A T-cell receptor targeting MAGE-A3 and its applications

Abstract

This invention relates to the field of biomedicine, and in particular to a T-cell receptor targeting MAGE-A3 and its applications. The T-cell receptor provided by this invention comprises six CDR regions: CDR1α, CDR2α, and CDR3α in the TCRα chain, and CDR1β, CDR2β, and CDR3β in the TCRβ chain, with amino acid sequences shown in SEQ ID Nos. 1-6, respectively. This invention utilizes reverse-locking engineering to modify wild-type MAGE-A3 TCR molecules, obtaining a highly efficient and non-toxic TCR targeting MAGE-A3, avoiding the toxic side effects caused by affinity maturation. This engineered TCR is then applied to the preparation of TCR-T cells for the treatment of solid tumors.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a T-cell receptor that targets MAGE-A3 and its uses. Background Technology

[0002] Cancer-testis antigens (CT antigens), also known as tumor-testis antigens, are antigens that are not expressed in normal tissues other than the testes and placenta, but are frequently expressed in various tumors, exhibiting high tumor specificity and strong immunogenicity. Among these, MAGE-A, MAGE-B, MAGE-C, and NY-ESO-1 family proteins have been extensively studied. Currently, CT antigens are a major target in TCR-T cell research.

[0003] MAGE-A3 is expressed in the tumor tissues of more than 76% of melanoma patients and some lung cancer patients. In addition, except for the placenta and testes, MAGE-A3 is not expressed in normal human cells. This provides a theoretical basis for tumor immunotherapy targeting MAGE-A3 and makes it an ideal target for TCR-T therapy. Summary of the Invention

[0004] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a T cell receptor targeting MAGE-A3 and its uses, in order to solve the problems in the prior art.

[0005] To achieve the above and other related objectives, the present invention provides a T-cell receptor targeting MAGE-A3, wherein the T-cell receptor comprises the following six CDR regions: CDR1α, CDR2α, and CDR3α in the TCRα chain, and CDR1β, CDR2β, and CDR3β in the TCRβ chain: wherein,

[0006] 1) The amino acid sequence of CDR1α is shown in SEQ ID No. 1;

[0007] 2) The amino acid sequence of CDR2α is shown in SEQ ID No. 2;

[0008] 3) The amino acid sequence of CDR3α is shown in SEQ ID No. 3;

[0009] 4) The amino acid sequence of CDR1β is shown in SEQ ID No. 4;

[0010] 5) The amino acid sequence of CDR2β is shown in SEQ ID No. 5;

[0011] 6) The amino acid sequence of CDR3β is shown in SEQ ID No. 6.

[0012] Wherein, the first X in SEQ ID No. 1 is selected from D or H; and / or, the second X in SEQ ID No. 1 is selected from S or H; and / or, the third X in SEQ ID No. 1 is selected from A or H; and / or, the fourth X in SEQ ID No. 1 is selected from N or H.

[0013] Wherein, the first X in SEQ ID No. 2 is selected from S or H; and / or, the second X in SEQ ID No. 2 is selected from R or H.

[0014] In SEQ ID No. 3, X is selected from F or H.

[0015] Wherein, the first X in SEQ ID No. 5 is selected from F or H; and / or, the second X in SEQ ID No. 5 is selected from T or H.

[0016] Wherein, the first X in SEQ ID No. 6 is selected from S or H; and / or, the second X in SEQ ID No. 6 is selected from P or H; and / or, the third X in SEQ ID No. 6 is selected from Y or H.

[0017] The present invention also provides an antibody drug comprising the aforementioned T-cell receptor.

[0018] The present invention also provides an isolated polynucleotide encoding the aforementioned T-cell receptor.

[0019] The present invention also provides a nucleic acid construct comprising the aforementioned polynucleotides and plasmid backbone.

[0020] The present invention also provides a viral vector containing the aforementioned polynucleotides.

[0021] The present invention also provides an isolated T cell containing the aforementioned T cell receptor, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned viral vector.

[0022] The present invention also provides the use of the aforementioned T-cell receptor, the aforementioned antibody drug, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned viral vector in the preparation of tumor treatment products.

[0023] The present invention also provides a tumor treatment product comprising the aforementioned T-cell receptor, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned viral vector, and pharmaceutically acceptable excipients.

[0024] As described above, the T-cell receptor targeting MAGE-A3 of the present invention and its use have the following beneficial effects:

[0025] This invention utilizes reverse-locking engineering to modify wild-type MAGE-A3 TCR molecules, obtaining a highly efficient and non-toxic TCR targeting MAGE-A3, avoiding the toxic side effects caused by affinity maturation. This engineered TCR is then applied to TCR-T cell preparation for the treatment of solid tumors. Attached Figure Description

[0026] Figure 1 The display shows the CDR information for the wild-type MAGE-A3 T cell receptor in this invention.

