D103-CAR comprises the amino acid sequence shown in SEQ ID NO:159.

[0284] MGVKVLFALICIAVAEAQVQLVQSGAEVKKPGESLKISCKGSGYSFTNYWIGWVRQMPGKGLEWMGIIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARPQGAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSEIVMTQTPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPLTFGQGTKVEIKTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:159).

[0285] In some embodiments, the chimeric antigen receptor further comprises a cleavage site.

[0286] Specifically, the cleavage site is selected from cleavage sites such as BamHI, SpeI, SalI, KpnI, XhoI, SphI, XbaI, EcoRI.

[0287] Specifically, the cleavage site is located at the N-terminus of the hinge region and at the C-terminus of the extracellular antigen-binding structure. In some embodiments, the amino acid sequence of the EcoRI cleavage site is: EF.

[0288] Specifically, the chimeric antigen receptor D9-CAR comprising a cleavage site comprises the amino acid sequence shown in SEQ ID NO:125:

[0289] EVHLVESGGGVVQPGRSLRLSCAASGFTFSNYAMHWVRQAPGKGLEWVAVISYDGSKKYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCAKPAGSTSGFDYWGQGTLVTVSSGGGGSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVVFGGGTKLTVLEFTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:125), where the bold part is the amino acid sequence of the EcoRI cleavage site;

[0290] All the chimeric antigen receptors of the present invention can contain the amino acid sequence of the cleavage site as shown in the above specific examples. The cleavage site is located at the N-terminus of the hinge region and at the C-terminus of the extracellular antigen-binding structure.

[0291] In some embodiments, the chimeric antigen receptor further comprises a signal peptide covalently linked to the N-terminus of the extracellular antigen-binding domain.

[0292] In some embodiments, the signal peptide contains the amino acid sequence shown in SEQ ID NO:91.

[0293] MGVKVLFALICIAVAEA (SEQ ID NO:91).

[0294] On the other hand, the present invention provides an isolated nucleic acid molecule, which contains the nucleotide sequence encoding the chimeric antigen receptor of the present invention.

[0295] An isolated nucleic acid refers to a nucleic acid molecule that has been separated from the components of its natural environment.

[0296] In some embodiments, the nucleic acid molecules are intended to include DNA molecules and RNA molecules. The nucleic acid molecules can be single-stranded or double-stranded and can be cDNA.

[0297] In some embodiments, the nucleic acid molecule comprises the nucleotide sequence shown in any one of SEQ ID NO: 109-118 or SEQ ID NO: 160-163.

[0298] Specifically, the chimeric antigen receptor D9-CAR comprises the nucleotide sequence shown in SEQ ID NO: 109.

[0299]

[0300] Specifically, the chimeric antigen receptor D10-CAR comprises the nucleotide sequence shown in SEQ ID NO: 110.

[0301]

[0302] Specifically, the chimeric antigen receptor D28-CAR comprises the nucleotide sequence shown in SEQ ID NO: 111.

[0303]

[0304] Specifically, the chimeric antigen receptor D30-CAR comprises the nucleotide sequence shown in SEQ ID NO: 112.

[0305]

[0306] Specifically, the chimeric antigen receptor D32-CAR comprises the nucleotide sequence shown in SEQ ID NO: 113.

[0307]

[0308] Specifically, the chimeric antigen receptor D46-CAR comprises the nucleotide sequence shown in SEQ ID NO: 114.

[0309]

[0310] Specifically, the chimeric antigen receptor D59-CAR comprises the nucleotide sequence shown in SEQ ID NO: 115.

[0311]

[0312] Specifically, the chimeric antigen receptor D61-CAR comprises the nucleotide sequence shown in SEQ ID NO: 116.

[0313]

[0314] Specifically, the chimeric antigen receptor D70-CAR comprises the nucleotide sequence shown in SEQ ID NO: 117.

[0315]

[0316] Specifically, the chimeric antigen receptor D72-CAR comprises the nucleotide sequence shown in SEQ ID NO: 118.

[0317]

[0318] Specifically, the chimeric antigen receptor D76-CAR comprises the nucleotide sequence shown in SEQ ID NO: 160.

[0319]

[0320] Specifically, the chimeric antigen receptor D79-CAR comprises the nucleotide sequence shown in SEQ ID NO: 161.

[0321]

[0322] Specifically, the chimeric antigen receptor D85-CAR comprises the nucleotide sequence shown in SEQ ID NO: 162.

[0323]

[0324] Specifically, the chimeric antigen receptor D103-CAR comprises the nucleotide sequence shown in SEQ ID NO: 163.

[0325]

[0326] In some embodiments, the nucleotide sequence further comprises a restriction enzyme site.

[0327] Specifically, the restriction enzyme site is selected from: restriction enzyme sites such as BamHI, SpeI, SalI, KpnI, XhoI, SphI, XbaI, EcoRI, etc.

[0328] In some embodiments, the nucleotide sequence of the EcoRI restriction enzyme site is: GAATTC.

[0329] Specifically, the chimeric antigen receptor D9-CAR containing the restriction enzyme site contains the nucleotide sequence shown in SEQ ID NO: 126:

[0330]

[0331] All of the chimeric antigen receptors of the present invention may contain a nucleotide sequence with a cleavage site as shown in the above specific examples, and the cleavage site is located upstream of the nucleotide sequence of the hinge region and downstream of the nucleotide sequence of the extracellular antigen-binding structure.

[0332] On the other hand, the present invention provides a nucleic acid construct, which comprises the nucleic acid molecule of the present invention; further, the nucleic acid construct comprises one or more control sequences operably linked to the nucleic acid molecule, and the one or more control sequences direct the production of the antibody or its antigen-binding fragment of the present invention in a suitable host cell, and the one or more control sequences are selected from: promoters, enhancers, stop signals, signal peptides, leader sequences, transcription terminators, and any combination thereof.

[0333] In some embodiments, the signal peptide of the present invention has the nucleotide sequence shown in SEQ ID NO: 119.

[0334] ATGGGCGTCAAGGTCCTGTTCGCCCTGATCTGCATCGCCGTCGCCGAGGCC(SEQ ID NO: 119).

[0335] On the other hand, the present invention provides an expression vector, which carries the nucleic acid molecule of the present invention.

[0336] In some embodiments, the expression vector is a γ-retroviral vector or a lentiviral vector.

[0337] On the other hand, the present invention provides an immune effector cell, which comprises the chimeric antigen receptor of the present invention, the nucleic acid molecule of the present invention, or the expression vector of the present invention.

[0338] In some embodiments, the immune effector cells are selected from T cells, natural killer cells, cytotoxic T lymphocytes, regulatory T cells, human embryonic stem cells, lymphoid progenitor cells, T cell precursors, or pluripotent stem cells that can differentiate into lymphocytes, macrophages, B cells, and immune cells with cell killing ability derived from induced pluripotent stem cells.

[0339] On the other hand, the present invention provides a method for preparing immune effector cells, which comprises introducing the expression vector of the present invention into immune effector cells.

[0340] On the other hand, the present invention provides a composition, which comprises the immune effector cells of the present invention and a pharmaceutically acceptable carrier.

