Chimeric antigen receptor targeting ly6g6d and use thereof

Abstract

Provided are a chimeric antigen receptor targeting Ly6G6D and the use thereof. Specifically, provided is a CAR that can specifically bind to Ly6G6D, which contains an Ly6G6D binding domain, a transmembrane domain, a co-stimulatory domain and an intracellular signal transduction domain. Further provided is the use of the CAR in the treatment of diseases or conditions related to the expression of Ly6G6D.

Description

A chimeric antigen receptor targeting Ly6G6D and its use Technical Field

[0001] The present invention 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 the 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 artificial or synthetic receptors for antigens, which are called chimeric antigen receptors (CARs), which are specific for the selected antigens. Targeted T cell therapy using CARs has achieved clinical success in the treatment of 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. It encodes a 133-amino acid protein with a molecular weight of approximately 13.7 kDa. Like most family members, Ly6G6D is attached to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor. Numerous studies have demonstrated that members of the LAG6 family are crucial for regulating immune, nervous, and complement functions. In cancer, many LAG6 family members play a key role in numerous cancer types, including gastric, cervical, breast, ovarian, lung, and bladder cancers. Therefore, the LAG6 gene family has the potential to play a significant role in clinical practice, not only as a biomarker for disease prognosis but also as a key target for new drug development. In-depth understanding of their biological functions will be crucial for elucidating the functions of the LAG6 family, analyzing protein interactions within their structure, exploring disease pathogenesis, and discovering new therapeutic targets.

[0004] Colorectal cancer can be divided into mismatch repair-deficient (dMMR) and mismatch repair-proficient (pMMR) types. The dMMR type mainly includes microsatellite instability-high (MSI-H) type, and the pMMR type mainly includes microsatellite instability-low (MSI-L) and microsatellite stable (MSS) types. For MSI-H type metastatic colorectal cancer, immune checkpoint inhibitors have a good therapeutic effect; however, for MSI-L and MSS types, which account for approximately 85% of all metastatic colorectal cancer patients, immune checkpoint inhibitor response rates are low and patient survival is low, and more effective and safe treatment drugs are urgently needed. Studies have shown that Ly6G6D is highly expressed in pMMR type advanced colorectal cancer tissues 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 use of the immune effector cells for treating cancer.

[0006] Specifically, on the one hand, the present invention provides a chimeric antigen receptor targeting Ly6G6D, wherein 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, wherein 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 of 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] The antibody or antigen-binding fragment comprises a light chain variable region, wherein 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] In some embodiments, the complementarity determining regions of the heavy chain variable region: H-CDR1, H-CDR2 and H-CDR3 are selected from a combination of any of the following groups:

[0015] (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

[0016] (2) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 7, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 8, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 9; or

[0017] (3) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 13, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 14, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 15; or

[0018] (4) 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: 19, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 20; or

[0019] (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

[0020] (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

[0021] (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

[0022] (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

[0023] (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

[0024] (10) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 50, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 51, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 52; or

[0025] (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

[0026] (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

[0027] (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

[0028] (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.

[0029] In some embodiments, the complementarity determining regions of the light chain variable region: L-CDR1, L-CDR2 and L-CDR3 are selected from a combination of any of the following groups:

[0030] (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

[0031] (2) L-CDR1 has the amino acid sequence set forth in SEQ ID NO: 10, L-CDR2 has the amino acid sequence set forth in SEQ ID NO: 11, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO: 12; or

[0032] (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

[0033] (4) 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: 21; or

[0034] (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

[0035] (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

[0036] (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

[0037] (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

[0038] (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

[0039] (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

[0040] (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

[0041] (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

[0042] (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

[0043] (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.

[0044] 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 a combination of any of the following groups:

[0045] (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; and

[0046] 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

[0047] (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; and

[0048] 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

[0049] (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; and

[0050] 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: 17; or

[0051] (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; and

[0052] 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

[0053] (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; and

[0054] 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

[0055] (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; and

[0056] 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

[0057] (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; and

[0058] 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

[0059] (8) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 40, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 41, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 42; and

[0060] 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

[0061] (9) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 46, H-CDR2 has the amino acid sequence shown in SEQ ID NO: 47, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 48; and

[0062] 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

[0063] (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; and

[0064] L-CDR1 has the amino acid sequence shown in SEQ ID NO: 53, L-CDR2 has the amino acid sequence shown in SEQ ID NO: 54, and L-CDR3 has the amino acid sequence shown in SEQ ID NO: 45.

[0065] (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; and

[0066] L-CDR1 has the amino acid sequence shown in SEQ ID NO: 129, L-CDR2 has the amino acid sequence shown in SEQ ID NO: 130, and L-CDR3 has the amino acid sequence shown in SEQ ID NO: 131.

[0067] (12) 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: 29, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 132; and

[0068] L-CDR1 has the amino acid sequence shown in SEQ ID NO:37, L-CDR2 has the amino acid sequence shown in SEQ ID NO:133, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:134.

[0069] (13) 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: 29, and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 128; and

[0070] L-CDR1 has the amino acid sequence shown in SEQ ID NO: 135, L-CDR2 has the amino acid sequence shown in SEQ ID NO: 136, and L-CDR3 has the amino acid sequence shown in SEQ ID NO: 137.

[0071] (14) H-CDR1 has the amino acid sequence set forth in SEQ ID NO: 138, H-CDR2 has the amino acid sequence set forth in SEQ ID NO: 139, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO: 140; and

[0072] 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.

[0073] 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, wherein the framework sequence is derived from a murine antibody, a human antibody, a primate antibody, or a mutant thereof.

[0074] 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 NOs: 55-64 or SEQ ID NOs: 144-147.

[0075] 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 NOs: 65-76 or SEQ ID NOs: 148-151.

[0076] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO:55; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:65.

[0077] 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.

[0078] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO:56; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:66.

[0079] 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:56; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:66.

[0080] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO:57; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:67.

[0081] 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:57; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:67.

[0082] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO:58; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:68.

[0083] 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.

[0084] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO:59; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:69.

[0085] 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.

[0086] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO:60; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:70.

[0087] 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.

[0088] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO:61; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:71.

[0089] 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:61; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:71.

[0090] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO: 62; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 72 or 73.

[0091] 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: 62; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 72 or 73.

[0092] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO:63; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO:74.

[0093] 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.

[0094] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO: 64; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 75 or 76.

[0095] 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.

[0096] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO: 144; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 148.

[0097] 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.

[0098] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO: 145; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 149.

[0099] 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: 145; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 149.

[0100] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO: 146; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 150.

[0101] 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: 146; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 150.

[0102] In some embodiments, the heavy chain variable region of the antibody or antigen-binding fragment comprises an amino acid sequence that is 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% identical to SEQ ID NO: 147; and the light chain variable region comprises an amino acid sequence that is 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% identical to SEQ ID NO: 151.

[0103] 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: 147; and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 151.

[0104] In some embodiments, the heavy chain variable region and the light chain variable region of the extracellular antigen binding domain are connected by a linker.

[0105] The linker is a peptide connector used to connect the heavy chain variable region and the light chain variable region of an antibody. It can also be a peptide connector modified by researchers. An example of the amino acid sequence of the linker is: (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.

