Construction of CAR-T cells targeting TSHR, using the natural protein TSH as the antigen-binding site

Abstract

The present invention provides CAR-T cells constructed based on the natural protein TSH. The present invention also provides chimeric antigen receptor (TAR) CAR-T cells constructed based on the tandem structure of the TSH α and β subunits, and uses thereof. The TSHαβ-CAR-T cells of the present invention can target and kill TSHR-positive thyroid cancer cells and are non-immunogenic, making them promising candidates for the treatment of thyroid cancer.

Description

[Technical Field]

[0001] The present invention relates to the field of biomedicine. Specifically, the present invention relates to CAR-T cells constructed based on the natural protein TSH. [Background technology]

[0002] CAR-T cell immunotherapy has achieved breakthroughs in hematological malignancies, with a variety of cell-based therapeutics currently being used in clinical trials. However, in the treatment of solid tumors, issues such as a lack of effective antigen targets and short survival times for CAR-T cells result in poor therapeutic efficacy. The immunogenicity of CAR molecules, leading to elimination of CAR-T cells by the patient's immune system, is a problem that exists in both hematological and solid tumor treatments, significantly reducing the therapeutic efficacy and ability of CAR-T cells to prevent tumor recurrence. Therefore, to improve the therapeutic efficacy of CAR-T cells in solid tumors, it is important to identify new targets and construct non-immunogenic CAR molecules.

[0003] Thyroid cancer is a common malignant tumor of the endocrine system. Its incidence has been rapidly increasing worldwide in recent years. Conventional treatments, such as surgical and radioactive iodine therapy, produce favorable therapeutic outcomes in most patients, but approximately 10% of patients still die due to tumor metastasis or lack of response to conventional treatments. Cellular immunotherapy may offer new possibilities for these patients. Therefore, the discovery of antigen targets for thyroid cancer and the development of CAR-T cell therapy are of great clinical significance. The thyroid-stimulating hormone receptor (TSHR) is primarily expressed on the surface of thyroid cells, is highly expressed in thyroid cancer cells, and has been shown to be a potential therapeutic target for thyroid-related diseases. CAR-T cells targeting the TSHR can effectively kill thyroid tumor cells.

[0004] Recent studies have shown that CAR-T cells constructed based on the scFv sequence of TSHR monoclonal antibodies can effectively kill TSHR-positive thyroid cancer cells, demonstrating the feasibility of targeting TSHR to treat thyroid cancer. However, problems remain in CAR-T cell therapy, such as the inability of CAR-T cells to persist in the body due to the immunogenicity of CARs.

[0005] Therefore, there is a need in the art to develop non-immunogenic CAR immune cells that target the TSHR. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide non-immunogenic CAR immune cells that target the TSHR. [Means for solving the problem]

[0007] In a first aspect of the present invention, there is provided a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular antigen-binding domain that targets TSHR, and the extracellular antigen-binding domain has the structure shown in formula (I) below. Tα-L-Tβ (I) (where, each "-" is independently a linker peptide or a peptide bond; Tα is the α subunit of the TSH protein, or a fragment thereof; Tβ is the β subunit of the TSH protein, or a fragment thereof; L is either absent or a linker peptide.

[0008] In another preferred example, the registration number of the TSH protein alpha subunit is NM_001303177.1.

[0009] In another preferred example, the registration number of the TSH protein β subunit is NM_000549.5.

[0010] In another preferred example, the TSH protein α-subunit fragment is a fragment obtained by truncating 0 to 10 amino acids from the N-terminus and / or 0 to 10 amino acids from the C-terminus of the TSH protein α-subunit shown in SEQ ID NO: 2.

[0011] In another preferred example, the TSH protein α-subunit fragment is a fragment obtained by truncating 1 to 10 amino acids, preferably 1 to 6 amino acids, and more preferably 1 to 2 amino acids, from the C-terminus of the TSH protein α-subunit shown in SEQ ID NO: 2.

[0012] In another preferred example, the amino acid sequence of the Tα is set forth in SEQ ID NO: 2 or SEQ ID NO: 19, or has a sequence identity of ≥ 85% (preferably ≥ 90%, more preferably ≥ 95%, e.g. ≥ 96%, ≥ 97%, ≥ 98% or ≥ 99%) with the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 19, and has the same or substantially the same binding function.

[0013] In another preferred example, the TSH protein β-subunit fragment is a fragment obtained by truncating 0 to 10 amino acids from the N-terminus and / or 0 to 20 amino acids from the C-terminus of the TSH protein β-subunit shown in SEQ ID NO: 4.

[0014] In another preferred example, the TSH protein β-subunit fragment is a fragment obtained by truncating 1 to 20 amino acids, preferably 1 to 16, 1 to 14, or 1 to 12 amino acids, from the C-terminus of the TSH protein β-subunit shown in SEQ ID NO: 4.

[0015] In another preferred example, the amino acid sequence of the Tβ is set forth in SEQ ID NO: 4, 20, 21 or 22, or has a sequence identity of ≥85% (preferably ≥90%, more preferably ≥95%, e.g., ≥96%, ≥97%, ≥98% or ≥99%) to the sequence set forth in SEQ ID NO: 4, 20, 21 or 22, and has the same or substantially the same binding function.

[0016] In another preferred example, L is a flexible linker peptide.

[0017] In another preferred example, the L sequence is (G4S)n, where n is an integer selected from 1 to 6, and preferably n is 2, 3, or 4.

[0018] In another preferred embodiment, the L sequence is shown in SEQ ID NO:32.

[0019] In another preferred example, the extracellular antigen-binding domain of the CAR further comprises a second extracellular domain directed against an additional target.

[0020] In another preferred embodiment, said additional target is a tumor-specific target, preferably a thyroid cancer-specific target.

[0021] In another preferred embodiment, the structure of the CAR is shown in Formula II below: S-EB-FH-TM-C-CD3ζ (II) (In the formula, each "-" is independently a linker peptide or a peptide bond; S is either absent or a signal peptide sequence, EB is the extracellular antigen-binding domain; H is either absent or a hinge region; TM is the transmembrane domain, C is either absent or an intracellular costimulatory domain; CD3ζ is the intracellular domain coding sequence derived from CD3ζ, F is either none or a marker protein.)

[0022] In another preferred example, the marker protein F may be detected directly or indirectly. In another preferred example, the CAR may be detected by an antibody that specifically targets the marker protein F.

[0023] In another preferred embodiment, the amino acid sequence of the marker protein F is shown in SEQ ID NO:31.

[0024] In another preferred example, S is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0025] In another preferred example, the H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, or a combination thereof.

[0026] In another preferred example, the H is a hinge region derived from CD8α.

[0027] In another preferred example, the TM is a transmembrane region of a protein selected from the group consisting of CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0028] In another preferred embodiment, the TM is a transmembrane region derived from CD8α.

[0029] In another preferred embodiment, the amino acid sequence of the H-TM is shown in SEQ ID NO:6.

[0030] In another preferred example, C is an intracellular costimulatory domain of a protein selected from the group consisting of OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or a combination thereof.

[0031] In another preferred example, C is an intracellular costimulatory domain derived from 4-1BB or CD28.

[0032] In another preferred example, the amino acid sequence of C is shown in SEQ ID NO: 8 or 10.

[0033] In another preferred example, the amino acid sequence of the CD3ζ-derived intracellular domain coding sequence is shown in SEQ ID NO:12.

[0034] In another preferred example, the amino acid sequence of said chimeric antigen receptor CAR is set forth in SEQ ID NO: 14, 16, 24, 26, 28 or 30, or has ≥85% (preferably ≥90%, more preferably ≥95%, such as ≥96%, ≥97%, ≥98% or ≥99%) sequence identity to the sequence set forth in SEQ ID NO: 14, 16, 24, 26, 28 or 30.