[0341] On the other hand, the present invention provides the use of the chimeric antigen receptor, the nucleic acid molecule, the expression vector, the immune effector cell or the composition of the present invention in the preparation of a medicament for treating a disease or disorder related to the expression of Ly6G6D; the disease or disorder related to the expression of Ly6G6D is colorectal cancer, esophageal cancer, gastric cancer, small intestine cancer, large intestine cancer or adenocarcinoma (e.g., colorectal adenocarcinoma, gastric adenocarcinoma or pancreatic adenocarcinoma), or a metastatic cancer thereof, such as metastatic colorectal adenocarcinoma.

[0342] On the other hand, the present invention provides a kit for treating cancer, the kit comprising the immune effector cell of the present invention.

[0343] In some embodiments, the kit of the present invention further includes instructions for using the immune effector cell to treat a cancer subject.

[0344] In some embodiments, the cancer of the present invention is colorectal cancer, esophageal cancer, gastric cancer, small intestine cancer, large intestine cancer or adenocarcinoma (e.g., colorectal adenocarcinoma, gastric adenocarcinoma or pancreatic adenocarcinoma), or a metastatic cancer thereof, such as metastatic colorectal adenocarcinoma.

[0345] Definitions and general terms

[0346] In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Also, the operation steps such as cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology, etc. used herein are all conventional steps widely used in the corresponding fields. At the same time, to better understand the present invention, the definitions and explanations of related terms are provided below.

[0347] When the terms "for example", "such as", "such as", "including", "comprising" or their variants are used herein, these terms will not be considered restrictive terms and will be interpreted as meaning "but not limited to" or "not limited to".

[0348] Unless otherwise specified herein or clearly contradictory according to the context, the terms "a" and "an" and "the" and similar referents in the context of describing the present invention (especially in the context of the following claims) should be construed to cover the singular and the plural.

[0349] Chimeric antigen receptor

[0350] The term "Chimeric Antigen Receptor" (CAR) refers to an engineered receptor that grafts or confers specificity of interest onto immune effector cells. CARs can be used to graft the specificity of monoclonal antibodies onto T cells by facilitating the transfer of their coding sequences via retroviral vectors.

[0351] There are three generations of CARs. "First-generation" CARs are typically constructed by fusing an extracellular antigen-binding domain (e.g., single-chain variable fragment (scFv)) to a transmembrane domain, which is then fused to the cytoplasmic / intracellular domain of a T cell receptor chain. "First-generation" CARs typically have an intracellular domain from the CD3ζ chain, which is the major transducer of signal transduction from the endogenous TCR. "First-generation" CARs can provide de novo antigen recognition and lead to the activation of CD4+ and CD8+ T cells via their CD3ζ chain signaling domain in a single fusion molecule, independent of HLA-mediated antigen presentation. "Second-generation" CARs add intracellular domains from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the cytoplasmic region of the CAR to provide additional signals to T cells. "Second-generation" CARs include those that provide both costimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). Preclinical studies have shown that "second-generation" CARs can enhance the anti-tumor activity of T cells. For example, in patients with chronic lymphocytic leukemia (CLL) and acute lymphocytic leukemia (ALL), clinical trials targeting the CD19 molecule have confirmed the strong efficacy of "second-generation" CAR-modified T cells. "Third-generation" CARs include those that provide multiple costimulations (e.g., CD28 and 4-1BB) and activation (CD3ζ).

[0352] According to the subject matter disclosed by the present invention, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, wherein the extracellular antigen-binding domain binds to Ly6G6D. In certain embodiments, the extracellular antigen-binding domain is an scFv. In certain embodiments, the extracellular antigen-binding domain is an optionally cross-linked Fab. In certain embodiments, the extracellular binding domain is an F(ab)2. In certain embodiments, any of the foregoing molecules can be included in a fusion protein with a heterologous sequence to form the extracellular antigen-binding domain. In a specific non-limiting embodiment, the extracellular antigen-binding domain comprises a human-derived scFv that specifically binds to human Ly6G6D.

[0353] In certain non-limiting embodiments, the extracellular antigen-binding domain of the CAR has high binding specificity and high binding affinity for Ly6G6D. For example, in these embodiments, the extracellular antigen-binding domain of the CAR (such as those implemented as a human scFv or an analogue thereof) binds to Ly6G6D with a dissociation constant (KD) of about 1 x 10 -9 M or lower.

[0354] The term "transmembrane domain" generally refers to the domain in the CAR that traverses the cell membrane and is linked to the intracellular signal transduction domain, playing a role in signal transmission.

[0355] The term "co-stimulatory domain" generally refers to the intracellular domain that can provide a co-stimulatory molecule, which is a cell surface molecule required for the effective response of lymphocytes to an antigen. The co-stimulatory domain can include members of the immunoglobulin superfamily (IgSF), such as the co-stimulatory domains of CD28 and ICOS, and can also include members of the tumor necrosis factor receptor superfamily (TNFRSF), such as the co-stimulatory domains of CD27, OX40, and 4-1BB.

[0356] CDR domains are defined using the Kabat system (Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The subject matter disclosed in the present invention further provides an extracellular antigen-binding domain (such as an scFv) comprising a conservative modification of the antibody sequences disclosed herein. For example, without any limitation, the extracellular antigen-binding domain (such as an scFv) of the subject matter disclosed in the present invention includes: a heavy chain variable region comprising CDR1, CDR2, and CDR3 sequences and a light chain variable region comprising CDR1, CDR2, and CDR3 sequences, wherein one or more of these CDR sequences comprise a specific amino acid sequence disclosed herein or a conservative modification thereof, and wherein the extracellular antigen-binding domain retains the required functional properties.

[0357] The term "antibody" generally refers to a polypeptide molecule that can specifically recognize and / or neutralize a particular antigen. For example, an antibody can comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any that contains its antigen-binding domain. "Antibody" includes monoclonal antibodies, antibody fragments, or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single-domain antibodies (e.g., dAb), single-chain antibodies (e.g., scFv), and antibody fragments that bind to an antigen (e.g., Fab, Fab’, and (Fab)2 fragments). "Antibody" also includes all recombinant forms of an antibody, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antibody fragments and their derivatives that bind to an antigen as described in the present application. Each heavy chain can be composed of a heavy-chain variable region (VH) and a heavy-chain constant region. Each light chain can be composed of a light-chain variable region (VL) and a light-chain constant region. The VH and VL regions can be further divided into hypervariable regions called complementarity-determining regions (CDRs), which are interspersed in more conserved regions called framework regions (FRs). Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with an antigen. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, which include various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq).

[0358] The terms "binding domain", "extracellular domain", "extracellular antigen-binding domain", "extracellular binding domain", "antigen-specific binding domain", and "extracellular antigen-specific binding domain" are used interchangeably and provide a domain or fragment of a CAR that has the ability to specifically bind to a target antigen (e.g., Ly6G6D). Examples include antibodies and their antigen-binding fragments, single-chain scFv antibodies, various fusions and conjugates based on scFv, such as scFv-Fc antibodies, immunoconjugates, antibody-drug conjugates (ADCs), multi / bispecific antibodies, chimeric antigen receptors (CARs).

[0359] The term "single-chain antibody" (scFv) can be an antibody formed by linking the heavy-chain variable region and the light-chain variable region via a linker peptide.

[0360] In certain embodiments, no more than one, no more than two, no more than three, no more than four, or no more than five residues are altered within a particular sequence or CDR region. Exemplary conservative amino acid substitutions are shown in Table 1.

