T cell receptors, their production and uses

Abstract

The present invention provides a method for constructing a T cell receptor and its use. The method utilizes the mutations in the intracellular constant region of the α and β chains of the T cell receptor to inhibit the degradation of the T cell receptor after the TCR antigen signal is activated, maintain the TCR level on the cell surface, and enhance the effect of TCR-T cell therapy. The method is suitable for various TCR-T cell therapies.

Description

[Technical field]

[0001] This application relates to the field of T cell receptors, and in particular to the construction and uses of modified T cell receptors. [Background technology]

[0002] Malignant tumors seriously threaten human health and life, and are one of the leading causes of human death, with their incidence increasing year by year. Traditional cancer treatments are primarily surgical, with radiotherapy and chemotherapy as adjuncts. Compared with traditional treatments, tumor immunotherapy targets the immune system and stimulates a systemic response against tumors. In addition, clinical data show that tumor immunotherapy can significantly improve the prognosis of patients with certain tumor types. Adoptive transfer of antigen-specific T cells is one of the main methods of tumor immunotherapy and has been studied for the treatment of hematological and solid tumors. It is mainly based on the T cell receptor on the surface of T cells, which can recognize various tumor antigens, thereby achieving the killing and elimination of tumor cells.

[0003] Currently, the T cell adoptive therapies that are being further studied mainly include CAR-T cell therapy and TCR-T therapy. In CAR-T, the antigen-binding portion of an antibody that recognizes a specific tumor antigen is bound to the CD3-ζ chain in vitro to form a chimeric protein, which is then transferred to the patient's T cells by gene transfer to cause the patient's T cells to express the chimeric antigen receptor. When the patient's T cells are "recoded," a large number of tumor T cells are generated, which then perform tumor killing. In TCR-T therapy, T cells recognize antigens presented by the major histocompatibility complex (MHC) on the surface of target cells with their TCRs, thereby directly attacking and killing the target cells. This is a T cell therapy technology developed based on natural TCRs or by weakly modifying TCRs. Its ability to recognize tumor epitopes presented by major histocompatibility complex molecules on the surface of tumor cells makes it more applicable.

[0004] The target of TCR-T cell therapy depends on the antigen TCR expressed on the surface of T cells, TCR is a receptor on the surface of T cells, which binds to CD3 through non-covalent bonds to form a TCR-CD3 complex, activates T cells by recognizing and binding to antigens presented by MHC, and promotes T cell division and differentiation. Unlike CAR, most TCRs are heterodimers composed of α and β chains, and both α and β chains contain antigen-binding regions, constant regions, and transmembrane domains. These α and β chains are covalently linked by disulfide bonds between conserved cysteine ​​residues located within a given region of each chain. Compared with CAR-T therapy, TCR-T can target most intracellular antigens and is not limited to surface antigens, that is, TCR-T can target most tumor antigens, especially recognize intracellular antigens of tumor cells. Therefore, the scope of tumor recognition by T cell receptors is broader than that of antibody drugs and CAR-T, which relies on antibodies to recognize tumors. In addition, CAR-T has deficiencies in the treatment of solid tumors, and TCR-T technology is expected to solve this problem.

[0005] At present, there are various TCR-T optimization and improvement methods to improve or enhance the effect of TCR-T-based tumor therapy. These methods mainly aim to enhance the recognition process of T cells to tumor cells by modifying TCR, which is the "probe" that T cells bind to tumor antigens, and improve the affinity of T lymphocytes to tumor cells, so that T cells that originally did not have the ability to recognize tumors can efficiently recognize and kill tumor cells. Specifically, they include the following:

[0006] (1) Increase the expression of exogenous TCR and enhance the antitumor effect of TCR-T. a. Mouserization of TCR constant region: Mouserization of TCR constant region using genetic engineering techniques reduces or prevents endogenous and exogenous TCR mismatch, promotes preferential pairing of exogenous TCR, thereby improving the expression level of T cell surface antigens, and enhances the antitumor effect of TCR-T. b. Cysteine ​​insertion method in TCR constant region: Cysteine ​​is introduced into the constant regions of TCR α and β chains to promote preferential pairing of exogenous TCR, improve the surface expression level of exogenous TCR, and reduce mismatch with endogenous TCR chain. This method can improve the efficacy and safety of tumor-reactive T cells. c. Insertion of hydrophobic mutation into TCR transmembrane region: Increasing the hydrophobicity of the transmembrane region of TCR α chain improves the stability of TCR and improves TCR expression on the T cell surface, thereby improving cell affinity and antitumor TCR activity.

[0007] (2) Improving the affinity of exogenous TCR to tumor antigens. Synthetic T cell receptor and antigen receptor (STAR): By inserting the VH and VL of the antigen-binding domain of an antibody into the TCRαβ constant domain, it is possible to combine the high affinity of CAR with the high signal transduction ability of the TCR complex, thereby enhancing the antitumor effect of TCR-T.

[0008] Although modified TCR-T cell adoptive therapy has achieved good results in the treatment of metastatic melanoma and other malignancies, there are still many limitations in tumor treatment.

[0009] (1) T cell dysfunction: a. Continuous antigen stimulation in the tumor microenvironment leads to increased expression of inhibitory receptors such as PD-1, Lag-3, CD39 and TIM-3, resulting in CD8 T cell dysfunction. + b) A hypoxic and low pH tumor microenvironment leads to a progressive loss of T cell effector functions, including reduced secretion levels of proinflammatory cytokines such as IFN-γ and TNF-α, reduced proliferation and self-renewal capacity of T cells, and impaired metabolic activity.

[0010] (2) The expression level of TCR on the T cell surface is reduced. Antigen stimulation in the tumor microenvironment reduces the expression of CD8 + The expression of exogenous TCR on the surface of T cells is reduced or its degradation is accelerated, and there are not enough TCR-T cells to attack tumor cells.

[0011] (3) Treatment toxicity exists during clinical treatment. Currently, the antigens targeted by TCR-T rarely exist in tumor tissue, so extratumoral toxicity targeted to the tumor occurs during treatment. Summary of the Invention [Problem to be solved by the invention]

[0012] In response to some of the technical shortcomings of TCR-T cell therapy mentioned above, the objective of the present application is to provide a T cell receptor that can inhibit degradation and enhance the anti-tumor effect of TCR-T cells. [Means for solving the problem]

[0013] In one aspect, the present application provides an isolated TCR alpha chain or fragment thereof of a T cell receptor (TCR), the TCR alpha chain comprising a TCR alpha chain constant region, the TCR alpha chain constant region comprising, in sequence, a TCR alpha chain extracellular constant region, a TCR alpha chain transmembrane region, and a TCR alpha chain intracellular constant region, the TCR alpha chain intracellular constant region being a mutant TCR alpha chain intracellular constant region in which at least one serine in a wild-type TCR alpha chain intracellular constant region has been mutated to alanine.

[0014] In another aspect, the present application provides an isolated TCR β chain or fragment thereof of a T cell receptor (TCR), the TCR β chain comprising a TCR β chain constant region, the TCR β chain constant region comprising, in sequence, a TCR β chain extracellular constant region, a TCR β chain transmembrane region, and a TCR β chain intracellular constant region, the TCR β chain intracellular constant region being a mutant TCR β chain intracellular constant region in which at least one lysine in a wild-type TCR β chain intracellular constant region has been mutated to arginine or alanine.

[0015] In another aspect, the present application provides an isolated T cell receptor or fragment thereof comprising a TCR α chain and / or a TCR β chain as described above.

[0016] In another aspect, the present application provides one or more T cell receptors, such as F5-WT TCR, F5-SA TCR, F5-KR TCR or F5-dMUT TCR, such as 1G4-WT TCR, 1G4-SA TCR, 1G4-KR TCR or 1G4-dMUT TCR.

[0017] In another aspect, the present application provides an isolated nucleic acid or fragment thereof encoding any of the aforementioned isolated TCR alpha chain or fragment thereof, TCR beta chain or fragment thereof, or T cell receptor or fragment thereof.

[0018] In another aspect, the present application provides a nucleic acid construct comprising any of the aforementioned isolated nucleic acids or fragments thereof.

[0019] In another aspect, the present application provides a vector comprising any of the aforementioned nucleic acid constructs.

[0020] In another aspect, the present application provides an engineered cell comprising any of the aforementioned nucleic acid constructs or vectors.

[0021] In another aspect, the present application also provides a TCR complex produced in a T cell by any of the aforementioned TCR nucleic acid constructs of the present application.

[0022] In another aspect, the present application provides functional detection of in vitro cultures of TCR-T cells.

[0023] In another aspect, the present application provides a method for producing a method for treating a cancer cell comprising: (1) Inhibition of degradation of T cell receptors during antigen stimulation (2) Providing a tumor-killing effect or inhibiting tumor growth; (3) Maintenance of T cell proliferation; and (4) Use of any of the isolated TCR α chain or fragment thereof, TCR β chain or fragment thereof, T cell receptor or fragment thereof, nucleic acid or fragment thereof, nucleic acid construct, vector, or engineered cell described above in one or more applications of preparing anti-tumor immune drugs and cells.

[0024] In another aspect, the mutant TCR-T cell therapy according to the present application is used alone, in combination with other therapies, or in combination with PD-1 / PD-L1 antibodies, cytokine therapy, or radiation therapy and chemotherapy.

[0025] In another aspect, the present application provides a method for producing a method for treating a cancer cell comprising: (1) mutating at least one lysine in the wild-type TCR β chain intracellular constant region to arginine or alanine; and / or The present invention provides a method for modifying a T cell receptor, comprising the step (2) of mutating at least one serine in the wild-type TCR α chain intracellular constant region to an alanine. Effect of the Invention

[0026] In this study, we use mutations in the ubiquitination modification sites of the α and β chain intracellular constant regions of the T cell receptor to inhibit degradation of the T cell receptor after TCR antigen signal activation and maintain the TCR level on the cell surface. This method can be applied to various TCR-T cell therapies.

[0027] Meanwhile, after antigen stimulation, SA TCR, KR TCR, and dMUT TCR cells showed higher TCR expression on the cell surface compared to WT TCR cells (wild-type TCR cells), and during the course of in vitro culture, SA TCR, KR TCR, and dMUT TCR cells showed a differentiation phenotype towards central memory T cells, and an in vitro depletion model revealed lower depletion levels in KR TCR and dMUT TCR cells compared to WT TCR, and this phenotypic change was attributed to a healthy mitochondrial state in SA TCR, KR TCR, and dMUT TCR cells, resulting in lower mitochondrial membrane potential and ROS levels in mutant TCR-T cells cultured in vitro. Meanwhile, in this application, it was revealed that KR TCR and dMUT TCR cells have stronger antitumor effects than WT TCR using a mouse transplanted tumor-T cell adoptive transfer model, and that in the dMUT TCR mutant group, there were more TCR-T cells in the spleen and blood of mice, while the proportion of KR TCR and dMUT TCR cells in tumor tissue was significantly increased. Regarding cell phenotype, in the SA TCR, KR TCR, and dMUT TCR groups, central memory T cells were more abundant in the spleen of mice, and dMUT mutant TCR-T cells in tumor tissue showed a lower T cell exhaustion phenotype and proliferation ability. Therefore, KR TCR and dMUT TCR cells show a better tumor growth inhibition potential in a therapy in which the same amount of TCR-T cells were introduced.

