MAGE-a4-specific t cell receptor screening and anti-tumor use

Abstract

This invention relates to the screening of MAGE-A4-specific T-cell receptors and their anti-tumor applications. Specifically, this invention provides a T-cell receptor TCR or its antigen-binding fragment, wherein the TCR or its antigen-binding fragment is capable of binding to MAGE-A4. 146‑154 The TCR binds to the epitope and the HLA-A11 complex, and the TCR contains an α-chain variable region and a β-chain variable region. The TCR or its antigen-binding fragment contains the following α-chain complementarity-determining regions (CDRs): α-chain complementarity-determining region CDR1 as shown in SEQ ID NO:10, α-chain complementarity-determining region CDR2 as shown in SEQ ID NO:11, α-chain complementarity-determining region CDR3 as shown in SEQ ID NO:12, β-chain complementarity-determining region CDR1 as shown in SEQ ID NO:14, β-chain complementarity-determining region CDR2 as shown in SEQ ID NO:15, and β-chain complementarity-determining region CDR3 as shown in SEQ ID NO:16. The specific T-cell receptor and its antigen-binding fragment can act as immune effector activators to stimulate the body's immune response, thereby producing anti-tumor and other disease-fighting effects.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a T-cell receptor (TCR) or its antigen-binding fragment that can specifically recognize the HLA-A11-restricted T-cell epitope of cancer testis antigen MAGE-A4. Background Technology

[0002] Cancer is an immune-related disease, and with socioeconomic development, the incidence and mortality burden of cancer are rapidly increasing. In 2011, cancer surpassed heart disease to become the leading cause of death worldwide. The WHO announced in December 2013 that more than 14 million new cancer cases are diagnosed globally each year.

[0003] Traditional cancer therapies such as surgical resection, radiotherapy, chemotherapy, and targeted therapy are insufficient to comprehensively address the challenges of tumor metastasis or recurrence. In the early 1980s, Allison and other researchers identified the gene structure of the αβ T cell receptor (TCR), responsible for recognizing antigens on the surface of T cells. In the late 1980s, Boone, Rosenberg, Old, and others discovered that various cancer patients possess tumor-specific antigens that can be recognized by T cells and specifically kill tumor cells, rekindling hope for tumor immunotherapy. Extensive research has focused on the development of therapeutic vaccines for tumors, with adoptive T lymphocyte therapy gaining increasing attention due to its targeted killing of tumor cells and its durable responsiveness. In recent years, with the rapid development of stem cell biology, immunology, molecular technology, and tissue engineering, cell immunotherapy, as a safe and effective treatment, has played an increasingly prominent role in the treatment of cancer and other diseases. Currently, the research and development of novel cell therapy technologies has become a crucial research area for addressing cancer and related diseases. In 2013, cancer immunotherapy was ranked first among the top 10 scientific breakthroughs of the year by Science magazine.

[0004] Adoptive cell therapy (ACT) is a highly personalized cancer treatment that aims to fight tumors by rebuilding the weakened or missing immune system in cancer patients. ACT involves isolating immune-active cells from a cancer patient, expanding and functionally identifying them in vitro, and then reinfusing them into the patient. This aims to either directly kill tumor cells or stimulate the body's immune response to kill tumor cells. The limited availability of antigens expressed only on cancerous tissues, rather than on normal, essential tissues, is a limiting factor for ACT therapy.

[0005] Currently, ACT therapy is mainly divided into chimeric antigen receptor T-cell (CAR-T) therapy and T-cell receptor-engineered T-cell (TCR-T) therapy. The former involves introducing artificial receptors targeting tumor surface antigens into T cells to exert anti-tumor functions. In contrast, TCR-T therapy uses T cells edited with TCRs, which can recognize intracellular specific antigenic epitopes presented on the surface of tumor cells by major histocompatibility complex (MHC) molecules. Since the intracellular antigens presented by MHC molecules far exceed the specific proteins on the tumor surface, TCR-T has potentially broader applicability than CAR-T.

[0006] TCR-T cell immunotherapy is a newly developed cell therapy technology in recent years and a typical example of "precision medicine." Currently, this technology has shown promising therapeutic prospects in the treatment of myeloma, melanoma, esophageal cancer, liver cancer, and synovial sarcoma. TCR-T cell immunotherapy was first applied to the treatment of HIV in the late 20th century. Recent studies have found that autologous immune cells engineered based on tumor antigen-specific TCRs such as MART-1, MAGE-A4, NY-ESO-1, and WT1 show good development potential in the treatment of melanoma, esophageal cancer, multiple myeloma, and synovial cell sarcoma.

[0007] To ensure the safety and efficacy of TCR-T therapy, MHC antigens should either be absent on cancer cells or weakly expressed on normal cells; therefore, selecting appropriate antigens is crucial. MAGE-A4 (melanoma-associated gene A4) belongs to the MAGE family. Based on tissue expression patterns, human MAGE family members can be divided into two categories: Type II MAGEs are expressed in many tissues throughout the body; while Type I MAGEs (members of the MAGE-A, -B, and -C subfamilies) are located on the X chromosome and are considered cancer testis antigens (CTAs). Normal expression of CTAs is only found in the testes (occasionally in the ovaries and placenta), but they are highly expressed in tumors of various tissue types. T cells can recognize these expression differences and perform tumor-killing functions; therefore, MAGE-A4 is considered a promising antigen target.

[0008] Because TCR-T therapy is limited by HLA typing, combining promising antigens with common HLA typing can broaden the range of patients who can be treated. HLA-A*02:01, HLA-A*11:01, and HLA-A*24:02 are subtypes with a relatively wide distribution at HLA-A loci in the general population, but their distribution varies considerably across different populations. The HLA-A*11:01 (HLA-A11) allele has a high frequency of distribution in East and Southeast Asian populations, but no HLA-A11-restricted T-cell epitopes have been reported for the MAGE-A4 antigen, posing a significant challenge to immunotherapy research in individuals with an HLA-A11 genetic background. Therefore, it is necessary to identify HLA-A11-restricted epitopes for MAGE-A4 and screen for specific TCRs to provide a research foundation for tumor immunotherapy in individuals with an HLA-A11 genetic background.

[0009] The MAGE-A4 gene (melanoma-associated gene A4, MAGE-A4) is located on the X chromosome and encodes the MAGE-A4 protein, a member of the MAGE-A gene family. The MAGE-A4 protein has 317 amino acids and a molecular weight of 34.9 kDa. The biological function of MAGE-A4 is not yet fully understood, but some studies suggest that MAGE-A proteins are oncoprolactin proteins that promote tumor cell survival, for example, by preventing cell cycle arrest and inhibiting p53-mediated apoptosis, thereby promoting tumor growth.

[0010] MAGE-A4 belongs to the cancer-testis antigen (CTA). Under normal circumstances, its expression is limited to the testes (occasionally the ovaries and placenta), but it is abnormally upregulated in a variety of cancers. Upregulation of MAGE-A4 is found in 47% of ovarian cancers, 19-35% of lung cancers, 22% of colon cancers, as well as testicular cancer, head and neck cancer, and skin cancer.

[0011] Recent clinical trials have shown that immunotherapies targeting MAGE-A4 for tumor treatment are primarily TCR-related therapies, including peptide vaccines, infusion of specific TCR-engineered cells, and treatment with specific TCR-related protein drugs. In 2021, Adaptimmune announced Phase II clinical trial data for SPEARHEAD-1, a TCR-T therapy targeting MAGE-A4, showing that 40% of patients with advanced / metastatic synovial sarcoma or myxoid / round cell liposarcoma received partial remission (PR). Currently, among the numerous clinical trials involving TCR treatment, seven are TCR-related therapies targeting MAGE-A4. These clinical trials all target individuals with HLA-A2 or HLA-A24 subtypes; no studies have reported on HLA-A11-restricted T-cell epitopes and TCR-T cell therapies. Summary of the Invention

[0012] The MAGE-A4 gene is highly expressed in various solid tumors. Upregulation of MAGE-A4 is observed in 47% of ovarian cancers, 19-35% of lung cancers, 22% of colon cancers, as well as testicular cancer, head and neck cancer, and skin cancer. Highly expressed MAGE-A4 can be presented to the cell surface by MHC molecules in cells and recognized by T cells to stimulate a T cell immune response, thereby eliminating tumor cells carrying MAGE-A4.

[0013] Specifically, when MAGE-A4 peptide acts as an antigen, it can induce the body to produce CD8. + CTL (cytotoxic lymphocyte) response. In non-spermonic cells, MAGE-A4, which is highly expressed on tumor cells, can be presented by HLA molecules and recognized by T cells.

[0014] One embodiment of the present invention includes screening for MAGE-A4 that specifically targets the tumor MAGE-A4 gene using a specific T-cell receptor (TCR) single-cell screening technique. 146-154 Epitope peptides are specific T-cell receptors (TCRs).

[0015] One embodiment of the present invention includes providing MAGE-A4 targeting the MAGE-A4 gene. 146-154 The specific T-cell receptor of the epitope polypeptide and its antigen-binding fragment. Another embodiment of the invention includes the use of the above-described T-cell receptor and its antigen-binding fragment in the preparation of a medicament for treating tumors carrying the MAGE-A4 gene.

[0016] This invention is based on the above principles. The MAGE-A4 peptide-specific TCR or its antigen-binding fragment in this invention binds to HLA-A11 and MAGE-A4. 146-154 The specific binding of the polypeptide complex molecule stimulates T cell activation, induces T cells to secrete cytokines such as IFN-γ, and subsequently kills cells expressing MAGE-A4. 146-154 Tumor cells containing polypeptides.

[0017] In this invention, "MAGE-A4-specific TCR" refers to a CTL epitope peptide targeting the HLA-A11-restricted MAGE-A4 peptide (MAGE-A4). 146-154 In a specific embodiment of the present invention, the TCR is a mouse-derived TCR, referred to as A11M103J TCR, A11M141J TCR, A11M161J TCR, A11M162J TCR, and A11M163J TCR.

[0018] This application includes TCRs or derivatives that specifically bind to the complex molecule of the MAGE-A4 peptide and HLA-A11, as well as TCR fragments that exhibit substantially the same antigen specificity as the original TCR. "TCR fragment" or "antigen-binding fragment" refers to an antigen-binding fragment of a TCR and TCR analogs, which typically include at least a portion of the antigen-binding region or variable region of the parent TCR, such as one or more CDRs. The TCR fragment retains at least some of the binding specificity of the parent TCR.

[0019] When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of the protein, such as MAGE-A4, within a heterogeneous population of proteins and / or other biological reagents. 146-154 The binding reaction of peptides to HLA-A11 complex molecules. Therefore, under specified conditions, a specific ligand / antigen binds to a specific receptor / antibody, and does not bind to other proteins present in the sample in significant amounts.

[0020] This invention also provides MAGE-A4 containing the present invention. 146-154 A pharmaceutical composition of a peptide-specific TCR, or its antigen-binding fragment. The pharmaceutical composition may be a protein-based pharmaceutical composition or a protein-drug conjugate. To prepare the pharmaceutical composition, it can be prepared by using MAGE-A4... 146-154The polypeptide-specific TCR or its antigen-binding fragment is mixed with a pharmaceutical carrier or excipient to prepare various desired dosage forms. Examples of dosage forms for the pharmaceutical compositions of this invention include, for example, oral dosage forms such as tablets, powders, pills, granules, fine granules, soft / hard capsules, film-coated tablets, pellets, sublingual tablets, and ointments; and non-oral dosage forms such as injections, suppositories, transdermal preparations, ointments, plasters, and topical liquids. Those skilled in the art can select appropriate dosage forms based on the route of administration and the target population.

[0021] The dosage of the active ingredient in the pharmaceutical composition of the present invention varies depending on the target patient, the target organ, symptoms, method of administration, etc. It can be determined based on the doctor's judgment, taking into account the type of dosage form, method of administration, patient's age and weight, patient's symptoms, etc.

[0022] The pharmaceutical compositions of the present invention may also contain other agents, including but not limited to cytotoxic agents, cell growth inhibitors, anti-angiogenic or antimetabolite drugs, targeted tumor drugs, immunostimulants or immunomodulators, or TCRs combined with cytotoxic agents, cell growth inhibitors or other toxic drugs.

[0023] Specifically, the present invention provides the following solutions:

[0024] 1. A T-cell receptor (TCR) or its antigen-binding fragment, wherein the TCR or its antigen-binding fragment is capable of binding to MAGE-A4. 146-154 The epitope binds to the HLA-A11 complex, and the TCR contains an α-chain variable region and a β-chain variable region, wherein the TCR or its antigen-binding fragment contains the following α-chain complementarity-determining region (CDR) and β-chain complementarity-determining region (CDR):

[0025] As shown in SEQ ID NO: 10, the α-chain complementarity determination region CDR1,

[0026] As shown in SEQ ID NO: 11, the α-chain complementarity determination region CDR2,

[0027] As shown in SEQ ID NO: 12, the α-chain complementarity determination region CDR3,

[0028] As shown in SEQ ID NO: 14, the β-chain complementarity determination region CDR1,

[0029] As shown in SEQ ID NO: 15, the β-chain complementarity determination region CDR2, and

[0030] As shown in SEQ ID NO: 16, the β-chain complementarity determination region CDR3; or

[0031] As shown in SEQ ID NO: 26, the α-chain complementarity determination region CDR1,

[0032] As shown in SEQ ID NO: 27, the α-chain complementarity determination region CDR2,

[0033] As shown in SEQ ID NO: 28, the α-chain complementarity determination region CDR3,

[0034] As shown in SEQ ID NO: 30, the β-chain complementarity determination region CDR1,

[0035] As shown in SEQ ID NO: 31, the β-chain complementarity determination region CDR2, and

[0036] Such as the β-chain complementarity determination region CDR3 shown in SEQ ID NO: 32; or

[0037] As shown in SEQ ID NO: 38, the α-chain complementarity determination region CDR1,

[0038] As shown in SEQ ID NO: 39, the α-chain complementarity determination region CDR2,

[0039] As shown in SEQ ID NO: 40, the α-chain complementarity determination region CDR3,

[0040] As shown in SEQ ID NO: 42, the β-chain complementarity determination region CDR1,

[0041] As shown in SEQ ID NO: 43, the β-chain complementarity determination region CDR2, and

[0042] Such as the β-chain complementarity determination region CDR3 shown in SEQ ID NO: 44; or

[0043] As shown in SEQ ID NO: 50, the α-chain complementarity determination region CDR1,

[0044] As shown in SEQ ID NO: 51, the α-chain complementarity determination region CDR2,

[0045] As shown in SEQ ID NO: 52, the α-chain complementarity determination region CDR3,

[0046] As shown in SEQ ID NO: 54, the β-chain complementarity determination region CDR1,

[0047] As shown in SEQ ID NO: 55, the β-chain complementarity determination region CDR2, and

[0048] Such as the β-chain complementarity determination region CDR3 shown in SEQ ID NO: 56; or

[0049] As shown in SEQ ID NO: 62, the α-chain complementarity determination region CDR1,

[0050] As shown in SEQ ID NO: 63, the α-chain complementarity determination region CDR2,

[0051] As shown in SEQ ID NO: 64, the α-chain complementarity determination region CDR3,

[0052] As shown in SEQ ID NO: 66, the β-chain complementarity determination region CDR1,

[0053] As shown in SEQ ID NO: 67, the β-chain complementarity determination region CDR2, and

[0054] The β-chain complementarity determination region CDR3 is shown in SEQ ID NO: 68.

[0055] 2. The T-cell receptor TCR or its antigen-binding fragment as described in Project 1, comprising:

[0056] α-chain variable region as shown in SEQ ID NO: 9, and β-chain variable region as shown in SEQ ID NO: 13;

[0057] α-chain variable region as shown in SEQ ID NO: 25, and β-chain variable region as shown in SEQ ID NO: 29;

[0058] α-chain variable region as shown in SEQ ID NO: 37, and β-chain variable region as shown in SEQ ID NO: 41;

[0059] α-chain variable regions as shown in SEQ ID NO: 49, and β-chain variable regions as shown in SEQ ID NO: 53; or

[0060] α-chain variable region as shown in SEQ ID NO: 61, and β-chain variable region as shown in SEQ ID NO: 65.

[0061] 3. The T-cell receptor TCR or its antigen-binding fragment as described in Project 1 or 2, wherein the T-cell receptor TCR is a murine TCR, a human-mouse chimeric TCR, or a humanized TCR.

[0062] 4. A polynucleotide encoding the T-cell receptor TCR or its antigen-binding fragment as described in any one of items 1-3.

[0063] 5. An expression vector comprising the polynucleotides described in item 4; preferably, the expression vector is a lentiviral vector.

[0064] 6. A host cell comprising the expression vector described in item 5.

[0065] 7. A method for preparing the T-cell receptor TCR or its antigen-binding fragment as described in any one of items 1-3, the method comprising:

[0066] 1) Cultivate the host cells described in Project 6;

[0067] 2) Recover the T cell receptor TCR or its antigen-binding fragment from any one of items 1-3 from the host cell or its culture medium.

[0068] 8. A pharmaceutical composition comprising any one of items 1-3, a T-cell receptor TCR or an antigen-binding fragment thereof, and a pharmaceutically acceptable carrier.

[0069] 9. The T-cell receptor TCR or its antigen-binding fragment as described in any one of items 1-3, used in the preparation of a drug for detecting cancer testis antigen MAGE-A4. 146-154 Its use in reagents for detecting or diagnosing tumor cells, or in the preparation of reagents for detecting or diagnosing cancer testis antigen MAGE-A4. 146-154 Uses in reagents for treating tumors;

[0070] Optionally, the cancer testis antigen MAGE-A4 146-154 The sequence is shown in SEQ ID NO: 1.

[0071] 10. Use of any one of items 1-3, the T-cell receptor TCR or its antigen-binding fragment, in the preparation of a medicament for treating tumors, wherein the tumor is MAGE-A4. 146-154 Positive.