[0035] In a second aspect of the invention, there is provided a nucleic acid molecule encoding a chimeric antigen receptor according to the first aspect of the invention.

[0036] In another preferred embodiment, the nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 13, 15, 23, 25, 27 or 29.

[0037] In a third aspect of the invention, there is provided a vector containing a nucleic acid molecule according to the second aspect of the invention.

[0038] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, a retroviral vector, a transposon, or a combination thereof.

[0039] In another preferred embodiment, the vector is a retroviral vector, preferably an SFG retroviral vector.

[0040] In another preferred example, the vector further comprises one selected from the group consisting of a promoter, a transcription enhancer element WPRE, a long terminal repeat sequence LTR, and the like.

[0041] In another preferred embodiment, the vector comprises the nucleotide sequence shown in SEQ ID NO: 13, 15, 23, 25, 27 or 29.

[0042] In a fourth aspect of the invention, there is provided a host cell which contains a vector according to the third aspect of the invention, or which has an exogenous nucleic acid molecule according to the second aspect of the invention integrated into its chromosome, or which expresses a CAR according to the first aspect of the invention.

[0043] In a fifth aspect of the present invention, there is provided an engineered immune cell which contains a vector according to the third aspect of the invention, or has an exogenous nucleic acid molecule according to the second aspect of the invention integrated into its chromosome, or which expresses a CAR according to the first aspect of the invention.

[0044] In another preferred embodiment, the engineered immune cells are selected from the group consisting of T cells, NK cells, NKT cells, macrophages, or a combination thereof.

[0045] In another preferred example, the engineered immune cells are chimeric antigen receptor T cells (CAR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).

[0046] In another preferred embodiment, the engineered immune cells are CAR-T cells.

[0047] In a sixth aspect of the present invention there is provided a method of producing an engineered immune cell according to the fifth aspect of the invention, the method comprising the step of transducing an immune cell with a nucleic acid molecule according to the second aspect of the invention or a vector according to the third aspect of the invention to obtain said engineered immune cell.

[0048] In another preferred example, the method further comprises a step of detecting the function and efficacy of the resulting engineered immune cells.

[0049] In a seventh aspect of the invention, there is provided a pharmaceutical composition comprising a CAR according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, and / or an engineered immune cell according to the fifth aspect of the invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0050] In another preferred embodiment, the formulation is a liquid formulation.

[0051] In another preferred example, the dosage form of the preparation is an injection.

[0052] In another preferred embodiment, the concentration of the engineered immune cells in the formulation is 1 x 10 3 ~1×10 8 cells / ml, preferably 1 x 10 4 ~1×10 7 cells / ml.

[0053] In an eighth aspect of the present invention, there is provided the use of a CAR according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, or a host cell according to the fourth aspect of the invention, and / or an engineered immune cell according to the fifth aspect of the invention in the manufacture of a medicament or formulation for preventing and / or treating a disease associated with high TSHR expression.

[0054] In another preferred embodiment, the disease associated with high expression of TSHR includes tumors, Graves' disease, or a combination thereof.

[0055] In another preferred example, the disease associated with high expression of TSHR is cancer or tumor, for example, thyroid tumor.

[0056] In a ninth aspect of the present invention, there is provided use of the engineered immune cells according to the fifth aspect of the present invention or the pharmaceutical composition according to the seventh aspect of the present invention for the prevention and / or treatment of diseases associated with high expression of TSHR.

[0057] In another preferred embodiment, the disease associated with high expression of TSHR includes tumors, Graves' disease, or a combination thereof.

[0058] In another preferred example, the disease associated with high expression of TSHR is cancer or tumor, such as thyroid cancer.

[0059] In a tenth aspect of the present invention, there is provided a method for treating a disease associated with high TSHR expression, the method comprising administering to a subject in need thereof an effective amount of an engineered immune cell according to the fifth aspect of the present invention or a pharmaceutical composition according to the seventh aspect of the present invention.

[0060] In another preferred example, the disease associated with high expression of TSHR is cancer or tumor, Graves' disease, or a combination thereof.

[0061] In another preferred embodiment, the disease is cancer or a tumor, such as thyroid cancer.

[0062] In another preferred example, the engineered immune cells or CAR immune cells comprised in the pharmaceutical composition are cells derived from the subject (autologous cells).

[0063] In another preferred example, the engineered immune cells or CAR immune cells comprised in the pharmaceutical composition are cells derived from a healthy individual (allogeneic cells).

[0064] In another preferred embodiment, the method may be used in combination with other therapeutic methods.

[0065] In another preferred example, the other therapeutic method includes chemotherapy, radiotherapy, targeted therapy, and the like.

[0066] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features detailed below (such as in the Examples) can be combined with each other to form new or preferred technical solutions, but due to space limitations, they will not be listed one by one here. [Brief explanation of the drawings]

[0067] The following drawings are used to illustrate specific embodiments of the present invention and are not used to limit the scope of the present invention as defined by the claims. [Figure 1] 1 shows the results of constructing a thyroid cancer cell line that overexpresses TSHR. [Figure 2]Schematic diagrams of the structures of TSHαβ-CAR and TSHβα-CAR, and their killing activity against TSHR-overexpressing thyroid cancer cells KTC-1 are shown. (A) Schematic diagrams of the structures of TSHαβ-CD28ζ-CAR and TSHβα-CD28ζ-CAR. SP indicates the signal peptide sequence, L indicates the (G4S)3 linker peptide sequence, F indicates the Flag tag sequence, Hinge and TM indicate the hinge and transmembrane domains of CD8α, respectively, ICD indicates the intracellular costimulatory domain of CD28, and CD3ζ indicates the intracellular domain of CD3ζ. (B) Expression of TSHαβ-CAR and TSHβα-CAR on T cells was detected by flow cytometry after staining with anti-Flag antibody. (C) The ratio of T cells (CD3+) to residual tumor cells (GFP+) detected by flow cytometry is shown after NT, TSHαβ-CAR-T, and TSHβα-CAR-T cells were cocultured with the TSHR-overexpressing thyroid cancer cell line KTC-1 at an effector-target ratio of 1:5 for 5 days. [Figure 3] The schematic structure of TSHαβ-CAR and its T cell infection efficiency are shown. (A) Schematic structure of TSHαβ-CD28ζ-CAR and TSHαβ-4-1BBζ-CAR. SP indicates the signal peptide sequence, L indicates the (G4S)3 linker peptide sequence, F indicates the Flag tag sequence, Hinge and TM indicate the hinge and transmembrane domains of CD8α, respectively, ICD indicates the intracellular costimulatory domain of CD28 or 4-1BB, and CD3ζ indicates the intracellular domain of CD3ζ. (B) Expression of TSHαβ-CAR on T cells was detected by flow cytometry after staining with anti-Flag antibody. (C) Statistical results of the infection rate of T cells from various volunteers infected with viruses expressing TSHαβ-CAR. [Figure 4]This shows that TSHαβ-CAR-T cells specifically kill TSHR-overexpressing thyroid cancer cell lines. (A) NT cells and TSHαβ-CAR-T cells were co-cultured with control and TSHR-overexpressing thyroid cancer cell lines at an effector-target ratio of 1:5 for 5 days, and the ratio of T cells (CD3+) to residual tumor cells (GFP+) was detected by flow cytometry. (B) Statistical results of the percentage of residual tumor cells in each group after the co-culture experiment. [Figure 5] We demonstrate that TSHαβ-CAR-T cells release large amounts of the cytokines IFN-γ and IL-2 when killing TSHR-positive tumor cells. NT cells and TSHαβ-CAR-T cells were co-cultured with control and TSHR-overexpressing thyroid cancer cell lines at an effector-target ratio of 1:5 for 24 hours, respectively, and the co-culture supernatants were collected and the levels of IFN-γ and IL-2 secreted in the supernatants were detected by ELISA. [Figure 6] Schematic diagrams of the structures of various truncated TSHαβ-CAR mutants and their killing activity against TSHR-overexpressing thyroid cancer cells FTC-133 and KTC-1 are shown. (A) Schematic diagram of the structure of TSHαβ-CAR truncated mutants, where SP represents the signal peptide sequence, F represents the Flag tag sequence, Hinge and TM represent the CD8 hinge and transmembrane domains, respectively, ICD represents the intracellular costimulatory domain of CD28, and CD3ζ represents the intracellular domain of CD3ζ. The numbers in parentheses indicate the corresponding TSHα or TSHβ amino acid segments. (B) Expression of each truncated TSHαβ-CAR on T cells was detected by flow cytometry after staining with anti-Flag antibody. (C) T cells expressing truncated TSHαβ-CAR are shown. These T cells were co-cultured with thyroid cancer cell lines FTC-133 and KTC-1, which overexpress TSHR, at an effector-target ratio of 1:5 for 5 days, and then the ratio of T cells (CD3+) to residual tumor cells (GFP=) was detected using a flow cytometer. DETAILED DESCRIPTION OF THE INVENTION

