WO2025139979A1 - Chimeric antigen receptor targeting ly6g6d and use thereof - Google Patents

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

The subject matter disclosed in the present invention provides methods and compositions for treating cancer. Specifically, the present invention provides a chimeric antigen receptor targeting Ly6G6D, an immune effector cell comprising the chimeric antigen receptor, and a method of using the immune effector cells for treating cancer.

Background Art

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

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

Colorectal cancer can be divided into mismatch repair-deficient (dMMR) and mismatch repair-complete (pMMR), of which the dMMR type mainly includes microsatellite high instability (MSI-H) type, and the pMMR type mainly includes microsatellite low instability (MSI-L) and microsatellite stable (MSS). For MSI-H type metastatic colorectal cancer, immune checkpoint inhibitors have a good therapeutic effect; while for MSI-L and MSS types, which account for about 85% of all metastatic colorectal cancer patients, the response rate of immune checkpoint inhibitors is low, and the survival of patients is low, and more effective and safe therapeutic 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

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.

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,

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

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

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

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

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

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

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

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:

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

(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

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

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

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

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

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

(8) H-CDR1 has the amino acid sequence 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; or

(9) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:46, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:47, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:48; or

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

(11) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:127, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:128; or

(12) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:18, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:132; or

(13) H-CDR1 has the amino acid sequence 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; or

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

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:

(1) 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:5 and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:6; or

(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

(3) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:4, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:16, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:17; or

(4) L-CDR1 has the amino acid sequence 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

(5) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:25, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:26, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:27; or

(6) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:31, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:32, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:33; or

(7) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:39; or

(8) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:43, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:44, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:45; or

(9) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:49; or

(10) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:53, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:54, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:45; or

(11) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:129, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:130, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:131; or

(12) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:133, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:134; or

(13) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:135, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:136, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:137; or

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

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:

(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

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

(2) 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

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

(3) 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

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) 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

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) 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

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) 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

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) 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

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) 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

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) 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

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) 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

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.

(11) H-CDR1 has the amino acid sequence shown in SEQ ID NO: 127, 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

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.

(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

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.

(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

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.

(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; and

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.

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.

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

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 with any one of SEQ ID NO:65-76 or SEQ ID NO:148-151.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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

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.

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.

The linker is a connecting peptide for connecting the heavy chain variable region and the light chain variable region of an antibody, and may also be a connecting peptide linker modified by scientific researchers. An example of the amino acid sequence of the linker is: (GnS)m, n is a natural number of 3-5, preferably 4, and m is a natural number of 1-5, preferably 3.

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

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 SEQ ID NOs: 152-155.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In some embodiments, 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Specifically, the restriction enzyme cutting site is selected from the group consisting of BamHI, SpeI, SalI, KpnI, XhoI, SphI, XbaI, EcoRI and the like.

Specifically, the restriction 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 restriction site is: EF.

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

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

The chimeric antigen receptors of the present invention may all contain an amino acid sequence of a restriction site as shown in the above specific examples, wherein the restriction 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 chimeric antigen receptor further comprises a signal peptide covalently linked to the N-terminus of the extracellular antigen binding domain.

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

MGVKVLFALICIAVAEA(SEQ ID NO:91).

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.

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

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

In some embodiments, the nucleotide sequence of the EcoRI restriction site is: GAATTC.

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

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

The chimeric antigen receptors of the present invention may all include 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.

On the other hand, the present invention provides a nucleic acid construct, which comprises the nucleic acid molecule described in 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 described in the present invention in an appropriate host cell, and the one or more control sequences are selected from: a promoter, an enhancer, a stop signal, a signal peptide, a leader sequence, a transcription terminator and any group thereof.

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

In yet another aspect, the present invention provides an expression vector, wherein the expression vector carries the nucleic acid molecule described above in the present invention.

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

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

In some embodiments, the immune effector cells are selected from T cells, natural killer cells, cytotoxic T lymphocytes, regulatory T cells, human embryonic stem cells, lymphoid progenitor cells, T cell 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.

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

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

On the other hand, 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.

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

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

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.

Definitions and general terms

In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular biology, biochemistry, nucleic acid chemistry, immunology and other operating steps used herein are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

When the terms "for example," "such as," "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."