[0072] 11. According to the use described in Item 10, the tumor is selected from melanoma, ovarian cancer, bladder cancer, non-small cell lung cancer, head and neck cancer, or synovial sarcoma.

[0073] Advantages of the present invention

[0074] By utilizing the MAGE-A4 of the present invention 146-154 The specific TCR of the gene, used to prepare T lymphocytes expressing this TCR (TCR-T), can effectively recognize and kill MAGE-A4. 146-154 Positive tumor cells can be expected to inhibit the growth of tumors, especially solid tumors, thus achieving the effect of tumor treatment.

[0075] The present invention targets MAGE-A4 146-154Specific T-cell receptors for gene epitopes and T cells expressing them possess high infection efficiency and binding properties, thus they can be used for early-stage drug research and to develop drugs targeting mutations associated with tumor growth and progression in experimental models. Therefore, they can be used for the treatment of MAGE-A4 expression... 146-154 The diagnosis and treatment of various tumors at different stages based on genes, and the development of drugs for treatment, especially drugs for treating solid tumors with high mutation frequencies. Attached Figure Description

[0076] Figure 1 ELISPOT assay for MAGE-A4 146-154 Diagram showing the IFN-γ secretion response induced by peptides in mouse spleen cells. Mock represents the negative control without stimulation, and MAGE-A4 is also shown. 146-154 The wells are for peptide stimulation detection, phorbol ester (PMA / ION) is a positive control, and Mus-1 to Mus-12 are mouse numbers.

[0077] Figure 2 MAGE-A4 146-154 Molecular sieve chromatography and biotinylation level detection of peptide-HLA-A11 complex protein. Figure A shows the MAGE-A4 before biotinylation. 146-154 Molecular sieve chromatography of the HLA-A11 complex; Figure B is MAGE-A4 indicated by the asterisk in Figure A. 146-154 Image C shows the SDS-PAGE of the HLA-A11 protein complex; Image D shows the biotinylated MAGE-A4. 146-154 Molecular sieve chromatography of the HLA-A11 complex; Figure D is an SDS-PAGE image showing the biotinylation level.

[0078] Figure 3 MAGE-A4 cells were isolated from HLA-A*11:01 transgenic mice. 146-154 Specific T cells. MAGE-A4 in each mouse spleen cell. 146-154 The HLA-A11 tetramer-positive cells are the part circled in the box in the figure.

[0079] Figure 4 HEK-293T cells co-transfected with TCR and CD3-CD8 were used to detect the binding specificity of TCRs using experimental tetramer or control HLA-A11 tetramer.

[0080] HEK-293T specifically binds to MAGE-A4 at the levels of A11M103J TCR, A11M141J TCR, A11M161J TCR, A11M162J TCR, and A11M163J TCR. 146-154 / HLA-A11 verification. The first row (horizontal) shows the expression of each TCR, and the second row (horizontal) shows the expression of each TCR with MAGE-A4. 146-154 The binding of / HLA-A11 tetramer.

[0081] Figure 5 SPR detection of A11M103J and MAGE-A4 146-154 Affinity for HLA-A11 protein. Figure A shows the A11M103J TCR after refolding in vitro inclusion bodies purified by molecular sieves; Figure C shows the MAGE-A4 after refolding in vitro inclusion bodies purified by molecular sieves. 146-154 / HLA-A11 protein; E indicates surface plasmon resonance (SPR) determination of A11M103J TCR and MAGE-A4 146-154 Affinity to HLA-A11. Figures B and D show the purity of TCR and pMHC proteins as determined by SDS-PAGE, respectively.

[0082] Figure 6 Infection efficiency assay of A11M103J TCR-T cells. TCR-T cells prepared using PBMCs from D1 or D2 volunteers; the first row (horizontal) represents cells prepared using MAGE-A4. 146-154 The results of flow cytometry staining with the control peptide / HLA-A11 tetramer are shown in the first row. The values ​​in the quadrants indicate the positive rate of TCR expression. The second row (horizontal) shows the results of flow cytometry staining with the control peptide / HLA-A11 tetramer. The values ​​in the quadrants indicate the positive rate of TCR expression.

[0083] Figure 7 The reaction of A11M103J TCR-T cells with target cells was detected using the ELISPOT assay. TCR-T cells secreting IFN-γ after stimulation with K562-A11 loaded with a peptide, and A11M103J TCR-T cells secreting IFN-γ after incubation with three cell lines co-expressing HLA-A11 and MAGE-A4 (i.e., A375-A11, K562-A11-MAGE-A4, or PANC1-MAGE-A4) (all purchased from Peking Union Medical College Hospital), were also examined. A375-A11, K562-A11-MAGE-A4, and PANC1-MAGE-A4 represent positive target cell stimulation detection wells. Primary T cells from two volunteers, D1 and D2, were used to create TCR-T cells. The figure shows cells forming positive reaction spots (SFCs), with 2 × 10T TCR-T cells per well. 4 The blank control consists of wells containing only TCR-T cells.

[0084] Figure 8 A11M103J TCR-T and load MAGE-A4 146-154 Response of target cells to peptides. Figure A shows A11M103J TCR-T cells loaded with different concentrations of MAGE-A4. 146-154 ELISA statistical graph of IFN-γ levels produced after incubation of K562-A11 target cells with the peptide, and A11M103J TCR-T cells with the unrelated peptide KRAS-G12V. 8-16 Incubation was used as a negative control. Figure B shows A11M103J TCR-T cells and cells loaded with different concentrations of MAGE-A4. 146-154 ELISA statistical graph of IFN-γ levels generated after incubation of the peptide with PANC1 target cells.

[0085] Figure 9 The response of A11M103J TCR-T cells to tumor cell lines expressing HLA-A11 and MAGE-A4. Figure A shows a schematic diagram of the interaction between A11M103J TCR-T cells and target cells. Figures B, C, and D show ELISA histograms of IFN-γ secretion by A11M103J TCR-T cells after interaction with different numbers of tumor cells.

[0086] Figure 10 The in vitro killing effect of A11M103J TCR-T cells on target cells expressing HLA-A11 and MAGE-A4. Figure A shows the gating strategy of the cell killing experiment. Figure B shows the comparison of killing effect of A11M103J TCR-T cells or Mock-T cells on A375-A11 and MDA-MB-436-A11 target cells. A375-A11 and MDA-MB-436-A11 cells are tumor cell lines that simultaneously express A11 and MAGE-A4.

[0087] Figure 11 Intracellular factor staining was performed on A11M103J TCR-T cells after interaction with target cells expressing HLA-A11 and MAGE-A4. TCR-T cells prepared from primary T cells of D1 or D2 volunteers were incubated with target cells co-expressing HLA-A11 and MAGE-A4 or control target cells. Flow cytometry was used to detect the IFN-γ secretion population within the TCR-T cells, with the x-axis representing CD8 and the y-axis representing intracellular IFN-γ secretion.

[0088] Figure 12The inhibitory effect of A11M103J TCR-T cells on A375-A11 melanoma cells in vivo. After transplanting A375-A11-luciferase tumor cells into NCG-immunodeficient mice, they were randomly divided into groups and A11M103J TCR-T cells were infused via the tail vein. Mock-T cells were used as a negative control, and PBS was used as a blank control. Figure A shows in vivo imaging of luciferase in mice at different days. Figure B shows the total fluorescence volume of mice at different days. Figure C shows the tumor weight of tumor tissue collected from mice after euthanasia on day 26. Statistical differences between groups were calculated using t-tests, where **: p < 0.01, ****: p < 0.0001.

[0089] Figure 13 A strategy for humanizing the A11M103J TCR. The A11M103J TCR sequence was humanized and named A11M103H TCR.

[0090] Figure 14 A11M103J and A11M103H TCRs specifically bind to MAGE-A4. 146-154 / HLA-A11 tetramer. The first row (horizontal) shows the TCR expression level after transfection of HEK-293T cells with A11M103J TCR or A11M103H TCR; the second and third rows (horizontal) show the expression levels of negative control tetramer or MAGE-A4 after transient transfection of HEK-293T cells with A11M103J TCR or A11M103H TCR. 146-154 Results of flow cytometry staining of HLA-A11 tetramer.

[0091] Figure 15 A11M103J TCR or A11M103H TCR with MAGE-A4 146-154 The binding characteristics of peptides to HLA-A11. Figure A shows the binding of A11M103J TCR and MAGE-A4. 146-154 Affinity assay of peptides with HLA-A11 complex proteins. Figure B shows the affinity of A11M103H TCR and MAGE-A4. 146-154 Affinity detection of peptides to HLA-A11 complex proteins.

[0092] Figure 16Infection efficiency assay of A11M103J or A11M103H TCR-T cells. The first and second rows (horizontal) show the tetramer assay of Mock-T or TCR-T cells from Donor v88 and Donor v291 volunteers. The first column (vertical) shows the tetramer assay of T cells from Donor v88 and Donor v291 volunteers that were not infected with TCR lentivirus, serving as a negative control; the second column (vertical) shows the tetramer assay of TCR-T cells prepared by infecting T cells from Donor v88 and Donor v291 volunteers with A11M103J TCR lentivirus; the third column (vertical) shows the tetramer assay of TCR-T cells prepared by infecting T cells from Donor v88 and Donor v291 volunteers with A11M103H TCR lentivirus.

[0093] Figure 17 The in vitro killing effects of A11M103J TCR-T and A11M103H TCR-T cells on A375-A11 and NCI-H520 target cells. Figure A shows the killing effect of A11M103J TCR-T or A11M103H TCR-T cells prepared using primary T cells from Donor v88 volunteers on A375-A11-luci cells. Figure B shows the killing effect of A11M103J TCR-T or A11M103H TCR-T cells prepared using primary T cells from Donor v88 volunteers on NCI-H520-luci cells. Figure C shows the killing effect of A11M103J TCR-T or A11M103H TCR-T cells prepared using primary T cells from Donor v291 volunteers on A375-A11-luci cells. Figure D shows the killing effect of A11M103J TCR-T or A11M103H TCR-T cells prepared using primary T cells from Donor v291 volunteers on NCI-H520-luci cells. Mock-T cells uninfected with lentiviruses served as a negative control.

[0094] Figure 18The inhibitory effects of A11M103J TCR-T cells and A11M103H TCR-T cells on NCI-H520 lung cancer cells in vivo. After transplanting NCI-H520 tumor cells into NCG-immunodeficient mice, they were randomly divided into groups and TCR-T cells prepared from Donor v310 or Donor v311 primary T cells from healthy volunteers, including A11M103J TCR-T cells or different doses of A11M103H TCR-T cells, were infused via the tail vein. Mock-T cells were used as a negative control, and physiological saline was used as a blank control. Figure A shows the statistical change in subcutaneous tumor volume over time in different groups of mice after tumor transplantation. Figure B shows the comparison of tumor weight isolated at the end of the experiment between different treatment groups. Figure C shows the tumor volume growth of a single mouse within each group. Statistical differences between groups were calculated using t-tests, where **: p < 0.01, ***: p < 0.001. Detailed Implementation

[0095] The present invention further illustrates its technical solutions through specific embodiments and accompanying drawings. However, those skilled in the art will understand that the following specific embodiments and examples are intended to illustrate the present invention and should not be construed as limiting the present invention in any way. It is well known to those skilled in the art that many modifications can be made to the present invention without departing from its spirit, and such modifications also fall within the scope of the present invention.

[0096] Unless otherwise specified, the experimental methods described below are conventional experimental methods in this field, and the experimental materials used are all readily available from commercial companies.

[0097] Example 1. MAGE-A4 antigenic epitope prediction and identification

[0098] In this embodiment, four HLA-A11 restricted epitope peptides of MAGE-A4 were first predicted and synthesized. These peptides were then used to immunize mice, and T cell responses were screened using the ELISPOT experiment to select the peptides that were immunogenic.

[0099] 1. HLA-A11 restriction epitope prediction of MAGE-A4 peptide

[0100] The MAGE-A4 peptide was used to predict HLA-A11-restricted T-cell epitope peptides using the NetMHC-4.0 online prediction system. The results showed that MAGE-A4... 146-154 It has a strong affinity for HLA-A11.

[0101] The commissioned company (Zhongke Yaguang Company) synthesized MAGE-A4 146-154(The sequence is shown in SEQ ID NO: 1).

[0102] 2. Immunization of HLA-A11 transgenic mice with MAGE-A4 peptide

[0103] MAGE-A4 was used in this step. 146-154 Peptide immunization of HLA-A11 transgenic mice (hereinafter referred to as HLA-A*11:01 transgenic mice) (obtained from Beijing Biocytogen) induced the production of MAGE-A4 in the mice. 146-154 Peptide-specific T cells for further MAGE-A4 production 146-154 Peptide-specific TCR.

[0104] Specifically, chemically synthesized MAGE-A4 is used. 146-154 100 μg of the polypeptide (sequence shown in SEQ ID NO: 1) was dissolved in 50 μL of sterile PBS and mixed with 50 μL of Quick CTL cell adjuvant (Biodragon). The mixture was injected subcutaneously into the groin of HLA-A*11:01 transgenic mice 4 to 6 weeks old, with each mouse receiving 100 μL of the mixture. Booster immunizations were performed in the same manner on days 7 and 14. The mice were euthanized on day 21 and spleen cells were collected for culture and analysis.

[0105] Mouse spleen cells (100 μL, 2.5 × 10⁶ cells / well) were added to a 96-well ELISPOT plate pre-coated with anti-mouse-IFN-γ antibody (BD, Biosciences). 5 Cell count / well), add one type of MAGE-A4 to be tested to each well. 146-154 The polypeptide (100 μL, 40 μg / mL) was used as a negative control and a positive control stimulus, respectively, with RPMI 1640 medium and PMA / ION (Beijing Dakowei). After mixing, the ELISPOT plates were placed in an incubator at 37°C. After incubation for 18 hours, the cells were discarded, and the plates were incubated sequentially with biotinylated anti-mouse-IFN-γ (BD, Biosciences) and enzyme conjugate (SA-HRP) (BD, Biosciences) according to the manufacturer's instructions. Color development was then performed using horseradish peroxidase substrate (AEC) (BD, Biosciences), and finally, the color development was stopped with water. Spots were captured and counted using an automated ELISPOT reader and image analysis software (Cellular Technology Limited).

[0106] The results showed MAGE-A4 146-154The peptide can activate multiple mouse T cells, causing them to secrete IFN-γ and appear as positive spots on ELISPOT plates (shown on...). Figure 1 ELISPOT detection revealed MAGE-A4 146-154 The peptide induced a specific T cell response in 9 / 12 mice.

[0107] Example 2. MAGE-A4 146-154 Peptide-specific T cell sorting and TCR gene cloning

[0108] In this embodiment, MAGE-A4 was first prepared. 146-154 The peptide tetramer with HLA-A11 was then used to collect CD3 from the spleen cells of immunized mice by staining with CD3 and CD8 antibodies. + CD8 + T cells were then sorted to obtain MAGE-A4. 146-154 Peptide-specific T cells.

[0109] 1. MAGE-A4 146-154 Preparation of peptide / HLA-A11 tetramer

[0110] The prokaryotic codons of β2m (β2-microglobulin, the light chain gene of HLA-A11) (Uniprot: P61769) and the HLA-A11 heavy chain gene (IMGT / HLA Acc No: HLA00043) were optimized using conventional methods. The resulting β2m nucleic acid sequence is shown in SEQ ID NO: 8, and its encoded amino acid sequence is shown in SEQ ID NO: 7. The resulting HLA-A11 heavy chain gene is shown in SEQ ID NO: 6, and its encoded amino acid sequence is shown in SEQ ID NO: 5. For the HLA-A11 heavy chain gene, a biotin-specific binding polypeptide sequence (Biotin-tag, amino acid sequence shown in SEQ ID NO: 4) was added to the C-terminus of the heavy chain gene shown in SEQ ID NO: 6.

[0111] These DNA sequences were synthesized by a company (Nanjing Genscript Biotech Co., Ltd.), and Nde I and Xho I restriction enzyme sites were introduced into them, with Nde I located at the 5' end and Xho I located at the 3' end. Using Nde I and Xho I restriction sites, the synthesized DNA sequences of the β2m and HLA-A11 heavy chain genes were cloned into the expression vector pET-21a (Invitrogen Co., Ltd.), respectively, to establish prokaryotic recombinant expression plasmids β2m-pET 21a and HLA-A11-pET 21a for the β2m and HLA-A11 heavy chain proteins.

[0112] Two expression plasmids were heat-activated and transformed into E. coli BL21(DE3) competent cells (purchased from Tianenze Biotechnology). IPTG was added to induce expression. E. coli was broken and homogenized to extract inclusion bodies, obtaining inclusion body proteins of β2m and HLA-A11 heavy chain in inclusion body state.

[0113] (1) Purification of MAGE-A4 146-154 peptide / HLA-A11 complex protein

[0114] 0.5 ml of β2m inclusion body protein (i.e., HLA-A11 light chain inclusion body protein) (30 mg / ml dissolved in a solution containing 6 M Ga-HCl, 50 mM Tris pH 8.0, 100 mM NaCl, 10 mM EDTA, and 10 mM DTT) was slowly added dropwise to a solution containing 1.5 mg of the above MAGE-A4. 146-154 The polypeptide (sequence shown in SEQ ID NO: 1) (synthesized by Beijing Zhongke Yaguang Co., Ltd.) was placed in 300 mL of refolding solution (20 mM Tris-HCl, 400 mM L-arginine, 2 mM EDTA, 5 mM GSH / GSSG / 1 mM). After 1 hour, the heavy chain inclusion bodies of HLA-A11 were slowly added dropwise to the above refolding solution at a molar ratio of β2m:HLA-A11 heavy chain = 1:1. The refolding time was more than 8 hours.