[0068] After extensive and thorough research, the present inventors have developed the first CAR-T cells based on the natural protein TSH. The natural TSHR-based ligand of the present invention uses the TSH α and β subunits to construct non-immunogenic second-generation CAR-T cells (TSHαβ-CAR-T) with a specific structure. In vitro experiments have demonstrated that the TSHαβ-CAR-T cells of the present invention can target and kill TSHR-positive thyroid cancer cells. Furthermore, truncation mutations of TSHα and TSHβ (e.g., C-terminal truncation mutations) do not affect TSHR recognition and binding in the antigen-binding domain. CAR-T cells containing the truncated mutations can still recognize and kill tumor cells. Based on this, the present invention was completed. term

[0069] In order to make the present invention more readily understandable, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art. Before describing the present invention, it should be understood that the present invention is not limited to the particular methods and experimental conditions described, as these methods and conditions may vary. It should also be understood that the terms used herein are intended only to describe particular embodiments and are not limiting. The scope of the present invention is limited only by the appended claims.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art.As used herein, when referring to a specific value, the term "about" means that this value can vary within 1% from the value mentioned.For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (for example, 99.1, 99.2, 99.3, 99.4, etc.).

[0071] As used herein, the terms "optional" or "optionally" mean that the described event or circumstance may occur, but does not necessarily have to occur.

[0072] As used herein, the term "containing" or "comprising" can be either open, semi-closed, or closed. In other words, the term also includes "consisting essentially of" or "consisting of."

[0073] The term "about" may refer to a value or composition that is within an acceptable error range of a particular value or composition as determined by one of ordinary skill in the art, which depends in part on how the value or composition is measured or determined.

[0074] The term "administration" refers to the physical introduction of a product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration such as injection or infusion.

[0075] "Transduction," "transfection," and "transformation" refer to the process of introducing an exogenous polynucleotide into a host cell where it is transcribed and translated to produce a polypeptide product, and include the use of plasmids to introduce exogenous polynucleotides into host cells (e.g., E. coli).

[0076] "Gene expression" or "expression" refers to the process by which a gene is transcribed, translated, and post-translationally modified to produce either the RNA or protein product of the gene.

[0077] "Polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxynucleotides (DNA), ribonucleotides (RNA), hybrid sequences thereof, and analogs. Polynucleotides may also include modified nucleotides, such as methylated or capped nucleotides, or nucleotide analogs. As used herein, the term "polynucleotide" refers interchangeably to single-stranded and double-stranded molecules. Unless otherwise specified, the polynucleotides in any example described herein include double-stranded forms and two complementary single strands that are known or predicted to form a double-stranded form.

[0078] Conservative amino acid substitutions are known in the art. In some embodiments, the amino acids to be substituted are one or more of glycine, alanine; valine, isoleucine, leucine, and proline; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine, lysine, arginine, and histidine; and / or phenylalanine, tryptophan, and tyrosine; methionine, and cysteine. Furthermore, the present invention also provides non-conservative amino acid substitutions, which allow for the substitution of amino acids from different groups. TSHR and TSH

[0079] TSHR is a glycoprotein receptor expressed primarily on the cell membrane surface of thyroid cells and plays an important regulatory role in physiological processes such as thyroid growth, differentiation, and thyroid hormone secretion. In pathological conditions, TSHR is a potential therapeutic target for Graves' disease. TSHR is also consistently expressed in the majority of differentiated thyroid cancer cells. In vivo experiments using mice have shown that TSHR-mediated growth signals are required for metastasis of differentiated thyroid cancer cells. Clinical specimens have also shown that primary and metastatic thyroid cancer lesions are TSHR-positive. These results suggest that TSHR may be a potential therapeutic target for differentiated thyroid cancer.

[0080] As used herein, the terms "TSH protein alpha subunit" and "TSHα" both refer to the alpha subunit of the thyroid-stimulating hormone (TSH) protein. The terms "TSH protein beta subunit" and "TSHβ" both refer to the beta subunit of the thyroid-stimulating hormone (TSH) protein. Thyroid-stimulating hormone (TSH) is a hormone secreted by the pituitary gland. TSH is a glycoprotein consisting of two non-covalently linked subunits, the alpha subunit of which is common to such hormones, while the beta subunit is unique to each. Chimeric antigen receptor (CAR)

[0081] Chimeric antigen receptors (CARs) consist of an extracellular antigen recognition domain, a transmembrane domain, and an intracellular costimulatory signal transduction domain.

[0082] CAR design has progressed through the following process. First-generation CARs contained only CD3ζ or FcγRI molecules as intracellular signaling components. Because they contained only a single intracellular activation domain, they only induced short-term T cell proliferation and relatively low cytokine secretion, failing to provide long-term T cell proliferation signals and sustained antitumor effects in vivo, resulting in poor clinical efficacy. Second-generation CARs, based on the traditional CAR structure, incorporated costimulatory molecules such as CD28, 4-1BB, OX40, and ICOS. Compared to first-generation CARs, their functionality was significantly improved, further enhancing the persistence of CAR-T cells and their ability to kill tumor cells. Third- and fourth-generation CARs were developed based on second-generation CARs, with new immune costimulatory molecules such as CD27 and CD134 sequentially tandemly connected.

[0083] The extracellular segment of the CAR recognizes a specific antigen and transmits the corresponding signal via the intracellular domain, resulting in cell activation, proliferation, cytotoxicity, and cytokine secretion, thereby eliminating the target cell. First, the patient's own cells (or allogeneic donor) are isolated, activated, and genetically modified to produce CAR immune cells, which are then infused back into the patient. This method significantly reduces the risk of graft-versus-host disease, and the antigen is recognized by immune cells in a non-MHC-restricted manner.

[0084] CAR-immune cell therapy has achieved extremely high clinical response rates in the treatment of hematopoietic malignancies that are unmatched by any conventional therapeutic approach, and clinical studies are rapidly expanding worldwide.

[0085] Specifically, the chimeric antigen receptor (CAR) of the present invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain.

[0086] The extracellular domain contains a specific antigen-binding domain, which may be a natural sequence or a derivative thereof that is responsible for specific ligand-receptor binding.

[0087] The CAR-T cells of the present invention are constructed by tandemly expressing two TSH subunits, using TSH as the antigen-binding site instead of the TSHR monoclonal antibody scFv sequence. The present invention successfully constructed CAR-T cells expressing a CAR that tandemly expresses the TSH α and β subunits, and in vitro experiments confirmed that they can specifically target and kill TSHR-positive thyroid cancer cells. In conventional CAR molecules, the scFv sequence that recognizes and binds to antigens is typically the variable region sequence of a mouse monoclonal antibody, which is immunogenic in the human body. Therefore, when CAR-T cells are infused into a patient's body, they are easily eliminated by the patient's immune system, resulting in reduced therapeutic efficacy and tumor recurrence. In contrast, TSH, a naturally occurring protein in the human body, has no immunogenicity, effectively circumventing the immunogenicity issues of CAR molecules, better promoting CAR-T cell survival in the body, and providing a long-term monitoring role to prevent tumor recurrence.