[0361] Table 1

[0362]

[0363]

[0364] The terms "conservative sequence modification" and "conservative sequence substitution" are used interchangeably and refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the CARs (such as extracellular antigen-binding domains) disclosed in the present invention that include amino acid sequences. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the scFvs disclosed in the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be grouped according to their physicochemical properties such as charge and polarity. Conservative amino acid substitutions are those in which the amino acid residue is replaced with an amino acid from the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutral-charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. In addition, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Thus, one or more amino acid residues within a CDR can be replaced with other amino acid residues from the same group, and the function retained by the altered antibody can be detected using the functional assays described herein.

[0365] The terms "identity" and "sequence identity" are used interchangeably and are used to refer to the sequence match between two polypeptides or between two nucleic acids. When a position in both of the two sequences being compared is occupied by the same base or amino acid monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. "Percent identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if 6 of 10 positions in two sequences match, then the two sequences have 60% identity.

[0366] In certain non-limiting embodiments, the extracellular antigen-binding domain of the CAR can include a linker that connects the heavy-chain variable region and the light-chain variable region of the extracellular antigen-binding domain. As used herein, the term "linker" refers to a functional group (e.g., chemical or polypeptide) that covalently links two or more polypeptides or nucleic acids such that they are joined to each other. As used herein, a "peptide linker" refers to one or more amino acids used to couple two proteins together (e.g., couple the VH and VL domains). Non-limiting examples of peptide linkers are disclosed in Shen et al., Anal. Chem. 80(6):1910-1917 (2008) and WO2014 / 087010.

[0367] In addition, the extracellular antigen-binding domain can include a leader sequence or signal peptide that directs the nascent protein into the endoplasmic reticulum. A signal peptide or leader sequence may be necessary if the CAR is to be glycosylated and anchored in the cell membrane. The signal sequence or leader sequence can be a peptide sequence (about 4-30 amino acids in length) present at the N-terminus of the newly synthesized protein that directs the newly synthesized protein into the secretory pathway.

[0368] In certain non-limiting embodiments, the transmembrane domain of the CAR includes a hydrophobic α-helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stabilities. After antigen recognition, the receptors cluster and signals are transmitted to the cell. According to the subject matter disclosed herein, the transmembrane domain of the CAR can include a CD8 polypeptide, a CD28 polypeptide, a CD3ζ polypeptide, a CD4 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a CTLA-4 polypeptide, a PD-1 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide (not based on a protein related to the immune response), or a combination thereof.

[0369] In certain non-limiting embodiments, the intracellular domain of the CAR can include a CD3ζ polypeptide that can activate or stimulate a cell (e.g., a lymphoid lineage cell such as a T cell). CD3ζ includes three ITAMs that transmit an activation signal to the cell (e.g., a lymphoid lineage cell such as a T cell) after antigen binding.

[0370] In certain non-limiting embodiments, the intracellular domain of the CAR further includes at least one signaling region. The at least one signaling region can include a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, a PD-1 polypeptide, a CTLA-4 polypeptide, a LAG-3 polypeptide, a 2B4 polypeptide, a BTLA polypeptide, a synthetic peptide (not based on a protein related to the immune response), or a combination thereof.

[0371] In certain embodiments, the signaling region is a co-stimulatory signaling region. In certain embodiments, the co-stimulatory region includes at least one co-stimulatory molecule, which can provide optimal lymphocyte activation. As used herein, a "co-stimulatory molecule" refers to a cell surface molecule other than the antigen receptor or its ligand that is required for a lymphocyte to respond effectively to an antigen. At least one co-stimulatory signaling region can include a CD28 polypeptide, a 4-1BB polypeptide, an OX40 polypeptide, an ICOS polypeptide, a DAP-10 polypeptide, or a combination thereof. A co-stimulatory molecule can bind to a co-stimulatory ligand, which is a protein expressed on the cell surface that, upon binding to its receptor, generates a co-stimulatory response, which is an intracellular response that modulates the stimulatory effect provided when an antigen binds to its CAR molecule. Co-stimulatory ligands include, but are not limited to, CD80, CD86, CD70, OX40L, 4-1BBL, CD48, TNFRSF14, and PD-L1. As an example, the 4-1BB ligand (i.e., 4-1BBL) can bind to 4-1BB (also referred to as "CD137") to provide an intracellular signal that, in combination with CAR signaling, induces effector cell function in CAR+ T cells.

[0372] In certain embodiments, the CAR includes: an extracellular antigen-binding region including a human scFv that specifically binds to human Ly6G6D, a transmembrane domain including a CD8 polypeptide, and an intracellular domain including a CD3ζ polypeptide and a co-stimulatory signaling region including a combination of 4-1BB and ICOS. In certain embodiments, the CAR further includes a signal peptide or leader sequence covalently linked to the 5' end of the extracellular antigen-binding domain.

[0373] In certain embodiments, the CAR of the presently disclosed subject matter can further include an inducible promoter for expressing a nucleic acid sequence in a human cell. The promoter for expressing the CAR gene can be a constitutive promoter.

[0374] Immune effector cell

[0375] The subject matter disclosed herein provides immune effector cells that express a CAR comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular domain, as described above, wherein the extracellular antigen-binding domain specifically binds to Ly6G6D (such as human Ly6G6D). The immune effector cells can be transduced with the CARs disclosed herein such that the cells express the CAR. The subject matter disclosed herein also provides methods of using such cells to treat tumors such as rectal cancer. The immune effector cells of the subject matter disclosed herein can be lymphoid lineage cells, including lymphoid lineage cells such as B cells, T cells, and natural killer (NK) cells, providing antibody production, regulation of the cellular immune system, detection of foreign substances in the blood, detection of host foreign cells, and the like. Non-limiting examples of lymphoid lineage cells include T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, embryonic stem cells, and pluripotent stem cells (e.g., pluripotent stem cells that can differentiate into lymphoid cells). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject matter disclosed herein can be any type of T cell, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells (e.g., TEM cells and TEMRA cells), regulatory T cells (also referred to as suppressor T cells), natural killer T cells, mucosa-associated invariant T cells, and γδ T cells. In certain embodiments, the T cells expressing the CAR express Foxp3 to achieve and maintain a T regulatory phenotype. Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and play a role during the innate immune response. NK cells do not require prior activation in order to exert their cytotoxic effect on target cells. Cytotoxic T cells (CTLs or killer T cells) are a subtype of T lymphocytes capable of inducing the death of infected somatic cells or tumor cells.

[0376] Vector

[0377] Genetic modification of immune effector cells (e.g., T cells, CTL cells, NK cells) can be achieved by transducing a substantially homogeneous cell population with a recombinant DNA or RNA construct. The vector can be a retroviral vector (e.g., γ-retrovirus) that is used to introduce the DNA or RNA construct into the host cell genome. For example, a polynucleotide encoding a CAR targeting Ly6G6D can be cloned into a retroviral vector and expression can be driven by its endogenous promoter, the retroviral long terminal repeat, or an alternative internal promoter.

[0378] Non-viral vectors or RNAs can also be used. Random chromosomal integration or targeted integration can be used (e.g., using nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and / or clustered regularly interspaced short palindromic repeats (CRISPR)) or transgenic expression (e.g., using native or chemically modified RNAs).