[0028] As will be appreciated, the present application not only inhibits the degradation of TCR by mutating amino acids in the TCR intracellular constant region, but also enhances the efficacy of TCR-T cell therapy, and can effectively inhibit the growth of tumors, which may be, but are not limited to, solid tumors, and may also be hematological tumors or lymphomas. [Brief description of the drawings]

[0029] [Figure 1]Antigen stimulation promotes the degradation of cell surface TCR. (A) In tumor-bearing mice inoculated with K562-NYESO-1, the expression of TCR-T cell surface TCR is detected in the spleen and tumor 12 days after the transfer of 1G4-TCR-T cells. (B) All in vitro functional experiments were performed on human primary T cells. 1G4-TCR-T cells were cultured in vitro and co-incubated with K562-NYESO-1 and K562-MART-1 cells, respectively, for 12 hours to detect the degradation of TCR. (C) Activated F5-TCR-T cells were cultured in vitro and 12 hours after CD3 antibody stimulation to detect the degradation of TCR. (D) Activated 1G4-TCR-T cells were cultured in vitro and 12 hours after CD3 antibody stimulation to detect the degradation of TCR. [Diagram 2] Figure 1 shows the results of inhibition of TCR downregulation and degradation by mutations. All in vitro functional experiments were performed on human primary T cells. (A) Schematic diagram of TCR mutations that inhibit degradation. (B) Expression of 1G4-TCR in human primary T cells after mutation. (C) Degradation of each TCR is detected after 12 hours of in vitro activated mutant F5-TCR-T cells stimulated with CD3 antibody ("R" indicates no CD3 antibody stimulation, "S" indicates CD3 antibody stimulation). (D) Degradation of each TCR is detected after 12 hours of in vitro activated mutant 1G4-TCR-T cells stimulated with CD3 antibody ("R" indicates no CD3 antibody stimulation, "S" indicates CD3 antibody stimulation). (E) Expression of each TCR is detected after in vitro activated mutant 1G4-TCR-T cells are stimulated with CD3 antibody for various times. [Diagram 3]Figure 1. Effect of mutations on in vitro function of 1G4-TCR-T cells. All in vitro function experiments were performed on human primary T cells. (A) Flow cytometry detection of CD45RO and CD27 as memory T cell differentiation indicators was performed for in vitro activated TCR-T cells. (B) Memory T cell differentiation indicator CD62L was detected for activated 1G4-TCR-T cells. (C) Memory T cell differentiation indicator TCF-1 was detected for in vitro activated 1G4-TCR-T cells. (D) Expression of each mutant TCR-T cell depletion molecule LAG-3 was detected using an in vitro depletion model. (E) Expression of TOX, a key transcription factor for TCR-T cell depletion, was detected using an in vitro depletion model. (F) Production of mutant TCR-T cytokines was detected using an in vitro depletion model. [Figure 4] A diagram comparing the mitochondrial functional status of each mutant 1G4-TCR-T cell. All in vitro experiments were performed on human primary T cells. (A) 12 days after in vitro activation of each TCR-T cell, the number of mitochondria in the cells is detected. (B) Changes in mitochondrial membrane potential in 1G4-TCR-T cells after mutation are detected. (C) The ROS production level of the intracellular mitochondria of in vitro activated 1G4-TCR-T cells is detected. (D) 12 days after in vitro activation of each TCR-T cell, after 2-NBDG treatment, glucose uptake of TCR-T cells is detected by FACS flow cytometry analysis. (E) Bodipy FL C16 uptake experiment is used to detect the utilization efficiency of extracellular fatty acids of TCR-T cells of each mutant group. [Diagram 5] The figure shows the comparison of the antitumor effect of each mutant 1G4-TCR-T cell. (A) The figure shows the statistical analysis of the tumor size at various time points for K562-NYESO-1 NSG tumor-bearing mice inoculated with 1G4-TCR-T cells. After T cells were introduced into K562-NYESO-1 NSG tumor-bearing mice, the weight of tumor tissues of each mutant group was detected on the 12th day (B), and the proportion of TCR-T cells introduced into the blood (C), spleen (D), and tumor tissue (E) of the tumor-bearing mice was also detected. [Figure 6]Figure showing the effect of mutations on the function of 1G4-TCR-T cells in mice. (A) In the spleen of NSG tumor-bearing mice, CD45RO and CD27 were used as memory T cell differentiation indicators, and flow cytometry detection was performed for each mutation group. (B) In the tumor tissue of NSG tumor-bearing mice, the expression of memory T cell differentiation indicator CD62L was detected for each mutation group. (C) In the tumor tissue of NSG tumor-bearing mice, the expression of memory T cell differentiation indicator CCR7 was detected for each mutation group. (D) A figure showing the detection and statistics of the proportion of exhausted T cells PD-1+TIM-3+ double positive cells for each mutation group in the tumor tissue of NSG tumor-bearing mice. The expression of immunosuppressive molecules PD-1 (E), TIM-3 (F), LAG-3 (G), and CD39 (H) of TCR-T cells of each mutation group was detected in the tumor tissue of NSG tumor-bearing mice. (I) The expression of the transcription factor TOX for each mutation group was detected in the tumor tissue of NSG tumor-bearing mice. (J) Detect the percentage of IFN-γ+ positive CD8+ T cells in the tumor tissue of NSG tumor-bearing mice. (K) Detect the percentage of IFN-γ+ TNF-α+ double positive TCR- T cells in the tumor tissue of NSG tumor-bearing mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] In one aspect, the present application provides an isolated TCR alpha chain or fragment thereof of a T cell receptor (TCR), said TCR alpha chain comprising a TCR alpha chain constant region, said TCR alpha chain constant region comprising, in sequence, a TCR alpha chain extracellular constant region, a TCR alpha chain transmembrane region, and a TCR alpha chain intracellular constant region. Optionally, said TCR alpha chain further comprises a TCR alpha chain variable region.

[0031] In some embodiments, the TCR alpha chain constant region and its corresponding TCR alpha chain extracellular constant region, TCR alpha chain transmembrane region, and TCR alpha chain intracellular constant region are each derived from the constant region of a wild-type T cell receptor (TCR) of a human, mouse, or other mammalian species.

[0032] In some embodiments, the TCR alpha chain intracellular constant region is a wild-type TCR alpha chain intracellular constant region derived from a human, mouse or other mammalian species, e.g., a wild-type TCR alpha chain intracellular constant region that comprises or consists of the amino acid sequence shown in SEQ ID NO:3.

[0033] In some embodiments, the TCR alpha chain intracellular constant region is a mutated TCR alpha chain intracellular constant region in which at least one serine in a wild-type TCR alpha chain intracellular constant region has been mutated to alanine, for example derived from a mutated TCR alpha chain intracellular constant region in which at least one serine in a wild-type TCR alpha chain intracellular constant region of a human, mouse or other mammalian species has been mutated to alanine.

[0034] In some embodiments, the mutant TCR α chain intracellular constant region has at least one serine mutated to alanine in the wild-type TCR α chain intracellular constant region, and the wild-type TCR α chain intracellular constant region comprises or consists of the amino acid sequence shown in SEQ ID NO:3, for example a mutant TCR α chain intracellular constant region in which one or two (all) serines in the amino acid sequence shown in SEQ ID NO:3 have been mutated to alanine.

[0035] In some embodiments, the mutated TCR α chain intracellular constant region comprises or consists of the amino acid sequence shown in SEQ ID NO:4.

[0036] In some embodiments, the TCR alpha chain comprises a TCR alpha chain variable region that is capable of binding to and recognizing one or more antigens, including, but not limited to, polypeptide-based antigens (e.g., NYESO-1, AFP, and MART-1), lipid-based antigens (e.g., β-GlcCer, eLPA, and LPE), and polysaccharide-based antigens (e.g., CA199, CA72-4, and CA125).

[0037] In some embodiments, the antigen is a tumor antigen, a microbial antigen, or an autoantigen, such as BCMA, CA9, CTAG, CCL-1, CSPG4, EGFR, EPG-2, EPG-40, FCRL5, FBP, OGD2, GPC3, GPRC5D, HER3, HER4, HLA-A1, HLA-A2, LRRC8A, CMV, MUC1, MUC16, MART-1, NCAM, PRAME, PSCA, PSMA, ROR1, TPBG, TAG72, TRP1, TRP2, VEGFR, VEGFR2, WT-1, MAGE-A1 / A3 / A4 / A6 / A10 / C2, gp100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1 / 2, BRLF-1, BMLF-1, HPV-16E6 / E7, KRAS The antigens are one or more of the following: KRAS G12D, KRAS G12V, TP53 R175H, β-GlcCer, eLPA, LPE, CA199, CA72-4, or CA125.

[0038] In some embodiments, the antigen is a tumor antigen, such as one or more of MAGE-A1 / A3 / A4 / A6 / A10 / C2, gp100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1 / 2, BRLF-1, BMLF-1, HPV-16E6 / E7, KRAS G12D, KRAS G12V, TP53 R175H, β-GlcCer, eLPA, LPE, CA199, CA72-4, or CA125. The antigen may be a cell surface antigen or an intracellular antigen.

[0039] In some embodiments, the TCR alpha chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:13 and recognizes the tumor antigen MART-1, or comprises or consists of the amino acid sequence shown in SEQ ID NO:21 and recognizes the tumor antigen NYESO-1.

[0040] In some embodiments, the TCR alpha chain variable region comprises complementarity determining regions (CDRs), such as CDR1, CDR2, and CDR3, which are comprised in the amino acid sequence shown in SEQ ID NO:13, or the amino acid sequence shown in SEQ ID NO:21.

[0041] In some embodiments, the TCR alpha chain variable region comprises a CDR1 having an amino acid sequence represented by SEQ ID NO:14, a CDR2 having an amino acid sequence represented by SEQ ID NO:15, and a CDR3 having an amino acid sequence represented by SEQ ID NO:16, and recognizes the tumor antigen MART-1, or comprises a CDR1 having an amino acid sequence represented by SEQ ID NO:22, a CDR2 having an amino acid sequence represented by SEQ ID NO:23, and a CDR3 having an amino acid sequence represented by SEQ ID NO:24, and recognizes the tumor antigen NYESO-1.

[0042] In some embodiments, the TCR α chain extracellular constant region is a TCR α chain extracellular constant region derived from a human, mouse, or other mammalian species.

[0043] In some embodiments, the TCR alpha chain extracellular constant region comprises or consists of the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations compared to the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO:1.

[0044] In some embodiments, the TCR α chain transmembrane region is a TCR α chain transmembrane region derived from a human, mouse or other mammalian species.

[0045] In some embodiments, the TCR alpha chain transmembrane region comprises or consists of the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations compared to the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO:2.

[0046] In some embodiments, the TCR alpha chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6.

[0047] In one aspect, the present application provides an isolated TCR β chain or fragment thereof of a T cell receptor (TCR), said TCR β chain comprising a TCR β chain constant region, said TCR β chain constant region comprising, in sequence, a TCR β chain extracellular constant region, a TCR β chain transmembrane region, and a TCR β chain intracellular constant region. Optionally, said TCR β chain further comprises a TCR β chain variable region.

[0048] In some embodiments, the TCR β chain constant region and its corresponding TCR β chain extracellular constant region, TCR β chain transmembrane region, and TCR β chain intracellular constant region are each derived from a wild-type T cell receptor (TCR) constant region of a human, mouse, or other mammalian species.

[0049] In some embodiments, the TCR β chain intracellular constant region is a wild-type TCR β chain intracellular constant region derived from a human, mouse or other mammalian species, for example a wild-type TCR β chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:9, for example a wild-type TCR β chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:47, for example a wild-type TCR β chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:48.

[0050] In some embodiments, the TCR β chain intracellular constant region is a mutated TCR β chain intracellular constant region, the mutated TCR β chain intracellular constant region being a wild-type TCR β chain intracellular constant region in which at least one lysine has been mutated to arginine or alanine, for example a mutant TCR β chain intracellular constant region in which at least one lysine has been mutated to arginine or alanine in a wild-type TCR β chain intracellular constant region derived from a human, mouse or other mammalian species.