[0088] In the present invention, the antigen-binding domain of the chimeric antigen receptor comprises a TSH protein α subunit (or a fragment thereof) and a β subunit (or a fragment thereof) linked in tandem, and comprises the structure shown in formula (I) below. Tα-L-Tβ(I) (wherein each "-" is independently a linker peptide or a peptide bond; Tα is the α subunit of the TSH protein, or a fragment thereof; Tβ is the β subunit of the TSH protein, or a fragment thereof; L is either absent or a linker peptide.

[0089] In one embodiment, the TSHα and / or TSHβ in the antigen-binding domain may be truncated, e.g., by 1 to 20 amino acids from the C-terminus. Such truncation mutations do not affect or substantially affect the recognition and binding of TSHR by the antigen-binding domain. CAR-T cells constructed based on these truncated variants can still recognize and kill tumor cells.

[0090] In some embodiments, the TSHα fragment can be obtained by truncating 0 to 10 amino acids from the N-terminus and / or 0 to 10 amino acids from the C-terminus of the full-length TSHα sequence shown in SEQ ID NO: 2. For example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be truncated from the N-terminus and / or 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be truncated from the C-terminus.

[0091] In some embodiments, the TSHβ fragment can be obtained by truncating 0 to 10 amino acids from the N-terminus and / or 0 to 20 amino acids from the C-terminus of the full-length TSHβ sequence shown in SEQ ID NO: 4. For example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids can be truncated from the N-terminus and / or 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 amino acids can be truncated from the N-terminus.

[0092] The intracellular domain contains the costimulatory signaling region and the zeta chain portion. The costimulatory signaling region refers to the portion of the intracellular domain that contains the costimulatory molecule. Costimulatory molecules are not antigen receptors or their ligands, but are cell surface molecules required for lymphocytes to respond effectively to antigens.

[0093] A linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that serves to connect the transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain. The linker may contain 0 to 300 amino acids, preferably 2 to 100 amino acids, and most preferably 3 to 50 amino acids.

[0094] When expressed in T cells, the CAR of the present invention can recognize antigens based on antigen-binding specificity. When bound to its cognate antigen, it affects tumor cells, suppressing their growth, promoting their death, or otherwise affecting tumor cells, thereby reducing or eliminating the tumor burden in patients. The antigen-binding domain is preferably fused with one or more intracellular domains derived from costimulatory molecules and the zeta chain. Preferably, the antigen-binding domain is fused with the intracellular domain of a combination of the CD28 or 4-1BB costimulatory domain and the CD3 zeta signaling domain.

[0095] In the present invention, the subunit elements constituting the extracellular antigen-binding domain of the present invention also include conservative variants based on the sequence, which means that a polypeptide is formed in which, compared to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4, up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids are substituted with amino acids having similar or close properties.

[0096] In the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably 40% or less, more preferably 35% or less, more preferably 1 to 33%, more preferably 5 to 30%, more preferably 10 to 25%, and more preferably 15 to 20% of the total number of amino acids in the initial amino acid sequence.

[0097] In the present invention, the number of added, deleted, modified and / or substituted amino acids is usually 1, 2, 3, 4 or 5, preferably 1 to 3, more preferably 1 to 2, and most preferably 1.

[0098] Regarding the hinge region and transmembrane region (transmembrane domain), the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR. In one embodiment, a naturally associated transmembrane domain is used for one of the domains in the CAR. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interaction with other members of the receptor complex. Chimeric antigen receptor immune cells (CAR immune cells)

[0099] In accordance with the present invention, there is provided a chimeric antigen receptor immune cell comprising a chimeric antigen receptor according to the first aspect of the present invention.

[0100] The chimeric antigen receptor immune cells of the present invention may be CAR-T cells, CAR-NK cells, or CAR macrophages. Preferably, the chimeric antigen receptor immune cells of the present invention are CAR-T cells.

[0101] As used herein, the terms "CAR-T cells", "CAR-T", and "CAR-T cells of the invention" all refer to CAR-T cells according to the fifth aspect of the invention.

[0102] CAR-T cells have the following advantages over other T cell-based therapies: (1) The action of CAR-T cells is not restricted by MHC. (2) Because many tumor cells express the same tumor markers, once a CAR gene for a specific tumor marker is constructed, it can be widely used. (3) CARs can utilize tumor protein markers as well as glycolipid non-protein markers, broadening the range of tumor marker targets. (4) Because the patient's own cells are used, the risk of rejection is reduced. (5) CAR-T cells have immune memory function and can survive for a long period in the body.

[0103] As used herein, the terms "CAR-NK cells," "CAR-NK," and "CAR-NK cells of the present invention" all refer to the CAR-NK cells described in the fifth aspect of the present invention. The CAR-NK cells of the present invention can be used for TSHR-high-expressing tumors.

[0104] Natural killer (NK) cells are major immune effector cells that protect the body from viral infections and tumor cell infiltration through non-antigen-specific pathways. Engineered NK cells may acquire new functions, such as the ability to specifically recognize tumor antigens and improved antitumor cytotoxicity.

[0105] Compared to CAR-T cells, CAR-NK cells have the following advantages: (1) they directly kill tumor cells by releasing perforin and granzymes, but do not kill normal cells in the body; (2) they release very small amounts of cytokines, reducing the risk of cytokine storms; and (3) they are very easy to amplify outside the body, allowing them to be developed as "off-the-shelf" products. Other than these, they are similar to CAR-T cell therapy. vector

[0106] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, such as by screening libraries from cells expressing the gene, by obtaining the gene from a vector known to contain the gene, or by direct isolation from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be produced synthetically.

[0107] The present invention also provides vectors containing the nucleic acid molecules of the present invention. Retrovirus-derived vectors, such as lentiviruses, are suitable tools for long-term gene transfer because they allow long-term and stable integration of transgenes and their propagation in daughter cells. Lentivirus vectors have the advantage over oncoretrovirus-derived vectors, such as murine leukemia viruses, of being able to transduce non-proliferating cells, such as hepatocytes. Another advantage is their low immunogenicity.

[0108] Briefly, the expression cassette or nucleic acid sequence of the present invention is usually operably linked to a promoter and incorporated into an expression vector. Such vectors are suitable for replication and integration into eukaryotic cells. A typical cloning vector contains transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0109] The expression construct of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols.Gene delivery methods are known in the art.See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466.The entire texts of these patents are incorporated herein by reference.In another embodiment, the present invention provides a gene therapy vector.

[0110] The nucleic acid can be cloned into various types of vectors. For example, the nucleic acid may be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0111] Furthermore, the expression vector can be provided to cells in the form of a viral vector.Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.Suitable vectors usually contain an origin of replication that functions in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selection markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0112] Many virus-based systems have been developed for gene transfer into mammalian cells.For example, retrovirus provides a convenient platform for gene delivery system.Selected gene can be inserted into vector and packaged into retroviral particle using technology known in the art.The recombinant virus is then isolated and delivered to target cells in vivo or ex vivo.Many retroviral systems are known in the art.In some embodiments, adenoviral vectors are used.Many adenoviral vectors are known in the art.In one embodiment, lentiviral vectors are used.

[0113] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. These are typically located 30–110 bp upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. Spacing between promoter elements is often flexible, allowing promoter function to be maintained even if one element is inverted or moved relative to another. In the thymidine kinase (TK) promoter, spacing between promoter elements can be increased to 50 bp before activity begins to decline. It has been shown that individual elements can initiate transcription either cooperatively or independently, depending on the promoter.