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) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Chimeric Antigen Receptor

The term "Chimeric Antigen Receptor" (CAR) is an engineered receptor that transfers or confers a specificity of interest to an immune effector cell. CARs can be used to transfer the specificity of a monoclonal antibody to a T cell by facilitating the transfer of its coding sequence via a retroviral vector.

There are three generations of CAR. "First generation" CAR is usually 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 usually 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 signal domain in a single fusion molecule, without relying on HLA-mediated antigen presentation. "Second generation" CAR adds the intracellular domain 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 lymphocytic leukemia (ALL), clinical trials targeting the CD19 molecule have demonstrated the strong efficacy of "second-generation" CAR-modified T cells."Third-generation" CARs include those that provide multiple co-stimulations (such as CD28 and 4-1BB) and activation (CD3ζ).

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 a 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.

In certain non-limiting embodiments, the extracellular antigen binding domain of CAR has high binding specificity and high binding affinity to Ly6G6D. For example, in these embodiments, the extracellular antigen binding domain of CAR (e.g., implemented with human scFv or its analogs) binds to Ly6G6D with a dissociation constant (KD) of about 1x ^10-9 M or less.

The term "transmembrane domain" usually refers to the domain in CAR that passes through the cell membrane, which is connected to the intracellular signal transduction domain and plays a role in transmitting signals.

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.

The CDR domains are defined using the Kabat system (Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). The subject matter disclosed in the present invention further provides an extracellular antigen-binding domain (e.g., scFv) comprising conservative modifications of the antibody sequences disclosed herein. For example, without any limitation, the extracellular antigen-binding domain (e.g., scFv) of the subject matter disclosed in the present invention comprises: 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 comprises a specific amino acid sequence disclosed herein or a conservative modification thereof, wherein the extracellular antigen-binding domain retains the desired functional properties.

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 consisting 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., dAb), single chain antibodies (e.g., scFv), 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, unglycosylated antibodies, and any antibody fragments and derivatives thereof that bind to antigens 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 the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with 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 (Clq) of the classical complement system.

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).

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.

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 changed within a particular sequence or CDR region. Exemplary conservative amino acid substitutions are shown in Table 1.

Table 1

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 where 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. Therefore, one or more amino acid residues within the CDR region can be replaced by other amino acid residues from the same group, and the altered antibody can be tested for retained function using the functional assays described herein.

The terms "identity" and "sequence identity" are interchangeable and are used 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 (for example, 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 have 60% identity.

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.

In addition, the extracellular antigen binding domain may include a leader sequence or signal peptide that imports 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 a newly synthesized protein, which guides the newly synthesized protein into the secretory pathway.

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, receptor clusters and signals are transmitted to cells. 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.

In certain non-limiting embodiments, the intracellular domain of CAR may include a CD3 ζ polypeptide that can activate or stimulate cells (e.g., lymphoid lineage cells such as T cells). CD3 ζ includes 3 ITAMs that transmit activation signals to cells (e.g., lymphoid lineage cells such as T cells) after binding to antigens.

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.

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, which 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 combined with co-stimulatory ligands, which are proteins expressed on the cell surface that produce co-stimulatory responses once bound to their receptors, and the above-mentioned co-stimulatory responses are provided when antigens are bound to their CAR molecules. The intracellular response of the effect of stimulation. Co-stimulatory ligands include, but are not limited to CD80, CD86, CD70, OX40L, 4-1BBL, CD48, TNFRSF14 and PD-L1. As an example, 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 the effector cell function of CAR+T cells.

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

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.

Immune effector cells

The subject disclosed in the present invention provides an immune effector cell expressing a CAR including an extracellular antigen binding domain, a transmembrane domain, and an intracellular domain, as described above, wherein the extracellular antigen binding domain specifically binds to Ly6G6D (e.g., human Ly6G6D). Immune effector cells can be transduced with CAR disclosed in the present invention so that cells express CAR. The subject 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 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 antibody production, cellular immune system regulation, detection of foreign substances in blood, detection of host exogenous cells, etc. 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 (e.g., pluripotent stem cells that can differentiate into lymphoid cells). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells participate in the acquired immune system. The T cells of the presently disclosed subject matter can be any type of T cells, 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 need to be pre-activated in order to exert their cytotoxic effects on target cells. Cytotoxic T cells (CTL or killer T cells) are a subtype of T lymphocytes that can induce the death of infected somatic cells or tumor cells.