[0051] In some embodiments, the mutant TCR β chain intracellular constant region has at least one lysine of a wild-type TCR β chain intracellular constant region mutated to arginine or alanine, and the wild-type TCR β chain intracellular constant region comprises or consists of the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:47 or SEQ ID NO:48, for example a mutant TCR β chain intracellular constant region in which one, two or three (all) lysines of the amino acid sequence shown in SEQ ID NO:9 have been mutated to arginine or alanine, for example a mutant TCR β chain intracellular constant region in which one or two (all) lysines of SEQ ID NO:47 or SEQ ID NO:48 have been mutated to arginine or alanine.

[0052] In some embodiments, the mutated TCR β chain intracellular constant region comprises or consists of the amino acid sequence shown in SEQ ID NO:10.

[0053] In some embodiments, the TCR β chain comprises a TCR β chain variable region that is capable of binding to and recognizing one or more antigens, including, but not limited to, polypeptide-based antigens (e.g., NYESO-1, AFP, and MART-1), lipid-based antigens (e.g., β-GlcCer, eLPA, and LPE), and polysaccharide-based antigens (e.g., CA199, CA72-4, and CA125).

[0054] In some embodiments, the antigen is a tumor antigen, a microbial antigen, or an autoantigen, such as BCMA, CA9, CTAG, CCL-1, CSPG4, EGFR, EPG-2, EPG-40, FCRL5, FBP, OGD2, GPC3, GPRC5D, HER3, HER4, HLA-A1, HLA-A2, LRRC8A, CMV, MUC1, MUC16, MART-1, NCAM, PRAME, PSCA, PSMA, ROR1, TPBG, TAG72, TRP1, TRP2, VEGFR, VEGFR2, WT-1, MAGE-A1 / A3 / A4 / A6 / A10 / C2, gp100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1 / 2, BRLF-1, BMLF-1, HPV-16E6 / E7, KRAS The antigens are one or more of the following: KRAS G12D, KRAS G12V, TP53 R175H, β-GlcCer, eLPA, LPE, CA199, CA72-4, or CA125.

[0055] In some embodiments, the antigen is a tumor antigen, such as one or more of MAGE-A1 / A3 / A4 / A6 / A10 / C2, gp100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1 / 2, BRLF-1, BMLF-1, HPV-16E6 / E7, KRAS G12D, KRAS G12V, TP53 R175H, β-GlcCer, eLPA, LPE, CA199, CA72-4, or CA125. The antigen may be a cell surface antigen or an intracellular antigen.

[0056] In some embodiments, the TCR β chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:17 and recognizes the tumor antigen MART-1, or comprises or consists of the amino acid sequence shown in SEQ ID NO:25 and recognizes the tumor antigen NYESO-1.

[0057] In some embodiments, the TCR β chain variable region comprises a complementarity determining region (CDR), such as CDR1, CDR2, CDR3, which is comprised in the amino acid sequence set forth in SEQ ID NO: 17, or the amino acid sequence set forth in SEQ ID NO: 25.

[0058] In some embodiments, the TCR β chain variable region comprises a CDR1 having an amino acid sequence represented by SEQ ID NO:18, a CDR2 having an amino acid sequence represented by SEQ ID NO:19, and a CDR3 having an amino acid sequence represented by SEQ ID NO:20, and recognizes the tumor antigen MART-1, or comprises a CDR1 having an amino acid sequence represented by SEQ ID NO:26, a CDR2 having an amino acid sequence represented by SEQ ID NO:27, and a CDR3 having an amino acid sequence represented by SEQ ID NO:28, and recognizes the tumor antigen NYESO-1.

[0059] In some embodiments, the TCR β chain extracellular constant region is a TCR β chain extracellular constant region derived from a human, mouse, or other mammalian species.

[0060] In some embodiments, the TCR β chain extracellular constant region comprises or consists of the amino acid sequence of SEQ ID NO:7, an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations compared to the amino acid sequence of SEQ ID NO:7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO:7.

[0061] In some embodiments, the TCR β chain transmembrane region is a TCR β chain transmembrane region derived from a human, mouse, or other mammalian species.

[0062] In some embodiments, the TCR β chain transmembrane region comprises or consists of the amino acid sequence of SEQ ID NO:8, an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations compared to the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO:8.

[0063] In some embodiments, the TCR β chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:11 or SEQ ID NO:12.

[0064] In another aspect, the present application provides an isolated T cell receptor or fragment thereof, wherein the T cell receptor comprises any of the TCR α chain and / or TCR β chain described above.

[0065] In some embodiments, the T cell receptor comprises any of the aforementioned TCR alpha chains, and the TCR alpha chain intracellular constant region comprises: (1) a wild-type TCR α chain intracellular constant region derived from a human, mouse or other mammalian species; (2) a wild-type TCR α chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:3; (3) A mutant TCR α chain intracellular constant region in which at least one serine in the wild-type TCR α chain intracellular constant region according to (1) or (2) is mutated to alanine; or (4) A mutant TCR α chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:4.

[0066] In some embodiments, the T cell receptor comprises any of the aforementioned TCR β chains, and the TCR β chain intracellular constant region is (1) a wild-type TCR β chain intracellular constant region derived from a human, mouse or other mammalian species; (2) a wild-type TCR β chain intracellular constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO:9, SEQ ID NO:47, or SEQ ID NO:48; (3) A mutant TCR β chain intracellular constant region in which at least one lysine in the wild-type TCR β chain intracellular constant region according to (1) or (2) is mutated to arginine or alanine; or (4) A mutant TCR β chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:10.

[0067] In some embodiments, the T cell receptor comprises any of the above TCR α chains and TCR β chains, and the TCR α chain intracellular constant region and the TCR β chain intracellular constant region cannot be wild-type at the same time. For example, when the TCR α chain intracellular constant region is any of the above wild-type TCR α chain intracellular constant regions, the TCR β chain intracellular constant region is any of the above mutant TCR β chain intracellular constant regions, and when the TCR α chain intracellular constant region is any of the above mutant TCR α chain intracellular constant regions, the TCR β chain intracellular constant region is any of the above wild-type or mutant TCR β chain intracellular constant regions.

[0068] In some embodiments, the present application provides an isolated T cell receptor or fragment thereof, the T cell receptor comprising a TCR α chain and a TCR β chain, the TCR β chain comprising a TCR β chain variable region and a TCR β chain constant region, the TCR β chain constant region comprising, in sequence, a TCR β chain extracellular constant region, a TCR β chain transmembrane region, and a TCR β chain intracellular constant region, the TCR β chain intracellular constant region comprising: (1) a mutant TCR β chain intracellular constant region in which at least one lysine in the wild-type TCR β chain intracellular constant region is mutated to arginine or alanine; or (2) A mutant TCR β chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:10.

[0069] Furthermore, the wild-type TCR β chain intracellular constant region is any of the wild-type TCR β chain intracellular constant regions described above in the present application, for example, (1) a wild-type TCR β chain intracellular constant region derived from a human, mouse or other mammalian species; (2) It is selected from wild-type TCR β chain intracellular constant regions that contain or consist of the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:47, or SEQ ID NO:48.

[0070] Further, the TCR α chain comprises a TCR α chain constant region, the TCR α chain constant region comprising a TCR α chain extracellular constant region, a TCR α chain transmembrane domain, and a TCR α chain intracellular constant region, which are linked in order, and the TCR α chain intracellular constant region is any of the wild-type TCR α chain intracellular constant regions or mutant TCR α chain intracellular constant regions described above in the present application, for example: (1) a wild-type TCR α chain intracellular constant region derived from a human, mouse or other mammalian species; (2) a wild-type TCR α chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:3; (3) A mutant TCR α chain intracellular constant region in which at least one serine in the wild-type TCR α chain intracellular constant region according to (1) or (2) is mutated to alanine. (4) A mutant TCR α chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:4; Additionally, the TCR α chain comprises a TCR α chain variable region.

[0071] In some embodiments, the present application provides an isolated T cell receptor or fragment thereof comprising a TCR α chain and a TCR β chain, wherein the TCR α chain comprises a TCR α chain variable region and a TCR α chain constant region, the TCR α chain constant region comprising, in sequence, a TCR α chain extracellular constant region, a TCR α chain transmembrane region, and a TCR α chain intracellular constant region, the TCR α chain intracellular constant region comprising: (1) a mutant TCR α chain intracellular constant region in which at least one serine in the wild-type TCR α chain intracellular constant region is mutated to alanine; (2) The present invention provides an isolated T cell receptor or a fragment thereof, characterized in that it is selected from a mutant TCR α chain intracellular constant region comprising the amino acid sequence shown in SEQ ID NO:4 or consisting of said amino acid sequence.

[0072] Furthermore, the wild-type TCR α chain intracellular constant region may be any of the wild-type TCR α chain intracellular constant regions described above in the present application, for example: (1) a wild-type TCR α chain intracellular constant region derived from a human, mouse or other mammalian species; (2) It is selected from wild-type TCR α chain intracellular constant regions that contain or consist of the amino acid sequence shown in SEQ ID NO:3.

[0073] Further, the TCR β chain comprises a TCR β chain constant region, the TCR β chain constant region comprising a TCR β chain extracellular constant region, a TCR β chain transmembrane region, and a TCR β chain intracellular constant region, which are linked in order, and the TCR β chain intracellular constant region is any of the wild-type TCR β chain intracellular constant regions or mutant TCR β chain intracellular constant regions described above in the present application, for example, (1) a wild-type TCR β chain intracellular constant region derived from a human, mouse or other mammalian species; (2) a wild-type TCR β chain intracellular constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO:9, SEQ ID NO:47, or SEQ ID NO:48; (3) A mutant TCR β chain intracellular constant region in which at least one lysine in the wild-type TCR β chain intracellular constant region according to (1) or (2) is mutated to arginine or alanine. (4) A mutant TCR β chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:10.

[0074] Furthermore, the TCR β chain comprises a TCR β chain variable region.

[0075] In some embodiments, the T cell receptor comprises a wild-type TCR α chain intracellular constant region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3, and / or a mutated TCR β chain intracellular constant region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:10.

[0076] In some embodiments, the T cell receptor comprises a mutant TCR α chain intracellular constant region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:4, and / or a wild-type TCR β chain intracellular constant region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:9, SEQ ID NO:47 or SEQ ID NO:48.

[0077] In some embodiments, the T cell receptor comprises a mutant TCR α chain intracellular constant region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:4, and / or a mutant TCR β chain intracellular constant region comprising or consisting of the amino acid sequence set forth in SEQ ID NO:10.

[0078] In some embodiments, any of the aforementioned T cell receptors comprises any of the aforementioned TCR alpha chain variable regions and / or TCR beta chain variable regions that are capable of binding and recognizing one or more antigens, including, but not limited to, polypeptide-based antigens (e.g., NYESO-1, AFP, and MART-1), lipid-based antigens (e.g., β-GlcCer, eLPA, and LPE), and polysaccharide-based antigens (e.g., CA199, CA72-4, and CA125). The antigen may be a tumor antigen, a microbial antigen, or a self-antigen, such as BCMA, CA9, CTAG, CCL-1, CSPG4, EGFR, EPG-2, EPG-40, FCRL5, FBP, OGD2, GPC3, GPRC5D, HER3, HER4, HLA-A1, HLA-A2, LRRC8A, CMV, MUC1, MUC16, MART-1, NCAM, PRA. ME, PSCA, PSMA, ROR1, TPBG, TAG72, TRP1, TRP2, VEGFR, VEGFR2, WT-1, MAGE-A1 / A3 / A4 / A6 / A10 / C 2, gp100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1 / 2, BRLF-1, BMLF-1, HPV-16E6 / E7, KRAS G12D, KRAS The antigen may be one or more of the following antigens: MAGE-A1 / A3 / A4 / A6 / A10 / C2, gp100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1 / 2, BRLF-1, BMLF-1, HPV-16E6 / E7, KRAS G12D, KRAS G12V, TP53 R175H, β-GlcCer, eLPA, LPE, CA199, CA72-4, or CA125. The antigen may be a cell surface antigen or an intracellular antigen.