[0114] An example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of conferring high levels of expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is the elongation growth factor-1α (EF-1α) promoter. However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate-early promoter, Rous sarcoma virus promoter, and human gene promoters such as the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence operably linked to the inducible promoter when desired and turn off expression when undesired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0115] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may contain either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a cell population targeted for transfection or infection with a viral vector. In other embodiments, the selection marker may be carried on a separate DNA fragment and used in a cotransfection process. Both the selection marker gene and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selection markers include antibiotic resistance genes, such as neo.

[0116] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and encodes a polypeptide whose expression is clearly indicated by a readily detectable characteristic, such as enzymatic activity. After DNA is introduced into the recipient cells, reporter gene expression is measured at an appropriate time. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). In one embodiment of the present invention, the reporter gene is a gene encoding mKate2 red fluorescent protein. Suitable expression systems are well known and can be prepared using known techniques or commercially available. Typically, a promoter is identified as a construct with at least five flanking regions that produces the highest level of reporter gene expression. Such promoter regions can be linked to a reporter gene and used to assess the ability of agents to modulate promoter-driven transcription.

[0117] Methods for introducing genes into cells and expressing genes in cells are known in the art.Regarding expression vectors, they can be easily introduced into host cells such as mammalian cells, bacterial cells, yeast cells, insect cells, etc., by any method known in the art.For example, expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0118] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for preparing cells containing vectors and / or foreign nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0119] Biological methods for introducing target polynucleotides into host cells include the use of DNA vectors and RNA vectors.Virus vectors, especially retrovirus vectors, have become the most widely used method for gene insertion into mammalian, for example, human cells.Other virus vectors include lentivirus, poxvirus, herpes simplex virus type I, adenovirus, and adeno-associated virus.See, for example, U.S. Patent No. 5,350,674 and 5,585,362.

[0120] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes. An example of a colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle).

[0121] When a non-viral delivery system is used, an example of a delivery vehicle is a liposome. It is contemplated that lipid formulations are used to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with a lipid. The lipid-associated nucleic acid may be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with the lipid, bound to the lipid, contained as a suspension in the lipid, contained in or complexed with the micelle, or otherwise associated with the lipid. The lipids, lipid / DNA, or lipid / expression vectors associated with the present compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure as a micelle or a "collapsed" structure. Lipids may also be simply dispersed in solution, forming aggregates of heterogeneous size and shape. Lipids are fatty substances that can occur naturally or synthetically. For example, lipids include lipid droplets that occur naturally in the cytoplasm and are contained in compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0122] In a preferred embodiment of the invention, the vector is an adeno-associated virus vector. formulation

[0123] The present invention further provides a formulation containing a chimeric antigen receptor CAR according to the first aspect of the invention, a nucleic acid molecule according to the second aspect of the invention, a vector according to the third aspect of the invention, a host cell according to the fourth aspect of the invention, or an engineered immune cell according to the fifth aspect of the invention, and a pharmaceutically acceptable carrier, diluent, or excipient. In one embodiment, the formulation is a liquid formulation. Preferably, the formulation is an injectable formulation. Preferably, the concentration of the CAR-T cells in the formulation is 1 x 10 3 ~1×10 8 cells / ml, more preferably 1 x 10 4 ~1×10 7 cells / ml.

[0124] In one embodiment, the formulation may include a buffer such as neutral buffered saline or sulfate buffered saline; a carbohydrate such as glucose, mannose, sucrose, dextran, or mannitol; a protein; a polypeptide or amino acid such as glycine; an antioxidant; a chelating agent such as EDTA or glutathione; an adjuvant (e.g., aluminum hydroxide); and a preservative. The formulation of the present invention is preferably formulated for intravenous administration. therapeutic use

[0125] The present invention encompasses the therapeutic use of cells (e.g., T cells) transduced with a lentiviral vector (LV) encoding an expression cassette of the present invention. The transduced T cells target the tumor cell marker TSHR, synergistically activating T cells and inducing an immune response in immune cells, thereby significantly improving the efficiency of tumor cell killing.

[0126] Accordingly, the present invention also provides a method of stimulating a T cell-mediated immune response against a target cell population or tissue in a mammal, comprising administering to the mammal a CAR cell of the present invention.

[0127] In one embodiment, the present invention involves cell therapy in which a patient's autologous T cells (or allogeneic donor) are isolated, activated, and genetically modified to produce CAR-T cells, which are then infused back into the same patient. This approach greatly reduces the risk of graft-versus-host disease because the antigen is recognized by the T cells in an MHC-free manner. Furthermore, a single CAR-T cell can treat all cancers that express that antigen. Unlike antibody therapies, CAR-T cells can replicate in the body, providing long-term persistence leading to sustained tumor control.

[0128] In one embodiment, the CAR-T cells of the present invention can induce robust T cell proliferation in vivo and persist for a long period of time. CAR-mediated immune responses can also be used as part of adoptive immunotherapy, in which CAR-modified T cells induce immune responses specific to the antigen-binding domain of the CAR. For example, CAR-T cells targeting TSHR induce specific immune responses against cells expressing TSHR.

[0129] Although the data disclosed herein specifically discloses adeno-associated viral vectors comprising tandem TSH protein α subunits and TSH protein β subunits, hinge and transmembrane regions, and CD28 or 4-1BB and CD3ζ signaling domains, the invention should be construed to include any number of variations in the components of each construct.

[0130] The cancers to be treated include non-vascularized or substantially non-vascularized tumors, as well as vascularized tumors. Cancers include non-solid tumors (e.g., hematopoietic tumors such as leukemia and lymphoma) and solid tumors. The types of cancers that can be treated with the CAR of the present invention include, but are not limited to, carcinoma, blastoma, sarcoma, certain leukemias or lymphomas, benign and malignant tumors, and malignant tumors such as sarcoma, carcinoma, and melanoma. Also included are adult tumors / cancers and pediatric tumors / cancers.

[0131] The CAR-modified T cells of the present invention can also be used as vaccines for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0132] For ex vivo immunization, at least one of the following is performed in vitro before the cells are administered to a mammal: i) expansion of the cells, ii) introduction of a nucleic acid encoding a CAR into the cells, and / or iii) cryopreservation of the cells.

[0133] Ex vivo processes are well known in the art and will be discussed in more detail below. Briefly, cells are isolated from a mammal (preferably human) and genetically modified (i.e., in vitro transduction or transfection) with a vector expressing a CAR disclosed herein. The CAR-modified cells can be administered to a mammalian recipient to achieve a therapeutic effect. The mammalian recipient may be human, and the CAR-modified cells may be autologous to the recipient. Optionally, the cells may be allogeneic, syngeneic, or xenogeneic to the recipient.

[0134] In addition to using cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0135] The present invention provides a method of treating a tumor, comprising administering a therapeutically effective amount of a CAR-modified T cell of the present invention to a subject in need thereof.

[0136] The CAR-modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with other components, such as diluents and / or IL-2, IL-17, or other cytokines or cell populations. Briefly, the pharmaceutical compositions of the present invention may comprise a combination of the target cell populations described herein and one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline and sulfate buffered saline; carbohydrates such as glucose, mannose, sucrose, dextran, and mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0137] The pharmaceutical composition of the present invention can be administered in an appropriate manner depending on the disease to be treated (or prevented). The dosage and frequency of administration are determined by factors such as the condition of the patient and the type and severity of the patient's disease, and the appropriate dosage can be determined by clinical trials.