[0027] Figure 2 The results show the effects of the mutant MAGE-A3 T cell receptor modification in this invention.

[0028] Figure 3 The results show the verification of the reverse-locking properties of the mutant MAGE-A3 T cell receptor in this invention.

[0029] Figure 4 The results shown are flow cytometry findings of T cell receptor expression levels in MAGE-A3 TCR-T cells in this invention.

[0030] Figure 5 The results show the detection results of cytokine release and cell exhaustion in MAGE-A3 TCR-T cells in this invention.

[0031] Figure 6 The results show the target cell killing efficiency of MAGE-A3 TCR-T cells in this invention.

[0032] Figure 7 The results show the in vivo tumor killing detection results of MAGE-A3 TCR-T cells in this invention. Detailed Implementation

[0033] The present invention provides a T cell receptor (TCR) targeting MAGE-A3, wherein the T cell receptor comprises the following six CDR regions: CDR1α, CDR2α and CDR3α in the TCRα chain, and CDR1β, CDR2β and CDR3β in the TCRβ chain.

[0034] In this invention, the TCR targeting MAGE-A3 is highly efficient and non-toxic, avoiding the toxic side effects caused by affinity maturation.

[0035] In some specific embodiments, the amino acid sequence of CDR1α is shown in SEQ ID No. 1.

[0036] In some specific embodiments, the amino acid sequence of CDR2α is shown in SEQ ID No. 2.

[0037] In some specific embodiments, the amino acid sequence of CDR3α is shown in SEQ ID No. 3.

[0038] In some specific embodiments, the amino acid sequence of CDR1β is shown in SEQ ID No. 4.

[0039] In some specific embodiments, the amino acid sequence of CDR2β is shown in SEQ ID No. 5.

[0040] In some specific embodiments, the amino acid sequence of CDR3β is shown in SEQ ID No. 6.

[0041] Wherein, the first X in SEQ ID No. 1 is selected from D or H; and / or, the second X in SEQ ID No. 1 is selected from S or H; and / or, the third X in SEQ ID No. 1 is selected from A or H; and / or, the fourth X in SEQ ID No. 1 is selected from N or H.

[0042] Wherein, the first X in SEQ ID No. 2 is selected from S or H; and / or, the second X in SEQ ID No. 2 is selected from R or H.

[0043] In SEQ ID No. 3, X is selected from F or H.

[0044] Wherein, the first X in SEQ ID No. 5 is selected from F or H; and / or, the second X in SEQ ID No. 5 is selected from T or H.

[0045] Wherein, the first X in SEQ ID No. 6 is selected from S or H; and / or, the second X in SEQ ID No. 6 is selected from P or H; and / or, the third X in SEQ ID No. 6 is selected from Y or H.

[0046] A CDR (complementarity determining region) generally refers to a region in an antibody or T-cell receptor that is spatially complementary to the antigenic determinant. The variability of an antibody or T-cell receptor is usually not uniformly distributed throughout its variable region. The α-chain and β-chain variable regions of monoclonal antibodies or T-cell receptors typically have three hypervariable regions (HVRs). These regions are spatially complementary to the antigenic determinant, hence the term complementarity determining region (CDR). Specifically, the α-chain variable region typically includes three CDRs: CDRα1, CDRα2, and CDRα3, and the β-chain variable region typically includes three CDRs: CDRβ1, CDRβ2, and CDRβ3.

[0047] In some specific embodiments, the α-chain variable region and the β-chain variable region may further include a framework region, which may be located between complementarity-determining regions or at both ends of the complementarity-determining regions. In some specific embodiments of the present invention, the framework region sequence is a human monoclonal antibody variable region or a mouse monoclonal antibody variable region framework region sequence obtained by substitution, deletion, or addition of one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, and the framework region sequence may have 80%, 85%, 90%, 93%, 95%, 97%, or 99% or more homology with the framework region sequence of the human monoclonal antibody variable region sequence.

[0048] In some specific embodiments, the amino acid sequence of the α-chain variable region of the T-cell receptor targeting MAGE-A3 is shown in SEQ ID No. 7; and / or, the amino acid sequence of the β-chain variable region of the T-cell receptor targeting MAGE-A3 is shown in SEQ ID No. 8.