[0079] In some embodiments, any of the aforementioned T cell receptors comprises any of the aforementioned TCR β chain variable regions, e.g., (1) a TCR β chain variable region comprising three complementarity determining regions (CDRs) contained in the amino acid sequence represented by SEQ ID NO:17; (2) a TCR β chain variable region comprising three complementarity determining regions CDRs contained in the amino acid sequence represented by SEQ ID NO: 25; (3) a TCR β chain variable region comprising CDR1 having an amino acid sequence represented by SEQ ID NO: 18, CDR2 having an amino acid sequence represented by SEQ ID NO: 19, and / or CDR3 having an amino acid sequence represented by SEQ ID NO: 20; (4) a TCR β chain variable region comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 26, CDR2 having the amino acid sequence shown in SEQ ID NO: 27, and / or CDR3 having the amino acid sequence shown in SEQ ID NO: 28; (5) A TCR β chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 17; or (6) It comprises an amino acid sequence shown in SEQ ID NO:25 or comprises a TCR β chain variable region consisting of said amino acid sequence.

[0080] In some embodiments, any of the aforementioned T cell receptors comprises any of the aforementioned TCR α chain variable regions, e.g., (1) a TCR α chain variable region comprising three complementarity determining regions (CDRs) contained in the amino acid sequence shown in SEQ ID NO:13; (2) a TCR α chain variable region comprising three complementarity determining regions CDRs contained in the amino acid sequence shown in SEQ ID NO:21; (3) a TCR α chain variable region comprising CDR1 having the amino acid sequence shown in SEQ ID NO: 14, CDR2 having the amino acid sequence shown in SEQ ID NO: 15, and / or CDR3 having the amino acid sequence shown in SEQ ID NO: 16; (4) a TCR α chain variable region comprising CDR1 of the amino acid sequence shown in SEQ ID NO: 22, CDR2 of the amino acid sequence shown in SEQ ID NO: 23, and / or CDR3 of the amino acid sequence shown in SEQ ID NO: 24; (5) a TCR α chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 13; or (6) It comprises an amino acid sequence shown in SEQ ID NO:21 or a TCR α chain variable region consisting of said amino acid sequence.

[0081] In some embodiments, the TCR alpha chain variable region and the TCR beta chain variable region of any of the aforementioned T cell receptors are (1) The TCR α chain variable region comprises three complementarity determining region CDRs contained in the amino acid sequence shown in SEQ ID NO: 13, and the TCR β chain variable region comprises three complementarity determining region CDRs contained in the amino acid sequence shown in SEQ ID NO: 17; (2) The TCR α chain variable region comprises three complementarity determining region CDRs contained in the amino acid sequence shown in SEQ ID NO:21, and the TCR β chain variable region comprises three complementarity determining region CDRs contained in the amino acid sequence shown in SEQ ID NO:25; (3) The TCR α chain variable region comprises a CDR1 having an amino acid sequence shown in SEQ ID NO:14, a CDR2 having an amino acid sequence shown in SEQ ID NO:15, and a CDR3 having an amino acid sequence shown in SEQ ID NO:16, and the TCR β chain variable region comprises a CDR1 having an amino acid sequence shown in SEQ ID NO:18, a CDR2 having an amino acid sequence shown in SEQ ID NO:19, and a CDR3 having an amino acid sequence shown in SEQ ID NO:20; (4) The TCR α chain variable region comprises a CDR1 having an amino acid sequence shown in SEQ ID NO:22, a CDR2 having an amino acid sequence shown in SEQ ID NO:23, and a CDR3 having an amino acid sequence shown in SEQ ID NO:24, and the TCR β chain variable region comprises a CDR1 having an amino acid sequence shown in SEQ ID NO:26, a CDR2 having an amino acid sequence shown in SEQ ID NO:27, and a CDR3 having an amino acid sequence shown in SEQ ID NO:28; (5) The TCR α chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the TCR β chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17; or (6) The TCR α chain variable region is selected from those which include or consist of the amino acid sequence shown in SEQ ID NO:21, and the TCR β chain variable region is selected from those which include or consist of the amino acid sequence shown in SEQ ID NO:25.

[0082] In some embodiments, the T cell receptor comprises any of the above TCR α chain extracellular constant regions and / or TCR β chain extracellular constant regions.

[0083] In some embodiments, the T cell receptor comprises any of the TCR α chain transmembrane regions and / or TCR β chain transmembrane regions described above.

[0084] In some embodiments, the T cell receptor comprises any of the above TCR alpha chain constant regions and / or TCR beta chain constant regions. For example, the TCR alpha chain constant region and the TCR beta chain constant region are (1) the TCR α chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:5, and the TCR β chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:12; (2) the TCR α chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:6, and the TCR β chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:12; or (3) The TCR α chain constant region is selected from those comprising or consisting of the amino acid sequence of SEQ ID NO:6, and the TCR β chain constant region is selected from those comprising or consisting of the amino acid sequence of SEQ ID NO:11.

[0085] In another aspect, the present application provides one or more T cell receptors F5-TCR, including F5-WT TCR, F5-SA TCR, F5-KR TCR, or F5-dMUT TCR, wherein the F5-TCR variable region recognizes the tumor antigen MART-1, the F5-TCR alpha chain variable region has the amino acid sequence of SEQ ID NO:13, the F5-TCR beta chain variable region has the amino acid sequence of SEQ ID NO:17, Furthermore, the F5-WT TCR alpha chain constant region is the amino acid sequence of SEQ ID NO:5, and the F5-WT TCR beta chain constant region is the amino acid sequence of SEQ ID NO:11; Furthermore, the F5-SA TCR alpha chain constant region is the amino acid sequence of SEQ ID NO:6, and the F5-SA TCR beta chain constant region is the amino acid sequence of SEQ ID NO:11; Furthermore, the F5-KR TCR alpha chain constant region is the amino acid sequence of SEQ ID NO:5, and the F5-KR TCR beta chain constant region is the amino acid sequence of SEQ ID NO:12; Furthermore, the F5-dMUT TCR α chain constant region is the amino acid sequence of SEQ ID NO:6, and the F5-dMUT TCR β chain constant region is the amino acid sequence of SEQ ID NO:12.

[0086] In another aspect, the present application provides one or more T cell receptors 1G4-TCR, including 1G4-WT TCR, 1G4-SA TCR, 1G4-KR TCR or 1G4-dMUT TCR, wherein the 1G4-TCR variable region recognizes the tumor antigen NYESO-1, the 1G4-TCR alpha chain variable region is the amino acid sequence of SEQ ID NO:21, or the 1G4-TCR beta chain variable region is the amino acid sequence of SEQ ID NO:25, Furthermore, the 1G4-WT TCR alpha chain constant region is the amino acid sequence of SEQ ID NO:5, and the 1G4-WT TCR beta chain constant region is the amino acid sequence of SEQ ID NO:11; Furthermore, the 1G4-SA TCR alpha chain constant region is the amino acid sequence of SEQ ID NO:6, and the 1G4-SA TCR beta chain constant region is the amino acid sequence of SEQ ID NO:11; Furthermore, the 1G4-KR TCR α chain constant region is the amino acid sequence of SEQ ID NO:5, and the 1G4-KR TCR β chain constant region is the amino acid sequence of SEQ ID NO:12.

[0087] Furthermore, the 1G4-dMUT TCR α chain constant region is the amino acid sequence of SEQ ID NO:6, and the 1G4-dMUT TCR β chain constant region is the amino acid sequence of SEQ ID NO:12.

[0088] In another aspect, any of the above TCR α chain, TCR β chain or T cell receptor is further linked to a signal peptide, and the signal peptide forms a signal peptide-variable region structure with the TCR α chain variable region and / or the TCR β chain variable region.

[0089] In some embodiments, the signal peptide is selected from human growth hormone signal peptide, CD8α signal peptide, and immunoglobulin signal peptide.

[0090] In some embodiments, the signal peptide comprises or consists of the amino acid sequence of SEQ ID NO:29 and / or SEQ ID NO:30.

[0091] In some embodiments, the TCR alpha chain signal peptide amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO:29.

[0092] In some embodiments, the TCR β chain signal peptide amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO:30.

[0093] The above-mentioned portions forming any of the TCR α chain, TCR β chain or T cell receptor of the present application may be linked to each other directly or via a linker sequence, which is a sequence in which 1 to 5 repeats of motifs such as GGGS, GGGGS, GSGSA and GGSGG are adjacently linked, and has a length of 3 to 25 amino acid residues.

[0094] In another aspect, the present application provides an isolated nucleic acid or fragment thereof encoding any of the aforementioned isolated TCR alpha chain or fragment thereof, TCR beta chain or fragment thereof, or T cell receptor or fragment thereof.

[0095] In some embodiments, the nucleic acid sequence encoding the wild-type TCR alpha chain constant region comprises or consists of the nucleic acid sequence of SEQ ID NO:31.

[0096] In some embodiments, the nucleic acid sequence encoding the wild-type TCR β chain constant region comprises or consists of the nucleic acid sequence of SEQ ID NO:32.

[0097] In some embodiments, the nucleic acid sequence encoding the SA TCR alpha chain constant region comprises or consists of the nucleic acid sequence of SEQ ID NO:33.

[0098] In some embodiments, the nucleic acid sequence encoding the KR TCR β chain constant region comprises or consists of the nucleic acid sequence of SEQ ID NO:34.

[0099] In some embodiments, the nucleic acid sequence encoding the F5-TCR alpha chain variable region comprises or consists of the nucleic acid sequence of SEQ ID NO:35.

[0100] In some embodiments, the nucleic acid sequence encoding the F5-TCR β chain variable region comprises or consists of the nucleic acid sequence of SEQ ID NO:36.

[0101] In some embodiments, the nucleic acid sequence encoding the 1G4-TCR alpha chain variable region comprises or consists of the nucleic acid sequence of SEQ ID NO:37.

[0102] In some embodiments, the nucleic acid sequence encoding the 1G4-TCR β chain variable region comprises or consists of the nucleic acid sequence of SEQ ID NO:38.

[0103] In some embodiments, the nucleic acid sequence encoding F5-WT-TCR comprises or consists of the nucleic acid sequence of SEQ ID NO:39.

[0104] In some embodiments, the nucleic acid sequence encoding the F5-SA-TCR comprises or consists of the nucleic acid sequence of SEQ ID NO:40.

[0105] In some embodiments, the nucleic acid sequence encoding the F5-KR-TCR comprises or consists of the nucleic acid sequence of SEQ ID NO:41.

[0106] In some embodiments, the nucleic acid sequence encoding the F5-dMUT-TCR comprises or consists of the nucleic acid sequence of SEQ ID NO:42.

[0107] In some embodiments, the nucleic acid sequence encoding 1G4-WT-TCR comprises or consists of the nucleic acid sequence of SEQ ID NO:43.

[0108] In some embodiments, the nucleic acid sequence encoding 1G4-SA-TCR comprises or consists of the nucleic acid sequence of SEQ ID NO:44.

[0109] In some embodiments, the nucleic acid sequence encoding 1G4-KR-TCR comprises or consists of the nucleic acid sequence of SEQ ID NO:45.

[0110] In some embodiments, the nucleic acid sequence encoding 1G4-dMUT-TCR comprises or consists of the nucleic acid sequence of SEQ ID NO: 46. In another aspect, the present application provides a nucleic acid construct comprising any of the foregoing isolated nucleic acids or fragments thereof.

[0111] In some embodiments, the nucleic acid construct further comprises one or more regulatory sequences operably linked to the nucleic acid, including, but not limited to, a promoter sequence that exhibits transcriptional activity in a host cell, a transcription terminator sequence that is recognized in a host cell and terminates transcription and is operably linked to the 3' end of the coding sequence, a preamble sequence operably linked to the 5' end of the coding sequence that is important for host cell translation, and the like.