[0138] When the term "effective amount," "immunologically effective amount," "antitumor effective amount," "tumor-suppressing effective amount," or "therapeutic amount" is used, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences such as the age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). Typically, the pharmaceutical compositions comprising the T cells described herein are administered in an amount of 10 4 ~10 9 cells / kg body weight, preferably 10 5 ~10 6The T cell compositions can be administered at a dose of 1000 cells / kg body weight (including all integer values ​​within these ranges). The T cell compositions can also be administered multiple times at these doses. The cells can be administered using injection techniques well known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by a medical technician by monitoring the patient for signs of disease and adjusting treatment accordingly.

[0139] The compositions of the present invention can be administered by any convenient method, including nebulization, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously (i.v.), or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by intravenous injection. The T cell compositions can be injected directly into a tumor, lymph node, or site of infection.

[0140] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to patients in combination with (e.g., before, concurrently with, or after) any number of relevant therapeutic modalities, including, but not limited to, treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known as ARA-C), or natalizumab treatment for MS patients, efavirenz treatment for psoriasis patients, or other treatments for patients with certain tumors. In further embodiments, the T cells of the invention can be used in combination with chemotherapy, radiation therapy, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the invention are administered to patients in combination with (e.g., before, concurrently, or after) bone marrow transplantation, treatment with chemotherapeutic agents such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide. For example, in one embodiment, a subject may undergo standard treatment with high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, after transplant, the subject receives an infusion of expanded immune cells of the present invention. In additional embodiments, the expanded cells are administered before or after surgery.

[0141] The dosage of the above treatments administered to patients will vary depending on the exact nature of the condition being treated and the individual receiving the treatment. Dosage ratios for human administration can be determined according to art-accepted practices. Typically, 1 x 10 CAR-T cells of the present invention are administered in a single dose. 6 ~1×10 10 One can be administered to the patient for each treatment or course of treatment, for example, by intravenous infusion. The main advantages of the present invention are as follows:

[0142] 1) The CAR of the present invention is constructed based on the natural TSH protein and is non-immunogenic, effectively avoiding the immunogenicity problem of CAR molecules and better promoting the survival of CAR-T cells in the body.

[0143] 2) The present invention utilizes the tandem structure of specific TSH protein αβ subunits to construct a CAR that can specifically target TSHR and has excellent killing ability.

[0144] The present invention will be further described below with reference to specific examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. In the following examples, experimental methods for which specific conditions are not specified were generally carried out according to conventional conditions, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are by weight. material and method Plasmid construction

[0145] We synthesized the cDNA coding sequences for the two subunits of human TSH, TSHα (NM_001303177.1, aa 25-116) and TSHβ (NM_000549.5, aa 21-138), using gene synthesis technology. The two subunits were linked by a (G4S)3 sequence to obtain TSHαβ (TSHα-TSHβ ligation) or TSHβα (TSHβ-TSHα ligation). After digestion with NcoI and MluI, these were cloned into the SFG retroviral vector backbone to construct the SFG-TSHαβ-Flag-CD8-CD28-CD3ζ or SFG-TSHαβ-Flag-CD8-4-1BB-CD3ζ (TSHαβ-CAR) vectors, or the SFG-TSHβα-Flag-CD8-CD28-CD3ζ (TSHβα-CAR) vectors. Here, CD8 denotes the sequence encoding the hinge region and transmembrane domain of CD8α, CD28 denotes the sequence encoding the intracellular costimulatory domain of CD28, 4-1BB denotes the sequence encoding the intracellular costimulatory domain of 4-1BB, and CD3ζ denotes the sequence encoding the intracellular domain of CD3ζ. CAR expression was detected by adding Flag between the hinge region of TSHαβ or TSHβα and CD8α (Figures 2A and 3A). Construction of truncated mutant plasmids

[0146] Using the TSHαβ-CAR plasmid vector as a template, primers were designed to amplify the coding sequences of the TSHα or TSHβ subunits, removing the corresponding amino acids at the C-terminus. After digestion with NcoI and MluI, the sequences were assembled into the TSHαβ-CAR vector backbone to replace the full-length TSHα or TSHβ subunits, constructing the truncated mutant vectors. Preparation of retroviruses expressing TSHαβ-CAR or TSHβα-CAR

[0147] 2 x 10 the day before 6293T cells were seeded into a 10-cm dish and cultured for 16 hours. After the cells adhered, they were used for virus packaging. The specific steps were as follows: 30 μL of GeneJuice transfection reagent was added to 470 μL of serum-free IMDM medium, mixed thoroughly, and incubated at room temperature for 5 minutes. During this time, another 1.5 mL EP tube was prepared and mixed with 4 μg of TSHαβ-CAR or TSHβα-CAR plasmid, 4 μg of PeqPam-3, and 2 μg of RDF packaging plasmid. After incubation, IMDM containing GeneJuice transfection reagent was added to the plasmid tube, mixed thoroughly, and incubated at room temperature for 15 minutes. After incubation, the mixture was evenly added dropwise to the 293T cell culture dish and culture continued. After 48 hours, the virus-containing culture supernatant was collected, filtered through a 0.45 μm filter, aliquoted, and frozen at -80°C for further use. 12 mL of fresh IMDM complete medium was added to the culture dish, and the culture was continued. After 24 and 72 hours, the viral supernatant was collected, filtered, aliquoted, and frozen at -80°C for use. Preparation and in vitro expansion of CAR-T cells

[0148] PBMC lymphocytes were isolated from the peripheral blood of healthy individuals, and the isolated lymphocytes were plated in 24-well plates coated with 1 μg / mL of CD3 and CD28 antibodies at 1 × 10 6 The cells were added at a concentration of 1000 cells / well. After 24 hours of stimulation, IL7 (10 ng / mL) and IL15 (5 ng / mL) were added, and after another 24 hours of stimulation, the cells were harvested and counted. CAR-T cells were prepared by viral infection. The specific experimental steps are as follows: 1 mL of viral supernatant expressing TSHαβ-CAR or TSHβα-CAR was collected and added to a 24-well plate coated with RetroNectin the day before. The plate was centrifuged at 2000 g for 1.5 hours. After centrifugation, the supernatant was discarded, and 5 × 10 5Activated T cells were added to each well. The culture plate was placed in a 37°C incubator and cultured for 1 hour, then centrifuged at 1000 g for 10 minutes. After centrifugation, the plate was placed in a 37°C, 5% CO2 incubator and cultured. After 72 hours, the medium was replaced with fresh medium, and thereafter, the medium was replaced every two days. Four days after viral infection, 5 × 10 5 Cells were harvested and stained with anti-Flag antibody. Viral infection efficiency and CAR molecule expression were detected using a flow cytometer (Figures 2B-C and 3B-C). After 12 days, CAR-T cells were harvested and subjected to in vitro experiments to detect CAR-T cell killing function. Construction of target cells overexpressing TSHR

[0149] Existing literature has reported that TSHR is highly expressed in thyroid cancer tumor tissues, but its protein expression is barely detectable in existing thyroid cancer cell lines. Therefore, all current in vitro studies targeting TSHR have used overexpression to construct target cell models. We detected TSHR expression in the thyroid cancer cell lines KTC-1 and FTC-133, but not at the protein level. Therefore, it is believed that the exogenous TSHR gene was overexpressed in these cells by lentiviral infection. The cDNA coding sequence of the TSHR gene was amplified by PCR using tumor cell cDNA as a template. TSHR was then cloned into the pCDH-CMV-MCS-IRES-GFP vector after digestion with XbaI and EcoRI enzymes. 293T cells were used to package the virus and infect the thyroid cancer cell lines KTC-1 and FTC-133, respectively, to construct target cell lines stably overexpressing TSHR. A control cell line expressing only GFP was also constructed (Figure 1). In vitro co-culture detection of CAR-T cell killing function