[0115] The refolded sample was concentrated by passing it through a 10 kDa filter using an ultrafiltration cup, followed by a buffer change to 20 mM Tris-Cl, 50 mM NaCl, pH 8.0. Two buffer changes were performed: after concentrating the sample to approximately 20 ml, 200 ml of buffer containing 20 mM Tris-Cl, 50 mM NaCl, pH 8.0 was added; then, after further concentration to approximately 20 ml, 20 mM Tris-Cl, 50 mM NaCl, pH 8.0 buffer was added again to bring the volume to 100 ml, and finally concentrated to approximately 10-20 ml. The sample was then centrifuged at 12000 rpm for 10 min at 4°C. The supernatant was transferred to an ultrafiltration tube and concentrated to approximately 0.5-1 ml. MAGE-A4 was analyzed using a Superdex 200 molecular sieve (purchased from Cytiva Life Sciences) on an AKTA Pure instrument (purchased from Cytiva Life Sciences). 146-154 The peptide / HLA-A11 complex was purified. MAGE-A4 was collected based on the absorbance at 280 nm. 146-154 The HLA-A11 complex protein peak was observed at approximately 15.8 mL (shown in...). Figure 2 ).

[0116] MAGE-A4 purified by molecular sieve 146-154 The HLA-A11 complex protein sample was collected in an ultrafiltration concentration tube and concentrated to approximately 500 μL. The precipitate was then removed by centrifugation at 4°C to obtain unbiotinylated MAGE-A4. 146-154 / HLA-A11 complex protein sample.

[0117] (2) Biotinylation reaction

[0118] Use the MAGE-A4 obtained in step (1) 146-154 / HLA-A11 complex protein samples were prepared into biotinylation reaction systems (500 μl) as follows.

[0119] Biotinylation reaction system (purchased from AVIDITY): Total 500 μL

[0120] MAGE-A4 146-154 / HLA-A1 complex protein sample 5 mg / ml 200 μL,

[0121] Buffer A (N-di(hydroxyethyl)glycine buffer) 50 μL,

[0122] Buffer B (ATP, Biotin) 50 μL,

[0123] Buffer D (Biotin) 50 μL,

[0124] 200 μM biotin, Bir-A enzyme (3 mg / ml) 7 μL,

[0125] 20 mM Tris-Cl, 50 mM NaCl, pH8.0 150 μL.

[0126] After preparation, mix well, place on ice, and incubate overnight in a cold storage at 4°C.

[0127] The biotinylated reaction system sample was purified by passing it through a Superdex 200 molecular sieve to remove excess biotin. This process was repeated to obtain the biotinylated MAGE-A4. 146-154 The HLA-A11 complex protein is approximately 0.1-0.2 mg, including biotinylated MAGE-A4. 146-154 The peak value of the HLA-A11 complex protein was approximately 15.5 mL (shown in...). Figure 2 ).

[0128] Biotinylation efficiency assay:

[0129] The above biotinylated MAGE-A4 146-154 The HLA-A11 complex was concentrated to approximately 1 mg / mL, and samples were taken for SDS-PAGE shift assays to verify the biotinylation effect.

[0130] Set up one sample and two controls:

[0131] A. Biotinylated MAGE-A4 146-154 / HLA-A11 complex sample 8 μL + molecular sieve buffer 2 μL;

[0132] B. Biotinylated MAGE-A4 146-154 / HLA-A11 complex sample 8 μL + streptavidin 2 μL (20 mg / mL);

[0133] C. Streptavidin 2 μL + molecular sieve buffer 8 μL.

[0134] The above samples were incubated on ice for 2 hours and then analyzed by SDS-PAGE. The results are shown below. Figure 2 middle.

[0135] The results showed that the biotinylated MAGE-A4 peptide / HLA-A11 complex could bind to streptavidin to form a large molecule, resulting in a delayed band appearance in SDS-PAGE. By comparing the ratio of ((pMHC sample band grayscale) - (pMHC+SA sample band grayscale)) / pMHC sample band grayscale before and after peptide biotinylation, it can be determined that MAGE-A4... 146-154 The HLA-A11 complex can efficiently biotinylate (e.g., Figure 2 (As shown).

[0136] (3) MAGE-A4 146-154 Preparation of peptide / HLA-A11-PE tetramer

[0137] Biotinylated MAGE-A4 146-154 The HLA-A11 complex molecules were concentrated by ultrafiltration, following the streptavidin-PE:MAGE-A4 formula. 146-154 The HLA-A11 complex at a molar ratio of 1:5 will be used to treat biotinylated MAGE-A4. 146-154 The HLA-A11 complex was tetrameric by adding streptavidin-PE or streptavidin-APC, and then incubated overnight at 4°C to obtain MAGE-A4. 146-154 / HLA-A11-PE tetramer available for use.

[0138] 2. MAGE-A4 146-154Peptide / HLA-A11-PE tetramer-specific T cell sorting and single-cell TCR gene amplification and sequencing

[0139] After washing and resuspending with PBS, the sample obtained in Example 1 after MAGE-A4 was taken. 146-154 Approximately 1 x 10^ ... 7 Centrifuge at 200-250g for 10 min; wash three times with PBS containing 0.5% BSA, and centrifuge at 200-250g for 10 min. Add PerCP-Cy5-CD8 (purchased from BD), FITC-CD3 fluorescent antibody (purchased from BD), and the MAGE-A4 obtained above. 146-154 / HLA-A11-PE tetramer (obtained from the tetramer preparation in step 1 above) was incubated with mouse spleen cells at a molar ratio of 1:1:1 at 25°C for 20 minutes; washed three times with PBS containing 0.5% BSA, centrifuged at 200-250g for 10 minutes; and resuspended the cells in PBS containing 0.5% BSA.

[0140] The cells were then sorted using flow cytometry. Lymphocyte subsets were selected, with CD3+ being the most prominent. + CD8 + T cells were sorted to obtain MAGE-A4. 146-154 HLA-A11-PE tetramer positive cells (shown in) Figure 3 (The boxes show tetramer-positive cells and their proportions).

[0141] Single positive cells were sorted into 96-well plates containing cell lysis buffer (purchased from Tiangen Biotech) and RNase inhibitor (purchased from Kangwei Century Biotech). MAGE-A4 assays were then performed on each well. 146-154 Total RNA was extracted from HLA-A11-PE tetramer-positive T cells, and the 5' RACE TCR gene was amplified, as detailed below.

[0142] 5' RACE consists of three steps: reverse transcription (RT-PCR), first-round PCR amplification, and second-round PCR amplification. The following procedure uses the Takara D315-FullRACE Kit and follows the manufacturer's instructions.

[0143] (1) RT-PCR: The downstream primer used was the TCR gene constant region specific primer GSP1 (purchased from Takara), and the upstream primer was a target-switching primer with oligoguanine deoxyribonucleic acid (Oligo dG) at the 3' end (purchased from Takara).

[0144] (2) First round of PCR: Using the TCR cDNA obtained in (1) above as a template, the upstream primer is the outer adapter primer 1 (5'RACE outer Primer, Takara), and the downstream primer is a specific primer for the TCR constant region upstream of the constant region GSP1 (purchased from Takara), to obtain the first round of PCR product of the α chain or β chain of TCR.

[0145] (3) Second round PCR: Using the first round PCR product of the α or β chain of TCR obtained in (2) above as a template, the upstream primer is the inner adapter primer 2 (5'RACE inner Primer, Takara), and the downstream primer is a segment of TCR constant region specific primer upstream of the constant region GSP2 (purchased from Takara D315 -FullRACE Kit) to obtain the second round PCR product of the α or β chain of TCR respectively.

[0146] The second round of PCR amplification products containing the TCR α and β variable regions were subjected to agarose gel electrophoresis, and the target genes of the TCR α or β variable regions were obtained at the 500 bp position, respectively. The target bands were recovered, and the target gene fragments were ligated into the T vector (pMD18T, Takara) using T4 ligase. The ligation products were then transformed into DH5α cells (purchased from Tiangen Biotech), and single-clone gene sequencing was performed (commissioned by Ruiboxingke).

[0147] After the above process, the obtained MAGE-A4 146-154 Single-cell TCR gene amplification and sequencing were performed on peptide-specific T cells. After analysis of the results, the high-frequency α-chain and β-chain combinations were selected as new TCRs. MAGE-A4 was then used to analyze the TCRs. 146-154 The peptide-specific TCRs were named A11M103J TCR, A11M141J TCR, A11M161J TCR, A11M162J TCR, and A11M163J TCR, respectively, and their binding and functional verification were further performed.

[0148] The A11M103J TCR has an α-chain variable region as shown in the sequence of SEQ ID NO: 9, and a β-chain variable region as shown in the sequence of SEQ ID NO: 13.

[0149] The A11M141J TCR has an α-chain variable region as shown in the sequence of SEQ ID NO: 25, and a β-chain variable region as shown in the sequence of SEQ ID NO: 29.

[0150] The A11M161J TCR has an α-chain variable region as shown in the sequence of SEQ ID NO: 37, and a β-chain variable region as shown in the sequence of SEQ ID NO: 41.

[0151] The A11M162J TCR has an α-chain variable region as shown in the sequence of SEQ ID NO: 49, and a β-chain variable region as shown in the sequence of SEQ ID NO: 53.

[0152] The A11M163J TCR has an α-chain variable region as shown in the sequence SEQ ID NO: 61 and a β-chain variable region as shown in the sequence SEQ ID NO: 65.

[0153] Example 3. MAGE-A4 146-154 Cell binding assays of peptide / HLA-A11 tetramer to specific TCRs expressing various MAGE-A4 peptides

[0154] In this embodiment, the inventors further confirmed that the selected A11M103J TCR, A11M103J TCR, A11M141J TCR, A11M161J TCR, and A11M162J TCR have specific targeting properties for MAGE-A4. 146-154 Specific recognition of HLA-A11.

[0155] First, the variable region (V region) genes of the α and β chains of the TCR obtained in step 2 above were ligated with the constant region (C region) genes of the α and β chains of the human TCR (synthesized by Hongxun Biotechnology Co., Ltd.) to obtain chimeric TCR α and β chain sequences. The specific sequences of the chimeric sequences (taking A11M103J as an example) are shown in Table 1 below.

[0156] Table 1. Selected TCR chimeric TCR α and β chain sequences

[0157]

[0158] The α and β chains of the TCR were linked by a P2A sequence (the amino acid sequence of the P2A sequence is shown in SEQ ID NO: 3). Using the lentiviral expression plasmid pCDH (purchased from Invitrogen) as the starting plasmid, chimeric A11M103J TCR, A11M141J TCR, A11M161J TCR, A11M162J TCR, and A11M163J TCR lentiviral expression vectors were constructed, namely lentiviral expression vectors A11M103J-pCDH, A11M141J-pCDH, A11M161J-pCDH, A11M162J-pCDH, and A11M163J-pCDH.

[0159] The TCR-pCDH lentiviral expression vector described above was co-transfected with the CD3-CD8-pCDH plasmid (purchased from Nanjing Genscript Biotech) expressing CD3 and CD8 at a 1:1 ratio into HEK-293T cells (purchased from ATCC). Twenty-four hours after co-transfection, the cells were centrifuged at 200-250 g for 10 min; washed three times with PBS containing 0.5% BSA; and centrifuged at 200-250 g for 10 min to obtain HEK-293T cells expressing each TCR.

[0160] Then, in order to further verify the above TCR and its corresponding MAGE-A4, the inventors... 146-154 The binding of peptides will cause the MAGE-A4 prepared above to bind. 146-154 / HLA-A11-PE tetramer was stained and analyzed with 293T cells expressing A11M103J TCR, A11M141J TCR, A11M161JTCR, A11M162J TCR, and A11M163J TCR to evaluate its binding specificity.

[0161] Specifically, the co-transfected HEK-293T cells were collected and divided into two equal portions. One portion was co-incubated with PerCP-Cy5-TCR antibody (Biolegend); the other portion was incubated with PerCP-Cy5-CD3 (Biolegend) and FITC-CD8 antibody (Biolegend), as well as the MAGE-A4 prepared above. 146-154 HLA-A11-PE was incubated with each cell at a 1:1:1 molar ratio for 30 min; the cells were washed three times with PBS containing 0.5% BSA, centrifuged at 200-250g for 10 min; and resuspended in PBS containing 0.5% BSA to detect the frequency of TCR-positive or tetramer-positive cells. Flow cytometry was used for analysis (shown in...). Figure 4 ).

[0162] Figure 4 This is a flow cytometry image of HEK-293T cells transfected with CD3 alone or co-transfected with CD3 and TCR by PerCP-TCR. The first row (horizontal) shows the expression of A11M103J TCR, A11M141J TCR, A11M161J TCR, A11M162J TCR, and A11M163J TCR, demonstrating that 293T cells transfected with A11M103J TCR, A11M141J TCR, A11M161J TCR, A11M162J TCR, and A11M163J TCR can express TCR. The second row (horizontal) shows the expression of TCR in HEK-293T cells transfected with MAGE-A4. 146-154Flow cytometry analysis of HLA-A11-PE tetramer on HEK-293T cells co-transfected with CD3-CD8 and A11M103J TCR, A11M141J TCR, A11M161J TCR, A11M162JTCR, and A11M163J TCR; results showed that 293T cells transfected with A11M103J TCR, A11M141J TCR, A11M161J TCR, and A11M162J TCR specifically bound to MAGE-A4. 146-154 / HLA-A11 tetramer.

[0163] Example 4. MAGE-A4 146-154 Specific TCR and MAGEA-4 146-154 Analysis of peptide / HLA-A11 binding characteristics

[0164] To accurately measure MAGE-A4 146-154 Specific TCR and corresponding MAGEA-4 146-154 The binding properties and affinity of the peptide / HLA-A11 complex were further investigated using surface plasmon resonance (SPR) assays at the protein level. Since the binding regions of each TCR are extracellular regions, and extracellular regions without transmembrane regions are soluble proteins, the extracellular region of A11M103J was synthesized. The specific procedures are as follows.

[0165] 1. TCR protein expression and purification

[0166] The extracellular regions of the α and β chains of the A11M103J TCR were optimized according to prokaryotic codons to synthesize chimeric DNA sequences of the extracellular regions of the α and β chains of A11M103J (A11M103J: α chain, SEQ ID NO: 21; β chain, SEQ ID NO: 22). The A11M103J TCR possesses an α chain variable region as shown in SEQ ID NO: 9 and a β chain variable region as shown in SEQ ID NO: 13. Restriction sites Nde I and Xho I were introduced, with Nde I located at the 5' end and Xho I located at the 3' end. The DNA sequences of the extracellular regions of the synthesized A11M103J TCR α and β chains were cloned into the expression vector pET21a (Invitrogen) using the restriction sites Nde I and Xho I, respectively, to establish prokaryotic recombinant expression plasmids for the extracellular regions of the A11M103J TCR α and β chains.

[0167] The expression plasmid was transformed into E. coli BL21(DE3) competent cells (TransGen Biotech) using the heat shock method, and IPTG was added to induce expression, obtaining the extracellular regions of the A11M103J TCR α and β chains in inclusion body state.

[0168] Inclusion bodies of the extracellular regions of the α chain and β chain of A11M103J TCR (each inclusion body was dissolved at 30 mg / ml in a solution containing 6 M Gua-HCl, 50 mM Tris pH 8.0, 100 mM NaCl, 10 mM EDTA, and 10 mM DTT) were added dropwise at a mass ratio of 2:1 to 1 L of prepared refolding solution (5 M urea, 20 mM Tris-HCl, 400 mM L-arginine, 2 mM EDTA, 5 mM GSH / GSSG / 1 mM). The addition was done in three separate drops of 3 mL each, with an interval of at least 8 h between each addition. The solution was then concentrated using a concentrator (Millipore).

[0169] After concentration, the solutions were placed in 4 L of deionized water and 4 L of dialysis buffer (10 mM Tris, 10 mM NaCl, pH 8.0) for 24 h each. Preliminary purification was then performed using Source 15Q ion-exchange chromatography, and the target protein was identified by SDS-PAGE.

[0170] Specifically, the target protein was concentrated using a concentrator (Millipore), and the buffer was changed with a solution of 20 mM Tris-HCl, 50 mM NaCl, pH 8.0. After concentration, it was purified using a Source 15Q ion exchange column (GE Healthcare) to obtain approximately 3-4 mg of A11M103J TCR protein. The target protein was detected by reducing (containing dithiothreitol (DTT)) and non-reducing (without dithiothreitol (DTT)) SDS-PAGE. Figure 5 ).

[0171] The results showed that A11M103J TCR eluted at 22.2 mS / cm, and the target protein peak appeared in molecular sieve chromatography at an elution volume of 15 mL. SDS-PAGE analysis revealed that A11M103J TCR exhibited an αβ heterodimer. In non-reducing SDS-PAGE without DTT, the band size was approximately 52 KD. In reducing SDS-PAGE with DTT, the disulfide bonds between the α and β chains were broken, resulting in bands of approximately 24 KD and 28 KD, respectively. Figure 5 ).

[0172] 2. SPR detection and analysis

[0173] The A11M103J TCR protein prepared by the above in vitro refolding experiment, and the biotinylated MAGE-A4 prepared in Example 1 were used. 146-154 The HLA-A11 complex protein was transferred to SPR buffer (PBS, 0.005% Tween-20, pH 7.4). MAGE-A4 was then added. 146-154 The HLA-A11 complex protein was diluted to 20 μg / ml and immobilized onto different channels of an SA chip (GE Health). Then, gradient dilutions (0 μM, 0.78 μM, 1.56 μM, 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM) of the A11M103J TCR protein were passed through the corresponding channels of the SA chip. Binding kinetic parameters were analyzed using BIA evaluation software, and affinity constants were calculated. The binding of A11M103J TCR to MAGE-A4 was investigated. 146-154 / HLA-A11 complex protein affinity detection ( Figure 5 ).

[0174] The results showed that A11M103J TCR combined with MAGE-A4 146-154 The binding of (RCFPVIFGK) / HLA-A11 exhibits a fast binding and fast dissociation pattern, with a binding affinity (KD) of 6.56 μM.

