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 use of modified T cell receptors. [Background technology]

[0002] Malignant tumors pose a serious threat to human health and life, and are one of the leading causes of human death, with their incidence increasing year by year. Traditional cancer treatments primarily involve surgery, supported by radiation therapy and chemotherapy. Compared to conventional treatments, tumor immunotherapy targets the immune system and stimulates a systemic response against tumors. Furthermore, clinical data have shown 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 investigated for the treatment of hematologic and solid tumors. It is primarily based on T cell receptors on the surface of T cells, which can recognize various tumor antigens, thereby killing and eliminating tumor cells.

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

[0004] TCR-T cell therapy targets antigens expressed on the surface of T cells. TCRs are receptors on the surface of T cells that bind to CD3 via non-covalent binding to form a TCR-CD3 complex. They activate T cells by recognizing and binding antigens presented by MHC, promoting their division and differentiation. Unlike CARs, most TCRs are heterodimers composed of an α chain and a β chain, both of which contain an antigen-binding region, a constant region, and a transmembrane domain. These α and β chains are covalently linked by disulfide bonds between conserved cysteine ​​residues located within specific regions of each chain. Compared to CAR-T therapy, TCR-T can target most intracellular antigens and is not limited to surface antigens. TCR-T can target most tumor antigens, particularly those within tumor cells. Therefore, the range of tumor recognition by T cell receptors is broader than that of antibody drugs or CAR-T, which rely on antibodies to recognize tumors. Furthermore, CAR-T has limitations in the treatment of solid tumors, and TCR-T technology is expected to address this issue.

[0005] Currently, 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 T cell recognition process for tumor cells by modifying the TCR, which is the "probe" that T cells bind to tumor antigens, and improve the affinity of T lymphocytes for tumor cells, thereby enabling T cells that originally lack the ability to recognize tumors to efficiently recognize and kill tumor cells. Specifically, these methods include the following:

[0006] (1) Increasing the expression of exogenous TCRs and enhancing the anti-tumor effects of TCR-T. a. Mouserization of TCR constant regions: Mouserization of TCR constant regions using genetic engineering techniques reduces or prevents endogenous and exogenous TCR mismatches, promoting preferential pairing of exogenous TCRs and thereby increasing the expression level of T cell surface antigens and enhancing the anti-tumor effects of TCR-T. b. Cysteine ​​insertion in the TCR constant region: Introducing cysteines into the constant regions of the TCR α and β chains promotes preferential pairing of exogenous TCRs, increasing the surface expression level of exogenous TCRs and reducing mismatches with endogenous TCR chains. This method can improve the efficacy and safety of tumor-reactive T cells. c. Insertion of hydrophobic mutations in the TCR transmembrane region: Increasing the hydrophobicity of the transmembrane region of the TCR α chain improves TCR stability and enhances TCR expression on the T cell surface, thereby improving cell affinity and anti-tumor TCR activity.

[0007] (2) Improving the affinity of exogenous TCRs for 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 region, the high affinity of the CAR and the high signaling ability of the TCR complex are combined, enhancing the anti-tumor 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 function, including decreased secretion levels of proinflammatory cytokines such as IFN-γ and TNF-α, reduced T cell proliferation and self-renewal capacity, and impaired metabolic activity.

[0010] (2) The expression level of TCR on the surface of T cells is reduced. Antigen stimulation in the tumor microenvironment induces CD8 + The expression of exogenous TCRs on the surface of T cells is reduced or their degradation is accelerated, resulting in insufficient TCR-T cells to attack tumor cells.

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

[0012] In response to some of the technical drawbacks 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 α chain of a T cell receptor (TCR) or a fragment thereof, wherein the TCR α chain comprises a TCR α chain constant region, wherein the TCR α chain constant region comprises, in order, a TCR α chain extracellular constant region, a TCR α chain transmembrane region, and a TCR α chain intracellular constant region, and the TCR α chain intracellular constant region is a mutant TCR α chain intracellular constant region, wherein at least one serine in a wild-type TCR α chain intracellular constant region has been mutated to alanine.