Carrier

The genetic modification of immune effector cells (e.g., T cells, CTL cells, NK cells) can be achieved by transducing substantially homogeneous cell compositions with recombinant DNA or RNA constructs. The carrier can be a retroviral vector (e.g., γ retrovirus), which is used to introduce DNA or RNA constructs into the 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.

Non-viral vectors or RNA may 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) may be used.

Drug administration

The CAR targeting Ly6G6D and the immune effector cells expressing it of the disclosed subject of the present invention 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 the administration of cells and compositions to increase the production of T cells in vitro or in vivo.

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

The term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, e.g., 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 the like. 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), etc. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc. Stabilizers have the meanings generally understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc. In certain exemplary embodiments, the pharmaceutically acceptable carrier includes a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such sterile injectable liquid is 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 buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

Treatment

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 removal. 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 in 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.

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 functions, which are enhanced by helper T cells and inhibited by Tregs and suppressive macrophages.

Targeted T cell therapy using engineered T lymphocytes. T cell engineering is a groundbreaking strategy to potentially address many of the previously observed deficiencies of early immunotherapy approaches.

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, ineffectiveness and immunosuppression. Genetic targeting of T cells can also be improved to prevent undesired destruction of normal tissues.

Chimeric Antigen Receptor (CAR): Tumor-specific T cells can be generated by transferring the gene encoding the CAR. Second-generation CARs include a tumor antigen binding domain fused to an intracellular signaling domain capable of activating T cells, and a co-stimulatory domain designed to enhance T cell potency and persistence. The CAR design can therefore coordinate antigen recognition and signal transduction, two functions that are physiologically undertaken by two separate complexes, the TCR heterodimer and the CD3 complex. The extracellular antigen binding domain of the CAR is usually 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, so the use of the same CAR can be applied to any patient expressing the target antigen. Antigen binding by the CAR triggers phosphorylation of the immunoreceptor tyrosine-based activation motif (ITAM) in the intracellular domain, initiating the signal transduction 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.

T cell requirements for expansion and survival: Proliferation of tumor-specific T cells may be required ex vivo. T cell proliferation must be accompanied by T cell survival to allow absolute T cell expansion and persistence. In order to proliferate in response to antigen, T cells generally need to receive two signals. One is provided by TCR recognition of antigenic 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 T cell cytolytic activity generally does not require concomitant co-stimulation, as previously demonstrated, there is a critical need to provide co-stimulatory signals to maintain the anti-tumor function of adoptively transferred T cells.

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

For treatment, the amount administered is an amount effective to produce the desired effect. The effective amount can be provided in a single administration or in a series of administrations. The effective amount can be provided in a bolus or by continuous infusion.

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 terms of treatment, 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. The effective amount is usually determined by a physician based on the specific circumstances and is within the skill of the art. When determining an appropriate dose to achieve an effective amount, several factors are generally considered. 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 administered.

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

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, which includes an effective amount of a unit dose form of an immune effector cell containing 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 containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blisters or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil or other materials suitable for containing drugs.

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.

If necessary, 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 the following: a description of the therapeutic agent; a dosage regimen and method of administration for treating or preventing cancer or its symptoms; precautions; warnings; indications; contraindications; overdose information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions can be printed directly on the container (when present), or affixed to the container as a label, or provided in or with the container as a separate sheet, brochure, card, or folded printed matter.

Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. According to the following detailed description of the accompanying drawings and preferred embodiments, various objects and advantages of the present invention will become practicable to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

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

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

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

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

FIG5 is a graph showing the anti-tumor results of different CAR-T cells in mice in Example 5.