[0379] Drug administration

[0380] The CAR targeting Ly6G6D and immune effector cells expressing it of the presently disclosed subject matter can be provided to a subject systemically or directly for treating or preventing cancer. In certain embodiments, the CAR targeting Ly6G6D and immune effector cells expressing it are directly injected into the organ of interest. Additionally or alternatively, the CAR targeting Ly6G6D and immune effector cells expressing it are provided indirectly to the organ of interest, e.g., by administration to the circulatory system (e.g., the tumor vasculature). Expansion agents and differentiation agents can be provided before, during, or after administration of the cells and compositions to increase T cell production in vitro or in vivo.

[0381] The compositions of the presently disclosed subject matter include pharmaceutical compositions comprising immune effector cells expressing a CAR targeting Ly6G6D and a pharmaceutically acceptable carrier. Administration can be autologous or non-autologous. For example, immune effector cells expressing a CAR targeting Ly6G6D and compositions containing them can be obtained from one subject and administered to the same subject or a different compatible subject. The presently disclosed peripheral blood-derived T cells or their progeny (e.g., obtained in vivo, ex vivo then in vivo, or in vitro) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection, or parenteral administration. When administering the pharmaceutical compositions of the presently disclosed subject matter (e.g., pharmaceutical compositions comprising immune effector cells expressing a CAR targeting Ly6G6D), they can be formulated in unit dose injectable forms (solutions, suspensions, emulsions).

[0382] The term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well-known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes but is not limited to: pH regulators, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure-maintaining agents, absorption-delaying agents, preservatives. For example, pH regulators include but are not limited to phosphate buffers. Surfactants include but are not limited to cationic, anionic or non-ionic surfactants, such as Tween-80. Ionic strength enhancers include but are not limited to sodium chloride. Preservatives include but are not limited to various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure-maintaining agents include but are not limited to sugars, NaCl and its analogs. Absorption-delaying agents include but are not limited to monostearates and gelatin. Diluents include but are not limited to water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), etc. Preservatives include but are not limited to various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meaning commonly understood by those skilled in the art and can stabilize the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolyzate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (such as 0.9% (w / v) NaCl), glucose solution (such as 5% glucose), solution containing a surfactant (such as 0.01% polysorbate 20), pH buffer solution (such as phosphate buffer solution), Ringer's solution and any combination thereof.

[0383] Treatment method

[0384] Tumor microenvironment. Tumors have a microenvironment that is hostile to the host immune response, which involves a series of mechanisms in malignant cells to protect themselves from immune recognition and elimination. This "hostile tumor microenvironment" includes a variety of immunosuppressive factors, including infiltrating regulatory CD4+ T cells (Tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), immunosuppressive cytokines including IL-10 and TGF-β, and the expression of ligands that target immunosuppressive receptors expressed by activated T cells (CTLA-4 and PD-1). These immunosuppressive mechanisms play a role in maintaining tolerance and suppressing inappropriate immune responses. However, within the tumor microenvironment, these mechanisms prevent effective anti-tumor immune responses. In summary, these immunosuppressive factors can induce significant inactivation or apoptosis of adoptively transferred CAR-modified T cells upon encounter with targeted tumor cells.

[0385] Challenges in tumor immunology. Effective tumor immunity requires tumor antigen recognition and unopposed tumor clearance by immune effector cells. Tumor antigens must contain peptide epitopes presented by the tumor and recognizable by specific cytotoxic T lymphocytes (CTLs). The induced CTLs must expand to a sufficient number and migrate to the tumor site, where they mature into effectors to exert their function, which is enhanced by helper T cells and inhibited by Tregs and inhibitory macrophages.

[0386] Targeted T cell therapy with engineered T lymphocytes. T cell engineering is a pioneering strategy to potentially address many of the previously observed limitations of early immunotherapy approaches.

[0387] Principles of genetic approaches: Cell engineering can be used to redirect T cells to tumor antigens and enhance T cell function. One motivation for genetic T cell modification is to enhance T cell survival and expansion and counteract the potential for T cell death, inactivation, and immunosuppression. Genetic targeting of T cells can also be improved to prevent unwanted destruction of normal tissues.

[0388] Chimeric Antigen Receptor (CAR): By transferring the gene encoding CAR, tumor-specific T cells can be generated. Second-generation CARs include a tumor antigen-binding domain fused to an intracellular signal transduction domain capable of activating T cells, and a co-stimulatory domain designed to enhance T cell efficacy and persistence. Thus, CAR design can coordinate antigen recognition and signal transduction, two functions physiologically carried out by two separate complexes, the TCR heterodimer and the CD3 complex. The extracellular antigen-binding domain of CARs typically derives from murine monoclonal antibodies (mAbs), human monoclonal antibodies, camelid antibodies, or from receptors or their ligands. Thus, antigen recognition is MHC-unrestricted, and thus the same CAR can be applicable to any patient expressing the target antigen. Antigen binding by the CAR triggers phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the intracellular domain, initiating the signal transduction cascade required for cytolytic induction, cytokine secretion, and proliferation. Since the MHC restriction of antigen recognition is bypassed, the function of CAR-targeted T cells is not affected by HLA downregulation or defects in the antigen processing machinery.

[0389] Requirements for T cell expansion and survival: Proliferation of tumor-specific T cells may be required in vivo and ex vivo. T cell proliferation must be accompanied by T cell survival to allow for absolute T cell expansion and persistence. To proliferate in response to antigen, T cells generally require two signals. One is provided by TCR recognition of the antigen peptide / MHC complex displayed on the surface of antigen-presenting cells (APCs). The other is provided by T cell co-stimulatory receptors such as the CD28 or 4-1BB receptor. While the cytolytic activity of T cells generally does not require accompanying co-stimulation, as previously demonstrated, there is a critical need for co-stimulatory signals to maintain the anti-tumor function of adoptively transferred T cells.

[0390] Immune monitoring: Lymphocytes are multifunctional "drugs" that exhibit dynamic effects after infusion. Upon encountering antigen, tumor-specific T cells activate and / or release a variety of proteins that can trigger tumor killing, T cell proliferation, and the recruitment or immunomodulation of other immune cells. Thus, measuring which proteins are secreted from which cells in what quantities and at what time points can provide insights into why a particular patient responds or does not respond, and provide critical feedback for designing more effective trials. These assay systems will allow for direct and meaningful clinical approach comparisons, thus facilitating the design of rational next-generation treatment strategies.

[0391] For treatment, the amount administered is the amount effective to produce the desired effect. The effective amount can be provided in a single or series of administrations. The effective amount can be provided by bolus injection or by continuous perfusion.

[0392] "Effective amount" (or "therapeutically effective amount") is an amount sufficient to effect a beneficial or desired clinical result in treatment. The effective amount can be administered to a subject in one or more doses. In treatment, an effective amount is an amount sufficient to alleviate, ameliorate, stabilize, reverse, or slow the progression of a disease or otherwise reduce the pathological consequences of the disease. The effective amount is typically determined by a physician in view of the circumstances and is within the skill of the art. When determining a suitable dosage to achieve an effective amount, several factors are typically considered. These factors include the age, sex, and weight of the subject, the disorder being treated, the severity of the disorder, and the dosage form and effective concentration of the immune effector cells being administered.