[0106] In some embodiments, the extracellular antigen binding domain is a single chain antibody (scFv).

[0107] The extracellular antigen binding domain comprises a single-chain antibody (scFv) represented 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 to any one of SEQ ID NOs: 81-90 or 152-155.

[0108] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from 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.

[0109] In some embodiments, the transmembrane domain comprises a transmembrane domain derived from a CD8α protein.

[0110] In some embodiments, the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:92.

[0111] In some embodiments, the chimeric antigen receptor further comprises a hinge region.

[0112] The hinge region generally refers to the connecting region between the antigen-binding region and the immune cell Fc receptor (FcR)-binding region.

[0113] In some embodiments, the hinge region connects the extracellular antigen-binding structure and the transmembrane domain.

[0114] In some embodiments, the hinge region comprises the amino acid sequence shown in SEQ ID NO:93.

[0115] In some embodiments, the intracellular domain of the chimeric antigen receptor comprises a signaling domain derived from CD3ζ.

[0116] In some embodiments, the intracellular domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:94.

[0117] In some embodiments, the intracellular domain further comprises at least one costimulatory domain.

[0118] In some embodiments, the costimulatory domain comprises a costimulatory domain derived from CD28, 4-1BB, OX-40, ICOS, or a combination thereof.

[0119] In some embodiments, the costimulatory domain comprises a domain derived from 4-1BB.

[0120] In some embodiments, the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO:95.

[0121] In some embodiments, the costimulatory domain comprises a domain derived from ICOS.

[0122] In some embodiments, the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO:96.

[0123] In some embodiments, the costimulatory domain comprises a domain derived from a combination of 4-1BB and ICOS.

[0124] In some embodiments, the costimulatory domain comprises the amino acid sequence shown in SEQ ID NO:97.

[0125] In some embodiments, the intracellular domain comprises the amino acid sequence shown in SEQ ID NO:98.

[0126] 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.

[0127] Specifically, the chimeric antigen receptor D9-CAR comprises the amino acid sequence shown in SEQ ID NO:99.

[0128] Specifically, the chimeric antigen receptor D10-CAR comprises the amino acid sequence shown in SEQ ID NO:100.

[0129] Specifically, the chimeric antigen receptor D28-CAR comprises the amino acid sequence shown in SEQ ID NO:101.

[0130] Specifically, the chimeric antigen receptor D30-CAR comprises the amino acid sequence shown in SEQ ID NO:102.

[0131] Specifically, the chimeric antigen receptor D32-CAR comprises the amino acid sequence shown in SEQ ID NO:103.

[0132] Specifically, the chimeric antigen receptor D46-CAR comprises the amino acid sequence shown in SEQ ID NO:104.

[0133] Specifically, the chimeric antigen receptor D59-CAR comprises the amino acid sequence shown in SEQ ID NO:105.

[0134] Specifically, the chimeric antigen receptor D61-CAR comprises the amino acid sequence shown in SEQ ID NO:106.

[0135] Specifically, the chimeric antigen receptor D70-CAR comprises the amino acid sequence shown in SEQ ID NO:107.

[0136] Specifically, the chimeric antigen receptor D72-CAR comprises the amino acid sequence shown in SEQ ID NO:108.

[0137] Specifically, the chimeric antigen receptor D76-CAR comprises the amino acid sequence shown in SEQ ID NO:156.

[0138] Specifically, the chimeric antigen receptor D79-CAR comprises the amino acid sequence shown in SEQ ID NO:157.

[0139] Specifically, the chimeric antigen receptor D85-CAR comprises the amino acid sequence shown in SEQ ID NO:158.

[0140] Specifically, the chimeric antigen receptor D103-CAR comprises the amino acid sequence shown in SEQ ID NO:159.

[0141] In some embodiments, the chimeric antigen receptor further comprises an enzyme cleavage site.

[0142] Specifically, the restriction enzyme cutting site is selected from the restriction enzyme cutting sites such as BamHI, SpeI, SalI, KpnI, XhoI, SphI, XbaI, EcoRI and the like.

[0143] Specifically, the enzyme 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 enzyme cleavage site is: EF.

[0144] Specifically, the chimeric antigen receptor D9-CAR comprising an enzyme cleavage site comprises the amino acid sequence shown in SEQ ID NO: 125:

[0145] EVHLVESGGGVVQPGRSLRLSCAASGFTFSNYAMHWVRQAPGKGLEWVAVISYDGSKKYYADSVKGRFTISRDDSKNTLYLQMNSLRAEDTAVYYCAKPAGSTSGFDYWGQGTLVTVSSGGG GSGGGGSGGGGSQSALTQPASVSGSPGQSITISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVSNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYTSSSTLVVFGGGTKLTVL TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCCWLTKKKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 125), wherein the bold portion is the amino acid sequence of the EcoRI restriction site;

[0146] The chimeric antigen receptors of the present invention may all comprise an amino acid sequence of an enzyme cleavage site as shown in the above specific examples, wherein the enzyme cleavage site is located at the N-terminus of the hinge region and at the C-terminus of the extracellular antigen binding structure.

[0147] In some embodiments, the chimeric antigen receptor further comprises a signal peptide covalently linked to the N-terminus of the extracellular antigen binding domain.

[0148] In some embodiments, the signal peptide comprises the amino acid sequence shown in SEQ ID NO:91.

[0149] MGVKVLFALICIAVAEA (SEQ ID NO:91).

[0150] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the chimeric antigen receptor of the present invention.

[0151] An isolated nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment.

[0152] In some embodiments, the nucleic acid molecule is intended to include DNA molecules and RNA molecules. The nucleic acid molecule can be single-stranded or double-stranded and can be a cDNA.

[0153] In some embodiments, the nucleic acid molecule comprises the nucleotide sequence shown in any one of SEQ ID NOs: 109-118 or SEQ ID NOs: 160-163.

[0154] Specifically, the chimeric antigen receptor D9-CAR comprises the nucleotide sequence shown in SEQ ID NO:109.

[0155] Specifically, the chimeric antigen receptor D10-CAR comprises the nucleotide sequence shown in SEQ ID NO:110.

[0156] Specifically, the chimeric antigen receptor D28-CAR comprises the nucleotide sequence shown in SEQ ID NO:111.

[0157] Specifically, the chimeric antigen receptor D30-CAR comprises the nucleotide sequence shown in SEQ ID NO:112.

[0158] Specifically, the chimeric antigen receptor D32-CAR comprises the nucleotide sequence shown in SEQ ID NO:113.

[0159] Specifically, the chimeric antigen receptor D46-CAR comprises the nucleotide sequence shown in SEQ ID NO:114.

[0160] Specifically, the chimeric antigen receptor D59-CAR comprises the nucleotide sequence shown in SEQ ID NO:115.

[0161] Specifically, the chimeric antigen receptor D61-CAR comprises the nucleotide sequence shown in SEQ ID NO:116.

[0162] Specifically, the chimeric antigen receptor D70-CAR comprises the nucleotide sequence shown in SEQ ID NO:117.

[0163] Specifically, the chimeric antigen receptor D72-CAR comprises the nucleotide sequence shown in SEQ ID NO:118.