DETAILED DESCRIPTION

The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

Example 1: Design and construction of CAR targeting Ly6G6D antigen, and lentiviral packaging

1.1. Construction of CAR targeting Ly6G6D antigen

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

Table 1

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

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

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

The EcoRI restriction site comprises the nucleotide sequence shown by GAATTC;

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

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

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

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

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

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

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

a. Inoculate 2*10 ⁷ 293T cells into a 15 cm dish containing 25 mL of cell growth medium and culture in an incubator at 37°C with 5% CO ₂ overnight for later use;

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

Table 2

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.

c. Remove the culture medium of 293T cells in the 15 cm dish, add all the above transfection system to the culture dish, mix gently and place the cells in an incubator at 37°C with 5% CO _2. After 6 hours of transfection, remove the transfection system and add 30 mL of lentivirus packaging medium. Continue to culture for 48 hours and collect the supernatant of the culture medium. After concentration or purification, store it in an ultra-low temperature refrigerator for later use.

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

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 T cells. After 3 days of activation, the activation beads were removed and T cells were infected with lentivirus. Cells were replaced with medium 24 hours after infection to remove residual lentivirus. CAR positivity was detected by flow cytometry 2 days after infection.

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%.

Example 3: Detection of killing effects of different CAR-T cells

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. Inoculate in a flat-bottom 96-well plate, 100 μL per well, and the cell number is 2*10 ⁴ /well.

Co-incubation of effector cells and target cells: After using T cells to adjust the positive rate of each group to be consistent, the plates are plated according to the set effector-target ratio. In addition, a separate CAR-T cell group is set as a negative control for effector cells under each effector-target ratio; a group of negative control groups for separate target cells is set at the same time; and a group of target cells + lysate group is set as a positive control for target cell lysis. After co-incubation for 20-22 hours, the cell culture supernatant is collected and the CAR-T cell killing ability is detected using the LDH method.

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 effectively kill target cells at an effect-target ratio 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 on target cells when the effect-target ratio is 2.0, with the killing efficiency of D59-CAR-T being about 62%, the killing efficiency of D70-CAR-T being about 70%, and the killing efficiency of D72-CAR-T being about 66%. As shown in Figure 2B, D76-CAR-T, D79-CAR-T, D85-CAR-T, and D103-CAR-T can effectively kill target cells at effector-target ratios of 1:8, 1:4, and 1:2, and the effect is dose-dependent. Among them, D79-CAR-T and D85-CAR-T cells have relatively good effects.

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

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

The experimental results are shown in Figures 3 and 4. D10, D46, D59, D70, and D72 shown in Figure 3 refer to D10-CAR-T, D46-CAR-T, D59-CAR-T, D70-CAR-T, and D72-CAR-T, respectively. The attached figure shows the cytokine release of each group of CAR-T cells and the control group; Figure 4 shows the cytokine release of D76, D79, D85, and D103 CAR-T cells and each control group. The horizontal axis refers to the name of each group, and the vertical axis refers to the concentration of the relevant factor, in pg/ml;

As shown in Figure 3, after co-incubation with target cells, D10-CAR-T, D46-CAR-T, D59-CAR-T, D70-CAR-T, and D72-CAR-T can effectively release IFN-g, TNF-α, IL-2 and other cytokines that promote CAR-T cells to kill target cells and promote T cell proliferation. IL-4, IL-6, IL-10 and other cytokines are also released to varying degrees; the release levels of cytokines after co-incubation with target cells are different for different CAR-Ts, among which D70-CAR-T cells have the highest release levels of IFN-g, TNF-α, and IL-2, which are close to 40,000 pg/mL, more than 1,000 pg/mL, and close to 4,000 pg/mL, respectively; the release level of D59-CAR-T is second, reaching 30,000 pg/mL, close to 1,000 pg/mL, and close to 4,000 pg/mL, respectively. D10-CAR-T and D46-CAR-T had high levels of IFN-g and TNF-α self-release when not co-incubated with target cells, among which D46-CAR-T had the highest level of self-release, with the level of IFN-g being about 2000pg/mL and the level of TNF-α being more than 100pg/mL; while D59-CAR-T, D70-CAR-T, and D72-CAR-T had no obvious self-release of IFN-g and TNF-α. No obvious self-release of IL-2 was found in all groups.

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 that are not co-incubated with target cells have no obvious release; (2) after co-incubation with target cells, the four CAR-T cells All of them significantly released IL-4, among which the D103 group had the highest level; (3) Only D76 CAR-T cells significantly released IL-6 factors after co-incubation with target cells; (4) The background level of IL-10 released by D76 CAR-T cells was low, but its release amount increased significantly after co-incubation with target cells; D79, D85, and D103 CAR-T cells all had high levels of background IL-10 release, and the release level further increased after co-incubation with target cells.