[0393] The term "subject" refers to a mammal, such as a primate mammal, such as a human. In some embodiments, the subject (e.g., a human) has a disease associated with Ly6G6D.

[0394] The subject matter disclosed herein provides a kit for treating or preventing cancer. In certain embodiments, the kit includes a therapeutic or prophylactic composition that includes an effective amount of immune effector cells in unit dose form that comprise a CAR targeting Ly6G6D. In specific embodiments, the cells further express at least one co-stimulatory ligand. In some embodiments, the kit includes a sterile container containing a therapeutic or prophylactic vaccine; such containers can be in the form of a box, ampoule, bottle, vial, tube, bag, pouch, blister, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for containing a medicament.

[0395] The terms "cancer" and "cancerous" refer to or describe a physiological condition in a mammal that is typically characterized by uncontrolled cell growth / proliferation. Cancer aspects include solid tumor cancers and non-solid tumor cancers. Solid cancer tumors include, but are not limited to, colorectal cancer, esophageal cancer, gastric cancer, small intestine cancer, large bowel cancer, or adenocarcinoma (e.g., colorectal adenocarcinoma, gastric adenocarcinoma, or pancreatic adenocarcinoma), or metastatic cancers thereof, such as metastatic colorectal adenocarcinoma. The cancer may be a LY6G6D-positive cancer.

[0396] If desired, the immune effector cells are provided together with instructions for administering the cells to a cancer patient or a subject at risk of developing the same. The instructions typically include information about the use of the composition for treating or preventing cancer. In other embodiments, the instructions include at least one of the following: a description of the therapeutic agent; a dosage regimen and method of administration for treating or preventing cancer or its symptoms; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and / or references. The instructions can be printed directly on the container (when present), or affixed to the container as a label, or provided as a separate sheet, booklet, card, or folded printed matter, in or with the container.

[0397] The implementation scheme of the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments. However, those skilled in the art will understand that the following drawings and embodiments are only used to illustrate the present invention, rather than limiting the scope of the present invention. According to the following detailed description of the drawings and preferred implementation schemes, various objects and advantageous aspects of the present invention will become feasible for those skilled in the art. Description of the Drawings

[0398] Figure 1 It is a graph of the positive rate results of CAR-T cells obtained by measurement in Example 2;

[0399] Figure 2 It is a graph of the in vitro killing results of different CAR-T cells in Example 3;

[0400] Figure 3 It is a graph of the measurement results of the release levels of cytokines after co-incubation of different CAR-T cells (D10, D46, D59, D70, D72) with target cells in Example 4;

[0401] Figure 4 It is a graph of the measurement results of the release levels of cytokines after co-incubation of different CAR-T cells (D76, D79, D85, D103) with target cells in Example 4;

[0402] Figure 5 It is a graph of the anti-tumor results of different CAR-T cells in mice in Example 5. Detailed Implementation Modes

[0403] The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications. For reagents or instruments not specified as the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0404] Example 1: Design and construction of CAR targeting Ly6G6D antigen, and lentivirus packaging

[0405] 1.1 Construction of CAR targeting Ly6G6D antigen

[0406] The CAR structure targeting Ly6G6D includes: signal peptide (Single peptide) – scFv – hinge region (CD8 hinge) – CD8α transmembrane region (CD8TM) – ICOS – 4-1BB – CD3ζ. Among them, the CAR structure and numbering are shown in Table 1.

[0407] Table 1

[0408] Abbreviation Structure D9-CAR Single peptide–D9 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ D10-CAR Single peptide–D10 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ D28-CAR Single peptide–D28 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ D30-CAR Single peptide–D30 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ D32-CAR Single peptide–D32 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ D46-CAR Single peptide–D46 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ D59-CAR Single peptide–D59 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ D61-CAR Single peptide–D61 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ D70-CAR Single peptide–D70 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ D72-CAR Single peptide–D72 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ D76-CAR Single peptide–D76 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ D79-CAR Single peptide–D79 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ D85-CAR Single peptide–D85 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ D103-CAR Single peptide–D103 scFv-CD8hinge+CD8TM-ICOS-4-1BB-CD3ζ

[0409] The scFv targeting Ly6G6D has the amino acid sequence shown in any one of SEQ ID NO: 81 - 90 or SEQ ID NO: 152 - 155; wherein, the scFv of D9-CAR has the amino acid sequence shown in SEQ ID NO: 81; the scFv of D10-CAR has the amino acid sequence shown in SEQ ID NO: 82; the scFv of D28-CAR has the amino acid sequence shown in SEQ ID NO: 83; the scFv of D30-CAR has the amino acid sequence shown in SEQ ID NO: 84; the scFv of D32-CAR has the amino acid sequence shown in SEQ ID NO: 85; the scFv of D46-CAR has the amino acid sequence shown in SEQ ID NO: 86; the scFv of D59-CAR has the amino acid sequence shown in SEQ ID NO: 87; the scFv of D61-CAR has the amino acid sequence shown in SEQ ID NO: 88; the scFv of D70-CAR has the amino acid sequence shown in SEQ ID NO: 89; the scFv of D72-CAR has the amino acid sequence shown in SEQ ID NO: 90; the scFv of D76-CAR has the amino acid sequence shown in SEQ ID NO: 152; the scFv of D79-CAR has the amino acid sequence shown in SEQ ID NO: 153; the scFv of D85-CAR has the amino acid sequence shown in SEQ ID NO: 154; the scFv of D103-CAR has the amino acid sequence shown in SEQ ID NO: 155;

[0410] The CAR structure targeting Ly6G6D contains the nucleotide (DNA) sequence shown in any one of SEQ ID NO: 109 - 118 or SEQ ID NO: 160 - 163; and wherein,

[0411] The signal peptide contains the nucleotide sequence shown in SEQ ID NO: 119:

[0412] ATGGGCGTCAAGGTCCTGTTCGCCCTGATCTGCATCGCCGTCGCCGAGGCC (SEQ ID NO: 119);

[0413] The EcoRI cleavage site contains the nucleotide sequence shown in GAATTC;

[0414] The hinge region contains the nucleotide sequence shown in SEQ ID NO: 120:

[0415] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGC CCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAG GGGGCTGGACTTCGCCTGTGATATCTAC(SEQ ID NO:120);

[0416] The transmembrane domain of the CD8α protein comprises the nucleotide sequence shown in SEQ ID NO:121:

[0417] ATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACC CTTTACTGC(SEQ ID NO:121);

[0418] The ICOS domain comprises the nucleotide sequence shown in SEQ ID NO:122:

[0419] TGTTGGCTTACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATA CATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCTAGACTCACAGATGTGACCCTA(SEQ ID NO:122);

[0420] The 4-1BB domain comprises the nucleotide sequence shown in SEQ ID NO:123:

[0421] AAGCGCGGCCGCAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGT GCAGACCACCCAGGAGGAGGACGGCTGTAGCTGCCGCTTCCCCGAGGAGGAGGAGGG CGGCTGCGAGCTG(SEQ ID NO:123);

[0422] The signal transduction domain of CD3ζ comprises the nucleotide sequence shown in SEQ ID NO:124:

[0423] CGCGTGAAATTTAGCCGCAGCGCCGACGCCCCCGCCTACCAGCAGGGCCAGAACCAGCTGTACAACGAGCTGAACCTGGGCCGCCGCGAGGAGTACGACGTGCTGGACAAGCGCCGgGGCCGCGACCCCGAGATGGGCGGCAAGCCCCAGCGCCGCAAGAACCCCCAGGAGGGCCTGTACAACGAGCTGCAGAAGGACAAGATGGCCGAaGCCTACAGCGAGATCGGCATGAAGGGCGAGCGCCGCCGgGGCAAGGGCCACGACGGCCTGTACCAGGGCCTGAGCACCGCCACCAAGGACACCTACGACGCCCTGCACATGCAGGCCCTGCCCCCCCGC(SEQ ID NO:124);

[0424] The DNA sequence of the above CAR was artificially synthesized and constructed into the shuttle plasmid of the fourth-generation lentivirus.