[0175] Therefore, the SPR results indicate that A11M103J TCR can bind to MAGE-A4. 146-154 / HLA-A11. Therefore, the A11M103J TCR has good binding properties and affinity, and it can be inferred that when the A11M103J TCR is used in anti-tumor therapy, it can bind to the MAGE-A4 gene. 146-154 Tumor cells produce IFN-γ, which in turn kills tumor cells, thus achieving the effect of treating tumors.

[0176] Example 5. Preparation of TCR-T cells and their reaction with corresponding polypeptide epitopes

[0177] In this embodiment, the A11M103J TCR gene was introduced into peripheral blood mononuclear cells (PBMCs) isolated from healthy volunteers to serve as TCR-T effector cells. MAGE-A4 was used... 146-154 Peptides or expressions of HLA-A11 and MAGE-A4 146-154Target cells were co-cultured with the aforementioned TCR-T effector cell system, and the levels of IFN-γ secreted by effector cells and target cells presenting MAGE-A4 peptide were detected. The effects of A11M103J TCR and MAGE-A4 expression on the TCR were investigated. 146-154 The effects of the peptide / HLA-A11 on target cells were evaluated. The specific procedures are as follows.

[0178] 1. Expressing MAGE-A4 146-154 Preparation of lentiviruses with specific TCRs

[0179] The A11M103J TCR lentiviral expression plasmid (A11M103J-pCDH) from Example 3 was mixed with lentiviral packaging plasmids PLP1, PLP2, and VSVG (purchased from Addgene) at a ratio of PLP1: PLP2: VSVG: TCR-pCDH = 20: 13: 5:20. 20 μL of this mixture was diluted in 1.25 ml of DMEM medium to prepare the DNA solution. 20 μL of polyetherimide (PEI, 1 μg / μl) was added to 1.25 ml of DMEM, and the entire PEI / DMEM solution was added to the prepared DNA solution. After incubating at room temperature for 15 minutes, the mixture was added to HEK-293T cells (Shanghai Cell Bank) cultured in 15 cm dishes and mixed thoroughly. After 6 hours, the culture medium was carefully aspirated, and 25 ml of fresh culture medium was added for continued culturing. After 72 hours, the supernatant containing the virus was collected, which is the lentivirus supernatant expressing A11M103J TCR.

[0180] 2. MAGE-A4 146-154 Preparation of specific TCR-T cells and detection of TCR expression efficiency

[0181] Peripheral blood lymphocytes were collected from two healthy volunteers (D1 and D2) to obtain PBMCs. ThermoFisher microspheres coated with anti-CD3 / anti-CD28 were added to the PBMCs at a 1:1 ratio for activation and overnight culture. Then, A11M103J TCR lentivirus was added to the PBMCs at a 1:1 volume ratio, mixed thoroughly, and incubated at 37°C in a 5% CO2 incubator. After 24 hours, the culture medium was replaced with complete medium, and the cells were cultured until day 10.

[0182] The anti-CD3 / anti-CD28 microspheres were removed under magnetic field conditions and washed twice with the same culture medium as cell culture to obtain the A11M103J TCR-T cells of this embodiment.

[0183] The MAGE-A4 prepared using PBMC cells mentioned above146-154 Specific TCR-T cells (cultured for 10 days) were cultured, and A11M103J TCR-T cells were processed using MAGE-A4. 146-154 HLA-A11 tetramer staining was performed using flow cytometry, as in Example 2, to confirm the expression of A11M103J TCR. Figure 6 ).

[0184] The results showed that tetramer-positive T cells with different positive rates of 14%-18% could be detected in A11M103J TCR-T cells prepared from volunteers D1 and D2 using PBMC cells.

[0185] It is known that these A11M103J TCR-T effector cells of the present invention can specifically bind to the corresponding MAGE-A4. 146-154 / HLA-A11.

[0186] 3. MAGE-A4 146-154 Specific TCR-T cells and MAGE-A4 146-154 Immunoreactivity assay of peptides

[0187] The A11M103J TCR-T cells were compared with those loaded with MAGE-A4 using the T cell detection method (IFN-γ-ELISPOT). 146-154 The levels of IFN-γ secreted by the target cells of the peptide or after its action were detected.

[0188] A11M103J TCR-T cells prepared using PBMC cells from two volunteers, D1 and D2, were mixed with HLA-A11-expressing antigen-presenting cells (K562-A11) (from the Peking Union Medical College Hospital Cell Bank) at a cell ratio of 2:1, and then injected at a volume of 100 μL (1.5 × 10⁶ cells / mL). 4 Cells were added per well to ELISPOT plates pre-coated with anti-IFN-γ antibody (BD Biosciences), followed by the addition of MAGE-A4 to ELISPOT plates containing A11M103J TCR-T cells. 146-154 The peptide (100 μL volume, 20 μg / mL) was used as the experimental group, and KRAS-G12V was added simultaneously. 8-16 (SEQ ID NO:2) was used as a negative control (100 μL, 20 μg / mL), and RPMI-1640 medium was added as a blank control (100 μL). The ELISPOT plates were cultured in a 37°C, 100% humidity, 5% CO2 cell incubator for 18 hours. ELISPOT spot analysis was performed on IFN-γ-producing specific T cells, and the results are shown below. Figure 7 .

[0189] ELISPOT experimental results showed that A11M103J TCR-T cells prepared using PBMCs from D1 and D2 volunteers were able to resist MAGE-A4. 146-154 The peptide elicited a strong T-cell immune response; however, no T-cell response to the control peptide was detected. Figure 7 ).

[0190] Example 6. MAGE-A4 146-154 Validation of specific T cell receptor A11M103J

[0191] In this embodiment, the A11M103J TCR-T effector cells prepared in Example 5 were used to verify the effect of A11M103J TCR on MAGE-A4. 146-154 Peptide recognition sensitivity, expression of HLA-A11 and MAGE-A4 146-154 The in vitro function and in vivo tumor-suppressive activity of target cells were investigated. The specific procedures are as follows.

[0192] 1. A11M103H TCR-T versus MAGE-A4 146-154 Sensitivity of peptide reactions

[0193] Using IFN-γ-ELISA to compare A11M103J TCR-T cells with different concentrations of MAGE-A4 146-154 The level of IFN-γ secreted after the peptide acts on target cells was detected.

[0194] Using the method described in Example 5, A11M103JTCR-T cells prepared from PBMC cells of two volunteers, D1 and D2, were mixed with target cells expressing HLA-A11 (K562-A11 or PANC1) (from the Kyowa Cell Bank) at a cell ratio of 2:1, and then added at a volume of 100 μL (1.5 × 10⁻⁶ cells / mL). 4 Cells per well were added to 96-well flat-bottomed plates, and different concentrations of MAGE-A4 were added. 146-154 Peptide (final concentration 1 nM-100 μM), three replicates per well, with different concentrations (final concentration 1 nM-100 μM) of KRAS-G12V added. 8-16 The polypeptide (SEQ ID NO:2) was used as a negative control. The 96-well flat-bottomed plates were incubated at 37°C, 100% humidity, and 5% CO2 for 24 hours. The supernatant from the 96-well plates was collected and centrifuged at 500 g for 5 min to remove residual cells. The supernatant was then added to an ELISA plate (BD Biosciences) coated with anti-IFN-γ antibody (BD Biosciences) to detect the IFN-γ level in the supernatant. The results were presented as follows: Figure 8 .

[0195] ELISA results of IFN-γ levels showed that A11M103J TCR-T cells prepared using PBMC cells from D1 and D2 volunteers were able to inhibit MAGE-A4. 146-154 The peptide elicited a strong T-cell immune response, and the level of secreted IFN-γ increased with increasing peptide concentration, while no T-cell response was detected with the irrelevant control peptide. Figure 8 ).

[0196] 2. A11M103J TCR-T expression of HLA-A11 and MAGE-A4 146-154 Detection of immune response of target cells

[0197] Different T cell detection methods (IFN-γ-ELISPOT or IFN-γ-ELISA) were used to analyze A11M103J TCR-T cells expressing HLA-A11 and MAGE-A4. 146-154 The level of IFN-γ secreted after action on target cells was detected.

[0198] In the IFN-γ-ELISPOT assay, A11M103J TCR-T cells prepared from PBMC cells of two volunteers, D1 and D2, were compared with cells expressing HLA-A11 and MAGE-A4. 146-154 The target cells (A375-A11 or K562-A11-MAGE-A4 or PANC1-MAGE-A4) (from the Kyowa Cell Bank) were mixed at a 2:1 ratio and in 200 μL volumes at a concentration of 1.5 × 10⁻⁶. 4 Cells / well were added to ELISPOT plates pre-coated with anti-IFN-γ antibody, expressing either HLA-A11 or MAGE-A4 only. 146-154 Cells (A375, K562-A11, or PANC1) (from Kyowa Cell Bank) were added in parallel as a negative control. The ELISPOT plates were cultured for 18 hours in a 37°C, 100% humidity, 5% CO2 cell incubator. ELISPOT spot analysis was performed on IFN-γ-producing specific T cells, and the results are shown below. Figure 9 B.

[0199] Furthermore, A11M103J TCR-T cells (2 × 10⁻⁶) were prepared using PBMC cells from two volunteers, D1 and D2. 4 Cell count / well), and expression of HLA-A11 and MAGE-A4 146-154Target cells (A375-A11, K562-A11-MAGE-A4, or PANC1-MAGE-A4) were mixed in different ratios (target cells: effector cells from 2:1 to 0.06:1). The prepared cell suspension was spread into 96-well plates with a flat bottom, 200 μL of cell suspension per well, expressing HLA-A11 or MAGE-A4. 146-154 Cells (A375, K562-A11, or PANC1) were added in parallel at corresponding ratios as negative controls, with three replicates per well. After culturing at 37°C for 20 h, the supernatant from the 96-well plate was collected and centrifuged at 500 g for 5 min to remove residual cells. The supernatant was then added to an ELISA plate (BD) coated with anti-IFN-γ antibody (BD Biosciences) to detect the IFN-γ level in the supernatant. The results were displayed on [the image / image / etc.]. Figure 9 CD.

[0200] ELISPOT experimental results showed that A11M103J TCR-T cells prepared using PBMCs from D1 and D2 volunteers were able to target cells expressing HLA-A11 and MAGE-A4. 146-154 The expression of HLA-A11 or MAGE-A4 on target cells elicited a strong T-cell immune response; on the other hand, no response was detected against HLA-A11 or MAGE-A4 expression alone. 146-154 T cell response of target cells ( Figure 9 B).

[0201] ELISA results of IFN-γ levels showed that A11M103JTCR-T cells prepared using PBMCs from D1 and D2 volunteers were able to target cells expressing HLA-A11 and MAGE-A4. 146-154 The target cells generate a T-cell immune response, and the T-cell immune response increases with the increase of the number of target cells. Figure 9 CD).

[0202] Therefore, it can be concluded that A11M103J TCR-T cells can specifically recognize HLA-A11 and MAGE-A4 expression. 146-154 The target cells can specifically secrete the cytokine IFN-γ, suggesting that the A11M103J TCR-T cells of the present invention have potential target cell killing activity and tumor therapeutic value.

[0203] 3. A11M103J TCR-T expression of HLA-A11 and MAGE-A4 146-154 In vitro killing activity of target cells

[0204] Flow cytometry was used to analyze A11M103J TCR-T cells expressing HLA-A11 and MAGE-A4. 146-154After the gene acts on the target cells, the killing activity of the target cells is evaluated.

[0205] A11M103J TCR-T cells were prepared using PBMC cells from D1 and D2 volunteers, and were expressed with HLA-A11 and MAGE-A4. 146-154 The target cells (A375-A11 or MDA-MB-436-A11) (from the Kyowa Cell Bank) were mixed at a 4:1 ratio and dispensed at a volume of 200 μL (5 × 10⁶ cells / mL). 5 Cells were added per well to 96-well plates with a flat bottom. Mock-T cells not transfected with A11M103J TCR were mixed with target cells in the same proportion as a control group. After culturing at 37°C for 48 h, all cells in each well were collected and centrifuged at 200-250g for 10 min. The cells were washed three times with PBS containing 0.5% BSA and centrifuged at 200-250g for 10 min to obtain a mixed sample of cytotoxic T cells and target cells.

[0206] The obtained T cells and target cells were co-incubated with PerCP-Cy5-CD3 and FITC-CD8 antibodies (Biolegend) at a 1:1 molar ratio for 30 min; washed three times with PBS containing 0.5% BSA, centrifuged at 200-250 g for 10 min, and absolute quantitative beads (Invitrogen) were added as a quantitative indicator. Flow cytometry analysis was then performed (shown in...). Figure 10 A, B).

[0207] Flow cytometry results showed that A11M103J TCR-T expressed HLA-A11 and MAGE-A4. 146-154 After incubation with target cells, the number and proportion of remaining target cells were lower than those in the Mock-T group, indicating that A11M103J TCR-T cells can target cells expressing HLA-A11 and MAGE-A4. 146-154 The target cells (A375-A11 and MDA-MB-436-A11) exhibited strong in vitro killing activity, while the Mock-T cells could not kill the corresponding target cells. This suggests that the A11M103J TCR-T cells of the present invention have strong target cell killing activity and potential value in tumor therapy.

[0208] 4. Expression of MAGE-A4 146-154 Cell subset analysis of target cell activated A11M103J TCR-T cells

[0209] Intracellular cytokine staining using flow cytometry was used to identify A11M103J TCR-T cells expressing MAGE-A4. 146-154The level of intracellular IFN-γ secreted after the gene acts on target cells was detected.

[0210] A11M103J TCR-T cells were prepared using PBMC cells from D1 and D2 volunteers, and were expressed with HLA-A11 and MAGE-A4. 146-154 The target cells were mixed at a 1:1 ratio and administered at a volume of 200 μL (1 × 10⁻⁶). 6 Cells were added per well to a flat-bottomed 96-well plate. After 1 hour of cell incubation, 1 μl of Golgi inhibitor was added to each well. After 6 hours, the cells were centrifuged at 200-250 g for 5 min. The cells were washed three times with PBS containing 0.5% BSA and centrifuged at 200-250 g for 5 min to obtain A11M103JTCR-T cells expressing cytokines intracellularly.

[0211] The obtained A11M103J TCR-T cells were co-incubated with PerCP-Cy5-CD3 and FITC-CD8 antibodies (Biolegend) at a 1:1 molar ratio for 30 min; washed three times with PBS containing 0.5% BSA, and centrifuged at 200-250 g for 10 min; 250 μL of cell permeabilization and fixation solution (BD) was added to each sample, and the cells were incubated on ice for 30 min, then washed three times with PermWash washing buffer (BD), and centrifuged at 200-250 g at 4℃ for 10 min; the cells were co-incubated with IFN-γ antibody (Biolegend) for 30 min, then washed three times with PermWash washing buffer (BD), and centrifuged at 200-250 g at 4℃ for 10 min; the cells were resuspended with PermWash washing buffer. Flow cytometry was used for analysis (shown in...). Figure 11 ).

[0212] Flow cytometry results showed that A11M103J TCR-T interacted with expressions of HLA-A11 and MAGE-A4. 146-154 Incubation with target cells can induce TCR-T cells to express IFN-γ cytokine, and A11M103J TCR-T cells expressing IFN-γ are CD8-positive T cell subsets. On the other hand, A11M103J TCR-T cells do not express IFN-γ after incubation with control target cells.

[0213] Therefore, A11M103J TCR-T can express HLA-A11 and MAGE-A4. 146-154 The target cells are activated to secrete IFN-γ, and the cell group that performs the function of IFN-γ cytokines is CD8+ T.

[0214] 5. Tumor suppressive activity of A11M103J TCR-T cells in a mouse model of tumors

[0215] In this embodiment, the tumor-suppressive effect of A11M103J TCR-T cells was evaluated using the NCG immunodeficient mouse A375-A11-luciferase melanoma tumor model.

[0216] The steps of the NCG mouse tumor suppression experiment using TCR-T cells included:

[0217] (1) Establishment of A375-A11-luciferase tumor model in NCG mice

[0218] NCG mice were obtained from Nanjing University-Nanjing Institute of Biomedicine and Biotechnology. Each NCG mouse was subcutaneously inoculated with HLA-A11 and MAGE-A4. 146-154 And A375-A11-luciferase tumor cells (Peking Union Medical College Hospital Cell Resource Center) were used to establish a mouse model of melanoma in NCG mice:

[0219] a) Number of A375-A11-luciferase cells inoculated: 2 × 10 5 1 cell / 200 μL / animal;

[0220] b) Injection site: Subcutaneous tissue on the back;

[0221] (2) A11M103J TCR-T cell therapy

[0222] On day 3 after inoculation of NCG mice with A375-A11-luciferase tumor cells, A11M103J TCR-T cells prepared from PBMCs of two volunteers (D1 and D2) in Example 5 were injected into NCG mice via the tail vein.

[0223] a) Number of A11M103J TCR-T cells inoculated: 1×10⁻⁶ 7 Cells / 200μL / animal;

[0224] b) Injection site: tail vein;

[0225] (3) Grouping and processing:

[0226] Three days after tumor cell injection, mice with relatively uniform tumor formation were selected and grouped, and then treated with A11M103JTCR-T cells via tail vein injection. This example used T cells (1×10⁻⁶) that had not been transfected with TCR. 7The injection group served as a negative control, with 10 mice in each group. Five mice were in each of the TCR-T cell therapy groups prepared on days 1 and 2. The PBS injection group served as a blank control, with 5 mice in each group. The group information and treatments are shown in the table below:

[0227] Table 2. Mouse grouping and treatment

[0228]

[0229] After tumor formation, tumor size is assessed by in vivo imaging every 4-7 days until the total in vivo fluorescence in mice reaches 3e. 10 At the end of the experiment, the mice were euthanized and the tumors were separated and weighed.

[0230] (4) Observation of treatment effect:

[0231] 1) Tumor growth detection:

[0232] a) After TCR-T cell injection, mice were injected intraperitoneally with fluorescein substrate (15 mg / mL, 200 μL / mouse) (Perkin Elmer). Ten minutes after injection, the growth of subcutaneous tumors in mice was analyzed using an in vivo imaging system, and the total fluorescence of tumor tissue was calculated using Living Image software.