[0014] In another aspect, the present application provides an isolated TCR β chain of a T cell receptor (TCR) or a fragment thereof, wherein the TCR β chain comprises a TCR β chain constant region, wherein the TCR β chain constant region comprises, in order, a TCR β chain extracellular constant region, a TCR β chain transmembrane region, and a TCR β chain intracellular constant region, and the TCR β chain intracellular constant region is 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 any of the TCR α chain and / or TCR β chains described above.

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

[0023] In another aspect, the present application provides a method for producing a method of manufacturing a pharmaceutical composition comprising: (1) Inhibition of T cell receptor degradation 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 above-mentioned 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 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 of manufacturing a pharmaceutical composition 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 is provided, which includes the step (2) of mutating at least one serine in the wild-type TCR α chain intracellular constant region to alanine. [Effects of the Invention]

[0026] In this study, we utilize 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 TCR levels on the cell surface. This method can be applied to various TCR-T cell therapies.

[0027] On the other hand, 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). During in vitro culture, SA TCR, KR TCR, and dMUT TCR cells exhibited a central memory T cell differentiation phenotype. An in vitro depletion model revealed lower levels of depletion in KR TCR and dMUT TCR cells compared to WT TCR. This phenotypic change was attributed to healthy mitochondrial conditions in SA TCR, KR TCR, and dMUT TCR cells. As a result, mutant TCR-T cells cultured in vitro exhibited lower mitochondrial membrane potential and ROS levels. In this study, we demonstrated that KR TCR and dMUT TCR cells had stronger antitumor effects than WT TCR cells using a mouse tumor-transplanted T cell adoptive transfer model. Furthermore, in the dMUT TCR mutant group, higher numbers of TCR-T cells were present in the mouse spleen and blood, whereas the proportion of KR TCR and dMUT TCR cells was significantly increased in tumor tissue. Regarding cell phenotype, in the SA TCR, KR TCR, and dMUT TCR groups, more central memory T cells were accumulated in the mouse spleen, while dMUT mutant TCR-T cells in tumor tissue exhibited a lower T cell exhaustion phenotype and proliferative capacity. Therefore, KR TCR and dMUT TCR cells demonstrate superior tumor growth inhibition potential when administered with the same amount of TCR-T cells.

[0028] As can be seen, the present application not only inhibits TCR degradation 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 are not limited to solid tumors, but may also be hematological tumors or lymphomas. [Brief explanation of the drawings]