[0164] Specifically, the chimeric antigen receptor D76-CAR comprises the nucleotide sequence shown in SEQ ID NO:160.

[0165] Specifically, the chimeric antigen receptor D79-CAR comprises the nucleotide sequence shown in SEQ ID NO:161.

[0166] Specifically, the chimeric antigen receptor D85-CAR comprises the nucleotide sequence shown in SEQ ID NO:162.

[0167] Specifically, the chimeric antigen receptor D103-CAR comprises the nucleotide sequence shown in SEQ ID NO:163.

[0168] In some embodiments, the nucleotide sequence further comprises an enzyme cleavage site.

[0169] Specifically, the restriction enzyme cutting site is selected from: BamHI, SpeI, SalI, KpnI, XhoI, SphI, XbaI, EcoRI and other restriction enzyme cutting sites.

[0170] In some embodiments, the nucleotide sequence of the EcoRI cleavage site is: GAATTC.

[0171] Specifically, the chimeric antigen receptor D9-CAR comprising an enzyme cleavage site comprises the nucleotide sequence shown in SEQ ID NO: 126:

[0172] GAGGTGCATCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGCTATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATCTCATATGATGGAAGTAAAAAATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACGATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAACCCGCTGGTAGTACCAGTGGGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGCGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCGATCACCATCTCCTGCACTGGAACCAGCAGTGACGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAATCGGCCCTCAGGGGTTTCTAATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCAGCTCATATACAAGCAGCAGCACTCTCGTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTA (SEQ ID NO: 126); wherein the bold portion is the nucleotide sequence of the EcoRI restriction site;

[0173] The chimeric antigen receptors of the present invention may all comprise a nucleotide sequence of an enzyme cleavage site as shown in the above specific examples, wherein the enzyme 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.

[0174] In yet another aspect, the present invention provides a nucleic acid construct comprising 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, wherein the one or more control sequences direct the production of the antibody or antigen-binding fragment thereof of the present invention in a suitable host cell, and the one or more control sequences are selected from the group consisting of: a promoter, an enhancer, a stop signal, a signal peptide, a leader sequence, a transcription terminator, and any group thereof.

[0175] In some embodiments, the signal peptide of the present invention has the nucleotide sequence shown in SEQ ID NO:119.

[0176] In yet another aspect, the present invention provides an expression vector carrying the nucleic acid molecule described above.

[0177] In some embodiments, the expression vector is a γ-retroviral vector or a lentiviral vector.

[0178] In yet another aspect, the present invention provides an immune effector cell, comprising the chimeric antigen receptor of the present invention, the nucleic acid molecule of the present invention, or the expression vector of the present invention.

[0179] 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, 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.

[0180] In yet another aspect, the present invention provides a method for preparing immune effector cells, comprising introducing the expression vector of the present invention into immune effector cells.

[0181] In yet another aspect, the present invention provides a composition comprising the immune effector cells of the present invention and a pharmaceutically acceptable carrier.

[0182] In yet another aspect, the present invention provides a use of the chimeric antigen receptor of the present invention, the nucleic acid molecule of the present invention, the expression vector of the present invention, the immune effector cell of the present invention, or the composition of the present invention 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, 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.

[0183] In yet another aspect, the present invention provides a drug kit for treating cancer, comprising the immune effector cells of the present invention.

[0184] In some embodiments, the kits described herein further comprise instructions for using the immune effector cells to treat a subject with cancer.

[0185] In some embodiments, the cancer described herein 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.

[0186] Definitions and General Terms

[0187] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology, and the like used herein are conventional procedures widely used in the relevant fields. To facilitate a better understanding of the present invention, definitions and explanations of relevant terms are provided below.

[0188] When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

[0189] The terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0190] Chimeric antigen receptor

[0191] The term "chimeric antigen receptor" (CAR) is an engineered receptor that transfers or confers the specificity of interest to immune effector cells. CAR can be used to transfer the specificity of a monoclonal antibody to T cells by promoting the transfer of its coding sequence via a retroviral vector.

[0192] There are three generations of CAR. "First generation" CAR is generally composed of an extracellular antigen binding domain (such as a single-chain variable fragment (scFv)) fused with a transmembrane domain and then fused with the cytoplasm / intracellular domain of the T cell receptor chain. "First generation" CAR generally has an intracellular domain from the CD3ζ chain, which is the main transmitter of signal transduction from endogenous TCR. "First generation" CAR can provide de novo antigen recognition and lead to the activation of CD4+ and CD8+ T cells through its CD3ζ chain signaling domain in a single fusion molecule, without relying on HLA-mediated antigen presentation. "Second generation" CAR adds intracellular domains from various costimulatory molecules (such as CD28, 4-1BB, ICOS, OX40) to the cytoplasmic region of CAR to provide additional signals to T cells. "Second generation" CAR includes those CARs that provide both costimulation (such as CD28 or 4-1BB) and activation (CD3ζ). Preclinical studies have shown that "second generation" CAR can improve the anti-tumor activity of T cells. For example, in patients with chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL), clinical trials targeting CD19 molecules have demonstrated the strong efficacy of "second-generation" CAR-modified T cells."Third-generation" CARs include those that provide multiple costimulations (such as CD28 and 4-1BB) and activation (CD3ζ).

[0193] According to the subject matter disclosed in the present invention, CAR includes 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 scFv. In certain embodiments, the extracellular antigen binding domain is an optionally cross-linked Fab. In certain embodiments, the extracellular binding domain is F(ab)2. In certain embodiments, any of the foregoing molecules may be included in a fusion protein with a heterologous sequence to form an extracellular antigen binding domain. In a specific non-limiting embodiment, the extracellular antigen binding domain includes a human scFv that specifically binds to human Ly6G6D.

[0194] In certain non-limiting embodiments, the extracellular antigen binding domain of CAR has high binding specificity and high binding affinity for Ly6G6D. For example, in these embodiments, the extracellular antigen binding domain of CAR (e.g., a human scFv or its analogue) is about 1 x 10 -9 It binds to Ly6G6D with a dissociation constant (KD) of M or lower.

[0195] The term "transmembrane domain" generally refers to the domain in CAR that passes through the cell membrane and is connected to the intracellular signal transduction domain to play a role in transmitting signals.

[0196] The term "costimulatory domain" generally refers to an intracellular domain that can provide an immune co-stimulatory molecule, which is a cell surface molecule required for the effective response of lymphocytes to antigens. The co-stimulatory domain may include immunoglobulin superfamily (IgSF) members, such as CD28, ICOS co-stimulatory domains, and may also include tumor necrosis factor receptor superfamily (TNFRSF) members, co-stimulatory domains, such as CD27, OX40, 4-1BB co-stimulatory domains.

[0197] CDR domains are defined using the Kabat system (Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, US Department of Health and Human Services, NIH Publication No. 91-3242). The subject matter disclosed herein further provides extracellular antigen-binding domains (e.g., scFv) comprising conservative modifications of the antibody sequences disclosed herein. For example, without limitation, the extracellular antigen-binding domains (e.g., scFv) of the subject matter disclosed herein comprise: 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, wherein the extracellular antigen-binding domain retains the desired functional properties.