Example 5: Preferred evaluation of anti-tumor effects of CAR-T cells based on different scFvs in mice

CAR-T cell preparation:

T cells were revived and resuspended in CTS complete medium. Anti-CD3/CD28 activation beads were used to activate T cells. After 3 days of activation, the activation beads were removed and T cells were infected with lentivirus. Cells were replaced with medium 24 hours after infection to remove residual lentivirus. CAR positivity was detected by flow cytometry 2 days after infection. Before cell freezing, uninfected T cells were used to adjust the CAR positivity of each group of CAR-T cells to the same level. After the cells were resuspended in freezing medium, they were aliquoted and frozen, transferred to program cooling and placed in an ultra-low temperature refrigerator for freezing, and then transferred to liquid nitrogen for storage 1 day later.

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

Data processing:
Tumor volume (cubic millimeters) = long diameter (mm) * short diameter (mm) * short diameter (mm) * 0.5
Tumor inhibition rate = [1-average tumor volume of the group/average tumor volume of the T cell group] * 100%.

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 inhibitory efficiencies of D59-CAR-T, D70-CAR-T, and D72-CAR-T reached 95.4%, 96%, and 92.9%, respectively.

In another batch of animal experiments, after data processing, Figure 5B showed that 21 days after reinfusion, calculated by the 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. Different groups of CAR-T cells had different inhibitory effects on tumors, among which the inhibitory effect of D76 CAR-T was 88.34%, the inhibitory effect of D79 CAR-T was 94.46%, the inhibitory effect of D85 CAR-T was 97.28%, the inhibitory effect of D103CAR-T was 33.68%, and the inhibitory effect of D85 CAR-T cells was relatively good.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "an example", "some embodiments", "example", "specific example", or "some examples" etc. 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 the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims (42)