[0112] In another aspect, the present application provides a vector comprising any of the aforementioned nucleic acid constructs.

[0113] In some embodiments, the vector is an expression vector or a crisper gene editing vector, such as a retroviral vector, a lentiviral vector, a baculoviral vector, a herpes virus vector, an adenoviral vector, or an adeno-associated virus (AAV) vector.

[0114] In some embodiments, the retroviral vector construct essentially comprises an origin of replication, a 5'-LTR, a 3'-LTR, and any of the aforementioned nucleic acid sequences or nucleic acid constructs.

[0115] Typically, expression of the polynucleotide sequence encoding the T cell receptor is achieved by operably linking a promoter to the nucleic acid sequence encoding the T cell receptor and incorporating the nucleic acid construct into an expression vector. The nucleic acid sequence encoding the T cell receptor of the present application can be cloned into many types of vectors, including, but not limited to, plasmids, phages, animal viruses, and the like.

[0116] Suitable expression vectors contain one or more promoter sequences functional in vivo, an origin of replication, suitable enzyme cleavage sites, and a selectable marker.

[0117] Suitable promoters include, but are not limited to, constitutive promoter sequences, such as the immediate early cytomegalovirus (CMV) promoter, elongation factor-1a (EF-1a), simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV), long terminal repeat (LTR) promoters, which are capable of driving high level expression of any polynucleotide sequence linked thereto, and also include, but are not limited to, inducible promoters, such as the metallothionein promoter and tetracycline promoter, which open when expression of the polynucleotide sequence is expected and close when expression is not expected.

[0118] Selectable markers include marker genes and reporter genes to facilitate the identification of selectively expressing cells in a cell population infected via a viral vector. Suitable marker genes include, but are not limited to, antibiotic resistance genes, neo genes, etc. Suitable reporter genes include, but are not limited to, β-galactosidase, green fluorescent protein gene, luciferase, chloramphenicol acetyltransferase, etc.

[0119] In another aspect, the present application provides an engineered cell comprising any of the aforementioned nucleic acid constructs or vectors.

[0120] In some embodiments, the engineered cells are primary cells obtained from a subject, hi some embodiments, the subject is a mammalian subject, such as a human.

[0121] In some embodiments, the engineered cells are T cells, preferably human T cells.

[0122] In some embodiments, the engineered cells are NK cells, NKT cells, macrophages, γδT cells, human CD4 + T cells, CD8 + T cells, or CD4 + T / CD8 + It is a mixed population of T cells.

[0123] The TCR nucleic acid constructs of the present application, or vectors comprising said nucleic acid constructs, can be used by conventional recombinant DNA techniques to express or produce a TCR or an engineered cell comprising said TCR. Typically, the method comprises: (1) transforming or transducing a suitable host cell with the TCR nucleic acid construct of the present application or a vector comprising said nucleic acid construct; (2) culturing a host cell in a suitable medium; and (3) isolating and purifying the engineered cells or TCRs of the present application from the medium or cells.

[0124] Methods for introducing various nucleic acid sequences or vectors into engineered cells are now well known in the art, whereby a gene of interest can be introduced into a host cell by physical, chemical, and biological methods.

[0125] In some embodiments, the present application uses retroviruses to introduce a sequence of interest into a host cell. Retroviral infection is currently a very widely used method for infecting primary cells of human or mouse origin. In some embodiments, the present application constructs various mutant TCR nucleic acid sequences into retroviral vectors, which are then packaged with a preferred viral envelope or capsid protein to generate viral particles. The recombinant viral particles are then delivered to a host or in vitro cultured cells to achieve expression of the gene of interest in the host cell.

[0126] The viral particle comprises a TCR nucleic acid sequence, a retroviral vector, and a nucleic acid sequence for a packaging protein.

[0127] In another aspect, the present application also provides a TCR complex produced in a T cell by any of the aforementioned TCR nucleic acid constructs of the present application.

[0128] In another aspect, the present application provides functional detection of TCR-T cells in in vitro culture. Specifically, it includes detection of memory phenotype of TCR-T cells, detection of exhaustion phenotype of TCR-T cells in in vitro exhaustion model, and detection of mitochondrial phenotype of TCR-T cells. The above detection of TCR-T cell memory phenotype and mitochondrial phenotype are detected using cell flow cytometry after activation of human primary T cells. The above mutant TCR-T cell exhaustion detection uses an exhaustion model constructed in vitro. Specifically, CD3 antibody is used to stimulate TCR-T cells with various mutations multiple times in vitro, and then the expression of exhaustion-related molecules is detected by flow cytometry.

[0129] In another aspect, the present application provides a method for producing a method for treating a cancer cell comprising: (1) Inhibition of degradation of T cell receptors during antigen stimulation (2) Providing a tumor-killing effect or inhibiting tumor growth; (3) Maintenance of T cell proliferation; and (4) Use of the isolated TCR α chain or fragment thereof, TCR β chain or fragment thereof, T cell receptor or fragment thereof, nucleic acid or fragment thereof, nucleic acid construct, vector, or engineered cell described in any of the above in one or more applications of preparing anti-tumor immune drugs and cells.

[0130] Meanwhile, the mutant TCR-T cell therapy according to the present application can be used alone, in combination with other therapies, or in combination with PD-1 / PD-L1 antibodies, cytokine therapy, or radiation therapy and chemotherapy. When the above therapies are used alone or in combination, the dosage and frequency are determined by various factors such as the characteristics of the disease and the severity of the disease. In theory, the TCR-T cell therapy according to the present application can be used multiple times at a low dose, and the above dose range is 10 4 ~10 9The method for adoptive therapy of TCR-T cells according to the present application is carried out according to internationally recognized adoptive techniques, and in one embodiment of the present application, the cells are 5×10 6 This is accomplished by injecting 10 TCR-T cells into the tail vein. In principle, those used in conjunction with T cell therapy can be injected directly into the tumor tissue or the site of infection.

[0131] In another aspect, the present application provides a method for producing a method for treating a cancer cell comprising: (1) mutating at least one lysine in any of the wild-type TCR β chain intracellular constant regions to arginine or alanine; and / or and (2) mutating at least one serine in any of the wild-type TCR α chain intracellular constant regions to alanine.

[0132] In order to more readily understand this disclosure, a number of terms are defined below.

[0133] A T cell receptor (TCR) is a molecule present on the surface of a T cell that recognizes a peptide-MHC complex. In some embodiments, the TCR is a complete or full-length TCR. In some embodiments, the present application provides TCR fragments that are smaller than a full-length TCR but retain binding to a specific antigenic peptide of an MHC molecule, i.e., an MHC-peptide complex. In some embodiments, the TCR is a heterodimer consisting of an α and β chain. In some embodiments, the TCR may be a single chain TCR (scTCR).

[0134] The term "variable region" or "variable domain" refers to the domain of the TCR α or β chain that is involved in the binding of the TCR to the antigen-MHC complex. The variable regions of the α and β chains of native TCRs generally have a similar structure, with each structural region containing four conserved framework regions (FR) and three hypervariable or complementarity determining regions (CDRs). Of these, CDR3 of each variable region is the primary CDR responsible for recognition of engineered antigens. A single TCR α or β chain variable region may be sufficient to confer binding to a peptide-MHC complex.

[0135] It should be understood that in some embodiments, the TCR alpha chain of the present application can be a human TCR alpha chain, a humanized TCR alpha chain, a chimeric TCR alpha chain, or a mouse-derived TCR alpha chain. In some embodiments of the present application, the TCR alpha chain is a chimeric TCR alpha chain, which includes sequences from multiple species, such as sequences from human and mouse. For example, human TCR constant regions can be replaced with mouse counterparts to improve the function and expression levels of human T cells (see, e.g., Daniel Sommermeyer et al., J Immunol. 2010 Jun 1;184(11):6223-31., which is incorporated herein by reference). Thus, the TCR can include a variable region of human origin and a constant region of mouse origin.

[0136] Similarly, in some embodiments, the TCR β chain of the present application may be a human TCR β chain, a humanized TCR β chain, a chimeric TCR β chain, or a murine TCR β chain. A chimeric TCR β chain includes sequences from multiple species, such as sequences from human and mouse.

[0137] The TCR α chain constant region and / or TCR β chain constant region described in the present application comprises an extracellular constant region, a transmembrane region, and an intracellular constant region, which are linked in order, and the extracellular constant region may comprise the TCR α chain and the TCR β chain hinge region, and is involved in the formation of the TCR α chain-TCR β chain disulfide bond. The transmembrane region also belongs to the constant region, and its function includes interacting with the cell membrane anchor of the TCR α chain and the TCR β chain and the CD3 subunit to form the TCR-CD3 complex. The possible function of the intracellular constant region includes participating in the conformational change of the TCR-CD3 complex and signal transduction after TCR signal transduction.

[0138] The term "antigen" refers to a cell surface molecule or a molecule that can be presented intracellularly by MHC or MHC-like molecules and bound by an antibody or T cell receptor (TCR), including, but not limited to, polypeptide-based antigens (e.g., NYESO-1, AFP, and MART-1), lipid-based 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 tumor-specific antigen (TSA).

[0139] The term "isolated" refers to material that has been removed from its natural state or otherwise manipulated by art, such as the alpha chain, beta chain, T cell receptor, and nucleic acids described herein. An isolated material may be largely or substantially free of components that normally accompany it in its natural state, or it may be manipulated to be in an artificial state with components that normally accompany it in its natural state. An isolated material may be in natural, chemically synthesized, or recombinant form. An isolated material may alternatively be in enriched, partially purified, or purified form.

[0140] It should be understood that "wild type" in the present invention means a naturally occurring amino acid sequence or encoding nucleic acid sequence, or having at least 90% to 100% identity compared to a naturally occurring amino acid sequence or encoding nucleic acid sequence, or having no more than 1 to 10 or 1 to 5 amino acid mutations (particularly conservative amino acid substitutions) compared to a naturally occurring amino acid sequence or encoding nucleic acid sequence, but still having the same or similar activity and / or function as that region.

[0141] "Wild type" refers to a region that is introduced as a template for the corresponding mutation or combination of mutations herein, e.g., "wild type derived from human, mouse, or other mammalian species" refers to a region that has or consists of the amino acid sequence or encoding nucleic acid sequence of the region of a naturally occurring human, mouse, rat, or other mammalian species. A region "derived from" human, mouse, or other mammalian species is substantially identical to the amino acid sequence or encoding nucleic acid sequence of the region of a naturally occurring human, mouse, rat, or other mammalian species, e.g., has at least 90% to 100% identity compared to the naturally occurring amino acid sequence or encoding nucleic acid sequence, or includes a region that has 1 to 10 or 1 to 5 amino acid mutations (particularly conservative amino acid substitutions) compared to the naturally occurring amino acid sequence or encoding nucleic acid sequence, but still has the same or similar activity and / or function as the region.

[0142] The term "mutation" may be understood to mean the substitution, deletion, or addition of one or more amino acids or nucleic acids. For example, it may be a conservative substitution of amino acids, which is known in the art and includes amino acid substitutions in which one of the amino acids with a particular physical and / or chemical property is replaced by another amino acid with the same chemical or physical property. For example, the conservative amino acid substitution may be an acidic amino acid for another acidic amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain for another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid for another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain for another amino acid with a polar side chain (Asn, Cys, Gin, Ser, Thr, Tyr, etc.), etc., and the conservative substitution may be made based on, for example, similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathicity of the residues involved.

[0143] When referring to a "mutation" of the TCR α chain intracellular constant region and / or TCR β chain intracellular constant region of the present application, it is preferably an amino acid substitution. For example, a mutated TCR α chain intracellular constant region refers to one in which at least one serine in the wild-type TCR α chain intracellular constant region is substituted with an alanine. A mutated TCR β chain intracellular constant region refers to one in which at least one lysine in the wild-type TCR β chain intracellular constant region is substituted with an arginine or an alanine.