[0198] The term "antibody" generally refers to a polypeptide molecule that can specifically recognize and / or neutralize a specific antigen. For example, an antibody may comprise an immunoglobulin composed of at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and includes any antigen-binding domain comprising the same. "Antibodies" include monoclonal antibodies, antibody fragments, or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single-domain antibodies (e.g., dAbs), single-chain antibodies (e.g., scFvs), and antibody fragments that bind to antigens (e.g., Fab, Fab', and (Fab)2 fragments). "Antibodies" also include all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, non-glycosylated antibodies, and any antigen-binding antibody fragments and derivatives described herein. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region. Each light chain may 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 amino terminus to 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 antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0199] 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). Including, for example, antibodies and antigen-binding fragments thereof, single-chain scFv antibodies, various fusions and conjugates based on scFv construction, such as scFv-Fc antibodies, immunoconjugates, antibody drug conjugates (ADCs), multi- / bispecific antibodies, chimeric antigen receptors (CARs).

[0200] The term "single-chain antibody" (scFv) may be an antibody composed of the heavy chain variable region and the light chain variable region connected by a linker peptide.

[0201] In certain embodiments, no more than one, no more than two, no more than three, no more than four, no more than five residues are altered within a particular sequence or CDR region.Exemplary conservative amino acid substitutions are shown in Table 1.

[0202] Table 1

[0203] The terms "conservative sequence modification" and "conservative sequence substitution" are interchangeable and refer to amino acid modifications that do not significantly affect or change the binding characteristics of the CAR (e.g., extracellular antigen binding domain) disclosed in the present invention including the amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the scFv 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 the physicochemical properties of amino acids such as charge and polarity. Conservative amino acid substitutions are those in which amino acid residues are replaced by amino acids in the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, histidine, negatively charged amino acids include aspartic acid, glutamic acid, and neutrally 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 polarity), asparagine, aspartic acid (acidic polarity), glutamic acid (acidic polarity), glutamine, histidine (basic polarity), lysine (basic polarity), serine, threonine and tyrosine; non-polar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan and valine. Thus, one or more amino acid residues within a CDR region can be replaced by other amino acid residues from the same group, and the modified antibody can be tested for retained function using the functional assays described herein.

[0204] The terms "identity" and "sequence identity" are used interchangeably to refer to the matching of sequences between two polypeptides or between two nucleic acids. When a position in both compared sequences is occupied by the same base or amino acid monomer subunit (e.g., a position in each of the two DNA molecules is occupied by adenine, or a position in each of the two polypeptides is occupied by lysine), then the molecules are identical at that position. The "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 x 100. For example, if 6 out of 10 positions in two sequences match, then the two sequences are 60% identical.

[0205] In certain non-limiting embodiments, the extracellular antigen binding domain of CAR may include a linker connecting the heavy chain variable region and the light chain variable region of the extracellular antigen binding domain. The term "linker" as used herein refers to a functional group (e.g., chemical or polypeptide) that covalently connects two or more polypeptides or nucleic acids so that they are connected to each other. "Peptide linker" as used herein refers to one or more amino acids for coupling two proteins together (e.g., coupling 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.

[0206] In addition, the extracellular antigen binding domain may include a leader sequence or signal peptide for importing the nascent protein into the endoplasmic reticulum. If CAR is to be glycosylated and anchored in the cell membrane, a signal peptide or leader sequence may be necessary. A signal sequence or leader sequence may be a peptide sequence (about 4-30 amino acids in length) present at the N-terminus of the newly synthesized protein, which guides the newly synthesized protein into the secretory pathway.

[0207] In certain non-limiting embodiments, the transmembrane domain of CAR includes a hydrophobic alpha helix across at least a portion of the membrane. Different transmembrane domains lead to different receptor stabilities. After antigen recognition, the receptors are clustered and the signal is transmitted to the cell. According to the subject matter disclosed in the present invention, the transmembrane domain of CAR may include CD8 polypeptides, CD28 polypeptides, CD3ζ polypeptides, CD4 polypeptides, 4-1BB polypeptides, OX40 polypeptides, ICOS polypeptides, CTLA-4 polypeptides, PD-1 polypeptides, LAG-3 polypeptides, 2B4 polypeptides, BTLA polypeptides, synthetic peptides (non-based on proteins related to immune response) or combinations thereof.

[0208] In certain non-limiting embodiments, the intracellular domain of CAR can include a CD3 zeta polypeptide that can activate or stimulate cells (e.g., lymphoid lineage cells such as T cells). CD3 zeta includes three ITAMs that transmit activation signals to cells (e.g., lymphoid lineage cells such as T cells) after binding to an antigen.

[0209] In certain non-limiting embodiments, the intracellular domain of CAR further includes at least one signal region.At least one signal region may include CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, PD-1 polypeptide, CTLA-4 polypeptide, LAG-3 polypeptide, 2B4 polypeptide, BTLA polypeptide, synthetic peptide (non-based on proteins related to immune response) or a combination thereof.

[0210] In some embodiments, the signal region is a co-stimulatory signal region. In some embodiments, the co-stimulatory region includes at least one co-stimulatory molecule that can provide optimal lymphocyte activation. As used herein, "co-stimulatory molecule" refers to cell surface molecules other than antigen receptors or their ligands required for lymphocytes to effectively respond to antigens. At least one co-stimulatory signal region may include CD28 polypeptide, 4-1BB polypeptide, OX40 polypeptide, ICOS polypeptide, DAP-10 polypeptide, or a combination thereof. Co-stimulatory molecules can be bound to co-stimulatory ligands, which are proteins expressed on the cell surface that produce a co-stimulatory response upon binding to their receptors, and the co-stimulatory response is the intracellular response that affects stimulation provided when the antigen is bound 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, a 4-1BB ligand (ie, 4-1BBL) can bind to 4-1BB (also known as "CD137") to provide an intracellular signal that binds to the CAR signal to induce effector cell function of CAR+ T cells.

[0211] In certain embodiments, CAR includes: an extracellular antigen binding region comprising a human scFv that specifically binds to human Ly6G6D, a transmembrane domain comprising a CD8 polypeptide, and an intracellular domain comprising a CD3ζ polypeptide and a costimulatory signal region comprising a combination of 4-1BB and ICOS. In certain embodiments, CAR also includes a signal peptide or leader sequence covalently linked to the 5' end of the extracellular antigen binding domain.

[0212] In certain embodiments, the CAR of the present disclosure may further include an inducible promoter for expressing the nucleic acid sequence in human cells. The promoter for expressing the CAR gene may be a constitutive promoter.

[0213] immune effector cells

[0214] The subject matter disclosed in the present invention provides the immune effector cells expressing the CAR including extracellular antigen binding domain, transmembrane domain and intracellular domain, as described above, wherein the extracellular antigen binding domain is specifically bound to Ly6G6D (such as human Ly6G6D).Immune effector cells can be transduced with CAR disclosed in the present invention so that cells express CAR. The subject matter disclosed in the present invention also provides a method for treating tumors such as rectal cancer using such cells. The immune effector cells of the subject matter disclosed in the present invention can be lymphoid lineage cells, including lymphoid lineage cells of B cells, T cells and natural killer (NK) cells, providing the production of antibodies, the regulation of the cellular immune system, the detection of foreign substances in the blood, the detection of host exogenous cells, etc. The non-limiting examples of lymphoid lineage cells include T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, embryonic stem cells and pluripotent stem cells (for example, pluripotent stem cells that can differentiate into lymphoid cells). T cells can be lymphocytes matured in the thymus, and are primarily responsible for cell-mediated immunity. T cells participate in acquired immune system. The T cells of the presently disclosed subject matter 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 known as suppressor T cells), natural killer T cells, mucosal-associated constant T cells, and γδ T cells. In certain embodiments, the CAR-expressing T cells express Foxp3 to achieve and maintain the T regulatory phenotype. Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and play a role in the innate immune response process. 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 that can induce the death of infected somatic cells or tumor cells.