[0049] Wherein, the first X in SEQ ID No. 7 is selected from D or H; and / or, the second X in SEQ ID No. 7 is selected from S or H; and / or, the third X in SEQ ID No. 7 is selected from A or H; and / or, the fourth X in SEQ ID No. 7 is selected from N or H; and / or, the fifth X in SEQ ID No. 7 is selected from S or H; and / or, the sixth X in SEQ ID No. 7 is selected from R or H; and / or, the seventh X in SEQ ID No. 7 is selected from F or H.

[0050] Wherein, the first X in SEQ ID No. 8 is selected from F or H; and / or, the second X in SEQ ID No. 8 is selected from T or H; and / or, the third X in SEQ ID No. 8 is selected from S or H; and / or, the fourth X in SEQ ID No. 8 is selected from P or H; and / or, the fifth X in SEQ ID No. 8 is selected from Y or H.

[0051] In some specific embodiments, the α-chain variable region may further be a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID No. 7; the β-chain variable region may further be a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID No. 8.

[0052] In some specific embodiments, the T cell receptor targeting MAGE-A3 further includes a TCRα chain constant region or a TCRβ chain constant region.

[0053] In some specific embodiments, the amino acid sequence of the constant region of the TCRα chain is shown in SEQ ID No. 9; or, the amino acid sequence of the constant region of the TCRβ chain is shown in SEQ ID No. 10.

[0054] In some specific embodiments, the TCRα chain constant region may further be a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID No. 9; the TCRβ chain constant region may further be a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID No. 10.

[0055] In some specific embodiments, the amino acid sequence of the TCRα chain is as shown in any of SEQ ID No. 11 or 13-19; and / or, the amino acid sequence of the TCRβ chain is as shown in any of SEQ ID No. 12 or 20-24.

[0056] Further, the amino acid sequence of the α chain of the TCR is as shown in SEQ ID No. 11, and the amino acid sequence of the β chain is as shown in any one of SEQ ID No. 20-24; or, the amino acid sequence of the α chain of the TCR is as shown in SEQ ID No. 13, and the amino acid sequence of the β chain is as shown in SEQ ID No. 20; or, the amino acid sequence of the α chain of the TCR is as shown in any one of SEQ ID No. 13-19, and the amino acid sequence of the β chain is as shown in SEQ ID No. 12.

[0057] In some specific embodiments, when having the aforementioned α and β chain variable region CDR sequences, the TCR α chain may further be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID No. 11 or 13-19; the TCR β chain may further be: a peptide segment having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in SEQ ID No. 11 or 13-19; Peptides having at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence similarity to the amino acid sequence shown in No. 12 or 20-24.

[0058] In some specific embodiments, the TCRα chain is selected from human TCRα chain, humanized TCRα chain, chimeric TCRα chain, or mouse TCRα chain; the TCRβ chain is selected from human TCRβ chain, humanized TCRβ chain, chimeric TCRβ chain, or mouse TCRβ chain. Here, "chimeric TCRα chain" or "chimeric TCRβ chain" refers to a TCRα chain or TCRβ chain containing sequences derived from more than one species, such as sequences derived from humans and mice.

[0059] In some specific embodiments, the T-cell receptor targeting MAGE-A3 binds to the MAGE-A3 antigen but not to the TITIN antigen.

[0060] In some specific embodiments, the MAGE-A3 antigen comprises the following amino acid sequence: SEQ ID No. 26: peptide of EVDPIGHLY; and / or, the TITIN antigen comprises the following amino acid sequence: SEQ ID No. 27: peptide of ESDPIVAQY.

[0061] The present invention also provides an antibody drug comprising the aforementioned T-cell receptor targeting MAGE-A3.

[0062] In some specific embodiments, the antibody drug may be a TCR-BiTE (Bispecific T-cell Engagers) antibody protein drug.

[0063] Furthermore, the TCR-BiTE antibody protein drug comprises the aforementioned T cell receptor targeting MAGE-A3, and a single-chain variable fragment (scFv) against any one of CD3, CD4, CD5, CD6, CD7, CD8, CD28, or 4-1BB.

[0064] The present invention also provides an isolated polynucleotide that encodes the aforementioned T-cell receptor targeting MAGE-A3.

[0065] The present invention also provides a nucleic acid construct comprising the aforementioned polynucleotides and plasmid backbone.

[0066] In some specific embodiments, the plasmid backbone is a viral, adenovirus, or adeno-associated virus plasmid backbone.

[0067] The present invention provides a viral vector containing the aforementioned polynucleotides.

[0068] In some specific embodiments, the viral vector is selected from lentiviruses, adenoviruses, or adeno-associated viruses.

[0069] The present invention provides an isolated T cell containing the aforementioned T cell receptor targeting MAGE-A3, the aforementioned polynucleotide, the aforementioned nucleic acid construct or the aforementioned viral vector.