[0150] Tumor cells were inoculated the day before into a 24-well plate at 2.5 x 10 cells per well. 5 After overnight culture, the cells adhered to the CAR +5 x 10 TSHαβ-CAR-T cells 4 Each well was added with a 1:5 effector-target ratio. Non-transduced (NT) cells were added to the control group, and the number of cells was the same as the total number of cells in the experimental group. After 5 days of co-culture, all T cells and tumor cells were collected. T cells were labeled with CD3 and tumor cells with GFP. Dead cells were removed using ZombieAqua Dye (Biolegend). The ratio of T cells to residual tumor cells was measured using a flow cytometer. ELISA detection of cytokine release

[0151] In the above-mentioned coculture bactericidal experiments, the culture supernatant was collected 24 hours after coculture, and the amounts of released cytokines in the coculture supernatant were detected using an ELISA kit (Mabtech) for detecting IFN-γ and IL2 according to the kit's instructions. Flow cytometry

[0152] In a co-culture system, T cells were labeled with an APC-CD3 antibody and tumor cells with GFP. CAR expression levels were measured with an APC-anti-Flag antibody. Data were collected using a BD FACSCanto II flow cytometer, with 10,000 viable cells collected per sample. Data were analyzed using FlowJo 10 software. Example 1 Construction of TSHαβ-CAR-T cells and TSHβα-CAR-T cells and detection of killing activity

[0153] Based on the principle of specific receptor-ligand binding, we tandemly expressed the two subunits of TSH, TSHα(CGA) and TSHβ, via a (G4S)3 sequence, and linked them to the hinge and transmembrane regions of CD8α, the intracellular costimulatory domain of CD28 or the intracellular costimulatory domain of 4-1BB, and the intracellular domain of CD3ζ. We constructed second-generation CARs (TSHαβ-CAR: TSHαβ-CD28ζ-CAR and TSHβα-CAR: TSHβα-CD28ζ-CAR) that specifically target TSHR (Figure 2A). CAR-T cells were generated by viral packaging and infection of lymphocytes isolated from the peripheral blood of healthy volunteers. Flow cytometry results demonstrated that retroviruses expressing TSHαβ-CAR and TSHβα-CAR effectively infected T cells, expressed the CAR molecule, and successfully prepared CAR-T cells (Figure 2B). Co-culture results showed that TSHαβ-CAR-T could effectively kill tumor cells, while TSHβα-CAR-T had no obvious killing effect (Figure 2C). Example 2 TSHαβ-CAR-T cell killing capacity and specificity

[0154] To further verify the ability of TSHαβ-CAR-T cells to target and kill TSHR-positive tumor cells, control and TSHR-overexpressing KTC-1 and FTC-133 cell lines were co-cultured with NT cells and TSHαβ-CAR-T cells. + The T cell to tumor cell ratio was 1:5. After 5 days of co-culture, all T cells and tumor cells were collected, and the T cells were labeled with APC fluorescein-conjugated CD3 antibody. The percentage of remaining tumor cells was detected by flow cytometry. As shown in the figure, after 5 days of co-culture, TSHαβ-CAR-T cells were able to effectively eliminate TSHR-positive tumor cells (Figure 4), accompanied by the release of large amounts of cytokines IFN-γ and IL-2. However, control cells that do not express TSHR showed no killing effect and no detectable cytokine release (Figure 5). The results demonstrated that TSHαβ-CAR-T cells can specifically recognize and kill TSHR-positive thyroid cancer cells. Example 3 Killing ability of truncated mutant TSHαβ-CAR-T cells

[0155] Analysis of the structural data of native TSH binding to TSHR from NCBI revealed that both the TSHα and TSHβ subunits have multiple sites that interact with TSHR, and the C-termini of both subunits have interactions between multiple consecutive amino acids and also interact with TSHR. In this example, various truncation mutants were constructed to examine the effect of truncation on the killing function of TSHαβ-CAR-T. The structures of each truncated mutant are shown in Figure 6A. The results of CAR expression efficiency and killing activity are shown in Figures 6B and 6C. The truncated mutations of TSHα and TSHβ did not affect the function of the CGA and TSHβ subunits, indicating that they could form a functional complex, recognize TSHR, and kill tumor cells. Consider

[0156] Experimental studies have shown that the two subunits of human TSH, TSHα and TSHβ, cannot bind to TSHR when linked in the order TSHβ-TSHα. However, unexpectedly, when these two subunits are linked in the specific order TSHα-TSHβ, they can function as the extracellular antigen-binding domain of the chimeric antigen receptor (CAR), binding to TSHR, enabling CAR-T cells to specifically recognize and kill TSHR-positive target cells. Furthermore, truncated mutations of TSHα and TSHβ (e.g., C-terminal truncated mutations) do not affect the recognition and binding of the antigen-binding domain to TSHR, and CAR-T cells containing the truncated mutations can still recognize and kill tumor cells.

[0157] All documents mentioned in this specification are incorporated by reference into this application to the same extent as if each individual document were individually incorporated by reference. It should also be understood that, after reading the above teachings of the present invention, one skilled in the art may make various changes and modifications to the present invention, and equivalents thereof are also within the scope defined by the claims appended hereto. Sequences of the invention