A chimeric antigen receptor targeting Ly6G6D, characterized in that the chimeric antigen receptor comprises an extracellular antigen binding domain, a transmembrane domain and an intracellular domain that bind to Ly6G6D, the extracellular antigen binding domain comprises an antibody or antigen binding fragment that targets and binds to Ly6G6D, wherein the antibody or antigen binding fragment comprises a heavy chain variable region, and the heavy chain variable region comprises complementary determining regions: H-CDR1, H-CDR2 and H-CDR3; wherein, H-CDR1 has the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 13, SEQ ID NO: 18, SEQ ID NO: 22, SEQ ID NO: 28, SEQ ID NO: 34, SEQ ID NO: 40, SEQ ID NO: 46, SEQ ID NO: 50, SEQ ID NO: 127 or SEQ ID NO: 138; and H-CDR2 has the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 19, SEQ ID NO: 23, SEQ ID NO: 29, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 51 or SEQ ID NO: 139; and H-CDR3 has the amino acid sequence shown in SEQ ID NO: 3, SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 30, SEQ ID NO: 36, SEQ ID NO: 42, SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 128, SEQ ID NO: 132 or SEQ ID NO: 140; And the antibody or antigen-binding fragment comprises a light chain variable region, and the light chain variable region comprises complementarity determining regions: L-CDR1, L-CDR2 and L-CDR3; wherein, L-CDR1 has the amino acid sequence shown in SEQ ID NO:4, SEQ ID NO:10, SEQ ID NO:25, SEQ ID NO:31, SEQ ID NO:37, SEQ ID NO:43, SEQ ID NO:53, SEQ ID NO:129, SEQ ID NO:135 or SEQ ID NO:141; and L-CDR2 has the amino acid sequence shown in SEQ ID NO: 5, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 26, SEQ ID NO: 32, SEQ ID NO: 38, SEQ ID NO: 44, SEQ ID NO: 54, SEQ ID NO: 130, SEQ ID NO: 133, SEQ ID NO: 136 or SEQ ID NO: 142; and L-CDR3 has the amino acid sequence shown in SEQ ID NO:6, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:21, SEQ ID NO:27, SEQ ID NO:33, SEQ ID NO:39, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:131, SEQ ID NO:134, SEQ ID NO:137 or SEQ ID NO:143. The chimeric antigen receptor according to claim 1, characterized in that the complementarity determining regions of the heavy chain variable region: H-CDR1, H-CDR2 and H-CDR3 are selected from the following combinations: (1) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:1, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:2, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:3; or (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 (3) H-CDR1 has the amino acid sequence shown in SEQ ID NO:13, H-CDR2 has the amino acid sequence shown in SEQ ID NO:14 and H-CDR3 has the amino acid sequence shown in SEQ ID NO:15; or (4) H-CDR1 has the amino acid sequence shown in SEQ ID NO:18, H-CDR2 has the amino acid sequence shown in SEQ ID NO:19 and H-CDR3 has the amino acid sequence shown in SEQ ID NO:20; or (5) H-CDR1 has the amino acid sequence shown in SEQ ID NO:22, H-CDR2 has the amino acid sequence shown in SEQ ID NO:23 and H-CDR3 has the amino acid sequence shown in SEQ ID NO:24; or (6) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:28, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:30; or (7) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:34, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:35, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:36; or (8) H-CDR1 has the amino acid sequence 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; or (9) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:46, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:47, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:48; or (10) H-CDR1 has the amino acid sequence shown in SEQ ID NO:50, H-CDR2 has the amino acid sequence shown in SEQ ID NO:51 and H-CDR3 has the amino acid sequence shown in SEQ ID NO:52; or (11) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:127, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:128; or (12) H-CDR1 has the amino acid sequence set forth in SEQ ID NO:18, H-CDR2 has the amino acid sequence set forth in SEQ ID NO:29, and H-CDR3 has the amino acid sequence set forth in SEQ ID NO:132; or (13) H-CDR1 has the amino acid sequence 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; or (14) H-CDR1 has the amino acid sequence shown in SEQ ID NO:138, H-CDR2 has the amino acid sequence shown in SEQ ID NO:139 and H-CDR3 has the amino acid sequence shown in SEQ ID NO:140. The chimeric antigen receptor according to claim 1, characterized in that the complementarity determining regions of the light chain variable region: L-CDR1, L-CDR2 and L-CDR3 are selected from the following combinations: (1) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:4, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:5 and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:6; or (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 (3) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:4, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:16, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:17; or (4) L-CDR1 has the amino acid sequence 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 (5) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:25, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:26, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:27; or (6) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:31, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:32, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:33; or (7) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:39; or (8) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:43, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:44, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:45; or (9) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:38, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:49; or (10) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:53, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:54, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:45; or (11) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:129, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:130, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:131; or (12) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:37, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:133, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:134; or (13) L-CDR1 has the amino acid sequence set forth in SEQ ID NO:135, L-CDR2 has the amino acid sequence set forth in SEQ ID NO:136, and L-CDR3 has the amino acid sequence set forth in SEQ ID NO:137; or (14) L-CDR1 has the amino acid sequence shown in SEQ ID NO:141, L-CDR2 has the amino acid sequence shown in SEQ ID NO:142, and L-CDR3 has the amino acid sequence shown in SEQ ID NO:143. The chimeric antigen receptor according to any one of claims 1 to 3, characterized in that the