[0070] In some specific embodiments, the T cells are TCR-T cells.

[0071] In some specific embodiments, the T cells are selected from helper T cells, suppressor T cells, effector T cells, cytotoxic T cells, delayed-type hypersensitivity T cells, native T cells, or memory T cells. Helper T cells are T cells that assist in humoral and cellular immunity; suppressor T cells are T cells that suppress cellular and humoral immunity; effector T cells are T cells that release lymphokines; cytotoxic T cells are T cells that kill target cells; delayed-type hypersensitivity T cells are T cells that participate in type IV hypersensitivity reactions and can act on helper and suppressor T cells to amplify the immune effect; native T cells are undifferentiated T cells; and memory T cells are T cells that remember specific antigen stimulation.

[0072] The present invention also provides the use of the aforementioned T-cell receptor targeting MAGE-A3, the aforementioned antibody drug, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned viral vector, or the aforementioned T cell in the preparation of tumor treatment products.

[0073] In some specific embodiments, the tumor is selected from one or more of the following: adrenocortical carcinoma, bladder urothelial carcinoma, breast cancer, cervical squamous cell carcinoma, cervical endogenous adenocarcinoma, bile duct carcinoma, colonic adenocarcinoma, lymphoid tumor, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myeloid leukemia, low-grade glioma of the brain, hepatocellular carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma, paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymic carcinoma, endometrial cancer, chronic myeloid leukemia, lung cancer, anal cancer, and retinoblastoma.

[0074] The present invention also provides a tumor treatment product comprising the aforementioned T-cell receptor targeting MAGE-A3, the aforementioned antibody drug, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned viral vector or the aforementioned T-cell and pharmaceutically acceptable excipients.

[0075] In some specific embodiments, the excipients include various excipients and diluents, which are not essential active ingredients and do not cause excessive toxicity after application. The excipients contain sterile water or physiological saline, stabilizers, excipients, antioxidants (ascorbic acid, etc.), buffers (phosphate, citric acid, other organic acids, etc.), preservatives, surfactants (PEG, Tween, etc.), chelating agents (EDTA, etc.), or binders. The excipients also contain other low molecular weight peptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol, or sorbitol. When the excipients are used in an aqueous solution for injection, they are selected from physiological saline, isotonic glucose solution, D-sorbitol isotonic solution, D-mannose isotonic solution, D-mannitol or sugar alcohol isotonic solution. The aqueous solution for injection contains a solubilizer. The solubilizer is selected from alcohols (ethanol), polyols (propylene glycol or PEG), and / or nonionic surfactants (Tween 80 or HCO-50). In the tumor treatment product provided by this invention, the aforementioned TCR, polynucleotide, nucleic acid construct, or T cell is a single effective ingredient, or it can be combined with one or more other active components useful for tumor treatment to form a combined formulation. The active components are various other drugs used for tumor treatment. The content of the active ingredient in the pharmaceutical composition is a safe and effective amount, which should be adjustable by those skilled in the art. For example, the dosage of the active ingredient in the aforementioned TCR, the aforementioned polynucleotide, the aforementioned nucleic acid construct, or the aforementioned T cell and tumor treatment product depends on the patient's weight, the type of application, the condition and severity of the disease. For example, the dosage of the aforementioned tumor treatment product as an active ingredient is 1-1000 mg / kg / day, 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or greater than 500 mg / kg / day.

[0076] The present invention also provides a method for tumor treatment, wherein the method comprises administering the aforementioned T cell receptor MAGE-A3, the aforementioned antibody drug, the aforementioned polynucleotide, the aforementioned nucleic acid construct, the aforementioned viral vector, or the aforementioned T cells to a tumor patient.

[0077] In some specific embodiments, the dosage is 1-1000 mg / kg / day. Specifically, the dosage is 1-3 mg / kg / day, 3-5 mg / kg / day, 5-10 mg / kg / day, 10-20 mg / kg / day, 20-30 mg / kg / day, 30-40 mg / kg / day, 40-60 mg / kg / day, 60-80 mg / kg / day, 80-100 mg / kg / day, 100-200 mg / kg / day, 200-500 mg / kg / day, or 500 mg-1000 mg / kg / day.

[0078] In some specific embodiments, the object of the method can be a mammal; preferably, the object of the method is a human.

[0079] In this invention, the T-cell receptor (TCR) is a molecule present on the surface of T cells that is responsible for recognizing peptide-MHC complexes. In some embodiments, the TCR is a truncated or full-length TCR. In some embodiments, the TCR is a heterodimer composed of α and β chains. In some embodiments, the TCR may also be a single-chain TCR (scTCR).