[0144] Calculation of identity between sequences is performed as follows.

[0145] To determine the percentage identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal alignment (e.g., gaps can be introduced in one or both of the first and second amino acid or nucleic acid sequences, or non-homologous sequences can be discarded for comparison). In one preferred embodiment, the length of the reference sequence to be aligned for comparison is at least 30%, preferably at least 40%, more preferably at least 50%, 60%, even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence. The amino acid residues or nucleic acids at the corresponding amino acid or nucleic acid positions are then compared. If 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 this position.

[0146] The sequence comparison and calculation of the percentage identity between two sequences may be carried out using a mathematical algorithm. In a preferred embodiment, the percentage identity between two amino acid sequences is determined by the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm incorporated in the GAP program of the GCG software package (available at http: / / www.gcg.com) using a Blossum 62 matrix or a PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6. In yet another preferred embodiment, the percentage identity between two nucleic acid sequences is determined by the GAP program in the GCG package (available at http: / / www.gcg.com) using a NWSgapdna.CMP matrix with gap weights of 40, 50, 60, 70 or 80 and length weights of 1, 2, 3, 4, 5 or 6. A particularly preferred set of parameters (and that should be used unless otherwise specified) employs a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0147] The percentage identity between two amino acid or nucleic acid sequences can be determined using the E. Meyers and W. Miller algorithm ((1989) CABIOS, 4:11-17) incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0148] Additionally or alternatively, the nucleic acid and protein sequences described herein may be used as a "query sequence" to perform a search against a common database, e.g., to identify other family member or related sequences.

[0149] As known in the art, "nucleotide" or "nucleic acid," which may be used interchangeably herein, refer to a nucleic acid chain of any length, including DNA and RNA. A nucleic acid can be a deoxyribonucleic acid, ribonucleic acid, modified nucleic acid or base, and / or their analogs, or any substrate that can be incorporated into a chain by DNA or RNA polymerase.

[0150] The nucleic acid sequences described herein can be obtained by PCR amplification using primers designed based on the nucleotide sequences described herein, or by the skilled artisan preparing a cDNA library for PCR amplification.

[0151] In this application, "wild type" and "WT" can be used interchangeably. "SA" refers to at least one serine in the amino acid sequence of the α chain intracellular constant region being mutated to alanine. "KR" refers to at least one lysine in the amino acid sequence of the β chain intracellular constant region being mutated to arginine. "dMUT" should be understood to mean the simultaneous presence of an "SA" mutation in the α chain intracellular constant region and a "KR" mutation in the amino acid sequence of the β chain intracellular constant region.

[0152] As used herein, F5-TCR refers to a class of TCR that can recognize the tumor antigen MART-1, and it should be understood that the F5-WT TCR, F5-SA TCR, F5-KR TCR, and F5-dMUT TCR included therein have the same TCR α chain variable region and TCR β chain variable region that are divided differently based on mutations in the α chain intracellular constant region or the β chain intracellular constant region.

[0153] Similarly, in the present application, 1G4-TCR means a TCR capable of recognizing the tumor antigen NYESO-1, and includes 1G4-WT TCR, 1G4-SA TCR, 1G4-KR TCR, and 1G4-dMUT TCR, which have the same TCR α chain variable region and TCR β chain variable region, and are distinguished according to differences in mutations in the α chain intracellular constant region or the β chain intracellular constant region.

[0154] "Vector" means something that can deliver one or more genes or sequences of interest to a host cell, and preferably express said genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, plasmids, cosmids or phage vectors.

[0155] The term "host cell" refers to cells into which exogenous nucleic acid has been introduced, and includes the progeny of those cells. In gene cloning operations, it should be understood that the designed appropriate enzyme cleavage site introduces one or more irrelevant residues at both ends of the expressed amino acid sequence and does not affect the activity of the target sequence, for example, includes, but is not limited to, enzyme cleavage sites such as NotI, BamHI, and XhoI.

[0156] It should be understood that to construct a fusion protein, or to obtain a recombinant protein that automatically localizes on the host cell membrane, or to enhance expression of a recombinant protein, amino acid fragments can be added to the N-terminus, C-terminus, or any suitable region within the protein, including, but not limited to, a signal peptide, a leader peptide, a 2A peptide, a terminal extension, etc. of the recombinant protein.

[0157] It should be understood that in order to purify the constructed recombinant protein, the amino or carboxyl terminus of the constructed protein may contain one or more protein tags, including, but not limited to, FLAG, HA, c-Myc, Poly-His, and the like.

[0158] It should be understood that the above examples (embodiments) are all illustrative and are not used to include all possible embodiments that may be included in the scope of the claims. Various modifications and changes may be made based on the above examples without departing from the scope of the present disclosure. Similarly, various technical features of the above examples may be arbitrarily combined to form another embodiment of the present application that may not be explicitly described. Therefore, the above examples only represent some embodiments of the present application and do not limit the scope of protection of the patent of the present application. Sequence Listing and Notes of the Application

[0159] [Table 1] JPEG2025501358000003.jpg194147JPEG2025501358000004.jpg193147JPEG2025501358000005.jpg192146JPEG2025501358000006.jpg191144JPEG2025501358000007.jpg191145JPEG2025501358000008.jpg190146JPEG2025501358000009.jpg172148Example 1. Construction of TCR expression vector

[0160] F5-TCR and 1G4-TCR were constructed, respectively. F5-TCR contains F5-WT TCR, F5-SA TCR, F5-KR TCR, and F5-dMUT TCR, and 1G4-TCR contains 1G4-WT TCR, 1G4-SA TCR, 1G4-KR TCR, and 1G4-dMUT TCR.

[0161] As described above, each F5-TCR or 1G4-TCR includes a TCR α chain and a TCR β chain, and the TCR α chain is a combination of a human growth hormone signal peptide, a TCR α chain variable region, a TCR α chain extracellular constant region, a TCR α chain transmembrane region, and a TCR α chain intracellular region, and the TCR β chain is a combination of a human growth hormone signal peptide, a TCR β chain variable region, a TCR β chain extracellular constant region, a TCR β chain transmembrane region, and a TCR β chain intracellular region, respectively. The TCR α chain and the TCR β chain are linked in series via the F2A peptide. The amino acid or nucleotide sequences of the above TCR α chain and the TCR β chain are as described in the above embodiments of the present application.

[0162] The above amino acid sequences, as well as the amino acid sequences in which serine is mutated to alanine in the TCR α chain intracellular constant region and lysine is mutated to arginine in the TCR β chain constant region, were all converted into base sequences after codon optimization and synthesized by the company (GenScript).

[0163] The full-length nucleotide sequences of the F5-WT TCR, F5-SA TCR, F5-KR TCR, and F5-dMUT TCR, and the 1G4-WT TCR, 1G4-SA TCR, 1G4-KR TCR, and 1G4-dMUT TCR are as set forth in SEQ ID NOs:39 to 46, respectively.

[0164] All TCR sequences in this application were finally cloned into the pMSGV-LNGFR-P2A vector by enzymatic digestion and ligation with T4 ligase. Example 2. Retroviral packaging and preparation methods

[0165] The TCR expression vector (pMSGV-TCR) constructed in Example 1 was co-transmitted with the envelope plasmid pHIT60 / RD114 into HEK293T cells to package the retrovirus. Specifically, the ratio of the packaging system was MSGV-TCR: pHIT60: RD114 = 2: 2: 1.3. The mixture was mixed uniformly in OPTI-DMEM medium, 15.9 μl of TransIT293-Mirus transfection reagent (Mirusbio #MIR 2700) was added, and the mixture was mixed uniformly by pipetting, and then the mixture was left at room temperature for 25 minutes. The above mixed liposome solution was added to the HEK293T cell culture dish, and the mixture was placed in a 37 ° C incubator and cultured for 48 h. The virus solution was then collected, filtered through a 0.22 μm filter membrane, and stored in a refrigerator at -80 ° C. Example 3. Methods for preparing and culturing primary human mutant TCR-T cells

[0166] Culture of human primary T cells: Human primary T cells were purchased from a commercial company (ALLCELLS). Complete medium for culturing primary T cells included RPMI-1640 medium (Hyclone #SH30809.01) with 5% human serum (Gemini #100-512), dual antibodies (Gibco #15140-122) and 1xGlutMAX (Gibco #35050-061), and the medium was also supplemented with 100U / ml rhIL-2 (PeproTech #200-02). Human primary T cells were resuspended in 1 ml of the above complete medium and added to a 24-well plate (Nest #702001) coated with 1 μg / ml hCD3 (Biolegend #317347) and 1 μg / ml hCD28 antibody (Biolegend #102121). The plate was then cultured in a 37°C incubator for 48 hours before virus infection.

[0167] Infection of human primary T cells with retrovirus: 750 μl of human primary T cells resuspended in a 24-well plate were aspirated into an EP tube (Axygen #MCT-105C), centrifuged at 2500 rpm for 5 minutes, the supernatant was discarded, and 500 μl of the corresponding virus solution (i.e., mutant TCR retrovirus) was used to resuspend the T cells, 0.75 μl of Polybrene (SANTA CRUZ #SC-134220) was added, pipetted uniformly, and added to the corresponding 24-well plate. The infection system at that time was 750 μl. After centrifugation at 30°C and 2500 rpm for 90 minutes, 500 μl of the supernatant was aspirated from the 24-well plate into an EP tube, centrifuged at 2500 rpm for 5 minutes, the supernatant was discarded, and 750 μl of T cell complete medium (1.25 times rhIL-2) was added to the 24-well plate and cultured in a 37°C incubator for 24 hours. The next day, a second viral infection procedure was performed, and the specific steps were the same as those described above. After infection, the human primary T cells were supplemented with fresh RPMI-1640 complete medium every 1 to 2 days. Example 4. Cell flow cytometry analysis method

[0168] The cell flow cytometry analysis technique was performed using a BD LSR Fortessa instrument (BD Bioscience). Detection of cell surface molecules: 2 × 10 5 The cells were aspirated into a 96-well U-bottom plate and centrifuged at 1800 rpm for 5 min, then the supernatant was discarded, and flow cytometry antibodies prepared in FACS buffer (containing 2% serum 1x PBS) were added, stained on ice away from light for 25 min, washed once with FACS buffer, and detected by flow cytometer.

[0169] Cytokine staining: 2×10 5Cells were aspirated into 96-well U-bottom plates and centrifuged at 1800 rpm for 5 min, then the supernatant was discarded, flow cytometry antibodies prepared in FACS buffer (1x PBS containing 2% serum) were added, stained for 25 min on ice away from light, washed once with FACS buffer, cells were fixed with paraformaldehyde fixative (Biolegend #420801) for 20 min, washed once with FACS buffer, cytokine flow cytometry antibodies prepared in permeabilization buffer (Invitrogen #00-8333-56) were added, stained for 25 min on ice away from light, washed once with FACS buffer, and detected by flow cytometer.