[0215] carrier

[0216] The genetic modification of immune effector cells (for example, T cells, CTL cells, NK cells) can be achieved by transducing substantially homogeneous cell compositions with recombinant DNA or RNA constructs.Carrier can be a retroviral vector (for example, γ retrovirus), which is used to introduce DNA or RNA constructs into host cell genome.For example, the polynucleotides encoding the CAR targeting Ly6G6D can be cloned into a retroviral vector, and expression can be driven by its endogenous promoter, retroviral long terminal repeats or alternative internal promoters.

[0217] Non-viral vectors or RNA can also be used. Random chromosomal integration or targeted integration (e.g., using nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and / or clustered regularly spaced short palindromic repeats (CRISPR) or transgenic expression (e.g., using natural or chemically modified RNA) can be used.

[0218] Drug administration

[0219] The CAR targeting Ly6G6D of the disclosed subject of the present invention and the immune effector cells expressing it can be provided systemically or directly to the subject for the treatment or prevention of cancer. In certain embodiments, the CAR targeting Ly6G6D and the immune effector cells expressing it are directly injected into the organ of interest. Alternatively or additionally, the CAR targeting Ly6G6D and the immune effector cells expressing it are indirectly provided to the organ of interest by, for example, being administered to the circulatory system (e.g., tumor vascular system). Amplifiers and differentiation agents can be provided before, during, or after cell and composition administration to increase the generation of T cells in vitro or in vivo.

[0220] The composition of the present invention's disclosed subject includes a pharmaceutical composition, which includes expressing the immune effector cells and pharmaceutically acceptable carriers of the CAR targeting Ly6G6D. Administration can be autologous or non-autologous. For example, the immune effector cells of the CAR targeting Ly6G6D and the composition comprising it can be obtained from a subject and administered to the same subject or different compatible subjects. The T cells derived from peripheral blood of the present invention's disclosed subject can be given by local injection, including catheter administration, systemic injection, local injection, intravenous injection or parenteral administration, or its progeny (for example, in vivo, in vitro or in vitro). When the pharmaceutical composition of the present invention's disclosed subject (for example, including the pharmaceutical composition of the immune effector cells of the CAR expressing targeting Ly6G6D) is administered, it can be formulated into unit dose injectable form (solution, suspension, emulsion).

[0221] The term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and 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 adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic 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, and the like. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and their analogs. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). 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 generally understood by those skilled in the art, and are capable of stabilizing 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 hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier comprises 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 (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffered solution (e.g., phosphate buffered solution), Ringer's solution, and any combination thereof.

[0222] Treatment

[0223] Tumor microenvironment. Tumors have a microenvironment that is hostile to the host immune response, which involves a series of mechanisms of malignant cells to protect themselves from immune recognition and clearance. This "hostile tumor microenvironment" includes a variety of immunosuppressive factors, including infiltrating regulatory CD4+ T cells (Treg), bone marrow-derived suppressor cells (MDSC), tumor-associated macrophages (TAM), immunosuppressive cytokines including IL-10 and TGF-β, and targeting the expression of ligands of 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, but within the tumor microenvironment, these mechanisms prevent effective anti-tumor immune responses. In short, these immunosuppressive factors can induce significant invalidation or apoptosis of adoptively transferred CAR-modified T cells when encountering targeted tumor cells.

[0224] 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 recognized by specific cytotoxic T lymphocytes (CTLs). The initiated CTLs must be expanded to sufficient numbers and migrate to the tumor site, where they mature into effectors to exert their function, which is enhanced by helper T cells and suppressed by Tregs and suppressive macrophages.

[0225] Targeted T cell therapy using engineered T lymphocytes. T cell engineering is a groundbreaking strategy that potentially addresses many of the previously observed shortcomings of early immunotherapy approaches.

[0226] Principle of genetic approach: 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 offset the potential for T cell death, anergy, and immunosuppression. Genetic targeting of T cells can also be improved to prevent the undesirable destruction of normal tissues.

[0227] Chimeric Antigen Receptor (CAR): Tumor-specific T cells can be generated by transferring the gene encoding the CAR. Second-generation CARs consist of a tumor antigen-binding domain fused to an intracellular signaling domain capable of activating T cells, as well as a co-stimulatory domain designed to enhance T cell potency and persistence. The CAR design thus coordinates antigen recognition and signaling, functions typically performed by two separate complexes: the TCR heterodimer and the CD3 complex. The extracellular antigen-binding domain of the CAR is typically derived from a murine monoclonal antibody (mAb), a human monoclonal antibody, a camelid antibody, or from a receptor or its ligand. Therefore, antigen recognition is non-MHC-restricted, allowing the same CAR to be used in 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 signaling cascade required for cytolytic induction, cytokine secretion, and proliferation. Because the MHC restriction of antigen recognition is circumvented, the function of CAR-targeted T cells is not affected by HLA downregulation or defects in the antigen processing machinery.

[0228] T cell requirements for expansion and survival: The proliferation of tumor-specific T cells may be required in vitro and in vivo. T cell proliferation must be accompanied by the survival of T cells to allow absolute T cell expansion and persistence. In order to proliferate in response to antigens, T cells generally need to receive two signals. One is provided by TCR recognition of antigen peptide / MHC complexes displayed on the surface of antigen presenting cells (APCs). The other is provided by T cell co-stimulatory receptors such as CD28 or 4-1BB receptors. Although the cytolytic activity of T cells generally does not require accompanying co-stimulation, as previously demonstrated, there is a key need for providing co-stimulatory signals to maintain the anti-tumor function of adoptively transferred T cells.

[0229] Immune Monitoring: Lymphocytes are multifunctional "drugs" that exhibit dynamic effects after infusion. Upon encountering antigens, tumor-specific T cells activate and / or release a variety of proteins that can trigger tumor killing, T cell proliferation, and recruitment of other immune cells or immunomodulation. Therefore, measuring which proteins are secreted from which cells, in what amounts, and at what time points can provide a deep understanding of why specific patients respond or do not respond, and provide critical feedback for designing more effective trials. These test systems will allow direct and meaningful comparisons of clinical approaches, thereby facilitating the design of rational next-generation therapeutic strategies.

[0230] For treatment, the amount administered is an amount effective to produce the desired effect. The effective amount can be provided in a single dose or in a series of doses. The effective amount can be provided in a bolus injection or by continuous infusion.