antibody or antigen-binding fragment comprises a heavy chain variable region framework sequence and a light chain variable region framework sequence, and the framework sequence is derived from a murine antibody, a human antibody, a primate antibody or a mutant thereof. The chimeric antigen receptor according to any one of claims 1-4 is characterized in that 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 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with any one of SEQ ID NO:55-64 or SEQ ID NO:144-147. The chimeric antigen receptor according to any one of claims 1-5 is characterized in that the light chain variable region of the antibody or antigen binding fragment comprises an amino acid sequence having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity with any one of SEQ ID NO:65-76 or SEQ ID NO:148-151. The chimeric antigen receptor according to any one of claims 1 to 6, characterized in that the heavy chain variable region and the light chain variable region of the extracellular antigen binding domain are connected by a linker. The chimeric antigen receptor according to any one of claims 1-7 is characterized in that the linker comprises an amino acid sequence shown in any one of SEQ ID NO:77-80. The chimeric antigen receptor according to any one of claims 1 to 8, characterized in that the extracellular antigen binding domain is a single-chain antibody. The chimeric antigen receptor according to any one of claims 1-9 is 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 NO:81-90 or SEQ ID NO:152-155. The chimeric antigen receptor according to any one of claims 1 to 10, characterized in that the transmembrane domain comprises a transmembrane domain derived from the following proteins: α, β or ζ chain of 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. The chimeric antigen receptor according to any one of claims 1 to 11, characterized in that the transmembrane domain comprises a transmembrane domain derived from a CD8α protein. The chimeric antigen receptor according to any one of claims 1-12 is characterized in that the transmembrane domain comprises the amino acid sequence shown in SEQ ID NO:92. The chimeric antigen receptor according to any one of claims 1 to 13, characterized in that the chimeric antigen receptor further comprises a hinge region. The chimeric antigen receptor according to any one of claims 1 to 14, characterized in that the hinge region connects the extracellular antigen binding structure and the transmembrane domain. The chimeric antigen receptor according to any one of claims 1-15 is characterized in that the hinge region comprises the amino acid sequence shown in SEQ ID NO:93. The chimeric antigen receptor according to any one of claims 1 to 16, characterized in that the intracellular domain of the chimeric antigen receptor comprises a signaling domain derived from CD3ζ. The chimeric antigen receptor according to any one of claims 1-17 is characterized in that the intracellular domain of the chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID NO:94. The chimeric antigen receptor according to any one of claims 1 to 18, characterized in that the intracellular domain further comprises at least one co-stimulatory domain. The chimeric antigen receptor according to any one of claims 1 to 19, characterized in that the costimulatory domain comprises a costimulatory domain derived from the following proteins: CD28, 4-1BB, OX-40, ICOS or a combination thereof. The chimeric antigen receptor according to any one of claims 1 to 20, characterized in that the co-stimulatory domain comprises a domain derived from 4-1BB. The chimeric antigen receptor according to claim 21 is characterized in that the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:95. The chimeric antigen receptor according to any one of claims 1 to 20, characterized in that the co-stimulatory domain comprises a domain derived from ICOS. The chimeric antigen receptor according to claim 23 is characterized in that the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:96. The chimeric antigen receptor according to any one of claims 1 to 20, characterized in that the co-stimulatory domain comprises a domain derived from a combination of 4-1BB and ICOS. The chimeric antigen receptor according to claim 25 is characterized in that the co-stimulatory domain comprises the amino acid sequence shown in SEQ ID NO:97. 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. The chimeric antigen receptor according to any one of claims 1-27 is 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. The chimeric antigen receptor according to any one of claims 1 to 28, characterized in that the chimeric antigen receptor further comprises a signal peptide covalently linked to the N-terminus of the extracellular antigen binding domain. The chimeric antigen receptor according to any one of claims 1-29 is characterized in that the signal peptide comprises the amino acid sequence shown in SEQ ID NO:91. 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. The nucleic acid molecule according to claim 31 is characterized in that the nucleic acid molecule comprises a nucleotide sequence shown in any one of SEQ ID NO: 109-118 or SEQ ID NO: 160-163. An expression vector, characterized in that the expression vector carries the nucleic acid molecule according to claim 31 or 32. The expression vector according to claim 33 is characterized in that the expression vector is a γ-retroviral vector or a lentiviral vector. An immune effector cell, characterized in that the immune cell comprises the chimeric antigen receptor described in any one of claims 1-30, the nucleic acid molecule described in claim 31 or 32, or the expression vector described in claim 33 or 34. The immune effector cell according to claim 35 is characterized in that 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 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. A method for preparing immune effector cells, characterized in that the method comprises introducing the expression vector of claim 33 or 34 into the immune effector cells. A composition, characterized in that it comprises the immune effector cells according to claim 35 or 36 and a pharmaceutically acceptable carrier. Use of the chimeric antigen receptor according to any one of claims 1 to 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 treating a disease or condition associated with the expression of Ly6G6D; the disease or condition associated with the expression of Ly6G6D is colorectal cancer. A medicine kit for treating cancer, characterized in that the medicine kit comprises the immune effector cells according to claim 35 or 36. The drug kit according to claim 40 is characterized in that the drug kit also includes instructions for using the immune response cells to treat cancer subjects. The drug kit according to claim 41, characterized in that the cancer is colorectal cancer.