[0233] b) The experiment was terminated after the last observation, and the tumor tissue was separated and weighed directly;

[0234] In vivo imaging and tumor weighing results in mice showed that tumors in the A11M103J TCR-T cell therapy group were significantly suppressed compared to the negative control group, and tumor cells were completely eliminated in 4 out of 10 mice in the A11M103J TCR-T cell therapy group. Figure 12 A); The total tumor fluorescence and tumor weight in the A11M103J TCR-T cell therapy group were significantly lower than those in the negative control group (t-test, **: p < 0.01, ****: p < 0.0001). Figure 12 B, C). The results of this embodiment indicate that A11M103J TCR-T cells can effectively inhibit tumor growth and have potential value in tumor treatment (e.g., ...). Figure 12 ).

[0235] Example 7. MAGE-A4 146-154 Validation of specific T cell receptor A11M103H

[0236] In this embodiment, it was confirmed that the A11M103H TCR has the effect of targeting MAGE-A4. 146-154 The specific recognition of HLA-A11 was evaluated, and the effect of A11M103H TCR on the expression of HLA-A11 and MAGE-A4 was assessed.146-154 The in vitro toxicity and in vivo tumor-suppressive activity of target cells were determined through the following procedures:

[0237] The framework region (FR) in the variable regions of the α and β chains of the chimeric A11M103J TCR obtained in Example 3 was humanized and named A11M103H TCR, resulting in the sequences of the humanized A11M103H TCR α and β chain variable regions (SEQ ID NO: 73, SEQ ID NO: 76). The humanized variable regions were then spliced ​​with the human constant regions to obtain the full-length sequences of the A11M103H TCR α and β chains (SEQ ID NO: 74, SEQ ID NO: 77), and their encoded amino acid sequences (SEQ ID NO: 75, SEQ ID NO: 78). The humanization strategy and the humanized sequences are shown in [the table / document / etc.]. Figure 13 .

[0238] 1. MAGE-A4 146-154 / HLA-A11 tetramer binding assay with HEK-293T cells expressing A11M103H TCR

[0239] Similar to the experimental method in Example 3, the α and β chains of A11M103H TCR were linked by a P2A sequence (the amino acid sequence of the P2A sequence is shown in SEQ ID NO: 3), and the humanized A11M103H TCR lentiviral expression vector, namely lentiviral expression vector A11M103H-pCDH, was constructed using the lentiviral expression plasmid pCDH (purchased from Invitrogen) as the starting plasmid.

[0240] The A11M103H TCR-pCDH lentiviral expression vector described above was co-transfected with the CD3-CD8-pCDH plasmid (purchased from Nanjing Genscript Biotech) expressing CD3 and CD8 at a 1:1 ratio into HEK-293T cells (purchased from ATCC). HEK-293T cells transfected with A11M103H TCR were used as a positive control. Twenty-four hours after co-transfection, the cells were centrifuged at 200-250g for 10 min; washed three times with PBS containing 0.5% BSA; and centrifuged at 200-250g for 10 min to obtain HEK-293T cells expressing A11M103H TCR.

[0241] Subsequently, in order to further verify the relationship between A11M103H TCR and MAGE-A4, the inventors... 146-154 The binding of peptides will bind the MAGE-A4 prepared in Example 2. 146-154HLA-A11-PE tetramer was stained and analyzed in HEK-293T cells expressing A11M103H TCR to evaluate its binding specificity. Specifically, the co-transfected HEK-293T cells were collected and divided into two aliquots. One aliquot was co-incubated with PerCP-Cy5-TCR antibody (Biolegend); the other aliquot was co-incubated with PerCP-Cy5-CD3 and FITC-CD8 antibodies and MAGE-A4. 146-154 HLA-A11-PE tetramer was co-incubated at a 1:1:1 molar ratio for 30 min, with an irrelevant peptide HLA-A11-PE tetramer added as a negative control. Cells were washed three times with PBS containing 0.5% BSA and centrifuged at 200-250g for 10 min. Cells were resuspended in PBS containing 0.5% BSA for detection of TCR-positive cell frequency or MAGE-A4. 146-154 The frequency of HLA-A11-PE tetramer-positive cells was analyzed using flow cytometry (shown in...). Figure 14 ).

[0242] Flow cytometry results showed that A11M103H TCR could specifically bind to MAGE-A4. 146-154 / HLA-A11 complex.

[0243] 2. MAGE-A4 146-154 Specific A11M103H TCR and MAGE-A4 146-154 / HLA-A11 Binding Characteristics Analysis

[0244] To accurately measure MAGE-A4 146-154 Specific A11M103H TCR and MAGE-A4 146-154 The binding properties and affinity of the / HLA-A11 protein were investigated. The inventors used surface plasmon resonance (SPR) assays to detect the protein-level affinity. Since the binding regions of each TCR are extracellular, and extracellular regions without transmembrane regions are soluble proteins, the extracellular region of the A11M103H TCR was synthesized. The specific procedures are as follows:

[0245] (1) Expression and purification of A11M103H TCR protein

[0246] The extracellular regions of the A11M103H TCR α and β chains were optimized according to eukaryotic codons. Kozak sequences were added to the 5' ends of both the A11M103H α and β chains; a his tag was added to the 3' end of the A11M103H TCR α chain, resulting in the synthesis of the DNA sequence of the extracellular region of the A11M103H humanized TCR α chain (SEQ ID NO: 79); and a twin strep tag was added to the 3' end of the A11M103H TCR β chain, resulting in the synthesis of the DNA sequence of the extracellular region of the A11M103H humanized TCR β chain (SEQ ID NO: 80). Restriction sites Nde I and Xho I were introduced, with Nde I located at the 5' end and Xho I located at the 3' end. The extracellular DNA sequences of the synthesized A11M103H TCR α and β chains were cloned into the expression vector pCAGGS (Invitrogen) using the restriction sites Nde I and Xho I, respectively, to establish eukaryotic recombinant expression plasmids for the extracellular proteins of the A11M103H TCR α and β chains.

[0247] Using the same strategy as above, the extracellular regions of the chimeric A11M103J TCR α and β chains from Example 3 were optimized according to eukaryotic codons. Kozak sequences were added to the 5' ends of both the A11M103J TCR α and β chains; a his tag was added to the 3' end of the A11M103J TCR α chain, resulting in the DNA sequence of the extracellular region of the A11M103J TCR α chain (SEQ ID NO: 23). A twin strep tag was added to the 3' end of the A11M103J TCR β chain, resulting in the DNA sequence of the extracellular region of the A11M103J TCR β chain (SEQ ID NO: 24). Restriction sites Nde I and Xho I were introduced, with Nde I located at the 5' end and Xho I located at the 3' end. The extracellular DNA sequences of the synthesized A11M103J TCR α and β chains were cloned into the expression vector pCAGGS (Invitrogen) using the restriction sites Nde I and Xho I, respectively, to establish eukaryotic recombinant expression plasmids for the extracellular proteins of the A11M103J TCR α and β chains.

[0248] The A11M103H TCR has an α-chain variable region as shown in the sequence of SEQ ID NO: 73, and a β-chain variable region as shown in the sequence of SEQ ID NO: 76. The A11M103J TCR has an α-chain variable region as shown in the sequence of SEQ ID NO: 9, and a β-chain as shown in the sequence of SEQ ID NO: 13.

[0249] The α and β chains of the A11M103H TCR-pCAGGS or A11M103J TCR-pCAGGS eukaryotic protein expression vectors described above were co-transfected into 293F cells (purchased from Thermo Fisher) at a 1:1 mass ratio. The 293F cells were cultured in a constant-temperature shaker at 37°C, 5% CO2, and 150 rpm / min. The culture supernatant was collected on day 5 post-transfection and initially purified using a His affinity chromatography column. Subsequently, soluble A11M103H TCR protein or A11M103J TCR protein was further purified using a Superdex 200 molecular sieve.

[0250] (2) SPR detection and analysis

[0251] Similar to the experimental method in Example 4, the soluble A11M103H TCR protein or A11M103J TCR protein expressed in the eukaryotic system prepared in this example, as well as the MAGE-A4 prepared in Example 1, were used. 146-154 The HLA-A11 complex protein was transferred to SPR buffer (PBS, 0.005% Tween-20, pH 7.4). A11M103H TCR protein or A11M103J TCR protein was diluted to 20 μg / ml and immobilized onto different channels of a CM5 chip (GE Health). Then, MAGE-A4 was serially diluted (0 μM, 0.325 μM, 0.625 μM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM) 146-154 The HLA-A11 complex protein was passed through the corresponding channels of a CM5 chip, and binding kinetic parameters were analyzed using BIA evaluation software, and the affinity constant was calculated. The binding of A11M103H TCR protein to MAGE-A4 was also investigated. 146-154 Detection of HLA-A11 complex protein affinity; and A11M103J TCR protein and MAGE-A4 146-154 / HLA-A11 complex protein affinity detection ( Figure 15 ).

[0252] The results showed that the A11M103H TCR protein is associated with MAGE-A4. 146-154 The binding of the / HLA-A11 complex protein exhibits a rapid binding and dissociation pattern, with a binding affinity (KD) of 5.98 μM; the A11M103J TCR protein binds to MAGE-A4. 146-154 The binding of the HLA-A11 complex protein follows a rapid binding-rapid dissociation pattern, with a binding affinity (KD) of 5.95 μM. Figure 15).

[0253] Therefore, the SPR results indicate that the A11M103H TCR or A11M103J TCR proteins can interact with MAGE-A4. 146-154 The A11M103H TCR and A11M103J TCR bind to the HLA-A11 complex. Therefore, the A11M103H TCR and A11M103J TCR exhibit good binding properties and affinity, suggesting that when the A11M103H TCR is used in anti-tumor therapy, it can target the MAGE-A4 gene carrier. 146-154 Tumor cells produce IFN-γ, which in turn kills tumor cells, thus achieving the effect of treating tumors.

[0254] 3. Preparation of A11M103H TCR-T cells and their in vitro killing activity against target cells

[0255] (1) Preparation of A11M103H TCR-T cells and detection of TCR expression efficiency

[0256] Similar to the experimental method in Example 5, peripheral blood lymphocytes were collected from two healthy volunteers (Donor v88 and Donor v291) to obtain PBMCs. These PBMCs were then activated and cultured overnight using anti-CD3 / anti-CD28 microspheres (ThermoFisher) at a 1:1 ratio. Afterwards, step 1 of this example, "MAGE-A4", was performed. 146-154 In the experiment "HLA-A11 tetramer binding to HEK-293T cells expressing A11M103H TCR", the A11M103H TCR lentiviral expression vector A11M103H-pCDH constructed was added to PBMC cells at a 1:1 volume ratio, mixed well, and incubated at 37°C in a 5% CO2 incubator. After 24 hours, the medium was replaced with complete medium and cultured until day 10. The anti-CD3 / anti-CD28 microspheres were removed under magnetic field conditions, and the cells were washed twice with the same medium as those used for cell culture to obtain the A11M103H TCR-T cells of this example.

[0257] The A11M103H TCR-T cells (cultured for 10 days) prepared using PBMC cells were cultured and analyzed using MAGE-A4. 146-154 HLA-A11-APC tetramer staining was performed, and flow cytometry analysis was conducted in the same manner as in Example 2 to confirm the expression of A11M103H TCR. Figure 16 ).

[0258] The results showed that tetramer-positive T cells with varying positivity rates (13%–37%) could be detected in A11M103HTCR-T cells prepared from volunteers Donor v88 and Donor v291 using PBMC cells. This indicates that these A11M103HTCR-T effector cells of the present invention can specifically bind to MAGE-A4. 146-154 / HLA-A11.

[0259] (2) A11M103H TCR-T expression of HLA-A11 and MAGE-A4 146-154 In vitro killing activity of target cells

[0260] The expression of HLA-A11 and MAGE-A4 in A11M103H TCR-T cells was evaluated using luciferase assay. 146-154 After the gene acts on target cells, A11M103H TCR-T cells exhibit killing activity against target cells.

[0261] Expressing HLA-A11 and MAGE-A4 146-154 Target cells (A375-A11-luciferase or NCI-H520-luciferase (Kyoho Cell Bank), 1 × 10⁻⁶) 4 The cell suspension was prepared by mixing A11M103H TCR-T cells (effect cells: target cells from 8:1 to 0.25:1) with PBMC cells from two volunteers, Donor v88 and Donor v291, at different ratios. 200 μL of the prepared cell suspension was seeded into each well of a 96-well plate. Mock-T cells uninfected with A11M103HTCR lentivirus were added in parallel at the corresponding ratio as a negative control, with three replicates per well. Wells without T cells served as blank controls. After culturing at 37°C for 24 h, the cells were centrifuged at 500 g for 5 min, the culture supernatant was discarded, and the remaining cells were collected. 50 μL of cell lysis buffer was added per well, and the cells were lysed on ice for 30 min. Then, 100 μL of luciferase (Full Gold) was added per well, and the luciferase activity in the lysis buffer was detected. The killing efficiency of T cells against target cells was calculated. Figure 17 ).

[0262] The formula for lethality is as follows:

[0263] Cell killing level = (fluorescence intensity of blank group - fluorescence intensity of experimental group) / fluorescence intensity of blank group.

[0264] Cell killing results showed that A11M103H TCR-T cells could significantly kill cells expressing HLA-A11 and MAGE-A4.146-154 The target cells (A375-A11-luciferase and NCI-H520-luciferase) were targeted, while the negative control group Mock-T cells could not exert good specific killing activity against the target cells, suggesting that the A11M103H TCR-T cells of the present invention have strong target cell killing activity and potential tumor therapeutic value.

[0265] 4. Tumor suppressive activity of A11M103H TCR-T cells in a mouse model of tumors

[0266] In this embodiment, the tumor-suppressive effect of A11M103H TCR-T cells was evaluated using the NCG immunodeficient mouse NCI-H520 lung cancer model.

[0267] The steps of the NCG mouse tumor suppression experiment using TCR-T cells included:

[0268] (1) Establishment of NCG mouse NCI-H520 tumor model

[0269] NCG mice were obtained from Nanjing University-Nanjing Institute of Biomedicine and Biotechnology. Each NCG mouse was subcutaneously inoculated with naturally expressed HLA-A11 and MAGE-A4. 146-154 NCI-H520 tumor cells (ATCC) were used to establish a mouse model of cancer in NCG mice.

[0270] a) Number of NCI-H520 cells inoculated: 5 × 10 6 1 cell / 200 μL / animal;

[0271] b) Injection site: Subcutaneous tissue on the back;

[0272] (2) A11M103H TCR-T cell therapy

[0273] On day 9 after NCG mouse NCI-H520 tumor cell inoculation, the tumor volume grew to approximately 50-100 mm. 3 Using the same method as in Example 7, A11M103H TCR-T cells prepared from the PBMCs of two volunteers, Donor v310 and Donor v311, were injected into NCG mice via the tail vein.

[0274] a) Number of A11M103H TCR-T cells inoculated: 2 × 10⁻⁶ 7 4×10 6 8×10 5 Cells / 200μL / animal;

[0275] b) Injection site: tail vein;

[0276] (3) Grouping and processing:

[0277] Nine days after tumor cell injection, mice with relatively uniform tumor formation were selected and grouped, and then treated with A11M103H TCR-T cells via tail vein injection. This example used T cells (2 × 10⁻⁶) that had not been transfected with TCR. 7 The injection group served as a negative control, using A11M103H TCR-T cells (2×10⁻⁶) from Example 6 above. 7 The injection groups served as treatment controls, with 10 mice in each group. Five mice were in each of the Donorv310 and Donorv311 TCR-T cell therapy groups. Five mice were in each of the saline injection groups, which served as blank controls. The group information and treatments are shown in the table below:

[0278] Table 3. Mouse grouping and treatment

[0279]

[0280] After tumor formation, tumor size was measured every three to five days, up to a maximum tumor volume of 2000 mm in mice. 3 At the end of the experiment, the mice were euthanized and the tumors were separated and weighed.

[0281] (4) Observation of treatment effect:

[0282] 1) Tumor growth detection:

[0283] a) After TCR-T cell injection, the diameter is measured with calipers in mm. The calculation formula is: v = 1 / 2 × a × b × b (a is the major diameter and b is the minor diameter).

[0284] b) The experiment was terminated after the last observation, and the tumor tissue was separated and weighed directly;

[0285] The results showed that injecting 2×10 7 The tumor volume in the high-dose A11M103H TCR-T cell therapy group was significantly smaller than that in the negative control group (t-test, p < 0.001). Figure 18 A), and 4×10 6 Medium dose group and 8×10 5 There was no significant difference in tumor volume between the low-dose group and the negative control group (t-test, p > 0.05). Analysis of tumor weight at the end of the experiment showed that the tumor weight in the high-dose group was significantly lower than that in the negative control group. Figure 18 B). The results of this embodiment indicate that A11M103H TCR-T cells can effectively inhibit tumor growth, and the tumor-suppressive activity shows a significant dose-dependent effect with the number of TCR-T cells, suggesting potential value in tumor treatment (e.g., Figure 18 ).