[0425] 1.2 Lentiviral packaging of CAR vectors targeting Ly6G6D based on different scFvs

[0426] a. Seed 2*10 7 293T cells into a 15-cm dish containing 25 mL of cell growth medium, and place it in an incubator at 37 °C with 5% CO2 overnight for standby;

[0427] b. Configure the lentiviral packaging system according to Table 2 below:

[0428] Table 2

[0429]

[0430]

[0431] Mix solution A and solution B separately, then transfer all of solution A to solution B, mix well, let it stand at room temperature for 15 minutes, and then add 10 mL of lentiviral transfection medium and mix well.

[0432] c. Remove the medium from the 293T cells in the 15-cm dish, add all of the above transfection system to the dish, mix gently, and place the cells in an incubator at 37 °C with 5% CO2 for culture. After 6 hours of transfection, aspirate the transfection system, add 30 mL of lentiviral packaging medium, continue to culture for 48 hours, collect the supernatant of the medium, concentrate or purify it, and store it in a ultra-low temperature refrigerator for standby.

[0433] Example 2: Preparation of CAR-T Cells and Determination of Positive Rate

[0434] Preparation of CAR-T cells: Resuscitate T cells and resuspend them in CTS complete medium (containing serum substitute, IL-7, IL-15). Activate T cells using anti-CD3 / CD28 activation beads. After 3 days of activation, remove the activation beads, and then infect the T cells with lentivirus. Replace the medium 24 hours after infection to remove residual lentivirus. Start detecting the positive rate of CAR by flow cytometry 2 days after infection.

[0435] The experimental results are as Figure 1 shown. As Figure 1 shown in Figure 1 A and

[0436] Example 3: Detection of Killing Effects of Different CAR-T Cells

[0437] Seeding of target cells: Collect target cells in the logarithmic growth phase, centrifuge at 500g for 4 minutes, discard the supernatant, and then resuspend the cells in CTS complete medium to a density of 2x10 6 / mL suspension. Inoculate 100 μL per well on a flat-bottom 96-well plate, and the cell number is 2*10 4 / well.

[0438] Co-incubation of effector cells and target cells: After adjusting the positive rate of each group to be the same using T cells, plate according to the set effector-to-target ratio. In addition, set a separate CAR-T cell group as the negative control for effector cells under each effector-to-target ratio condition; at the same time, set a separate negative control group of target cells; set a group of target cells + lysis buffer group as the positive control for target cell lysis. After 20 - 22 hours of co-incubation, collect the cell culture supernatant and detect the killing ability of CAR-T cells using the LDH method.

[0439] The experimental results are as follows Figure 2 shown. As can be seen from Figure 2 A, D9-CAR-T, D10-CAR-T, D28-CAR-T, D30-CAR-T, D32-CAR-T, D46-CAR-T, D59-CAR-T, D70-CAR-T, and D72-CAR-T can all effectively kill target cells at effector-to-target ratios of 0.5:1, 1:1, and 2:1; among them, D59-CAR-T, D70-CAR-T, and D72-CAR-T have the strongest killing ability against target cells at an effector-to-target ratio of 2.0. The killing efficiency of D59-CAR-T is approximately 62%, the killing efficiency of D70-CAR-T is approximately 70%, and the killing efficiency of D72-CAR-T is approximately 66%. As can be seen from Figure 2 B, D76-CAR-T, D79-CAR-T, D85-CAR-T, and D103-CAR-T can all effectively kill target cells at effector-to-target ratios of 1:8, 1:4, and 1:2, and show a dose-dependent relationship. Among them, D79-CAR-T and D85-CAR-T cells have relatively better effects.

[0440] Example 4: Determination of the release levels of cytokines after co-incubation of CAR-T cells based on different scFvs with target cells

[0441] CAR-T cells and target cells were co-incubated according to the method of Example 3. In the first batch of CAR-T cells, D10-CAR-T, D46-CAR-T, D59-CAR-T, D70-CAR-T, and D72-CAR-T were co-incubated with the NCI-H716-Ly6G6D stable cell line expressing Ly6G6D; in the second batch of CAR-T cells, D76, D79, D85, and D103 CAR-T were co-incubated with the colo320DM-Ly6G6D stable cell line expressing Ly6G6D. The groups incubated with only CAR-T cells without adding target cells and the group incubated with only target cells were used as control groups. Cell supernatants were collected while measuring cell killing and diluted 5-fold for use. The release amounts of six cytokines, including IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ, in the samples were detected according to the BD cytometric beads array human Th1 / Th2 cytokine Kit II kit (product number: 551809).

[0442] The experimental results are as follows Figure 3 and Figure 4 shown. Figure 3As shown, D10, D46, D59, D70, and D72 respectively refer to D10-CAR-T, D46-CAR-T, D59-CAR-T, D70-CAR-T, and D72-CAR-T. The accompanying drawings show the cytokine release levels of each group of CAR-T cells and the control group; Figure 4 Shown are the cytokine release levels of D76, D79, D85, and D103 CAR-T cells and each control group. The abscissa refers to the names of each group, and the ordinate refers to the concentration of the relevant factor, with the unit pg / ml;

[0443] From Figure 3 It can be seen that after co-incubation with target cells, D10-CAR-T, D46-CAR-T, D59-CAR-T, D70-CAR-T, and D72-CAR-T can all effectively release cytokines such as IFN-g, TNF-α, and IL-2, which promote the killing of target cells by CAR-T cells and the proliferation of T cells. Cytokines such as IL-4, IL-6, and IL-10 are also released to varying degrees; the cytokine release levels are different after different CAR-T cells are co-incubated with target cells. Among them, the release levels of IFN-g, TNF-α, and IL-2 of D70-CAR-T cells are the highest, approaching 40000 pg / mL, exceeding 1000 pg / mL, and approaching 4000 pg / mL respectively; the release levels of D59-CAR-T are the second highest, reaching 30000 pg / mL, approaching 1000 pg / mL, and approaching 4000 pg / mL respectively. D10-CAR-T and D46-CAR-T have relatively high levels of IFN-g and TNF-α self-release under the condition of not being co-incubated with target cells. Among them, the self-release level of D46-CAR-T is the highest, with the self-release level of IFN-g being approximately 2000 pg / mL and the self-release level of TNF-α exceeding 100 pg / mL; while D59-CAR-T, D70-CAR-T, and D72-CAR-T have no obvious IFN-g and TNF-α self-release. There is no obvious IL-2 self-release in all groups.