[0158] CGA (aa25-116) coding sequence: GCTCCTGATGTGCAGGATTGCCCAGAATGCACGCTACAGGAAAACCCATTCTTCTCCCAGCCGGGTGCCCCAATACTTCAGTGCATGGGCTGCTGCTTCTCTAGAGCATATCCCACTCCACTAAGGTCCAAGAAGACGATGTTGGTCCAAAAGAACGTCACCTCAGAGTCCACTTGCTGTGTAGCTAAATCATATAACAGGGTCACAGTAATGGGGGGTTTCAAAGTGGAGAACCACACGGCGTGCCACTGCAGTACTTGTTATTATCACAAATCT(SEQ ID NO: 1) CGA (aa25-116) amino acid sequence: APDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKS(SEQ ID NO: 2) TSHβ(aa21-138) coding sequence: TTTTGTATTCCAACTGAGTATACAATGCACATCGAAAGGAGAGAGTGTGCTTATTGCCTAACCATCAACACCACCATCTGTGCTGGATATTGTATGACACGGGATATCAATGGCAAACTGTTTCTTCCCAAATATGCTCTGTCCCAGGATGTTTGCACATATAGAGACTTCATCTACAGGACTGTAGAAATACCAGGATGCCCACTCCATGTTGCTCCCTATTTTTCCTATCCTGTTGCTTTAAGCTGTAAGTGTGGCAAGTGCAATACTGACTATAGTGACTGCATACATGAAGCCATCAAGACAAACTACTGTACCAAACCTCAGAAGTCTTATCTGGTAGGATTTTCTGTC(SEQ ID NO: 3) TSHβ(aa21-138) amino acid sequence: FCIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQDVCTYRDFIYRTVEIPGCPLHVAPYFSYPVALSCKCGKCNTDYSDCIHEAIKTNYCTKPQKSYLVGFSV(SEQ ID NO: 4) CD8α hinge and transmembrane domain coding sequence: ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC(SEQ ID NO: 5) CD8α hinge and transmembrane domain amino acid sequence: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO: 6) CD28 intracellular costimulatory domain coding sequence: AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC(SEQ ID NO: 7) CD28 intracellular costimulatory domain amino acid sequence: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS(SEQ ID NO: 8) 4-1BB intracellular costimulatory domain coding sequence: AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO: 9) 4-1BB intracellular costimulatory domain amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO: 10) CD3ζ intracellular domain coding sequence: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGC CTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID NO: 11) CD3ζ intracellular domain amino acid sequence: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 12) TSHαβ-CD8α-CD28-CD3ζ coding sequence: TSHαβ-CD8α-CD28-CD3ζ amino acid sequence: MEFGLSWLFLVAILKGVQCAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKSGGGGSGGGS GGGGSFCIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQDVCTYRDFIYRTVEIPGCPLHVAPYFSYPVALSCKCGKCNTDYSDCIHEAIKTNYCTKPQKSYLVGF SVTRGSDYKDDDDKGGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 14) TSHαβ-CD8α-4-1BB-CD3ζ coding sequence: TSHαβ-CD8α-4-1BB-CD3ζ amino acid sequence: MEFGLSWLFLVAILKGVQCAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKSGGGGSGGGS GGGGSFCIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQDVCTYRDFIYRTVEIPGCPLHVAPYFSYPVALSCKCGKCNTDYSDCIHEAIKTNYCTKPQKSYLVGF SVTRGSDYKDDDDKGGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE EGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 16) TSHβα-CD8α-CD28-CD3ζ coding sequence: TSHβα-CD8α-CD28-CD3ζ amino acid sequence: MEFGLSWLFLVAILKGVQCFCIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQDVCTYRDFIYRTVEIPGCPLHVAPYFSYPVALSCKCGKCNTDYSDCIHEAIKT NYCTKPQKSYLVGFSVGGGGSGGGGSGGGGSAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYH KSTRGSDYKDDDDKGGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF AAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 18) Truncated TSHα (25-114) amino acid sequence: APDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYH(SEQ ID NO: 19) Truncated TSHβ(21-126) amino acid sequence: FCIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQDVCTYRDFIYRTVEIPGCPLHVAPYFSYPVALSCKCGKCNTDYSDCIHEAIKTNYCT(SEQ ID NO: 20) Truncated TSHβ(21-124) amino acid sequence: FCIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQDVCTYRDFIYRTVEIPGCPLHVAPYFSYPVALSCKCGKCNTDYSDCIHEAIKTNY(SEQ ID NO: 21) Truncated TSHβ(21-122) amino acid sequence: FCIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQDVCTYRDFIYRTVEIPGCPLHVAPYFSYPVALSCKCGKCNTDYSDCIHEAIKT(SEQ ID NO: 22) TSHαβ(21-126) CAR coding sequence: TSHαβ(21-126) CAR amino acid sequence: MEFGLSWLFLVAILKGVQCAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKSGGGGSGG GGSGGGGSFCIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQDVCTYRDFIYRTVEIPGCPLHVAPYFSYPVALSCKCGKCNTDYSDCIHEAIKTNYCTTRGS DYKDDDDKGGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY RSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 24) TSHαβ(21-124) CAR coding sequence: TSHαβ(21-124) CAR amino acid sequence: MEFGLSWLFLVAILKGVQCAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKSGGGGSG GGGSGGGGSFCIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQDVCTYRDFIYRTVEIPGCPLHVAPYFSYPVALSCKCGKCNTDYSDCIHEAIKTNYTRGSD YKDDDDKGGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAY RSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 26) TSHαβ(21-122) CAR coding sequence: TSHαβ(21-122) CAR amino acid sequence: MEFGLSWLFLVAILKGVQCAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHKSGGGGSG GGGSGGGGSFCIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQDVCTYRDFIYRTVEIPGCPLHVAPYFSYPVALSCKCGKCNTDYSDCIHEAIKTTRGSDY KDDDDKGGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYR SRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 28) TSHα(25-114)β CAR coding sequence: TSHα(25-114)β CAR amino acid sequence: MEFGLSWLFLVAILKGVQCAPDVQDCPECTLQENPFFSQPGAPILQCMGCCFSRAYPTPLRSKKTMLVQKNVTSESTCCVAKSYNRVTVMGGFKVENHTACHCSTCYYHGGGGSGGGS GGGGSFCIPTEYTMHIERRECAYCLTINTTICAGYCMTRDINGKLFLPKYALSQDVCTYRDFIYRTVEIPGCPLHVAPYFSYPVALSCKCGKCNTDYSDCIHEAIKTNYCTKPQKSYTR GSDYKDDDDKGGTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAA YRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 30) Marker proteins TRGSDYKDDDDKGG(SEQ ID NO: 31) Linker peptide GGGGSGGGGSGGGGS(SEQ ID NO: 32)

Claims

1. A chimeric antigen receptor (CAR), comprising an extracellular antigen-binding domain that targets TSHR, and wherein the extracellular antigen-binding domain has a structure shown in formula (I) below. Tα-L-Tβ (I) (where, each "-" is independently a linker peptide or a peptide bond; Tα is the α subunit of the TSH protein, or a fragment thereof; Tβ is the TSH protein β subunit, or a fragment thereof; L is either absent or a linker peptide.

2. The CAR according to claim 1, wherein the TSH protein α-subunit fragment is a fragment obtained by truncating 1 to 10 amino acids from the C-terminus of the TSH protein α-subunit shown in SEQ ID NO: 2, preferably 1 to 6 amino acids, more preferably 1 to 2 amino acids.

3. The CAR according to claim 1 or 2, wherein the amino acid sequence of the Tα is set forth in SEQ ID NO: 2 or SEQ ID NO: 19, or has a sequence identity of ≥ 85% (preferably ≥ 90%, more preferably ≥ 95%, e.g. ≥ 96%, ≥ 97%, ≥ 98% or ≥ 99%) with the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 19, and has the same or substantially the same binding function.

4. The CAR according to claim 1, wherein the TSH protein β subunit fragment is a fragment obtained by truncating 1 to 20 amino acids from the C-terminus of the TSH protein β subunit shown in SEQ ID NO: 4, preferably 1 to 16, 1 to 14, or 1 to 12 amino acids.

5. The CAR according to claim 1 or 4, wherein the amino acid sequence of the Tβ is set forth in SEQ ID NO: 4, 20, 21 or 22, or has a sequence identity of ≥85% (preferably ≥90%, more preferably ≥95%, e.g., ≥96%, ≥97%, ≥98% or ≥99%) to the sequence set forth in SEQ ID NO: 4, 20, 21 or 22, and has the same or substantially the same binding function.

6. The L sequence is (G ４ S) n, where n is an integer selected from 1 to 6. The chimeric antigen receptor according to any one of claims 1 to 5.

7. The chimeric antigen receptor according to any one of claims 1 to 6, wherein the structure of the CAR is represented by the following formula II: S-EB-F-H-TM-C-CD3ζ (II) (In the formula, each "-" is independently a linker peptide or a peptide bond; S is either absent or a signal peptide sequence; EB is the extracellular antigen-binding domain; H is either absent or a hinge region; TM is the transmembrane domain; C is either absent or an intracellular costimulatory domain; CD3ζ is a sequence encoding the intracellular domain derived from CD3ζ, F is none or a marker protein.)

8. The chimeric antigen receptor according to any one of claims 1 to 7, wherein the amino acid sequence of the chimeric antigen receptor CAR is set forth in SEQ ID NO: 14, 16, 24, 26, 28, or 30, or has a sequence identity of ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98%, or ≥99%) to the sequence set forth in SEQ ID NO: 14, 16, 24, 26, 28, or 30.

9. A nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 1 to 8.

10. A vector comprising the nucleic acid molecule of claim 9.

11. A host cell, characterized in that it contains the vector according to claim 10, or has an exogenous nucleic acid molecule according to claim 9 integrated into its chromosome, or expresses the CAR according to any one of claims 1 to 8.

12. An engineered immune cell, characterized in that it contains the vector according to claim 10, or has an exogenous nucleic acid molecule according to claim 9 integrated into its chromosome, or expresses the CAR according to any one of claims 1 to 8.

13. The engineered immune cell of claim 12, wherein the engineered immune cell is a CAR-T cell.

14. A pharmaceutical composition comprising the CAR of any one of claims 1 to 8, the nucleic acid molecule of claim 9, the vector of claim 10, the host cell of claim 11, and / or the engineered immune cell of claim 12 or 13, and a pharmaceutically acceptable carrier, diluent, or excipient.

15. Use of the engineered immune cells of claim 12 or 13 or the pharmaceutical composition of claim 14 for the prevention and / or treatment of diseases associated with high TSHR expression.

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