[0286] sequence

[0287] SEQ ID NO: 1, MAGE-A4 146-154 polypeptide

[0288] RCFPVIFGK

[0289] SEQ ID NO: 2: Control KRAS-G12V 8-16 polypeptide

[0290] VVGAVGVGK

[0291] SEQ ID NO: 3: Amino acid sequence of P2A sequence

[0292] ATNFSLLKQAGDVEENPGP

[0293] SEQ ID NO: 4: Amino acid sequence of the biotin-tag

[0294] GLNDIFEAQKIEWH

[0295] SEQ ID NO: 5: HLA-A11 heavy chain amino acid sequence

[0296] GSHSMRYFYTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEY

[0297] WDQETRNVKAQSQTDRVDLGTLRGYYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYD

[0298] GKDYIALNEDLRSWTAADMAAQITKRKWEAAHAAEQQRAYLEGRCVEWLRRYLENGKETL

[0299] QRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDG

[0300] TFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWELGSGGGLNDIFEAQKIEWH

[0301] SEQ ID NO: 6: Optimized nucleic acid sequence of HLA-A11 heavy chain prokaryotic codon

[0302] ATGGGCTCTCACTCCATGAGGTATTTCTACACCTCCGTGTCCCGGCCCGGCCGCGGGGAGCCCCGCTTCATCGCCGTGGGCTACGTGGACGACACGCAGTTCGTGCGGTTCGACAGCGACGCCGCGAGCCAGAGGATGGAGCCGCGGGCGCCGTGGATAGAGCAGGAGGGGCCGGAGTATTGGGACCAGGAGACACGGAATGTGAAGGCCCAGTCACAGACTGACCGAGTGGACCTGGGGACCCTGCGCGGCTACTACAACCAGAGCGAGGACGGTTCTCACACCATCCAGATAATGTATGGCTGCGACGTGGGGCCGGACGGGCGCTTCCTCCGCGGGTACCGGCAGGACGCCTACGACGGCAAGGATTACATCGCCCTGAACGAGGACCTGCGCTCTTGGACCGCGGCGGACATGGCAGCTCAGATCACCAAGCGCAAGTGGGAGGCGGCCCATGCGGCGGAGCAGCAGAGAGCCTACCTGGAGGGCCGGTGCGTGGAGTGGCTCCGCAGATACCTGGAGAACGGGAAGGAGACGCTGCAGCGCACGGACCCCCCCAAGACACATATGACCCACCACCCCATCTCTGACCATGAGGCCACCCTGAGGTGCTGGGCCCTGGGCTTCTACCCTGCGGAGATCACACTGACCTGGCAGCGGGATGGGGAGGACCAGACCCAGGACACGGAGCTCGTGGAGACCAGGCCTGCAGGGGATGGAACCTTCCAGAAGTGGGCGGCTGTGGTGGTGCCTTCTGGACAGGAGCAGAGATACACCTGCCATGTGCAGCATGAGGGTTTGCCCAAGCCCCTCACCCTGAGATGGGAGCTGGGATCCGGTGGTGGTCTGAACGATATTTTTGAAGCTCAGAAAATCGAATGGCATTAA

[0303] SEQ ID NO: 7, Amino acid sequence of the light chain of HLA - A11

[0304] IQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM

[0305] SEQ ID NO: 8: Optimized nucleic acid sequence of HLA-A11 light chain prokaryotic codon

[0306] ATGATCCAGCGTACTCCAAAGATTCAGGTTTACTCACGTCATCCAGCAGAGAATGGAAAGTCAAATTTCCTGAATTGCTATGTGTCTGGGTTTCATCCATCCGACATTGAAGTTGACTTACTGAAGAATGGAGAGAGAATTGAAAAAGTGG AGCATTCAGACTTGTCTTTCAGCAAGGACTGGTCTTTCTATCTCTTGTACTACACTGAATTCACCCCCACTGAAAAAGATGAGTATGCCTGCCGTGTGAACCATGTGACTTTGTCACAGCCCAAGATAGTTAAGTGGGATCGAGACATGTAA

[0307] SEQ ID NO: 9: Amino acid sequence of the α-chain variable region of the A11M103J TCR

[0308] MNSSPGFMTVMLLIFTRAHGDSVTQTEGQVALSEEDFLTIHCNYSASGYPALFWYVQYPGEGPQFLFRASRDKEKGSSRGFEATYDKGTTSFHLRKASVQESDSAVYYCALGSASSGSWQLIFGSGTQLTVMPD

[0309] SEQ ID NO: 10: α-chain CDR1 amino acid sequences of A11M103J TCR and A11M103H TCR

[0310] ASGYPA

[0311] SEQ ID NO: 11: α-chain CDR2 amino acid sequences of A11M103J TCR and A11M103H TCR

[0312] ASRDKEK

[0313] SEQ ID NO: 12: α-chain CDR3 amino acid sequences of A11M103J TCR and A11M103H TCR

[0314] ALGSASSGSWQLI

[0315] SEQ ID NO: 13: Amino acid sequence of the β-chain variable region of the A11M103J TCR

[0316] MGSRLFFVLSSLLCSKHMEAAVTQSPRNKVAVTGGKVTLSCNQTNNHNNMYWYRQDTGHGLRLLIHYSYGAGSTEKGDIPDGYKASRPSQENFSLILELATPSQTSVYFCASGDADFYAEQFFGPGTRLTVL

[0317] SEQ ID NO: 14: β-chain CDR1 amino acid sequences of A11M103J TCR and A11M103H TCR

[0318] NNHNN

[0319] SEQ ID NO: 15: β-chain CDR2 amino acid sequences of A11M103J TCR and A11M103H TCR

[0320] SYGAGS

[0321] SEQ ID NO: 16: β-chain CDR3 amino acid sequences of A11M103J TCR and A11M103H TCR

[0322] ASGDADFYAEQF

[0323] SEQ ID NO: 17: Full-length amino acid sequence of the α chain

[0324] MNSSPGFMTVMLLIFTRAHGDSVTQTEGQVALSEEDFLTIHCNYSASGYPALFWYVQYPGEGPQFLFRASRDKEKGSSRGFEATYDKGTTSFHLRKASVQESDSAVYYCALGSASSGSWQLIFGSGTQLTVMPDIQN PDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0325] SEQ ID NO: 18: Nucleic acid sequence of the full-length α chain

[0326] ATGAACTCTTCTCCAGGCTTCATGACTGTGATGCTCCTCATATTCACAAGGGCCCATGGAGACTCAGTGACTCAGACGGAAGGTCAAGTGGCCCTCTCAGAAGAGGACTTTCTTACGATACACTGCAACTACTCAGCCTCAGGGTACCCAGCTCTGTTCTGGTATGTGCAGTATCCCGGAGAAGGTCCACAGTTCCTCTTTAGAGCCTCAAGGGACAAAGAGAAAGGAAGCAGCAGAGGTTTTGAAGCTACATATGATAAAGGGACCACCTCCTTCCACTTGCGGAAAGCCTCAGTGCAAGAGTCAGACTCGGCTGTGTACTACTGTGCTCTGGGTTCAGCATCTTCTGGCAGCTGGCAACTCATCTTTGGATCTGGAACCCAACTGACAGTTATGCCTGATATCCAGAACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAATGCGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGAAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTGATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGC

[0327] SEQ ID NO: 19: Amino acid sequence of the full-length β chain

[0328] MGSRLFFVLSSLLCSKHMEAAVTQSPRNKVAVTGGKVTLSCNQTNNHNNMYWYRQDTGHGLRLIHYSYGAGSTEKGDIPDGYKASRPSQENFSLILELATPSQTSVYFCASGDADFYAEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0329] SEQ ID NO: 20: Nucleic acid sequence of the full-length β-chain

[0330] ATGGGCTCCAGGCTCTTCTTCGTGCTCTCCAGTCTCCTGTGTTCAAAACACATGGAGGCTGCAGTCACCCAAAGCCCAAGAAACAAGGTGGCAGTAACAGGAGGAAAGGTGACATTGAGCTGTAATCAGACTAATAACCACAACAACATGTACTGGTATCGGCAGGACACGGGGCATGGGCTGAGGCTGATCCATTATTCATATGGTGCTGGCAGCACTGAGAAAGGAGATATCCCTGATGGATACAAGGCCTCCAGACCAAGCCAAGAGAACTTCTCCCTCATTCTGGAGTTGGCTACCCCCTCTCAGACATCAGTGTACTTCTGTGCCAGCGGTGATGCGGACTTCTATGCTGAGCAGTTCTTCGGACCAGGGACACGACTCACCGTCCTAGAGGACCTGAAAAACGTGTTCCCACCCGAGGTCGCTGTGTTTGAGCCATCAGAAGCAGAGATCTCCCACACCCAAAAGGCCACACTGGTGTGCCTGGCCACAGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAATGGGAAGGAGGTGCACAGTGGGGTCTGCACAGACCCGCAGCCCCTCAAGGAGCAGCCCGCCCTCAATGACTCCAGATACTGCCTGAGCAGCCGCCTGAGGGTCTCGGCCACCTTCTGGCAGAACCCCCGCAACCACTTCCGCTGTCAAGTCCAGTTCTACGGGCTCTCGGAGAATGACGAGTGGACCCAGGATAGGGCCAAACCTGTCACCCAGATCGTCAGCGCCGAGGCCTGGGGTAGAGCAGACTGTGGCTTCACCTCCGAGTCTTACCAGCAAGGGGTCCTGTCTGCCACCATCCTCTATGAGATCTTGCTAGGGAAGGCCACCTTGTATGCCGTGCTGGTCAGTGCCCTCGTGCTGATGGCCATGGTCAAGAGAAAGGATTCCAGAGGC

[0331] SEQ ID NO: 21: Nucleic acid sequence with prokaryotic codon optimization for the extracellular region of the α chain of A11M103J

[0332] ATGGGCGATAGCGTGACGCAGACCGAAGGCCAAGTGGCGCTGAGCGAAGAAGATTTTCTGACCATTCATTGCAACTATAGCGCGAGCGGCTATCCGGCGCTGTTTTGGTATGTGCAGTATCCGGGCGAAGGCCCGCAGTTTCTGTTTCGCGCGAGCCGCGATAAAGAAAAAGGCAGCAGCCGCGGCTTTGAAGCGACCTATGATAAAGGCACCACGAGCTTTCATCTGCGCAAAGCGAGCGTGCAAGAAAGCGATAGCGCGGTGTATTATTGCGCGCTGGGCAGCGCGAGCAGCGGCAGCTGGCAGCTGATTTTTGGCAGCGGCACGCAGCTGACCGTGATGCCGGATATTCAGAACCCGGATCCGGCGGTGTATCAGCTGCGCGATAGCAAAAGCAGCGATAAAAGCGTGTGCCTGTTTACCGATTTTGATAGTCAGACCAACGTGAGTCAGAGCAAAGATAGCGATGTGTATATTACCGATAAATGCGTGCTGGATATGCGCAGCATGGATTTTAAAAGCAACAGCGCGGTGGCGTGGAGCAACAAAAGCGATTTTGCGTGCGCGAACGCGTTTAACAACAGCATTATTCCGGAAGATACCTTTTTTCCGAGCCCGGAAAGCAGC

[0333] SEQ ID NO: 22: Nucleic acid sequence with prokaryotic codon optimization for the extracellular region of the β-chain of A11M103J

[0334] ATGGAAGCGGCGGTGACGCAGAGCCCGCGCAACAAAGTGGCGGTGACCGGCGGCAAAGTGACCCTGAGCTGCAATCAGACCAACAACCATAACATGTATTGGTATCGCCAAGATACCGGCCATGGCCTGCGCCTGATTCATTATAGCTATGGCGGGCAGCAGAGCGAAAAGGGCGATTATTT CCGGATGGCTATAAAGCGAGCCGCCCGAGCCAAGAAAACTTTAGCCTGATTCTGGAACTGGCGACCCCGAGTCAGACGAGCGTGTATTTGCGCGAGCGGCGATGCGGATTTTTATGCGGAACAGTTTTGGCCCGGGCACCCGCCTGACCGTGCTGGAAGATTTGGCCCGGGCACCCCTGACCGTGCTGGAAGATTTTTGGCCCGGGCACCCCTGACCGTGCTGGAAGATTTGGCGAGCGGCGATGCTGCT CCGGAAGTGGCGGTGTTTGAACCGAGCGAAGCGGAAATTAGCCATACGCAGAAAGCGACCCTGGTGTGCCTGGCGACCGGCTTTTACCCGGATCATGTGGAACTGAGCTGGTGGGTGAACGGCAAAGAAGTGCATAGCGGCGTGCACCGATCCAGCCGCTGAAGAACGACCAGGGCGGG AACGATAGCCGCTATGCGCTGAGCAGCCGCCTGCGCGTGAGCGCGACCTTTTGGCAAGATCCGCGCAACCATTTTCGCTGCCAAGTGCAGTTTTATGGCCTGAGCGAAAACGATGAATGGACCCAAGATCGCGCGAAACCGGTTACGCAGATTGAGCGGAAGGGGGGGCCGCCGGAT

[0335] SEQ ID NO: 23:A11M103J TCR α-synthetic DNA binding site (SEQ IDNO: 23)

[0336] ATGGGATGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACAGGAGTTCACTCTGGAGACTCCGTGACACAGACCGAGGGCCAAGTGGCTCTGAGCGAAGAGGATTTTCTGACAATCCACTGTAACTACAGCGCCTCCGGCTACCCTGCCCTGTTTTGGTACGTGCAGTACCCCGGCGAGGGCCCTCAGTTCCTGTTTAGAGCCTCCAGAGACAAGGAGAAGGGCTCCTCCAGAGGCTTTGAGGCCACCTACGACAAGGGCACAACCTCCTTCCACCTGAGGAAGGCCAGCGTGCAGCTGTCTGACTCCGCTGTTTACTACTGCGCCCTGGGCTCCGCTTCCTCTGGAAGCTGGCAACTGATCTTCGGCTCCGGCACCCAGCTGACAGTGATGCCTGATATCCAGAATCCTGATCCTGCCGTGTACCAGCTGAGGGATTCCAAGAGCTCCGATAAGAGCGTGTGCCTGTTTACAGACTTTGATTCCCAGACCAATGTGAGCCAGTCCAAGGACTCCGATGTGTACATCACCGACAAGTGCGTGCTGGACATGAGGTCCATGGACTTCAAGAGCAATAGCGCCGTGGCCTGGAGCAATAAGTCCGATTTCGCCTGCGCCAATGCCTTCAACAACTCCATCATCCCTGAGGATACATTTTTCCCTTCCCCCGAGAGCAGCCACCACCATCATCACCAC

[0337] SEQ ID NO: 24: Eukaryotic codon-optimized DNA sequence of the extracellular region of the A11M103J TCR β chain (SEQ ID NO: 24)

[0338] ATGGGATGGAGCTGCATCATCCTGTTTCTGGTGGCCACAGCCACCGGCGTTCACAGCGAGGCTGCTGTGACACAGTCCCCAAGGTACAAGGTGGCCGTGACCGGAGGAAAGGTGACACTGAGCTGCAATCAGACAAACAACCACAACAACATGTACTGGTACAGACAGGATACAGGCCACGGCCTGAGGCTGATCCACTACTCTTACGGCGCCGGCAGCACAGAGAAGGGAGATATCCCCGACGGCTACAAGGCCAGCAGGCCTAGCCAAGAGAATTTCAGCCTGATCCTGGAGCTGGCCACACCTAGCCAGACCTCTGTGTACTTTTGTGCCAGCGGCGACGCCGATTTTTACGCCGAACAGTTCTTCGGCCCTGGCACCAGACTGACCGTGCTGGAGGATCTGAAGAATGTGTTTCCCCCCGAGGTGGCCGTGTTCGAACCAAGCGAAGCCGAGATCAGCCACACCCAGAAGGCCACACTGGTGTGCCTGGCTACCGGATTTTACCCTGATCACGTGGAGCTGAGCTGGTGGGTGAACGGAAAGGAGGTGCACTCCGGCGTGTGCACAGATCCTCAGCCACTGAAGGAGCAGCCTGCCCTGAATGATTCCAGGTACGCCCTGAGCTCCAGGCTGAGAGTGTCCGCTACATTCTGGCAGGACCCCAGAAACCACTTTAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAATGATGAGTGGACCCAGGATAGGGCCAAGCCTGTGACACAGATCGTGAGCGCCGAGGCTTGGGGAAGAGCTGATGGATCCTGGTCCCACCCCCAGTTCGAGAAGGGAGGAGGATCCGGAGGCGGATCTGGAGGATCTGCTTGGTCCCACCCACAGTTTGAGAAG

[0339] SEQ ID NO: 25: Variable region of the α-chain of the A11M141J TCR

[0340] MNMRPVTSSVLVLLLMLRRSNGDSVTQTEGLVTVTEGLPVKLNCTYQTTYLTIAFFWYVQYLNEAPQVLLKSSTDNKRTEHQGFHATLHKSSSSFHLQKSSAQLSDSALYYCALSDSAGGYKVVFGSGTRLLVSPD

[0341] SEQ ID NO: 26: α-chain CDR1 of A11M141J TCR

[0342] TTYLTIA

[0343] SEQ ID NO: 27: α-chain CDR2 of A11M141J TCR

[0344] SSTDNKR

[0345] SEQ ID NO: 28: α-chain CDR3 of A11M141J TCR

[0346] ALSDSAGGYKVV

[0347] SEQ ID NO: 29: β-chain variable region of A11M141J TCR

[0348] MWTFLLLLWSQGSVFSVLLYQKPNRDICQSGTSLKIQCVADSQVVSMFWYQQFQEQSLMLMATANEGSEATYESGFTKDKFPISRPNLTFSTLTVNNARPGDSSIYFCSSTGYYAEQFFGPGTRLTVL

[0349] SEQ ID NO: 30: CDR1 of the β chain of A11M141J TCR

[0350] SQVVS

[0351] SEQ ID NO: 31: CDR2 of the β chain of A11M141J TCR

[0352] ANEGSEA

[0353] SEQ ID NO: 32: CDR3 of the β chain of A11M141J TCR

[0354] SSTGYYAEQF

[0355] SEQ ID NO: 33: Full-length amino acid sequence of the α chain of the A11M141J TCR

[0356] MNMRPVTSSVLVLLLMLRRSNGDSVTQTEGLVTVTEGLPVKLNCTYQTTYLTIAFFWYVQYLNEAPQVLLKSSTDNKRTEHQGFHATLHKSSSSFHLQKSSAQLSDSALYYCALSDSAGGYKVVFGSGTRLLVSPDIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVPCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0357] SEQ ID NO: 34: Full-length nucleotide sequence of the α-chain of the A11M141J TCR