[0231] An "effective amount" (or "therapeutically effective amount") is an amount sufficient to affect a beneficial or desired clinical outcome in treatment. An effective amount can be administered to a subject in one or more doses. In therapeutic terms, an effective amount is an amount sufficient to alleviate, improve, stabilize, reverse, or slow the progression of a disease or otherwise reduce the pathological consequences of a disease. An effective amount is typically determined by a physician based on the specific circumstances and is within the skill of the art. Several factors are typically considered when determining an appropriate dose to achieve an effective amount. These factors include the age, sex, and weight of the subject, the condition being treated, the severity of the condition, and the dosage form and effective concentration of the immune effector cells being administered.

[0232] The term "subject" refers to a mammal, such as a primate mammal, such as a human. In some embodiments, the subject (eg, human) suffers from a disease associated with Ly6G6D.

[0233] The subject matter disclosed in the present invention provides a medicine box for treating or preventing cancer. In certain embodiments, the medicine box includes a therapeutic or preventive composition comprising an effective amount of a unit dose form of an immune effector cell comprising a CAR targeting Ly6G6D. In a specific embodiment, the cell further expresses at least one co-stimulatory ligand. In some embodiments, the medicine box includes a sterile container containing a therapeutic or preventive vaccine; such a container can be a box, an ampoule, a bottle, a vial, a tube, a bag, a pouch, a 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 accommodating drugs.

[0234] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth / proliferation. Aspects of cancer 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 intestine 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.

[0235] 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 cancer. 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: 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 in or with the container as a separate sheet, brochure, card, or foldout.

[0236] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only intended to illustrate the present invention and are not intended to limit the scope of the invention. Based on the following detailed description of the drawings and preferred embodiments, the various objects and advantages of the present invention will become apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0237] FIG1 is a graph showing the CAR-T cell positive rate results obtained by measuring the CAR-T cell positive rate in Example 2;

[0238] FIG2 is a graph showing the in vitro killing results of different CAR-T cells in Example 3;

[0239] FIG3 is a graph showing the results of measuring cytokine release levels after co-incubation of different CAR-T cells (D10, D46, D59, D70, and D72) with target cells in Example 4;

[0240] FIG4 is a graph showing the results of measuring cytokine release levels after co-incubation of different CAR-T cells (D76, D79, D85, and D103) with target cells in Example 4;

[0241] FIG5 is a graph showing the anti-tumor effects of different CAR-T cells in mice according to Example 5. DETAILED DESCRIPTION

[0242] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0243] Example 1: Design and construction of CAR targeting Ly6G6D antigen and lentiviral packaging

[0244] 1.1. Construction of CAR targeting Ly6G6D antigen

[0245] The CAR structure targeting Ly6G6D includes: signal peptide (Single peptide)–scFv–hinge region (CD8hinge)–CD8α transmembrane region (CD8TM)–ICOS–4-1BB–CD3ζ. The CAR structure and number are shown in Table 1.

[0246] Table 1

[0247] The scFv targeting Ly6G6D has an amino acid sequence as shown in any one of SEQ ID NOs: 81-90 or 152-155; wherein, the scFv of D9-CAR has an amino acid sequence as shown in SEQ ID NO: 81; the scFv of D10-CAR has an amino acid sequence as shown in SEQ ID NO: 82; the scFv of D28-CAR has an amino acid sequence as shown in SEQ ID NO: 83; the scFv of D30-CAR has an amino acid sequence as shown in SEQ ID NO: 84; the scFv of D32-CAR has an amino acid sequence as shown in SEQ ID NO: 85; the scFv of D46-CAR has an amino acid sequence as shown in SEQ ID NO: 86; the scFv of D59-CAR has an amino acid sequence as shown in SEQ ID NO: 87; the scFv of D61-CAR has an amino acid sequence as shown in SEQ ID NO: 88; the scFv of D70-CAR has an amino acid sequence as shown in SEQ ID NO: 89; the scFv of D72-CAR has an amino acid sequence as shown in SEQ ID NO: 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; and the scFv of D103-CAR has the amino acid sequence shown in SEQ ID NO: 155.

[0248] The Ly6G6D-targeting CAR structure comprises the nucleotide (DNA) sequence shown in any one of SEQ ID NOs: 109-118 or SEQ ID NOs: 160-163; and wherein,

[0249] The signal peptide comprises the nucleotide sequence shown in SEQ ID NO: 119:

[0250] The EcoRI restriction site comprises the nucleotide sequence shown in GAATTC;

[0251] The hinge region comprises the nucleotide sequence shown in SEQ ID NO: 120:

[0252] The transmembrane domain of the CD8α protein comprises the nucleotide sequence shown in SEQ ID NO: 121:

[0253] The ICOS domain comprises the nucleotide sequence shown in SEQ ID NO: 122:

[0254] The 4-1BB domain comprises the nucleotide sequence shown in SEQ ID NO: 123:

[0255] The signaling domain of CD3ζ comprises the nucleotide sequence shown in SEQ ID NO: 124:

[0256] The DNA sequence of the above CAR was artificially synthesized and constructed into the shuttle plasmid of the fourth-generation lentivirus.

[0257] 1.2 Lentiviral packaging of Ly6G6D-targeting CAR vectors based on different scFvs

[0258] a. 2*10 7 293T cells were inoculated into a 15 cm dish containing 25 mL of cell growth medium and cultured overnight in an incubator at 37°C with 5% CO2.

[0259] b. Configure the lentiviral packaging system according to Table 2 below:

[0260] Table 2

[0261] Mix solution A and solution B separately, then transfer all of solution A to solution B. After fully mixing, let it stand at room temperature for 15 minutes, then add 10 mL of lentiviral transfection medium and mix well.

[0262] c. Remove the culture medium from the 293T cells in the 15 cm dish and add all of the transfection system to the dish. Gently mix and incubate the cells in a 37°C, 5% CO2 incubator. Six hours after transfection, remove the transfection system and add 30 mL of lentiviral packaging medium. Continue incubating for another 48 hours, then collect the supernatant, concentrate or purify it, and store in a freezer until needed.

[0263] Example 2: Preparation of CAR-T cells and determination of positive rate

[0264] CAR-T cell preparation: T cells were revived and resuspended in CTS complete medium (containing serum replacement, IL-7, and IL-15). Anti-CD3 / CD28 activation beads were used to activate the T cells. After 3 days of activation, the beads were removed and the T cells were infected with lentivirus. Cell culture medium was changed 24 hours after infection to remove residual lentivirus. CAR positivity was measured by flow cytometry starting 2 days after infection.

[0265] The experimental results are shown in Figure 1. As shown in Figures 1A and 1B, D9-CAR, D10-CAR, D28-CAR, D30-CAR, D32-CAR, D46-CAR, D59-CAR, D61-CAR, D70-CAR, D72-CAR, D85-CAR, D103-CAR, D76-CAR, and D79-CAR can all be effectively expressed on the surface of CAR-T cells. The positive rates of different CAR molecules are not exactly the same. The expression rates of D61-CAR and D103-CAR are about 20%, the expression rate of D79-CAR is about 25%, the expression rate of D46-CAR, D76-CAR, and D85-CAR is about 30%; the expression rate of D9-CAR is about 35%; the expression rates of seven CAR molecules, including D10-CAR, D28-CAR, D30-CAR, D32-CAR, D59-CAR, D70-CAR, and D72-CAR, are between 40% and 50%.