[0170] Transcription factor staining: 2×10 5 Cells were aspirated into 96-well U-bottom plates and centrifuged at 1800 rpm for 5 min, the supernatant was discarded, flow cytometry antibodies prepared in FACS buffer (1x PBS with 2% serum) were added, stained for 25 min on ice away from light, washed once with FACS buffer, cells were fixed with FOXP3 transcription factor fixative (Invitrogen #00-5523-00) for 20 min, washed once with FACS buffer, cytokine flow cytometry antibodies prepared in permeabilization buffer were added, stained for 25 min on ice away from light, washed once with FACS buffer, and detected by flow cytometer. Flow cytometry data were analyzed using FlowJo software (Tree Star). Example 5. Degradation of TCR by antigen stimulation

[0171] Experiment on downregulation of TCR expression by in vivo tumor antigen stimulation: For tumor-bearing mice inoculated with K562-NYESO-1, TCR expression on the surface of TCR-T cells in the spleen and tumor was detected 12 days after the introduction of 1G4-TCR-T cells. As shown in Figure 1(A), in tumor tissue, tumor antigen stimulation caused downregulation of TCR expression. Experiment on downregulation and degradation of TCR by in vitro target cell antigen stimulation: 1G4-TCR-T cells and K562-NYESO-1 target cells or K562-MART-1 non-target cells were mixed in a 24-well plate at a 1:1 ratio and co-incubated in a 37°C incubator for 12 hours (the specific time is determined by the experiment), and the cells were aspirated and subjected to flow FACS detection. Detection of TCR downregulation was measured by staining T cell surface TCR, while analysis of TCR degradation level was detected by staining intracellular TCR after fixing and permeabilizing the cells. As a result, as shown in Figure 1 (B), target cell antigen stimulation promoted the downregulation and degradation of TCR. Experiment on TCR downregulation and degradation by hCD3 antibody stimulation: After resuspending 1G4-TCR-T cells and F5-TCR-T cells after activation, they were added to a 24-well plate coated with hCD3 antibody and co-incubated in an incubator at 37 °C for 12 hours (the specific time is determined by the experiment), and the cells were aspirated and subjected to flow FACS detection. Detection of TCR downregulation was measured by staining T cell surface TCR, and analysis of TCR degradation level was detected by staining intracellular TCR after fixing and permeabilizing cells. As shown in Figure 1 (C and D), in vitro CD3 antibody stimulation promoted TCR downregulation and degradation in humanized T cells. Experiment to compare the expression of each mutant TCR: After resuspending each mutant 1G4-TCR-T cells after activation, the expression of different TCRs was detected by FACS staining. As shown in FIG. 2(B), there was no clear difference in the expression of each mutant TCR compared to the wild-type TCR.Comparative experiment of downregulation and degradation of each mutant TCR by hCD3 antibody stimulation: 1G4-TCR-T cells and F5-TCR-T cells after activation were resuspended and added to a 24-well plate coated with hCD3 antibody, and co-incubated in an incubator at 37 °C for the time required for the experiment. Detection of TCR downregulation was measured by staining T cell surface TCR, and analysis of TCR degradation level was detected by staining intracellular TCR after fixing and permeabilizing cells. As a result, as shown in Figure 2 (C and D), compared with WT TCR, in the mutant group, SA TCR, KR TCR, and dMUT TCR showed more T cell membrane retention and could significantly resist downregulation and degradation by antibody stimulation, which was especially obvious in the dMUT TCR group. As shown in Figure (E), the experiment of detecting surface TCR expression at various time points by hCD3 antibody stimulation showed that dMUT TCR has the strongest resistance to degradation compared with other groups. Example 6. In vitro detection of T cell function and construction of depletion model

[0172] In vitro functional detection of T cells: In vitro activated human primary T cells were infected with each mutant 1G4-TCR retrovirus, and then cultured in RPMI-1640 complete medium for 12 days. 2 × 10 5 The cells were aspirated into a 96-well plate and subjected to flow FACS detection. The detection indicators were the expression of T cell memory molecules such as CD62L, TCF1, CD27, and CD45RO. As a result, as shown in Figure 3 (A-C), compared with the WT TCR group, SA TCR, KR TCR, and dMUT TCR-T cells showed a significantly higher expression of CD27 + CD45RO + The proportion of central memory T cells (DMT T cells) was significantly increased in the mutant group, and the mutant group had a strong tendency to differentiate into memory T cells. The expression of the T cell memory molecules CD62L and TCF1 was also increased, most notably in dMUT TCR-T cells.

[0173] Construction of an in vitro depletion model of T cells: 48 hours after in vitro activated human primary T cells were infected with each mutant 1G4-TCR retrovirus, the T cells were removed and centrifuged at 2500 rpm for 5 min, the TCR-T cells were resuspended in complete medium, and plated on a 24-well plate (10 per well) pre-coated with 2 μg / ml hCD3 antibody. 6 The TCR-T cells were then stimulated for 48 hours, and the above steps were then repeated twice. After the culture was completed, the TCR-T cells were harvested and subjected to flow cytometry detection. The construction of this in vitro exhaustion model was based on related documents known in the art (Santosha A. Vardhana et al., Nat Immunol. 2020 Sep;21(9):1022-1033.), and the detection indicators were exhaustion-related molecules such as PD-1, TIM3, LAG-3, TOX, etc., and cytokines TNF-α and IFN-γ. As a result, as shown in Figure 3 (D-F), the expression of exhaustion-related molecules LAG-3 and TOX was obviously decreased in KR TCR-T cells and dMUT TCR-T cells, and more dMUT TCR-T cells produced the cytokines TNF-α and IFN-γ, suggesting that KR TCR-T cells and dMUT TCR-T cells are more resistant to the occurrence of T cell exhaustion. Example 7. Mitochondrial function detection and metabolic analysis of T cells

[0174] Detection of mitochondrial indicators: In vitro activated human primary T cells were infected with each mutant 1G4-TCR retrovirus, after which T cells were cultured in RPMI-1640 complete medium for 12 days and 2 × 10 5 The cells were absorbed into an EP tube, stained by adding MitoTracker Green (Invitrogen #M7514) to a final concentration of 50 nM, and stained for 1 hour at 37 °C away from light, and then flow FACS detection was performed. As a result, as shown in Figure 4 (A), there was no obvious change in the number of mitochondria in each mutant TCR-T cell compared with the WT TCR cell. As a method for detecting mitochondrial membrane potential, 2 × 10 5The cells were aspirated into an EP tube, stained by adding TMRE (Invitrogen #T669) to a final concentration of 200 nM, and stained for 1 hour at 37°C away from light, followed by flow FACS detection. As a result, as shown in Figure 4(B), compared with WT TCR cells, SA TCR, KR TCR, and dMUT TCR-T cells, especially dMUT TCR-T cells, had significantly reduced mitochondrial membrane potential. As a method for detecting mitochondrial reactive oxygen species, 2 × 10 5 The cells were aspirated into an EP tube and stained with MitoSOX (Invitrogen #M36008) at a final concentration of 5 μM for 1 hour at 37 °C away from light, followed by flow FACS detection. As shown in Figure 4 (C), mitochondrial ROS production was significantly reduced in SA TCR, KR TCR, and mutant TCR-T cells compared to WT TCR cells. To summarize the results of the above examples, each TCR mutation does not affect the number of mitochondria in T cells, but in mutant TCR cells, especially dMUT TCR-T cells, mitochondria show a healthier state, which is associated with the memory phenotype during in vitro culture of dMUT TCR-T cells and resistance to T cell exhaustion.

[0175] Detection of metabolic indicators: Human primary T cells infected with mutant 1G4-TCR retrovirus were cultured in vitro for 12 days in RPMI-1640 complete medium in a 37°C incubator, and 2 × 10 5The cells were aspirated into EP tubes and centrifuged at 2500 rpm for 5 min, the supernatant was discarded, and then the T cells were resuspended using glucose-free and serum-free medium and placed in a 24-well plate. The cells were treated with 2-NBDG (Invitrogen #N13195) at a final concentration of 50 μM or Bodipy FL C16 (Invitrogen #D3821) at 1 μM, and stained at 37 °C for 30 min in the dark, followed by flow FACS detection. Before detection by flow cytometer, they may be labeled with other labeling molecules according to the requirements of the experiment. The results in Figure 4 (D and E) showed that compared with WT TCR cells, the glucose uptake was obviously reduced in SA TCR, KR TCR, and dMUT TCR-T cells, and there was no obvious change in the uptake of extracellular fatty acids. Example 8. Detection of antitumor function of mutant TCR-T cells

[0176] Antitumor experiments of mutant TCR-T cells were performed in 4- to 8-week-old immunodeficient NSG mice. To confirm that mutant TCR-T cells have a superior antitumor effect to wild-type TCR-T cells, we first used 8 × 10 5 K562-NYESO-1 cells were inoculated subcutaneously into the axilla of the right hind leg of NSG mice, and after the target cells had grown subcutaneously in the mice for approximately 8–12 days, the tumor volume was measured using a caliper. 3 ~120mm 3 In the case of 6 Human primary T cells were injected into the tail vein of NSG mice. Then, the tumor volume was measured every 2 days, and about 14 days later (the specific operation time is determined according to the experimental phenomenon), the blood, spleen and tumor tissues of NSG mice were collected. First, the tumor tissue was weighed, and then the red blood cells in the blood and spleen of the mouse were dissolved using an erythrocyte lysing agent (Biolegend #420301). The mouse tumor tissue was crushed and then subjected to Percoll density gradient centrifugation (Cytiva #17089110), and the human primary T cells introduced through the tail vein were collected from the blood, spleen and tumor tissue of the mouse and analyzed by flow cytometry staining.

[0177] Specific results are shown in Figures 5 and 6. First, in Figure 5(A), the tumor growth volume was statistically analyzed, and it was found that the tumor growth rate was significantly slower in the experimental group into which KR TCR-T cells and dMUT TCR-T cells were introduced, compared to the WT TCR group. The tumor tissue was weighed, and as shown in Figure 5(B), the tumor weight was significantly reduced in the experimental group into which KR TCR-T cells and dMUT TCR-T cells were introduced. In addition, in this example, the amount of introduced CD8 in the spleen, blood, and tumor tissue of tumor-bearing NSG mice was significantly reduced. + The percentage of TCR-T cells was calculated. As a result, as shown in Figure 5 (C-E), the percentage of dMUT TCR-T cells was significantly increased in the spleen and blood of mice compared with WT TCR-T cells. Meanwhile, the percentage of KR TCR-T cells and dMUT TCR-T cells was significantly increased in the tumor tissues of mice compared with WT TCR-T cells. This indicated that KR TCR-T cells and dMUT TCR-T cells have stronger antitumor persistence in the NSG mouse adoptive immunity model. Further analysis of the cell phenotype showed that, as shown in Figure 6 (A), the CD27 expression level in SA TCR, KR TCR, and dMUT TCR-T cells in the spleen was significantly higher than that in wild-type TCR-T cells. + CD45RO + It was revealed that the proportion of central memory T cells (CMCs) was significantly increased. Furthermore, through the analysis of TCR-T cells in the tumor tissue, it was found that dMUT TCR-T cells expressed higher T cell memory molecules CD62L and CCR7 compared to WT TCR-T cells, as shown in Figure 6 (B and C). Figure 6 (D) shows the expression of PD-1 in the SA TCR, KR TCR, and dMUT TCR groups in the tumor tissue. + TIM-3 +The proportion of double positive cells was significantly decreased compared to the WT TCR group. Furthermore, by detecting the fluorescence intensity of the inhibitory molecules PD-1, TIM-3, LAG-3, and CD39, it was found that the expression of inhibitory molecules was significantly decreased in the mutant TCR-T cells compared to the WT TCR-T cells, as shown in Figure 6 (E-H). In addition, as shown in Figure 6 (I), it was found that the expression of the transcription factor TOX, which is important for exhaustion, was significantly decreased in the dMUT TCR-T cells. Thus, the above indicated that the mutant TCR-T cells could resist the occurrence of T cell exhaustion in an in vivo antitumor model. In addition, the production of cytokines by TCR-T cells in tumor tissues was detected, and as shown in Figure 6 (J and K), the IFN-γ expression was significantly decreased in the adoptive KR TCR-T cell group and the dMUT TCR-T cell group. + CD8 + The percentage of CD8 T cells was significantly increased. This indicates that KR TCR cells and dMUT TCR-T cells have stronger antitumor effects and are CD8 + KR TCR-T cells and CD8 + TNF-α in dMUT TCR-T + IFN-γ + A higher proportion of double-positive cells was shown. KR TCR-T cells produced more cytokines than WT TCR-T cells, but less cytokines than dMUT TCR-T cells, indicating that dMUT TCR-T cells had stronger effector functions in tumor tissues.