[0358] ATGAACATGCGTCCTGTCACCTCCTCAGTTCTCGTGCTCCTCCTAATGCTCAGAAGGAGCAATGGAGACTCCGTGACCCAGACAGAAGGCCTGGTCACTGTCACCGAGGGGTTGCCTGTGAAGCTGAACTGCACCTATCAGACTACTTATTTAACTATTGCCTTTTTCTGGTATGTGCAATATCTCAACGAAGCCCCTCAGGTACTCCTGAAGAGCTCCACAGACAACAAGAGGACCGAGCACCAAGGGTTCCACGCCACTCTCCATAAGAGCAGCAGCTCCTTCCATCTGCAGAAGTCCTCAGCGCAGCTGTCAGACTCTGCCCTGTACTACTGTGCTCTGAGTGATTCGGCTGGAGGCTATAAAGTGGTCTTTGGAAGTGGGACTCGATTGCTGGTAAGCCCTGACATTCAGAACCCCGACCCCGCCGTGTATCAGCTGAGAGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAAACCAACGTGAGCCAAAGCAAGGACAGCGACGTGTACATCACCGACAAGTGCGTGCTGGACATGAGAAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCTAGCAGCGACGTGCCCTGCGACGTGAAGCTGGTGGAGAAGAGCTTCGAGACCGACACCAACCTGAACTTTCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTCCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGGAGCAGC

[0359] SEQ ID NO: 35: Full-length amino acid sequence of the β-chain of A11M141J TCR

[0360] MWTFLLLLWSQGSVFSVLLYQKPNRDICQSGTSLKIQCVADSQVVSMFWYQQFQEQSLMLMATANEGSEATYESGFTKDKFPISRPNLTFSTLTVNNARPGDSSIYFCSSTGYYAEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSKAEIAHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0361] SEQ ID NO: 36: Full-length nucleotide sequence of the β-chain of A11M141J TCR

[0362] ATGTGGACATTCCTGCTACTTCTTTGGAGCCAAGGTTCTGTATTCAGTGTCCTCCTCTACCAAAAGCCAAACAGGGACATCTGTCAAAGTGGCACTTCACTGAAAATCCAGTGTGTGGCTGACAGTCAAGTTGTTTCGATGTTTTGGTACCAACAGTTCCAGGAACAGAGCTTGATGCTCATGGCAACTGCAAATGAAGGCTCTGAAGCCACATACGAGAGTGGATTCACCAAGGACAAGTTTCCAATCAGCCGGCCAAACCTAACATTCTCAACGTTGACAGTGAACAATGCAAGGCCTGGAGACAGCAGTATCTATTTCTGTAGTTCCACAGGGTACTATGCTGAGCAGTTCTTCGGACCAGGGACACGACTCACCGTCCTAGAGGACCTGAAGAACGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCTAGCAAGGCCGAGATCGCCCACACACAGAAGGCTACCCTGGTGTGCCTGGCCACCGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGCAAAGAGGTGCACAGCGGCGTGTGCACCGACCCTCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCAGATACTGCCTGAGCAGCAGACTGAGAGTGAGCGCCACCTTCTGGCAGAACCCTAGAAACCACTTCAGATGCCAAGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAAGACAGAGCTAAGCCCGTGACACAGATCGTGAGCGCCGAGGCCTGGGGGAGAGCCGACTGCGGCATCACAAGCGCTAGCTACCACCAAGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGAGCGCCCTGGTGCTGATGGCCATGGTGAAGAGAAAGGACAGCAGAGGC

[0363] SEQ ID NO: 37: Variable region of the α chain of A11M161J TCR

[0364] MRPVTCSVLVLLLMLRRSNGDSVTQTEGLVTVTEGLPVMLNCTYQTAYSDVAFFWYVQYLNEAPKLLLRSSTDNKRTEHQGFHATLHKSSSSFHLQKSSVQLSDSALYYCALSETSNMGYKLTFGGTSLLVDPN

[0365] SEQ ID NO: 38: α-chain CDR1 of A11M161J TCR

[0366] TAYSDVA

[0367] SEQ ID NO: 39: α-chain CDR2 of A11M161J TCR

[0368] SSTDNKR

[0369] SEQ ID NO: 40: α-chain CDR3 of A11M161J TCR

[0370] ALSETSNMGYKLT

[0371] SEQ ID NO: 41: β-chain variable region of A11M161J TCR

[0372] MAPRLFCLALCFLRAEPTNAGVIQTPRHKVTGKGQEATLWCEPISGHSAVFWYRQTIVQGLEFLTYFRNQAPIDDSGMPKERFSAQMPNQSHSTLKIQSTQPQDSAVYLCASSLDWGGAYTGQLYFGEGSKLTVL

[0373] SEQ ID NO: 42: CDR1 of the β chain of A11M161J TCR

[0374] SGHSA

[0375] SEQ ID NO: 43: CDR2 of the β chain of A11M161J TCR

[0376] FRNQAP

[0377] SEQ ID NO: 44: CDR3 of the β chain of A11M161J TCR

[0378] ASSLDWGGAYTGQLY

[0379] SEQ ID NO: 45: Full-length amino acid sequence of the α chain of the A11M161J TCR

[0380] MRPVTCSVLVLLLMLRRSNGDSVTQTEGLVTVTEGLPVMLNCTYQTAYSDVAFFWYVQYLNEAPKLLLRSSTDNKRTEHQGFHATLHKSSSSFHLQKSSVQLSDSALYYCALSETSNMGYKLTFGTGTSLLVDPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVPCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0381] SEQ ID NO: 46: Full-length nucleotide sequence of the α-chain of the A11M161J TCR

[0382] ATGCGTCCTGTCACCTGCTCAGTTCTTGTGCTCCTCCTAATGCTCAGAAGGAGCAATGGAGACTCCGTGACCCAGACAGAAGGCCTGGTCACTGTCACAGAAGGGTTGCCTGTGATGCTGAACTGCACCTATCAGACTGCTTACTCAGATGTTGCCTTTTTCTGGTATGTGCAATATCTCAACGAAGCCCCTAAACTACTCCTGCGGAGCTCCACAGACAACAAGAGGACCGAGCACCAAGGGTTCCACGCCACTCTCCATAAGAGCAGCAGCTCCTTCCATCTGCAGAAGTCCTCAGTGCAGCTGTCAGACTCTGCCCTGTACTACTGTGCTCTGAGTGAGACTAGCAACATGGGCTACAAACTTACCTTCGGGACAGGAACAAGCTTGTTGGTTGATCCAAACATTCAGAACCCCGACCCCGCCGTGTATCAGCTGAGAGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAAACCAACGTGAGCCAAAGCAAGGACAGCGACGTGTACATCACCGACAAGTGCGTGCTGGACATGAGAAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCTAGCAGCGACGTGCCCTGCGACGTGAAGCTGGTGGAGAAGAGCTTCGAGACCGACACCAACCTGAACTTTCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTCCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGGAGCAGC

[0383] SEQ ID NO: 47: Full-length amino acid sequence of the β-chain of the A11M161J TCR

[0384] MAPRLLFCLALCFLRAEPTNAGVIQTPRHKVTGKGQEATLWCEPISGHSAVFWYRQTIVQGLEFLTYFRNQAPIDDSGMPKERFSAQMPNQSHSTLKIQSTQPQDSAVYLCASSLDWGGAYTGQLYFGEGSKLTVLEDLKNVFPPEVAVFEPSKAEIAHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0385] SEQ ID NO: 48: Full-length nucleotide sequence of the β-chain of the A11M161J TCR

[0386] ATGGCCCCCAGGCTCCTTTTCTGTCTGGCTCTTTGCTTCTTGAGAGCAGAACCAACAAATGCTGGTGTCATCCAAACACCTAGGCACAAGGTGACAGGGAAGGGACAAGAAGCAACTCTGTGGTGTGAGCCAATTTCAGGACATAGTGCTGTTTTCTGGTACAGACAGACCATTGTGCAGGGCCTGGAGTTCCTGACTTACTTTCGAAATCAAGCTCCTATAGATGATTCAGGGATGCCCAAGGAACGATTCTCAGCTCAGATGCCCAATCAGTCGCACTCAACTCTGAAGATCCAGAGCACGCAACCCCAGGACTCAGCGGTGTATCTTTGTGCAAGCAGCCTCGACTGGGGGGGCGCATACACCGGGCAGCTCTACTTTGGTGAAGGCTCAAAGCTGACAGTGCTGGAGGACCTGAAGAACGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCTAGCAAGGCCGAGATCGCCCACACACAGAAGGCTACCCTGGTGTGCCTGGCCACCGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGCAAAGAGGTGCACAGCGGCGTGTGCACCGACCCTCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCAGATACTGCCTGAGCAGCAGACTGAGAGTGAGCGCCACCTTCTGGCAGAACCCTAGAAACCACTTCAGATGCCAAGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAAGACAGAGCTAAGCCCGTGACACAGATCGTGAGCGCCGAGGCCTGGGGGAGAGCCGACTGCGGCATCACAAGCGCTAGCTACCACCAAGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGAGCGCCCTGGTGCTGATGGCCATGGTGAAGAGAAAGGACAGCAGAGGC

[0387] SEQ ID NO: 49: α-chain variable region of A11M162J TCR

[0388] MKSFSISLVVLWLQLNWVNSQQKVQQSPESLIVPEGGMASLNCTSSDRNVDYFWWYRQHSGKSPKMLMSIFSNGEKEEGRFTVHLNKASLHTSLHIRDSQPSDSALYLCAATSFNTEGADRLTFGKGTQLIIQPY

[0389] SEQ ID NO: 50: α-chain CDR1 of A11M162J TCR

[0390] DRNVDY

[0391] SEQ ID NO: 51: α-chain CDR2 of A11M162J TCR

[0392] IFSNGE

[0393] SEQ ID NO: 52: α-chain CDR3 of A11M162J TCR

[0394] AATSFNTEGADRLT

[0395] SEQ ID NO: 53: β-chain variable region of A11M162J TCR

[0396] MGCRLLSCVAFCLLGIGPLETAVFQTPNYHVTQVGNEVSFNCKQTLGHDTMYWYKQDSKKLLKIMFSYNNKQLIVNETVPRRFSPQSSDKAHLNLRIKSVEPEDSAVYLCASSWGLGSSYEQYFPGGTRLTVL

[0397] SEQ ID NO: 54: CDR1 of the β chain of A11M162J TCR

[0398] LGHDT

[0399] SEQ ID NO: 55: CDR2 of the β chain of A11M162J TCR

[0400] YNNKQL

[0401] SEQ ID NO: 56: CDR3 of the β chain of A11M162J TCR

[0402] ASSWGLGSSYEQY

[0403] SEQ ID NO: 57: Full-length amino acid sequence of the α chain of the A11M162J TCR

[0404] MKSFSISLVVLWLQLNWVNSQQKVQQSPESLIVPEGGMASLNCTSSDRNVDYFWWYRQHSGKSPKMLMSIFSNGEKEEGRFTVHLNKASLHTSLHIRDSQPSDSALYLCAATSFNTEGADRLTFGKGTQLIIQPYIQ NPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVPCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0405] SEQ ID NO: 58: Full-length α-chain nucleotide sequence of A11M162J TCR

[0406] ATGAAATCCTTTAGTATTTCCCTAGTGGTCCTGTGGCTTCAGCTAAACTGGGTGAACAGCCAACAGAAGGTGCAGCAGAGCCCAGAATCCCTCATTGTTCCAGAGGGAGGCATGGCCTCTCTCAACTGCACTTCCAGTGATCGTAATGTTGACTACTTCTGGTGGTACAGACAGCACTCTGGGAAAAGCCCCAAGATGCTGATGTCTATCTTCTCCAATGGTGAAAAGGAAGAAGGCAGATTCACAGTTCACCTCAATAAAGCCAGCCTGCATACTTCCCTGCACATCAGAGACTCCCAGCCCAGTGACTCTGCTCTCTACCTCTGTGCAGCCACCTCTTTTAATACAGAAGGTGCAGATAGACTCACCTTTGGGAAAGGAACTCAGCTGATCATCCAGCCCTACATTCAGAACCCCGACCCCGCCGTGTATCAGCTGAGAGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAAACCAACGTGAGCCAAAGCAAGGACAGCGACGTGTACATCACCGACAAGTGCGTGCTGGACATGAGAAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCTAGCAGCGACGTGCCCTGCGACGTGAAGCTGGTGGAGAAGAGCTTCGAGACCGACACCAACCTGAACTTTCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTCCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGGAGCAGC

[0407] SEQ ID NO: 59: Full-length amino acid sequence of the β-chain of A11M162J TCR

[0408] MGCRLLSCVAFCLLGIGPLETAVFQTPNYHVTQVGNEVSFNCKQTLGHDTMYWYKQDSKKLLKIMFSYNNKQLIVNETVPRRFSPQSSDKAHLNLRIKSVEPEDSAVYLCASSWGLGSSYEQYFGPGTRLTVLEDLKNVFPPEVAVFEPSKAEIAHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0409] SEQ ID NO: 60: Full-length nucleotide sequence of the β-chain of the A11M162J TCR

[0410] ATGGGCTGTAGGCTCCTAAGCTGTGTGGCCTTCTGCCTCTTGGGAATAGGCCCTTTGGAGACGGCTGTTTTCCAGACTCCAAACTATCATGTCACACAGGTGGGAAATGAAGTGTCTTTCAATTGTAAGCAAACTCTGGGCCACGATACTATGTATTGGTACAAGCAAGACTCTAAGAAATTGCTGAAGATTATGTTTAGCTACAATAATAAGCAACTCATTGTAAACGAAACAGTTCCAAGGCGCTTCTCACCTCAGTCTTCAGATAAAGCTCATTTGAATCTTCGAATCAAGTCTGTAGAGCCGGAGGACTCTGCTGTGTATCTCTGTGCCAGCAGCTGGGGACTGGGGAGCTCCTATGAACAGTACTTCGGTCCCGGCACCAGGCTCACGGTTTTAGAGGACCTGAAGAACGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCTAGCAAGGCCGAGATCGCCCACACACAGAAGGCTACCCTGGTGTGCCTGGCCACCGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGCAAAGAGGTGCACAGCGGCGTGTGCACCGACCCTCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCAGATACTGCCTGAGCAGCAGACTGAGAGTGAGCGCCACCTTCTGGCAGAACCCTAGAAACCACTTCAGATGCCAAGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAAGACAGAGCTAAGCCCGTGACACAGATCGTGAGCGCCGAGGCCTGGGGGAGAGCCGACTGCGGCATCACAAGCGCTAGCTACCACCAAGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGAGCGCCCTGGTGCTGATGGCCATGGTGAAGAGAAAGGACAGCAGAGGC

[0411] SEQ ID NO: 61: Variable region of the α chain of the A11M163J TCR

[0412] MKTVTGPLFLCFWLQLNCVSRGEQVEQRPPHLSVREGDSAVITCTYTDPNSYYFFWYKQEPGASLQLLMKVFSSTEINEGQGFTVLLNKKDKRLSLNLTAAHPGDSAAYFCAVRDNNNAPRFGAGTKLSVKPN

[0413] SEQ ID NO: 62: α-chain CDR1 of A11M163J TCR

[0414] DPNSYY

[0415] SEQ ID NO: 63: α-chain CDR2 of A11M163J TCR

[0416] VFSSTEI

[0417] SEQ ID NO: 64: α-chain CDR3 of A11M163J TCR

[0418] AVRDNNNAPR

[0419] SEQ ID NO: 65: β-chain variable region of A11M163J TCR

[0420] MSNTVLADSAWGITLLSWVTVFLLGTSSADSGVVQSPRHIIKEKGGRSVLTCIPISGHSNVVWYQQTLGKELKFLIQHYEKVERDKGFLPSRFSVQQFDDYHSEMNMSALELEDSAMYFCASSPWDERLFFGHGTKLSVL

[0421] SEQ ID NO: 66: CDR1 of the β chain of A11M163J TCR

[0422] SGHSN

[0423] SEQ ID NO: 67: CDR2 of the β chain of A11M163J TCR

[0424] HYEKVE

[0425] SEQ ID NO: 68: CDR3 of the β chain of A11M163J TCR

[0426] ASSPWDERLF

[0427] SEQ ID NO: 69: Full-length amino acid sequence of the α chain of the A11M163J TCR

[0428] MKTVTGPLFLCFWLQLNCVSRGEQVEQRPPHLSVREGDSAVITCTYTDPNSYYFFWYKQEPGASLQLLMKVFSSTEINEGQGFTVLLNKKDKRLSLNLTAAHPGDSAAYFCAVRDNNNAPRFGAGTKLSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVPCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0429] SEQ ID NO: 70: Full-length nucleotide sequence of the α-chain of A11M163J TCR

[0430] ATGAAGACAGTGACTGGACCTTTGTTCCTGTGCTTCTGGCTGCAGCTGAACTGTGTGAGCAGAGGCGAGCAGGTGGAGCAGCGCCCTCCTCACCTGAGTGTCCGGGAGGGAGACAGTGCCGTTATCACCTGCACCTACACAGACCCTAACAGTTATTACTTCTTCTGGTACAAGCAAGAGCCGGGGGCAAGTCTTCAGTTGCTTATGAAGGTTTTCTCAAGTACGGAAATAAACGAAGGACAAGGATTCACTGTCCTACTGAACAAGAAAGACAAACGACTCTCTCTGAACCTCACAGCTGCCCATCCTGGGGACTCAGCCGCGTACTTCTGCGCAGTCAGGGATAACAACAATGCCCCACGATTTGGAGCGGGAACCAAATTATCAGTAAAACCAAACATTCAGAACCCCGACCCCGCCGTGTATCAGCTGAGAGACAGCAAGAGCAGCGACAAGAGCGTGTGCCTGTTCACCGACTTCGACAGCCAAACCAACGTGAGCCAAAGCAAGGACAGCGACGTGTACATCACCGACAAGTGCGTGCTGGACATGAGAAGCATGGACTTCAAGAGCAACAGCGCCGTGGCCTGGAGCAACAAGAGCGACTTCGCCTGCGCCAACGCCTTCAACAACAGCATCATCCCCGAGGACACCTTCTTCCCTAGCAGCGACGTGCCCTGCGACGTGAAGCTGGTGGAGAAGAGCTTCGAGACCGACACCAACCTGAACTTTCAGAACCTGAGCGTGATCGGCTTCAGAATCCTGCTCCTGAAGGTGGCCGGCTTCAACCTGCTGATGACCCTGAGACTGTGGAGCAGC