[0266] Example 3: Detection of the killing effect of different CAR-T cells

[0267] Target cell plating: Collect target cells in logarithmic growth phase, centrifuge at 500g for 4 minutes, discard the supernatant, and resuspend the cells in CTS complete medium to a density of 2x10 6 / mL suspension was inoculated into a flat-bottom 96-well plate, 100 μL per well, and the number of cells was 2*10 4 / hole.

[0268] Co-incubation of effector and target cells: After adjusting the positive rate of each group using T cells, plate the cells according to the set effector-target ratio. Additionally, a separate CAR-T cell group was established at each effector-target ratio as a negative control for effector cells; a separate target cell group was also established as a negative control; and a target cell + lysis buffer group was established as a positive control for target cell lysis. After 20-22 hours of co-incubation, the cell culture supernatant was collected and the CAR-T cell killing ability was assayed using the LDH assay.

[0269] The experimental results are shown in Figure 2. As shown in Figure 2A, 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-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-target ratio of 2.0, with the killing efficiency of D59-CAR-T being approximately 62%, the killing efficiency of D70-CAR-T being approximately 70%, and the killing efficiency of D72-CAR-T being approximately 66%. As shown in Figure 2B, D76-CAR-T, D79-CAR-T, D85-CAR-T, and D103-CAR-T cells were able to effectively kill target cells at effector-to-target ratios of 1:8, 1:4, and 1:2, respectively, in a dose-dependent manner. Among them, D79-CAR-T and D85-CAR-T cells were relatively effective.

[0270] Example 4: Determination of cytokine release levels after co-incubation of CAR-T cells based on different scFvs with target cells

[0271] CAR-T cells were co-incubated with target cells according to the method of Example 3. The first batch of CAR-T cells included D10-CAR-T, D46-CAR-T, D59-CAR-T, D70-CAR-T, and D72-CAR-T cells co-incubated with the NCI-H716-Ly6G6D stably transfected cell line expressing Ly6G6D; the second batch of CAR-T cells included D76, D79, D85, and D103 CAR-T cells co-incubated with the colo320DM-Ly6G6D stably transfected cell line expressing Ly6G6D. Groups in which only CAR-T cells were added without target cells and groups incubated with only target cells served as control groups. Cell supernatants were collected while measuring cell killing and diluted 5-fold for use. The release of six cytokines, including IL-2, IL-4, IL-6, IL-10, TNF-α, and IFN-γ, in the samples was detected using the BD cytometric beads array human Th1 / Th2 cytokine Kit II kit (Cat. No. 551809).

[0272] The experimental results are shown in Figures 3 and 4. D10, D46, D59, D70, and D72 in Figure 3 refer to D10-CAR-T, D46-CAR-T, D59-CAR-T, D70-CAR-T, and D72-CAR-T, respectively. The figures show the cytokine release levels of each group of CAR-T cells and the control group. Figure 4 shows the cytokine release levels of D76, D79, D85, and D103 CAR-T cells and each control group. The horizontal axis indicates the names of the groups, and the vertical axis indicates the concentration of the relevant factors, in pg / ml.

[0273] Figure 3 shows that after co-incubation with target cells, D10-CAR-T, D46-CAR-T, D59-CAR-T, D70-CAR-T, and D72-CAR-T cells all effectively released cytokines such as IFN-g, TNF-α, and IL-2, which promote CAR-T cell cytotoxicity and T cell proliferation. Cytokines such as IL-4, IL-6, and IL-10 were also released to varying degrees. Cytokine release levels varied among different CAR-T cells after co-incubation with target cells, with D70-CAR-T cells releasing the highest levels of IFN-g, TNF-α, and IL-2, reaching nearly 40,000 pg / mL, over 1,000 pg / mL, and nearly 4,000 pg / mL, respectively. D59-CAR-T cells released the next highest levels, reaching 30,000 pg / mL, nearly 1,000 pg / mL, and nearly 4,000 pg / mL, respectively. D10-CAR-T and D46-CAR-T cells spontaneously released high levels of IFN-g and TNF-α without co-incubation with target cells. D46-CAR-T cells had the highest spontaneous release levels, with IFN-g levels of approximately 2000 pg / mL and TNF-α levels exceeding 100 pg / mL. However, D59-CAR-T, D70-CAR-T, and D72-CAR-T cells showed no significant spontaneous release of IFN-g or TNF-α. No significant spontaneous release of IL-2 was observed in any of the groups.

[0274] As shown in Figure 4, (1) after co-incubation with colo320DM-Ly6G6D cells, D76, D79, D85, and D103 CAR-T cells can effectively release IL-2, IFN-γ, TNF-α and other factors, among which D76 CAR-T cells release the highest and D85 CAR-T cells release the lowest; CAR-T cells not co-incubated with target cells have no obvious release; (2) after co-incubation with target cells, all four CAR-T cells significantly release IL-4, among which D103 group has the highest level; (3) only D76 CAR-T cells significantly release IL-6 factors after co-incubation with target cells; (4) the background level of IL-10 released by D76 CAR-T cells is low, but its release amount increases significantly after co-incubation with target cells; D79, D85, D103 CAR-T cells all released high levels of background IL-10, and the release level was further increased after co-incubation with target cells.

[0275] Example 5: Evaluation of the anti-tumor effect of CAR-T cells based on different scFvs in mice

[0276] CAR-T cell preparation:

[0277] T cells were resuspended in complete CTS medium and activated with anti-CD3 / CD28 activating beads. After 3 days of activation, the beads were removed and the T cells were infected with lentivirus. Cell culture medium was changed 24 hours after infection to remove residual lentivirus. CAR positivity was assessed by flow cytometry starting 2 days after infection. Before freezing, uninfected T cells were used to adjust the CAR positivity of each CAR-T cell group to the same level. Cells were resuspended in freezing medium, aliquoted, and cryopreserved. The cells were then transferred to a cryogenic freezer and stored in liquid nitrogen after 1 day.

[0278] Recovery and administration: After cell recovery, resuspend in pre-cooled PBS before administration. In the first batch of experiments, the dose of D10, D46, D59, D70, and D72 CAR-T cell groups was set at 3.6*10 per mouse. 6 CAR+ cells; in the second batch of experiments, the D79, D85, and D103 CAR-T cell groups were administered at a dose of 2.0*10 per mouse. 6 Each mouse was administered a 200 μL injection volume. When the tumor volume reached approximately 150 cubic millimeters, CAR-T cells were infused back through the tail vein. Tumor volume was measured twice a week after infusion.

[0279] Data processing: Tumor volume (mm3) = major diameter (mm) * minor diameter (mm) * minor diameter (mm) * 0.5 Tumor inhibition rate = [1 - average tumor volume of the group / average tumor volume of the T cell group] * 100%.

[0280] The experimental results are shown in Figure 5. After data processing, the results shown in Figure 5A show that the five groups of CAR-T cells, D10-CAR, D46-CAR, D59-CAR, D70-CAR, and D72-CAR, all showed tumor inhibitory effects relative to T cells. Different groups of CAR-T cells had different inhibitory effects on NCI-H508-Ly6G6D tumors, among which D46-CAR-T had an inhibitory effect of 30.5%; D10-CAR-T had an inhibitory effect of 59%; 21 days after CAR-T transfusion, the tumor inhibition efficiencies of D59-CAR-T, D70-CAR-T, and D72-CAR-T reached 95.4%, 96%, and 92.9%, respectively.