[0444] From Figure 4It can be seen that: (1) After co-incubation with colo320DM-Ly6G6D cells, D76, D79, D85, and D103 CAR-T cells can all effectively release factors such as IL-2, IFN-γ, and TNF-α. Among them, D76 CAR-T cells have the highest release, and D85 CAR-T cells have the lowest release; CAR-T cells not co-incubated with target cells show no obvious release. (2) After co-incubation with target cells, all 4 types of CAR-T cells significantly release IL-4, and the level in the D103 group is the highest. (3) Only D76 CAR-T cells significantly release IL-6 factor after co-incubation with target cells. (4) The background release level of IL-10 in D76 CAR-T cells is relatively low, but its release amount increases significantly after co-incubation with target cells; D79, D85, and D103 CAR-T cells all have a relatively high background release of IL-10, and after co-incubation with target cells, the release level further increases.

[0445] Example 5: Evaluation of the anti-tumor effect of CAR-T cells based on different scFvs in mice

[0446] Preparation of CAR-T cells:

[0447] Resuscitate T cells, resuspend them in CTS complete medium, and activate the T cells using anti-CD3 / CD28 activation beads. After 3 days of activation, remove the activation beads, and then perform lentiviral infection on the T cells. Replace the medium 24 hours after infection to remove residual lentivirus. Start detecting the CAR positive rate using a flow cytometer 2 days after infection. Before cryopreserving the cells, adjust the CAR positive rate of each group of CAR-T cells to be the same using uninfected T cells. After resuspending the cells in cryopreservation solution, aliquot and cryopreserve them, transfer them to a programmable freezer and store them in an ultra-low temperature freezer. Transfer them to liquid nitrogen for storage 1 day later.

[0448] Resuscitation and administration: After resuscitating the cells, resuspend them in pre-cooled PBS before administration. In the first batch of experiments, the administration dose for the D10, D46, D59, D70, and D72 CAR-T cell groups was set at 3.6×10 6 CAR+ cells per mouse; in the second batch of experiments, the administration dose for the D79, D85, and D103 CAR-T cell groups was set at 2.0×10 6 CAR+ cells per mouse. The administration volume for each mouse was 200 μL. When the tumor volume was about 150 cubic millimeters, CAR-T cells were infused through the tail vein, and the tumor volume was measured twice a week after infusion.

[0449] Data processing:

[0450] Tumor volume (cubic millimeters) = major axis (millimeters) × minor axis (millimeters) × minor axis (millimeters) × 0.5

[0451] Tumor inhibition rate = [1 - average tumor volume of this group / average tumor volume of T cell group] * 100%.

[0452] The experimental results are as Figure 5 shown, Figure 5 As shown in A, after data processing of the results, it can be seen that the CAR-T cells of the five groups of D10-CAR, D46-CAR, D59-CAR, D70-CAR, and D72-CAR all showed tumor inhibition effects compared with T cells; the CAR-T cells of different groups had different inhibitory effects on NCI-H508-Ly6G6D tumors. Among them, the inhibitory effect of D46-CAR-T was 30.5%; the inhibitory effect of D10-CAR-T was 59%; 21 days after the CAR-T infusion, the inhibitory efficiencies of D59-CAR-T, D70-CAR-T, and D72-CAR-T on tumors reached 95.4%, 96%, and 92.9% respectively.

[0453] In another batch of animal experiments, after data processing, it was obtained that Figure 5 as shown in B, it can be seen that 21 days after the infusion, calculated by the average tumor volume, the CAR-T cells of the groups of D76-CAR, D79-CAR, D85-CAR, and D103-CAR all showed tumor inhibition effects compared with T cells. The CAR-T cells of different groups had different inhibitory effects on tumors. Among them, the inhibitory effect of D76 CAR-T was 88.34%, the inhibitory effect of D79 CAR-T was 94.46%, the inhibitory effect of D85 CAR-T was 97.28%, the inhibitory effect of D103CAR-T was 33.68%, and the inhibitory effect of D85 CAR-T cells was relatively good.

[0454] In the description of this specification, the description with reference to terms such as "one embodiment", "some embodiments", "one example", "some examples", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0455] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A chimeric antigen receptor targeting Ly6G6D, characterized in that The chimeric antigen receptor comprises an extracellular antigen binding domain that binds to Ly6G6D, a transmembrane domain and an intracellular domain, wherein the extracellular antigen binding domain comprises an antibody or antigen binding fragment that targets and binds to Ly6G6D, wherein the antibody or antigen binding fragment comprises a heavy chain variable region, and the heavy chain variable region comprises complementary determining regions: H-CDR1, H-CDR2 and H-CDR3; wherein, H-CDR1 has the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 127 or SEQ ID NO: 138; and H-CDR2 has the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO:14, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:29, SEQ ID NO:35, SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:51 or SEQ ID NO:139; and H-CDR3 has the amino acid sequence shown in SEQ ID NO:3, SEQ ID NO:9, SEQ ID NO:15, SEQ ID NO:20, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:36, SEQ ID NO:42, SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:128, SEQ ID NO:132 or SEQ ID NO:140; And the antibody or antigen-binding fragment comprises a light chain variable region, and the light chain variable region comprises complementarity determining regions: L-CDR1, L-CDR2 and L-CDR3; wherein, L-CDR1 has the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:53, SEQ ID NO:129, SEQ ID NO:135 or SEQ ID NO:141; and L-CDR2 has the amino acid sequence shown in SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:26, SEQ ID NO:32, SEQ ID NO:38, SEQ ID NO:44, SEQ ID NO:54, SEQ ID NO:130, SEQ ID NO:133, SEQ ID NO:136 or SEQ ID NO:142; and L-CDR3 has the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:131, SEQ ID NO:134, SEQ ID NO:137 or SEQ ID NO:

143.

2. The chimeric antigen receptor according to claim 1, characterized in that The complementary determining regions of the heavy chain variable region: H-CDR1, H-CDR2 and H-CDR3 are selected from the following combinations: (1) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 1, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 3; or (2) H-CDR1 has the amino acid sequence shown in SEQ ID NO:7, H-CDR2 has the amino acid sequence shown in SEQ ID NO:8, and H-CDR3 has the amino acid sequence shown in SEQ ID NO:9; or (3) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 13, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 14, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 15; or (4) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 18, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 19, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 20; or (5) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:22, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:23, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:24; or (6) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:28, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:30; or (7) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:34, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:35, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:36; or (8) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:40, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:41, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:42; or (9) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:46, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:47, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:48; or (10) H-CDR1 has the amino acid sequence shown in SEQ ID NO:50, H-CDR2 has the amino acid sequence shown in SEQ ID NO:51, and H-CDR3 has the amino acid sequence shown in SEQ ID NO:52; or (11) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 127, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 128; or (12) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:18, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:132; or (13) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:18, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:128; or (14) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 138, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 139, and H-CDR3 has the amino acid sequence shown in SEQ ID NO:

140.