[0080] In this invention, the term "TCRα chain constant region" or "TCRβ chain constant region" comprises an extracellular constant region, a transmembrane region, and an intracellular constant region connected in sequence. The extracellular constant region may include the hinge regions of the TCRα and TCRβ chains, participating in the formation of disulfide bonds between the TCRα and TCRβ chains. The transmembrane region is also a constant region, and its functions include participating in the cell membrane anchoring of the TCRα and TCRβ chains and interacting with the CD3 subunit to form the TCR-CD3 complex. The possible functions of the intracellular constant region include participating in the conformational change of the TCR-CD3 complex and signal transduction after TCR signal transduction.

[0081] In this invention, the term "variable region" or "variable domain" refers to the domain of an immunoglobulin superfamily binding protein (e.g., the α-chain or β-chain of a TCR) involved in the binding of an immunoglobulin superfamily binding protein (e.g., a TCR) to an antigen. The variable domains (Vα and Vβ, respectively) of the α-chain and β-chain of a native TCR typically have similar structures, each containing four conserved frame regions (FRs) and three core regions (CDRs). The Vα domain is encoded by two independent DNA segments, a variable gene segment and a linker gene segment (VJ); the Vβ domain is encoded by three independent DNA segments, namely, a variable gene segment, a diversity gene segment, and a linker gene segment (VDJ). A single Vα or Vβ domain may be sufficient to confer antigen-binding specificity. Furthermore, TCRs binding to specific antigens can be isolated from antigen-binding TCRs using either the Vα or Vβ domain to screen libraries of complementary Vα or Vβ domains, respectively.

[0082] In this invention, the term "antigen" refers to a cell surface molecule or an intracellular molecule presented by MHC molecules or MHC-like molecules that can be bound by antibodies or T-cell receptors (TCRs), including but not limited to polypeptide antigens (e.g., NYESO-1, AFP, and MART-1), lipid antigens (e.g., β-GlcCer, eLPA, and LPE), or polysaccharide antigens (e.g., CA199, CA72-4, and CA125). The antigen may be a tumor antigen, such as a tumor-associated antigen (TAA) or a tumor-specific antigen (TSA).

[0083] In this invention, the term "isolated" refers to material that has been removed from its natural state or otherwise artificially manipulated, such as the α-chain, β-chain, T-cell receptor, and nucleic acid described herein. The isolated material may be substantially or substantially free of the components normally accompanying it in its natural state, or may be manipulated into an artificial state in conjunction with the components normally accompanying it in its natural state. The isolated material may be in a natural, chemically synthesized, or recombinant form. The isolated material may also, or alternatively, be in an enriched, partially purified, or purified form.

[0084] In this invention, the term "polynucleotide" is also called "nucleotide" or "nucleic acid" and refers to a nucleic acid chain consisting of deoxyribonucleic acid, ribonucleic acid, modified nucleic acid or base, and / or analogs thereof, or any substrate that can be incorporated into the chain by DNA or RNA polymerase.

[0085] In this invention, the term "nucleic acid construct," also known as "vector," refers to a device capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing said genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, plasmids, granules, or phage vectors.

[0086] In this invention, the term "host cell" refers to a cell in which exogenous nucleic acids have been introduced, including the progeny of these cells.

[0087] In this invention, to determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal alignment, or non-homologous sequences may be discarded for comparison purposes). In a preferred embodiment, for comparison purposes, the length of the reference sequence being aligned is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the reference sequence length. The amino acid residues or nucleic acids at the corresponding amino acid or nucleic acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleic acid at the corresponding position in the second sequence, the molecules are identical at that position.

[0088] Mathematical algorithms can be used to compare sequences and calculate the percentage of identity between two sequences. In a preferred embodiment, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at http: / / www.gcg.com) is used in the GAP program integrated into the GCG software package, employing a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two amino acid sequences. In yet another preferred embodiment, the GAP program in the GCG software package (available at http: / / www.gcg.com) is used, employing an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6, to determine the percentage of identity between two nucleic acid sequences. The particularly preferred set of parameters (and unless otherwise specified, a set of parameters to be used) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a shift vacancy penalty of 5.

[0089] Alternatively, the PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4 can be used to determine the percentage of identity between two amino acid sequences or nucleic acid sequences using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0).

[0090] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0091] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated in the text, the singular forms "a", "an" and "this" include the plural forms.

[0092] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the present invention, both endpoints of each numerical range and any value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, apparatus, and materials used in the embodiments, based on the knowledge of the prior art possessed by one of ordinary skill in the art and the description of this invention, any prior art methods, apparatus, and materials similar to or equivalent to those described, apparatus, and materials in the embodiments of this invention may be used to implement the present invention.