[0431] SEQ ID NO: 71: Full-length amino acid sequence of the β-chain of the A11M163J TCR

[0432] MSNTVLADSAWGITLLSWVTVFLLGTSSADSGVVQSPRHIIKEKGGRSVLTCIPISGHSNVVWYQQTLGKELKFLIQHYEKVERDKGFLPSRFSVQQFDDYHSEMNMSALELEDSAMYFCASSPWDERLFFGHGTKLSVLEDLKNVFPPEVAVFEPSKAEIAHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0433] SEQ ID NO: 72: Full-length nucleotide sequence of the β-chain of A11M163J TCR

[0434] ATGTCTAACACTGTCCTCGCTGATTCTGCCTGGGGCATCACCCTGCTATCTTGGGTTACTGTCTTTCTCTTGGGAACAAGTTCAGCAGATTCTGGGGTTGTCCAGTCTCCAAGACACATAATCAAAGAAAAGGGAGGAAGGTCCGTTCTGACGTGTATTCCCATCTCTGGACATAGCAATGTGGTCTGGTACCAGCAGACTCTGGGGAAGGAATTAAAGTTCCTTATTCAGCATTATGAAAAGGTGGAGAGAGACAAAGGATTCCTACCCAGCAGATTCTCAGTCCAACAGTTTGATGACTATCACTCTGAAATGAACATGAGTGCCTTGGAACTGGAGGACTCTGCTATGTACTTCTGTGCCAGCTCCCCCTGGGACGAAAGATTATTTTTCGGTCATGGAACCAAGCTGTCTGTCCTGGAGGACCTGAAGAACGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCTAGCAAGGCCGAGATCGCCCACACACAGAAGGCTACCCTGGTGTGCCTGGCCACCGGCTTCTACCCCGACCACGTGGAGCTGAGCTGGTGGGTGAACGGCAAAGAGGTGCACAGCGGCGTGTGCACCGACCCTCAGCCCCTGAAGGAGCAGCCCGCCCTGAACGACAGCAGATACTGCCTGAGCAGCAGACTGAGAGTGAGCGCCACCTTCTGGCAGAACCCTAGAAACCACTTCAGATGCCAAGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAAGACAGAGCTAAGCCCGTGACACAGATCGTGAGCGCCGAGGCCTGGGGGAGAGCCGACTGCGGCATCACAAGCGCTAGCTACCACCAAGGCGTGCTGAGCGCCACCATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCCGTGCTGGTGAGCGCCCTGGTGCTGATGGCCATGGTGAAGAGAAAGGACAGCAGAGGC

[0435] SEQ ID NO: 73: Amino acid sequence of the variable region of the TCR α chain of A11M103H

[0436] MNSSPGFMTVMLLIFTRAHGNSVTQMEGPVTLSEEAFLTINCTYSASGYPALFWYVQYPGEGPQFLFRASRDKEKGSNKGFEATYRKETTSFHLEKGSVQVSDSAVYYCALGSASSGSWQLIFGSGTKLQVIPD

[0437] SEQ ID NO: 74: A11M103H TCR α chain full-length amino acid sequence

[0438] MNSSPGFMTVMLLIFTRAHGNSVTQMEGPVTLSEEAFLTINCTYSASGYPALFWYVQYPGEGPQFLFRASRDKEKGSNKGFEATYRKETTSFHLEKGSVQVSDSAVYYCALGSASSGSWQLIFGSGTKLQVIPDIQNPDPA VYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVPCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSSRAKRSGSG

[0439] SEQ ID NO: 75: A11M103H TCR α nucleic acid sequence

[0440] ATGAACAGCTCCCCTGGCTTTATGACCGTGATGCTGCTGATCTTCACAAGGGCCCACGGCAACAGCGTGACCCAAATGGAGGGCCCTGTGACCCTGAGCGAGGAAGCTTTTCTGACCATCAATTGCACCTACTCCGCCTCCGGCTACCCTGCTCTGTTTTGGTACGTGCAGTACCCTGGCGAGGGCCCTCAATTTCTGTTTAGGGCCTCCAGAGATAAGGAGAAGGGCAGCAATAAGGGCTTCGAGGCCACCTACAGAAAGGAGACCACAAGCTTTCACCTGGAGAAGGGCTCCGTGCAGGTGTCTGATAGCGCCGTTTACTACTGTGCCCTGGGCAGCGCTTCCAGCGGAAGCTGGCAGCTGATCTTTGGCAGCGGCACCAAGCTGCAGGTCATTCCTGACATCCAGAACCCTGATCCCGCCGTGTACCAGCTGAGAGACAGCAAGTCCTCCGACAAGAGCGTGTGTCTGTTCACCGACTTTGACAGCCAGACCAACGTGAGCCAGAGCAAGGACTCCGATGTGTACATCACCGACAAGTGCGTGCTGGACATGAGAAGCATGGATTTTAAGTCCAACAGCGCCGTGGCCTGGAGCAATAAGAGCGATTTCGCCTGCGCCAACGCCTTTAACAACAGCATCATCCCTGAGGACACCTTTTTCCCCTCCTCCGACGTGCCCTGTGATGTGAAGCTGGTGGAGAAGTCCTTTGAGACAGATACCAACCTGAATTTTCAGAATCTGCTGGTCATTGTGCTGAGAATCCTGCTGCTGAAGGTGGCCGGCTTCAATCTGCTGATGACCCTGAGACTGTGGAGCAGCAGAGCCAAGAGAAGCGGCTCCGGA

[0441] SEQ ID NO: 76: Amino acid sequence of the variable region of the A11M103H TCR β chain

[0442] MGSRLFFVLSSLLCSKHMEAGVTQTPRFRVLKTGQSMTLLCAQTNNHNNMYWYRQDTGHGLRLIYYSYGAGSTEKGDVPDGYNVSRLKKENFLLGLESAAPSQTSVYFCASGDADFYAEQFFGPGTRLTVL

[0443] SEQ ID NO: 77: Amino acid sequence of the full-length A11M103H TCR β chain

[0444] MGSRLFFVLSSLLCSKHMEAGVTQTPRFRVLKTGQSMTLLCAQTNNHNNMYWYRQDTGHGLRLIYYSYGAGSTEKGDVPDGYNVSRLKKENFLLGLESAAPSQTSVYFCASGDADFYAEQFFGPGTRLTVLEDLKNVFPPEVAVFEPSKAEIAHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0445] SEQ ID NO: 78: A11M103H TCR β nucleic acid sequence

[0446] ATGGGAAGCAGACTGTTTTTCGTGCTGTCCTCCCTGCTGTGCTCCAAGCACATGGAGGCCGGAGTGACACAGACCCCTAGATTCAGGGTGCTGAAGACCGGCCAGAGCATGACACTGCTGTGCGCTCAGACCAATAACCACAACAACATGTACTGGTACAGACAGGATACAGGCCACGGCCTGAGACTGATCTACTACAGCTACGGCGCCGGCAGCACAGAGAAGGGAGATGTTCCCGATGGCTACAACGTGAGCAGGCTGAAGAAGGAGAACTTTCTGCTGGGCCTGGAGTCCGCCGCTCCTAGCCAGACCAGCGTGTATTTCTGCGCCAGCGGCGACGCTGATTTCTACGCTGAGCAGTTCTTCGGCCCCGGCACAAGACTGACCGTGCTGGAGGATCTGAAGAACGTGTTCCCTCCTGAGGTGGCCGTGTTCGAGCCTTCTAAGGCCGAGATCGCCCACACACAGAAGGCCACACTGGTGTGCCTGGCCACAGGATTCTACCCCGACCACGTGGAGCTGTCCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGCGTGTGCACAGATCCACAGCCTCTGAAGGAGCAGCCTGCCCTGAACGATAGCAGGTACTGTCTGAGCAGCAGGCTGAGAGTGAGCGCCACATTCTGGCAGAACCCTAGAAACCACTTCAGGTGCCAGGTGCAGTTCTACGGCCTGAGCGAGAATGACGAGTGGACACAGGATAGAGCCAAGCCCGTGACACAGATCGTGAGCGCTGAGGCTTGGGGCAGAGCTGATTGCGGAATCACATCCGCCAGCTACCACCAGGGCGTGCTGTCCGCTACAATCCTGTACGAGATCCTGCTGGGCAAGGCCACCCTGTACGCTGTTCTGGTGAGCGCTCTGGTGCTGATGGCCATGGTGAAGAGGAAGGATTCCAGAGGC

[0447] SEQ ID NO: 79: DNA sequence of the extracellular region of humanized TCR α-chain with eukaryotic codon optimization

[0448] ATGGGATGGTCCTGTATCATCCTGTTCCTGGTGGCCACCGCCACAGGAGTTCACTCTGGAAATAGCGTGACACAGATGGAGGGCCCTGTGACCCTGTCCGAAGAGGCTTTCCTGACAATCAATTGTACCTACTCCGCCTCCGGCTACCCTGCTCTGTTCTGGTACGTGCAGTACCCCGGCGAGGGACCTCAATTTCTGTTCAGGGCCAGCAGAGACAAGGAGAAGGGCAGCAATAAGGGCTTCGAGGCCACATACAGGAAGGAGACCACCAGCTTTCACCTGGAGAAGGGCTCCGTGCAGGTGTCTGACAGCGCTGTTTACTACTGTGCCCTGGGCTCCGCCAGCTCTGGAAGCTGGCAGCTGATCTTCGGCTCCGGAACCAAGCTGCAGGTCATTCCCGATATCCAGAACCCTGATCCCGCCGTGTACCAGCTGAGGGATAGCAAGTCCTCCGACAAGAGCGTGTGCCTGTTTACCGACTTCGACTCCCAGACCAACGTGAGCCAGTCCAAGGATTCCGATGTGTACATCACCGACAAGTGTGTGCTGGATATGAGATCCATGGACTTTAAGAGCAATTCCGCCGTGGCCTGGAGCAATAAGTCCGACTTTGCCTGCGCCAATGCCTTTAATAACTCCATCATCCCCGAGGATACCTTCTTCCCCTCCCCCGAATCCAGCCACCACCATCATCACCAC

[0449] SEQ ID NO: 80: DNA sequence of the extracellular region of humanized TCR β-chain with eukaryotic codon optimization

[0450] ATGGGATGGTCCTGTATCATCCTGTTTCTGGTGGCCACCGCCACCGGAGTTCACAGCGAGGCTGGAGTTACCCAGACACCTAGGTTTAGGGTGCTGAAGACCGGCCAGAGCATGACCCTGCTGTGTGCTCAGACCAATAATCACAATAATATGTACTGGTACAGGCAGGACACAGGCCACGGCCTGAGACTGATCTACTACTCCTACGGCGCCGGCTCCACCGAAAAGGGAGATGTTCCTGATGGCTACAATGTGAGCAGGCTGAAGAAGGAGAACTTTCTGCTGGGCCTGGAGAGCGCCGCTCCTAGCCAGACCAGCGTGTATTTTTGCGCCAGCGGCGACGCCGACTTCTACGCTGAGCAATTCTTTGGCCCCGGCACCAGACTGACAGTGCTGGAGGATCTGAAGAATGTGTTCCCCCCCGAGGTGGCCGTGTTCGAGCCTAGCGAGGCTGAAATCAGCCACACCCAGAAGGCCACCCTGGTGTGTCTGGCTACCGGATTCTACCCTGATCACGTGGAGCTGTCCTGGTGGGTGAACGGAAAGGAGGTGCACAGCGGCGTGTGCACAGACCCTCAACCACTGAAGGAGCAGCCCGCTCTGAATGACTCCAGATACGCCCTGAGCAGCAGGCTGAGAGTGAGCGCTACCTTCTGGCAGGATCCCAGAAACCACTTTAGATGCCAGGTGCAGTTCTACGGCCTGAGCGAGAACGACGAGTGGACCCAAGATAGGGCCAAGCCTGTGACCCAGATCGTGAGCGCTGAGGCTTGGGGAAGAGCCGATGGATCCTGGAGCCACCCACAGTTCGAGAAGGGCGGAGGATCCGGAGGAGGATCTGGAGGAAGCGCTTGGTCCCACCCTCAGTTTGAGAAG

[0451] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. T cell receptor TCR or its antigen-binding fragment, wherein, The TCR or its antigen-binding fragment can bind to MAGE-A4. 146-154 The epitope binds to the HLA-A11 complex, and the TCR contains an α-chain variable region and a β-chain variable region, wherein the TCR or its antigen-binding fragment contains the following α-chain complementarity-determining region (CDR) and β-chain complementarity-determining region (CDR): As shown in SEQ ID NO: 10, the α-chain complementarity determination region CDR1, As shown in SEQ ID NO: 11, the α-chain complementarity determination region CDR2, As shown in SEQ ID NO: 12, the α-chain complementarity determination region CDR3, As shown in SEQ ID NO: 14, the β-chain complementarity determination region CDR1, As shown in SEQ ID NO: 15, the β-chain complementarity determination region CDR2, and As shown in SEQ ID NO: 16, the β-chain complementarity determination region CDR3; or As shown in SEQ ID NO: 26, the α-chain complementarity determination region CDR1, As shown in SEQ ID NO: 27, the α-chain complementarity determination region CDR2, As shown in SEQ ID NO: 28, the α-chain complementarity determination region CDR3, As shown in SEQ ID NO: 30, the β-chain complementarity determination region CDR1, As shown in SEQ ID NO: 31, the β-chain complementarity determination region CDR2, and Such as the β-chain complementarity determination region CDR3 shown in SEQ ID NO: 32; or As shown in SEQ ID NO: 38, the α-chain complementarity determination region CDR1, As shown in SEQ ID NO: 39, the α-chain complementarity determination region CDR2, As shown in SEQ ID NO: 40, the α-chain complementarity determination region CDR3, As shown in SEQ ID NO: 42, the β-chain complementarity determination region CDR1, As shown in SEQ ID NO: 43, the β-chain complementarity determination region CDR2, and Such as the β-chain complementarity determination region CDR3 shown in SEQ ID NO: 44; or As shown in SEQ ID NO: 50, the α-chain complementarity determination region CDR1, As shown in SEQ ID NO: 51, the α-chain complementarity determination region CDR2, As shown in SEQ ID NO: 52, the α-chain complementarity determination region CDR3, As shown in SEQ ID NO: 54, the β-chain complementarity determination region CDR1, As shown in SEQ ID NO: 55, the β-chain complementarity determination region CDR2, and Such as the β-chain complementarity determination region CDR3 shown in SEQ ID NO: 56; or As shown in SEQ ID NO: 62, the α-chain complementarity determination region CDR1, As shown in SEQ ID NO: 63, the α-chain complementarity determination region CDR2, As shown in SEQ ID NO: 64, the α-chain complementarity determination region CDR3, As shown in SEQ ID NO: 66, the β-chain complementarity determination region CDR1, As shown in SEQ ID NO: 67, the β-chain complementarity determination region CDR2, and The β-chain complementarity determination region CDR3 is shown in SEQ ID NO:

68.

2. The T-cell receptor TCR or its antigen-binding fragment according to claim 1, comprising: α-chain variable region as shown in SEQ ID NO: 9, and β-chain variable region as shown in SEQ ID NO: 13; α-chain variable region as shown in SEQ ID NO: 25, and β-chain variable region as shown in SEQ ID NO: 29; α-chain variable region as shown in SEQ ID NO: 37, and β-chain variable region as shown in SEQ ID NO: 41; α-chain variable regions as shown in SEQ ID NO: 49, and β-chain variable regions as shown in SEQ ID NO: 53; or α-chain variable region as shown in SEQ ID NO: 61, and β-chain variable region as shown in SEQ ID NO:

65.

3. The T-cell receptor TCR or its antigen-binding fragment according to claim 1 or 2, wherein the T-cell receptor TCR is a murine TCR, a human-mouse chimeric TCR, or a humanized TCR.

4. A polynucleotide encoding the T-cell receptor TCR or its antigen-binding fragment as described in any one of claims 1-3.

5. An expression vector comprising the polynucleotide of claim 4; preferably, the expression vector is a lentiviral vector.

6. A host cell comprising the expression vector of claim 5.

7. A method for preparing the T-cell receptor TCR or its antigen-binding fragment according to any one of claims 1-3, the method comprising: 1) Culturing the host cells as described in claim 6; 2) Recover the T cell receptor TCR or its antigen-binding fragment as described in any one of claims 1-3 from the host cell or its culture medium.

8. A pharmaceutical composition comprising any one of claims 1-3, the T-cell receptor TCR or its antigen-binding fragment, and a pharmaceutically acceptable carrier.

9. The T-cell receptor TCR or its antigen-binding fragment as described in any one of claims 1-3, used in the preparation of a drug for detecting cancer testis antigen MAGE-A4. 146-154 Its use in reagents for detecting or diagnosing tumor cells, or in the preparation of reagents for detecting or diagnosing cancer testis antigen MAGE-A4. 146-154 Uses in reagents for treating tumors; Optionally, the cancer testis antigen MAGE-A4 146-154 The sequence is shown in SEQ ID NO:

1.

10. Use of the T-cell receptor TCR or its antigen-binding fragment according to any one of claims 1-3 in the preparation of a medicament for treating tumors, wherein, The tumor is MAGE-A4. 146-154 Positive.

11. The use according to claim 10, wherein the tumor is selected from melanoma, ovarian cancer, bladder cancer, non-small cell lung cancer, head and neck cancer, or synovial sarcoma.

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