3. The chimeric antigen receptor according to claim 1, characterized in that The complementary determining regions of the light chain variable region: L-CDR1, L-CDR2 and L-CDR3 are selected from the following combinations: (1) L-CDR1 has the amino acid sequence shown in SEQ ID NO:4, L-CDR2 has the amino acid sequence shown in SEQ ID NO:5, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:6; or (2) L-CDR1 has the amino acid sequence shown in SEQ ID NO: 10, L-CDR2 has the amino acid sequence shown in SEQ ID NO: 11, and L-CDR3 has the amino acid sequence shown in SEQ ID NO: 12; or (3) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:4, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:16, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:17; or (4) L-CDR1 has the amino acid sequence shown in SEQ ID NO:4, L-CDR2 has the amino acid sequence shown in SEQ ID NO:16, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:21; or (5) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:25, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:26, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:27; or (6) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:31, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:32, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:33; or (7) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:39; or (8) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:43, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:44, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:45; or (9) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:49; or (10) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:53, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:54, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:45; or (11) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 129, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 130, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 131; or (12) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:133, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:134; or (13) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 135, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 136, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 137; or (14) L-CDR1 has the amino acid sequence shown in SEQ ID NO: 141, L-CDR2 has the amino acid sequence shown in SEQ ID NO: 142, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:

143.

4. The chimeric antigen receptor according to any one of claims 1 to 3, characterized in that The antibody or antigen-binding fragment comprises a heavy chain variable region framework sequence and a light chain variable region framework sequence, wherein the framework sequence is derived from a murine antibody, a human antibody, a primate antibody or a mutant thereof.

5. The chimeric antigen receptor according to any one of claims 1 to 4, characterized in that The heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs:55-64 or SEQ ID NOs:144-147.

6. The chimeric antigen receptor according to any one of claims 1 to 5, characterized in that The light chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs: 65-76 or SEQ ID NOs: 148-151.

7. The chimeric antigen receptor according to any one of claims 1 to 6, characterized in that The heavy chain variable region and the light chain variable region of the extracellular antigen binding domain are connected by a linker.

8. The chimeric antigen receptor according to any one of claims 1 to 7, characterized in that The linker comprises an amino acid sequence shown in any one of SEQ ID NOs: 77-80.

9. The chimeric antigen receptor according to any one of claims 1 to 8, characterized in that The extracellular antigen binding domain is a single chain antibody.

10. The chimeric antigen receptor according to any one of claims 1 to 9, characterized in that The extracellular antigen binding domain comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs: 81-90 or SEQ ID NOs: 152-155.

11. The chimeric antigen receptor according to any one of claims 1 to 10, characterized in that The transmembrane domain comprises a transmembrane domain derived from the α, β or ζ chain of the T cell receptor, CD28, CD3ζ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, 4-1BB, CD154 or a combination thereof.

12. The chimeric antigen receptor according to any one of claims 1 to 11, characterized in that The transmembrane domain comprises a transmembrane domain derived from CD8α protein.

13. The chimeric antigen receptor according to any one of claims 1 to 12, characterized in that The transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:

92.

14. The chimeric antigen receptor according to any one of claims 1 to 13, characterized in that The chimeric antigen receptor also comprises a hinge region.

15. The chimeric antigen receptor according to any one of claims 1 to 14, characterized in that The hinge region connects the extracellular antigen binding structure and the transmembrane domain.

16. The chimeric antigen receptor according to any one of claims 1 to 15, characterized in that The hinge region comprises the amino acid sequence shown in SEQ ID NO:

93.

17. The chimeric antigen receptor according to any one of claims 1 to 16, characterized in that The intracellular domain of the chimeric antigen receptor comprises a signaling domain derived from CD3ζ.

18. The chimeric antigen receptor according to any one of claims 1 to 17, characterized in that The intracellular domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:

94.

19. The chimeric antigen receptor according to any one of claims 1 to 18, characterized in that The intracellular domain also includes at least one co-stimulatory domain.

20. The chimeric antigen receptor according to any one of claims 1 to 19, characterized in that The costimulatory domain comprises a costimulatory domain derived from the following proteins: CD28, 4-1BB, OX-40, ICOS, or a combination thereof.

21. The chimeric antigen receptor according to any one of claims 1 to 20, characterized in that The co-stimulatory domain comprises a domain derived from 4-1BB.

22. The chimeric antigen receptor according to claim 21, characterized in that The co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:

95.

23. The chimeric antigen receptor according to any one of claims 1 to 20, characterized in that The co-stimulatory domain comprises a domain derived from ICOS.

24. The chimeric antigen receptor according to claim 23, characterized in that The co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:

96.

25. The chimeric antigen receptor according to any one of claims 1 to 20, characterized in that The co-stimulatory domain comprises domains derived from a combination of 4-1BB and ICOS.

26. The chimeric antigen receptor according to claim 25, characterized in that The co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:

97.

27. The chimeric antigen receptor according to any one of claims 1 to 26, characterized in that The intracellular domain comprises the amino acid sequence shown in SEQ ID NO:

98.

28. The chimeric antigen receptor according to any one of claims 1 to 27, characterized in that The chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NOs: 99-108 or SEQ ID NOs: 156-159.

29. The chimeric antigen receptor according to any one of claims 1 to 28, characterized in that The chimeric antigen receptor further comprises a signal peptide covalently linked to the N-terminus of the extracellular antigen binding domain.

30. The chimeric antigen receptor according to any one of claims 1 to 29, characterized in that The signal peptide comprises the amino acid sequence shown in SEQ ID NO:

91.

31. An isolated nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding the chimeric antigen receptor according to any one of claims 1-30.

32. The nucleic acid molecule according to claim 31, characterized in that The nucleic acid molecule comprises a nucleotide sequence shown in any one of SEQ ID NOs: 109-118 or SEQ ID NOs: 160-163.

33. An expression vector, characterized in that The expression vector carries the nucleic acid molecule of claim 31 or 32.

34. The expression vector according to claim 33, characterized in that The expression vector is a γ-retroviral vector or a lentiviral vector.

35. An immune effector cell, characterized in that The immune cell comprises the chimeric antigen receptor according to any one of claims 1 to 30, the nucleic acid molecule according to claim 31 or 32, or the expression vector according to claim 33 or 34.

36. The immune effector cell according to claim 35, characterized in that The immune effector cells are selected from T cells, natural killer cells, cytotoxic T lymphocytes, regulatory T cells, human embryonic stem cells, lymphocyte progenitor cells, T cell precursor cells, or pluripotent stem cells from which lymphocytes can be differentiated, macrophages, B cells, and immune cells with cell killing ability derived from induced pluripotent stem cells.

37. A method for preparing immune effector cells, characterized in that: The method comprises introducing the expression vector of claim 33 or 34 into immune effector cells.

38. A composition, characterized in that The composition comprises the immune effector cells of claim 35 or 36 and a pharmaceutically acceptable carrier.

39. Use of the chimeric antigen receptor described in any one of claims 1-30, the nucleic acid molecule described in claim 31 or 32, the expression vector described in claim 33 or 34, the immune effector cell described in claim 35 or 36, or the composition described in claim 38 in the preparation of a medicament for treating a disease or condition associated with the expression of Ly6G6D; the disease or condition associated with the expression of Ly6G6D is colorectal cancer.

40. A drug kit for treating cancer, characterized in that: The kit comprises the immune effector cells of claim 35 or 36.

41. The kit according to claim 40, characterized in that The kit also includes instructions for using the immune response cells to treat a subject with cancer.

42. The kit according to claim 41, characterized in that The cancer is colorectal cancer.

Images