[0093] The sequence information used in this application is as follows:

[0094] SEQ ID No.1

[0095] XXXIYX

[0096] SEQ ID No.2

[0097] IQSXQXE

[0098] SEQ ID No. 3

[0099] AVRPGGAGSYQLTX

[0100] SEQ ID No. 4

[0101] SGHRS

[0102] SEQ ID No.5

[0103] YXSEXQ

[0104] SEQ ID No.6

[0105] AXSXNMADEQXF

[0106] SEQ ID No.7

[0107] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTXXXIYXLQWFRQDPGKGLTSLLLIQSXQXEQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPGGAGSYQLTXGKGTKLSVIPN

[0108] SEQ ID No.8

[0109] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYXSEXQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCAXSXNMADEQXFGPGTRLTVTE

[0110] SEQ ID No.9

[0111] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0112] SEQ ID No.10

[0113] DLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0114] Wild-type α-chain of SEQ ID No.11

[0115] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPGGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0116] Wild-type β-chain of SEQ ID No.12

[0117] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSPNMADEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0118] SEQ ID No.13 MAGEα-D28H

[0119] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTHSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPGGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0120] SEQ ID No.14 MAGEα-S29H

[0121] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDHAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPGGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0122] SEQ ID No.15 MAGEα-A30H

[0123] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSHIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPGGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0124] SEQ ID No.16 MAGEα-N33H

[0125] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYHLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPGGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0126] SEQ ID No.17 MAGEα-S54H

[0127] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSHQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPGGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0128] SEQ ID No.18 MAGEα-R56H

[0129] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQHEQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPGGAGSYQLTFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0130] SEQ ID No.19 MAGEα-F105H

[0131] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPGGAGSYQLTHGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0132] SEQ ID No.20 MAGEβ-F51H

[0133] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYHSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSPNMADEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0134] SEQ ID No.21 MAGEβ-T54H

[0135] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSEHQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSPNMADEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0136] SEQ ID No.22 MAGEβ-S94H

[0137] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCAHSPNMADEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0138] SEQ ID No.23 MAGEβ-P96H

[0139] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSHNMADEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0140] SEQ ID No.24MAGEβ-Y103H

[0141] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSPNMADEQHFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0142] SEQ ID No.25

[0143] MKWVTFISLLFLFSSSSRAEVDPIGHLYGGGGSGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQKMEPRAPWIEQEGPEYWDQETRNMKAHSQTDRANLGTLRGAYNQSEDGSHTIQIMYGCDVGPDGRFLRGYRQDAYDGKDYIALNEDLRSWTAADMAAQITKRKWEAVHAAEQRRVYLEGRCVDGLRRYLENGKETLQRTDPPKTHMTHHPISDHEATLRCWALGFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPKPLTLRWELSSGGGGSGLNDIFEAQKIEWHEHHHHHHHHHH

[0144] Example 1 Screening of T cell receptors targeting MAGE - A3

[0145] The known wild - type MAGE - A3 TCR sequence is:

[0146] TCRα chain:

[0147] METLLGLLILWLQLQWVSSKQEVTQIPAALSVPEGENLVLNCSFTDSAIYNLQWFRQDPGKGLTSLLLIQSSQREQTSGRLNASLDKSSGRSTLYIAASQPGDSATYLCAVRPGGAGSYQLTFGKGTKLSVIPNIQN PDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS

[0148] TCRβchain:

[0149] MGSRLLCWVLLCLLGAGPVKAGVTQTPRYLIKTRGQQVTLSCSPISGHRSVSWYQQTPGQGLQFLFEYFSETQRNKGNFPGRFSGRQFSNSRSEMNVSTLELGDSALYLCASSPNMADEQYFPGGTRLTVTEDLKNVFPPEVAVFEPSEAEISHT QKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

[0150] Among them, the CDR region (α chain and β chain) of the corresponding pMHC (MAGE-A3 antigenic epitope peptide-HLA-A*01:01) is as follows: Figure 1 As shown.

[0151] Based on the CDR prediction results, suitable amino acid sites in the α-chain and β-chain CDR regions were selected and mutated to histidine (H). The modified sequence was then integrated into the lentiviral vector pHR via molecular cloning. Subsequently, the MAGE-A3 TCR sequence was introduced into the genome of the human leukemia T cell line SKW-3 via lentiviral infection, enabling stable expression of the corresponding TCR molecules: MAGE-A3 WT (wild-type), MAGE-A3 A3A (high affinity), and MAGEα / β-mutants (histidine mutants).