[0281] In another batch of animal experiments, data processing yielded Figure 5B, which showed that 21 days after infusion, calculated by average tumor volume, CAR-T cells in the D76-CAR, D79-CAR, D85-CAR, and D103-CAR groups all showed tumor inhibitory effects relative to T cells. CAR-T cells in different groups had different inhibitory effects on tumors, with D76 CAR-T having an inhibitory effect of 88.34%, D79 CAR-T having an inhibitory effect of 94.46%, D85 CAR-T having an inhibitory effect of 97.28%, and D103CAR-T having an inhibitory effect of 33.68%. The inhibitory effect of D85 CAR-T cells was relatively good.

[0282] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "an embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and features of different embodiments or examples, unless they are mutually inconsistent.

[0283] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify 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 Ly6G6D, a transmembrane domain, and an intracellular domain. The extracellular antigen-binding domain comprises an antibody or antigen-binding fragment that targets and binds Ly6G6D. The antibody or antigen-binding fragment comprises a heavy-chain variable region that comprises complementarity-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 that 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 The L-CDR2 has an 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 The L-CDR3 has an 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, wherein 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: (1) H-CDR1 has an amino acid sequence shown in SEQ ID NO:1, H-CDR2 has an amino acid sequence shown in SEQ ID NO:2 and H-CDR3 has an amino acid sequence shown in SEQ ID NO:3; or (2) H-CDR1 has an amino acid sequence shown in SEQ ID NO:7, H-CDR2 has an amino acid sequence shown in SEQ ID NO:8 and H-CDR3 has an amino acid sequence shown in SEQ ID NO:9; or (3) H-CDR1 has an amino acid sequence shown in SEQ ID NO:13, H-CDR2 has an amino acid sequence shown in SEQ ID NO:14 and H-CDR3 has an amino acid sequence shown in SEQ ID NO:15; or (4) H-CDR1 has an amino acid sequence shown in SEQ ID NO:18, H-CDR2 has an amino acid sequence shown in SEQ ID NO:19 and H-CDR3 has an amino acid sequence shown in SEQ ID NO:20; or (5) H-CDR1 has an amino acid sequence shown in SEQ ID NO:22, H-CDR2 has an amino acid sequence shown in SEQ ID NO:23 and H-CDR3 has an amino acid sequence shown in SEQ ID NO:24; or (6) H-CDR1 has an amino acid sequence shown in SEQ ID NO:28, H-CDR2 has an amino acid sequence shown in SEQ ID NO:29 and H-CDR3 has an amino acid sequence shown in SEQ ID NO:30; or (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; or (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 (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 (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 (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 (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 (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 (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.

3. The chimeric antigen receptor according to claim 1, wherein 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: (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 (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 (3) 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: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 shown in SEQ ID NO:25, L-CDR2 has the amino acid sequence shown in SEQ ID NO:26, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:27; or (6) L-CDR1 has the amino acid sequence shown in SEQ ID NO:31, L-CDR2 has the amino acid sequence shown in SEQ ID NO:32, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:33; or (7) L-CDR1 has the amino acid sequence shown in SEQ ID NO:37, L-CDR2 has the amino acid sequence shown in SEQ ID NO:38, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:39; or (8) L-CDR1 has the amino acid sequence shown in SEQ ID NO:43, L-CDR2 has the amino acid sequence shown in SEQ ID NO:44, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:45; or (9) L-CDR1 has the amino acid sequence shown in SEQ ID NO:37, L-CDR2 has the amino acid sequence shown in SEQ ID NO:38, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:49; or (10) L-CDR1 has the amino acid sequence shown in SEQ ID NO:53, L-CDR2 has the amino acid sequence shown in SEQ ID NO:54, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:45; or (11) L-CDR1 has the amino acid sequence shown in SEQ ID NO:129, L-CDR2 has the amino acid sequence shown in SEQ ID NO:130, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:131; or (12) L-CDR1 has the amino acid sequence shown in SEQ ID NO:37, L-CDR2 has the amino acid sequence shown in SEQ ID NO:133, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:134; or (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 (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.

4. The chimeric antigen receptor according to any one of claims 1-3, characterized in that, The antibody or antigen-binding fragment thereof comprises a heavy chain variable region framework sequence and a light chain variable region framework sequence, and 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 1-4, wherein The heavy chain variable region of the antibody or antigen-binding fragment thereof 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 with 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 1-5, characterized in that, The light chain variable region of the antibody or antigen-binding fragment thereof 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 with 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 1-6, characterized in that, The heavy chain variable region and the light chain variable region of the extracellular antigen-binding domain are linked by a linker.

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

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

10. The chimeric antigen receptor according to any one of claims 1-9, 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 with 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 1-10, characterized in that, The transmembrane domain comprises a transmembrane domain derived from the following proteins: 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 1-11, characterized in that, The transmembrane domain comprises a transmembrane domain derived from the CD8α protein.

13. The chimeric antigen receptor according to any one of claims 1-12, 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 1-13, characterized in that, The chimeric antigen receptor further comprises a hinge region.

15. The chimeric antigen receptor according to any one of claims 1-14, 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 1-15, 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 1-16, characterized in that, The intracellular domain of the chimeric antigen receptor comprises a signal transduction domain derived from CD3ζ.

18. The chimeric antigen receptor according to any one of claims 1-17, 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 1-18, characterized in that, The intracellular domain further comprises at least one co-stimulatory domain.

20. The chimeric antigen receptor according to any one of claims 1-19, characterized in that, The co-stimulatory domain comprises a co-stimulatory domain derived from a protein selected from CD28, 4-1BB, OX-40, ICOS, or a combination thereof.

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

22. The chimeric antigen receptor according to claim 21, wherein 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 1-20, wherein The co-stimulatory domain comprises a domain derived from ICOS.

24. The chimeric antigen receptor according to claim 23, wherein 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 1-20, characterized in that, The co-stimulatory domain comprises a domain derived from a combination of 4-1BB and ICOS.

26. The chimeric antigen receptor according to claim 25, wherein 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 1-26, 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 1-27, characterized in that, The chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NO:99-108 or SEQ ID NO:156-159.

29. The chimeric antigen receptor according to any one of claims 1-28, 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 1-29, 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, wherein The nucleic acid molecule comprises the nucleotide sequence shown in any one of SEQ ID NO:109-118 or SEQ ID NO:160-163.

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

34. The expression vector according to claim 33, wherein, 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 1-30, 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, wherein The immune effector cell is 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 from which lymphocytes can be differentiated, macrophages, B cells, 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 according to claim 33 or 34 into an immune effector cell.

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

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

40. A medicine box for treating cancer, characterized in that, The kit comprises the immune effector cell according to claim 35 or 36.

41. The medicine box according to claim 40, characterized in that, The kit further includes instructions for using the immune response cell to treat a cancer subject.

42. The medicine box according to claim 41, wherein, The cancer is colorectal cancer.

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