[0178] In T cell adoptive immunotherapy, the current TCR-T cell therapy faces the challenges of collecting limited T cells from patients to expand TCR-T cells in large quantities, and the fact that tumor patients face persistent decline in T cell function and the occurrence of T cell exhaustion after adoptive therapy. The method of modifying mutant TCR-T cells according to the present application, particularly the modification of dMUT TCR-T, allows TCR-T cells to exhibit more sustained antitumor effects after being expanded in vitro and reinjected into tumor models, and also resists the occurrence of T cell exhaustion. This suggested that TCR-T cells modified by dMUT TCR mutation are expected to achieve sustained and efficient antitumor function in clinical production and treatment.

Claims

1. 1. An isolated T cell receptor or fragment thereof comprising a TCR α chain and a TCR β chain, The TCR β chain comprises a TCR β chain variable region and a TCR β chain constant region, the TCR β chain constant region comprising a TCR β chain extracellular constant region, a TCR β chain transmembrane region, and a TCR β chain intracellular constant region, which are linked in order, and the TCR β chain intracellular constant region comprises (1) a mutant TCR β chain intracellular constant region in which at least one lysine in the wild-type TCR β chain intracellular constant region is mutated to arginine or alanine; or (2) A T cell receptor or a fragment thereof, characterized in that it is selected from a mutant TCR β chain intracellular constant region comprising the amino acid sequence shown in SEQ ID NO: 10 or consisting of said amino acid sequence.

2. The wild-type TCR β chain intracellular constant region is (1) a wild-type TCR β chain intracellular constant region derived from a human, mouse, or other mammalian species; or (2) The isolated T cell receptor according to claim 1, characterized in that it is selected from a wild-type TCR β chain intracellular constant region that comprises or consists of the amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 47 or SEQ ID NO:

48.

3. The TCR α chain comprises a TCR α chain constant region, the TCR α chain constant region comprising, in order, a TCR α chain extracellular constant region, a TCR α chain transmembrane domain, and a TCR α chain intracellular constant region, the TCR α chain intracellular constant region comprising: (1) a wild-type TCR α chain intracellular constant region derived from a human, mouse or other mammalian species; (2) A wild-type TCR α chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 3; (3) A mutant TCR α chain intracellular constant region in which at least one serine in the wild-type TCR α chain intracellular constant region according to (1) or (2) is mutated to alanine; or (4) A mutant TCR α chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 4; 3. The isolated T cell receptor of claim 1, wherein optionally, the TCR alpha chain further comprises a TCR alpha chain variable region.

4. 1. An isolated T cell receptor or fragment thereof comprising a TCR α chain and a TCR β chain, The TCR α chain comprises a TCR α chain variable region and a TCR α chain constant region, the TCR α chain constant region comprising a TCR α chain extracellular constant region, a TCR α chain transmembrane region, and a TCR α chain intracellular constant region, which are linked in order, and the TCR α chain intracellular constant region comprises (1) a mutant TCR α chain intracellular constant region in which at least one serine in the wild-type TCR α chain intracellular constant region is mutated to alanine; or (2) A T cell receptor or a fragment thereof, characterized in that it is selected from a mutant TCR α chain intracellular constant region comprising the amino acid sequence shown in SEQ ID NO: 4 or consisting of said amino acid sequence.

5. The wild-type TCR α chain intracellular constant region is (1) a wild-type TCR α chain intracellular constant region derived from a human, mouse, or other mammalian species; or (2) The isolated T cell receptor described in claim 4, characterized in that it is selected from a wild-type TCR α chain intracellular constant region that includes or consists of the amino acid sequence shown in SEQ ID NO:

3.

6. The TCR β chain comprises a TCR β chain constant region, the TCR β chain constant region comprising a TCR β chain extracellular constant region, a TCR β chain transmembrane region, and a TCR β chain intracellular constant region, which are linked in order, and the TCR β chain intracellular constant region comprises (1) a wild-type TCR β chain intracellular constant region derived from a human, mouse or other mammalian species; (2) a wild-type TCR β chain intracellular constant region comprising or consisting of an amino acid sequence represented by SEQ ID NO: 9, SEQ ID NO: 47, or SEQ ID NO: 48; (3) A mutant TCR β chain intracellular constant region in which at least one lysine in the wild-type TCR β chain intracellular constant region according to (1) or (2) is mutated to arginine or alanine; or (4) A mutant TCR β chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 10; 6. The isolated T cell receptor of any one of claims 4 to 5, wherein optionally, the TCR β chain further comprises a TCR β chain variable region.

7. 2. The isolated T cell receptor of claim 1 , wherein the isolated T cell receptor recognizes a polypeptide-based antigen, a lipid-based antigen, or a polysaccharide-based antigen.

8. 2. The isolated T cell receptor of claim 1 , wherein the isolated T cell receptor recognizes a tumor antigen, a microbial antigen, or an autoantigen.

9. The isolated T cell receptors include BCMA, CA9, CTAG, CCL-1, CSPG4, EGFR, EPG-2, EPG-40, FCRL5, FBP, OGD2, GPC3, GPRC5D, HER3, HER4, HLA-A1, HLA-A2, LRRC8A, CMV, MUC1, MUC16, MART-1, NCAM, PRAME, PSC A, PSMA, ROR1, TPBG, TAG72, TRP1, TRP2, VEGFR, VEGFR2, WT-1, MAGE-A1 / A3 / A4 / A6 / A10 / C2, g p100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1 / 2, BRLF-1, BMLF-1, HPV-16E6 / E7, KRAS G12D, KRAS G12V, TP53 R175H, β-GlcCer, eLPA, LPE, CA199, CA72-4, or CA125, preferably one or more antigens of MAGE-A1 / A3 / A4 / A6 / A10 / C2, gp100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1 / 2, BRLF-1, BMLF-1, HPV-16E6 / E7, KRAS G12D, KRAS G12V, TP53 9. The isolated T cell receptor of claim 8, which recognizes one or more of the following antigens: R175H, β-GlcCer, eLPA, LPE, CA199, CA72-4, or CA125.

10. The TCR β chain variable region is (1) a TCR β chain variable region comprising three complementarity determining regions (CDRs) contained in the amino acid sequence represented by SEQ ID NO: 17; (2) a TCR β chain variable region comprising three complementarity determining regions (CDRs) contained in the amino acid sequence represented by SEQ ID NO: 25; (3) a TCR β chain variable region comprising CDR1 of the amino acid sequence shown in SEQ ID NO: 18, CDR2 of the amino acid sequence shown in SEQ ID NO: 19, and / or CDR3 of the amino acid sequence shown in SEQ ID NO: 20; (4) A TCR β chain variable region comprising CDR1 of the amino acid sequence represented by SEQ ID NO: 26, CDR2 of the amino acid sequence represented by SEQ ID NO: 27, and / or CDR3 of the amino acid sequence represented by SEQ ID NO: 28; (5) A TCR β chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 17; or (6) An isolated T cell receptor according to any one of the preceding claims, characterized in that it is selected from a TCR β chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO:

25.

11. The TCR α chain variable region is (1) a TCR α chain variable region comprising three complementarity determining regions (CDRs) contained in the amino acid sequence represented by SEQ ID NO: 13; (2) a TCR α chain variable region comprising three complementarity determining regions (CDRs) contained in the amino acid sequence represented by SEQ ID NO: 21; (3) a TCR α chain variable region comprising CDR1 of the amino acid sequence shown in SEQ ID NO: 14, CDR2 of the amino acid sequence shown in SEQ ID NO: 15, and / or CDR3 of the amino acid sequence shown in SEQ ID NO: 16; (4) A TCR α chain variable region comprising CDR1 of the amino acid sequence shown in SEQ ID NO: 22, CDR2 of the amino acid sequence shown in SEQ ID NO: 23, and / or CDR3 of the amino acid sequence shown in SEQ ID NO: 24; (5) A TCR α chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO: 13; or (6) An isolated T cell receptor according to any one of the preceding claims, characterized in that it is selected from a TCR α chain variable region comprising or consisting of the amino acid sequence shown in SEQ ID NO:

21.

12. 10. The isolated T cell receptor of any one of the preceding claims, comprising one or more of the following features: (1) The TCR α chain extracellular constant region is derived from the TCR α chain extracellular constant region of a human, mouse, or other mammalian species. (2) The TCR α-chain extracellular constant region comprises or consists of the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations compared to the amino acid sequence of SEQ ID NO:1, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO:

1. (3) The TCR α-chain transmembrane region is derived from the TCR α-chain transmembrane region of a human, mouse, or other mammalian species. (4) The TCR α-chain transmembrane domain comprises or consists of the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations compared to the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO:

2. (5) The TCR β chain extracellular constant region is derived from a TCR β chain extracellular constant region of a human, mouse, or other mammalian species. (6) The TCR β chain extracellular constant region comprises or consists of the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations compared to the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO:

7. (7) The TCR β chain transmembrane region is derived from a TCR β chain transmembrane region of a human, mouse, or other mammalian species. (8) The TCR β chain transmembrane region comprises or consists of the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutations compared to the amino acid sequence of SEQ ID NO:8, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence of SEQ ID NO:

8.

13. The TCR α chain constant region and the TCR β chain constant region are: (1) the TCR α chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 5, and the TCR β chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 12; (2) the TCR α chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 6, and the TCR β chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 12, or (3) The isolated T cell receptor according to any one of the preceding claims, characterized in that the TCR α chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 6, and the TCR β chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:

11.

14. 2. The isolated T cell receptor of any one of the preceding claims, further comprising a signal peptide, said signal peptide forming a signal peptide-variable region structure with the TCR α chain variable region and / or the TCR β chain variable region.

15. 15. The isolated T cell receptor of claim 14, wherein the signal peptide is a human growth hormone signal peptide, a CD8α signal peptide, or an immunoglobulin signal peptide.

16. 10. An isolated nucleic acid or a fragment thereof encoding the isolated T cell receptor or a fragment thereof according to any one of the preceding claims.

17. A nucleic acid construct comprising the isolated nucleic acid or a fragment thereof of claim 16.

18. A vector comprising the nucleic acid construct of claim 17.

19. 19. The vector of claim 18, which is an expression vector or a crisper gene editing vector, preferably a retroviral vector.

20. An engineered cell comprising a nucleic acid construct according to claim 17 or a vector according to any one of claims 18 to 19.

21. 21. The engineered cell of claim 20, which is a T cell, e.g. a human T cell.

22. 1. A method for producing an engineered cell, comprising: (1) transforming or transducing a host cell with a nucleic acid construct according to claim 17 or a vector according to any one of claims 18 to 19; (2) culturing a host cell in a suitable medium; and (3) isolating and purifying the engineered cells from the medium or cells.

23. (1) Inhibition of degradation of T cell receptors during antigen stimulation; (2) providing a tumor killing effect or inhibiting tumor growth; (3) Maintenance of T cell proliferation; and (4) Use of the isolated T cell receptor or fragment thereof, nucleic acid or fragment thereof, nucleic acid construct, vector or engineered cell according to any one of the preceding claims in one or more of the following applications: preparation of anti-tumor immune medicines and cells.

24. A method for modifying a T cell receptor, comprising: (1) mutating at least one lysine in the wild-type TCR β chain intracellular constant region to arginine or alanine; and / or A method for modifying a T cell receptor, comprising the step (2) of mutating at least one serine in the wild-type TCR α chain intracellular constant region to alanine.

25. (1) The wild-type TCR β chain intracellular constant region is selected from the wild-type TCR β chain intracellular constant regions according to claim 2; and / or (2) The method for modifying a T cell receptor according to claim 24, characterized in that the wild-type TCR α-chain intracellular constant region is selected from the wild-type TCR α-chain intracellular constant regions described in claim 5.

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