[0152] Subsequently, a series of SKW-3 cells overexpressing TCR were co-incubated at a 1:1 ratio with HEK-293T cells (expressing HLA-A*01:01) loaded with MAGE-A3 (EVDPIGHLY) and TITIN (ESDPIVAQY) antigenic epitope peptides in 96-well plates for 14 hours. SKW-3 cell activation (CD69 MFI) was then assessed to evaluate the effect of the mutation. The data are as follows: Figure 2 As shown.

[0153] Based on the test results, TCR molecules that can be activated by the MAGE-A3 antigenic epitope peptide and do not respond to the TITIN antigenic epitope peptide (a highly efficient and non-toxic qualitative indicator) were screened. Two effective mutant TCR molecules were obtained: MAGEα-D28H (amino acid D at position 28 of the αchain is mutated to H, including SEQ ID No. 13 and 12) and MAGEβ-F51H (amino acid F at position 51 of the βchain is mutated to H, including SEQ ID No. 11 and 20). Furthermore, combining the two yielded a dual-modified TCR molecule (MAGEα-D28H & β-F51H, including SEQ ID No. 13 and 20).

[0154] Example 2: Optical tweezers detection of the bond lifetime between screened TCR molecules and pMHC

[0155] In this embodiment, optical tweezers technology is used to detect the bond lifetime of the bond formed between the screened MAGE-A3 TCR histidine mutant and pMHC, thereby determining whether a reverse-locking bond is formed between the two.

[0156] First, a biotinylation site was added to the MAGE-A3 pMHC protein sequence, and the protein was cloned into the pD649 plasmid, transfected with Expi293F, and the pMHC protein was produced. After purification with Ni-NTA, the pMHC protein was biotinylated using BirA and purified by molecular sieve chromatography to obtain pure biotinylated pMHC protein (amino acid sequence shown in SEQ ID No. 25). Subsequently, SKW-3 cells expressing the TCR molecule to be detected and the pMHC protein were loaded onto an M-Trap optical tweezers instrument to detect the maintenance time of the OP and anti-OP bonds under different stress conditions, obtaining bond lifetime curves. The bond lifetime curves are shown below. Figure 3 As shown.

[0157] According to the test results, the bond lifetime between the two TCR mutants, MAGEα-D28H and MAGEβ-F51H, and the pMHC protein first increases and then decreases with the increase of external force, which is consistent with the reverse-locked bond property.

[0158] Example 3: Preparation of MAGE-A3 TCR-T cells

[0159] The MAGE-A3 TCR sequence was introduced into the genome of human primary T cells via lentiviral infection, enabling stable expression of the corresponding TCR molecule. The TCR expression level detection results are as follows: Figure 4 As shown.

[0160] Based on the flow cytometry results of the above experiments, the TCR positivity rate of each MAGE-A3 TCR-T cell line was approximately 95%. The high positivity rate and small differences indicate that these cells can be used for subsequent in vitro experiments.

[0161] Example 4: In vitro functional activity verification of MAGE-A3 TCR-T cells

[0162] MAGE-A3 TCR-T cells were co-incubated with target cells HCT-116 (human colon cancer cell line) at a 1:1 ratio in 96-well plates for 6 h and 48 h, respectively. At 6 h, cell samples were collected for flow cytometry analysis to detect the release of inflammatory cytokines (IFNγ, TNF) from TCR-T cells. At 48 h, cell samples were collected for flow cytometry analysis to detect TCR-T cell exhaustion (PD-1). The results are as follows: Figure 5 As shown.

[0163] According to flow cytometry results, the anti-locked TCR-T cells exhibited better activation than wild-type TCR-T cells (MAGEWT), with the dual anti-locked TCR-T cells (MAGEα-D28H & β-F51H) showing activation effects approaching those of high-affinity TCR-T cells (A3A). Furthermore, the exhaustion level of the anti-locked TCR-T cells was significantly lower than that of high-affinity TCR-T cells, similar to that of wild-type TCR-T cells. These results indicate that anti-locked TCR-T cells, especially the dual anti-locked cells, possess excellent target cell recognition and activation capabilities, while effectively avoiding cell exhaustion caused by over-activation.

[0164] Example 6: Detection of Target Cell Killing Efficiency

[0165] MAGE-A3 TCR-T cells were co-incubated with target cells HCT-116Luc (an HCT-116 cell line overexpressing luciferase) at different ratios (10:1, 5:1, and 1:1) in 96-well plates for 24 h. After co-incubation, luciferin (luciferin, a substrate for luciferase degradation) solution was added for further incubation. The luminescence intensity was detected using an Envision chemiluminescence analyzer (quantitative substrate degradation, allowing calculation of the number of surviving target cells) to assess target cell killing efficiency. The results are as follows: Figure 6As shown, the results indicate that the mutant TCR is more effective at killing target cells than the wild type.

[0166] Example 7: In vivo tumor killing detection

[0167] Prepare 6-week-old NSG immunodeficient mice, 5 mice per group, and subcutaneously inject 5×10 6 HCT-116 cells were used to create a subcutaneous tumor model, and tumor volume was measured every 2-3 days. The tumor was allowed to grow to 100-200 mm. 3 At approximately 7 days, administer 5×10 via tail vein injection. 6 Treatment with TCR-T cells included a saline group and five MAGE TCRs (MAGE WT, MAGEα-D28H, MAGEβ-F51H, MAGEα-D28H & β-F51H, and A3A). Tumor volume was measured every 2–3 days, and tumor growth curves were plotted. Results are as follows: Figure 7 As shown, MAGEα-D28H & β-F51H significantly slowed tumor growth compared to the saline group and the wild-type group.

[0168] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications and variations of the methods listed herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. A T-cell receptor targeting MAGE-A3, characterized in that, The T cell receptor comprises the following six CDR regions: CDR1α, CDR2α, and CDR3α in the TCRα chain, and CDR1β, CDR2β, and CDR3β in the TCRβ chain: wherein, 1) The amino acid sequence of CDR1α is shown in SEQ ID No. 1; 2) The amino acid sequence of CDR2α is shown in SEQ ID No. 2; 3) The amino acid sequence of CDR3α is shown in SEQ ID No. 3; 4) The amino acid sequence of CDR1β is shown in SEQ ID No. 4; 5) The amino acid sequence of CDR2β is shown in SEQ ID No. 5; 6) The amino acid sequence of CDR3β is shown in SEQ ID No. 6; Wherein, the first X in SEQ ID No. 1 is selected from D or H; and / or, the second X in SEQ ID No. 1 is selected from S or H; and / or, the third X in SEQ ID No. 1 is selected from A or H; and / or, the fourth X in SEQ ID No. 1 is selected from N or H; And / or, the first X in SEQ ID No. 2 is selected from S or H; and / or, the second X in SEQ ID No. 2 is selected from R or H; And / or, in SEQ ID No. 3, X is selected from F or H; And / or, the first X in SEQ ID No. 5 is selected from F or H; and / or, the second X in SEQ ID No. 5 is selected from T or H; And / or, the first X in SEQ ID No. 6 is selected from S or H; and / or, the second X in SEQ ID No. 6 is selected from P or H; and / or, the third X in SEQ ID No. 6 is selected from Y or H.

2. The T cell receptor according to claim 1, characterized in that, The amino acid sequence of the α-chain variable region of the T-cell receptor targeting MAGE-A3 is shown in SEQ ID No. 7; and / or, the amino acid sequence of the β-chain variable region of the T-cell receptor targeting MAGE-A3 is shown in SEQ ID No.

8.

3. The T-cell receptor according to claim 1, characterized in that, The amino acid sequence of the TCRα chain is as shown in any of SEQ ID No. 11 or 13-19; and / or, the amino acid sequence of the TCRβ chain is as shown in any of SEQ ID No. 12 or 20-24.

4. An antibody drug, characterized in that, The antibody drug comprises the T-cell receptor as described in any one of claims 1-3.

5. The antibody drug according to claim 4, characterized in that, The antibody drug is a TCR-BiTE antibody protein drug.

6. An isolated polynucleotide, characterized in that, The polynucleotide encodes the T-cell receptor as described in any one of claims 1-3.

7. A nucleic acid construct, characterized in that, The nucleic acid construct comprises the polynucleotide and plasmid backbone as described in claim 6.

8. A viral vector, characterized in that, The viral vector contains the polynucleotide as described in claim 6.

9. An isolated T cell, characterized in that, The T cell contains the T cell receptor as described in any one of claims 1-3, the polynucleotide as described in claim 6, the nucleic acid construct as described in claim 7, or the viral vector as described in claim 8.

10. Use of the T-cell receptor of any one of claims 1-3, the antibody drug of claim 4 or 5, the polynucleotide of claim 6, the nucleic acid construct of claim 7, or the viral vector of claim 8 in the preparation of tumor therapeutic products.

11. A tumor treatment product, characterized in that, The tumor treatment product comprises the T-cell receptor of any one of claims 1-3, the antibody drug of claim 4 or 5, the polynucleotide of claim 6, the nucleic acid construct of claim 7, or the viral vector of claim 8, and pharmaceutically acceptable excipients.

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