[0029] [Figure 1]Antigen stimulation promotes cell surface TCR degradation. (A) In tumor-bearing mice inoculated with K562-NYESO-1, TCR expression on TCR-T cells was detected in the spleen and tumor 12 days after 1G4-TCR-T cell transfer. (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 TCR degradation. (C) Activated F5-TCR-T cells were cultured in vitro and TCR degradation was detected 12 hours after CD3 antibody stimulation. (D) Activated 1G4-TCR-T cells were cultured in vitro and TCR degradation was detected 12 hours after CD3 antibody stimulation. [Figure 2] These figures show the results of mutations that inhibit TCR downregulation and degradation. 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) In vitro activated mutant F5-TCR-T cells were stimulated with CD3 antibody for 12 hours, and then degradation of each TCR was detected ("R" indicates no CD3 antibody stimulation, "S" indicates CD3 antibody stimulation). (D) In ​​vitro activated mutant 1G4-TCR-T cells were stimulated with CD3 antibody for 12 hours, and then degradation of each TCR was detected ("R" indicates no CD3 antibody stimulation, "S" indicates CD3 antibody stimulation). (E) In vitro activated mutant 1G4-TCR-T cells were stimulated with CD3 antibody for various times, and then expression of each TCR was detected. [Figure 3]Figure 1 shows the effect of mutations on the in vitro function of 1G4-TCR-T cells. All in vitro functional experiments were performed on human primary T cells. (A) Flow cytometry was performed on in vitro activated TCR-T cells to detect CD45RO and CD27 as memory T cell differentiation indicators. (B) Activated 1G4-TCR-T cells were examined for CD62L, a memory T cell differentiation indicator. (C) In vitro activated 1G4-TCR-T cells were examined for TCF-1, a memory T cell differentiation indicator. (D) Expression of LAG-3, a TCR-T cell depletion molecule, was detected in each mutant TCR-T cell using an in vitro depletion model. (E) Expression of TOX, a transcription factor critical 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] This figure compares 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 was measured. (B) Changes in mitochondrial membrane potential in 1G4-TCR-T cells after mutation were measured. (C) The level of ROS production in the intracellular mitochondria of in vitro activated 1G4-TCR-T cells was measured. (D) 12 days after in vitro activation of each TCR-T cell, after 2-NBDG treatment, glucose uptake in TCR-T cells was measured by FACS flow cytometry analysis. (E) Bodipy FL C16 uptake experiment was performed to measure the utilization efficiency of extracellular fatty acids in TCR-T cells of each mutant group. [Figure 5] (A) Comparison of the antitumor effects of each mutant 1G4-TCR-T cell. (B) Statistical analysis of tumor size at various time points in K562-NYESO-1 NSG tumor-bearing mice inoculated with 1G4-TCR-T cells. After T cell transfer into K562-NYESO-1 NSG tumor-bearing mice, the weight of tumor tissue (B) was measured for each mutant group on day 12. The proportion of transferred TCR-T cells in the blood (C), spleen (D), and tumor tissue (E) of tumor-bearing mice was also measured. [Figure 6]This figure shows the effect of mutations on the function of 1G4-TCR-T cells in mice. (A) Flow cytometry was performed on the spleens of NSG tumor-bearing mice to detect CD45RO and CD27, which are indicators of memory T cell differentiation, for each mutation group. (B) Expression of CD62L, an indicator of memory T cell differentiation, was detected in tumor tissue of NSG tumor-bearing mice for each mutation group. (C) Expression of CCR7, an indicator of memory T cell differentiation, was detected in tumor tissue of NSG tumor-bearing mice for each mutation group. (D) Statistical analysis of the percentage of PD-1+TIM-3+ double-positive exhausted T cells in tumor tissue of NSG tumor-bearing mice for each mutation group. Expression of the immunosuppressive molecules PD-1 (E), TIM-3 (F), LAG-3 (G), and CD39 (H) was detected on TCR-T cells in tumor tissue of NSG tumor-bearing mice for each mutation group. (I) Expression of the transcription factor TOX was detected in tumor tissue of NSG tumor-bearing mice for each mutation group. (J) Detecting the percentage of IFN-γ+ positive CD8+ T cells in the tumor tissue of NSG tumor-bearing mice. (K) Detecting 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 INVENTION

[0030] In one aspect, the present application provides an isolated TCR alpha chain of a T cell receptor (TCR), or a fragment thereof, wherein the TCR alpha chain comprises 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. Optionally, the 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 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:3.

[0033] In some embodiments, the TCR alpha chain intracellular constant region is 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, for example, derived from a mutant 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 mutant 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 antigens (e.g., NYESO-1, AFP, and MART-1), lipid antigens (e.g., β-GlcCer, eLPA, and LPE), and polysaccharide 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: 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 contained in the amino acid sequence set forth in SEQ ID NO:13 or the amino acid sequence set forth in SEQ ID NO:21.

[0041] In some embodiments, the TCR alpha chain variable region comprises a CDR1 having the amino acid sequence shown in SEQ ID NO:14, a CDR2 having the amino acid sequence shown in SEQ ID NO:15, and a CDR3 having the amino acid sequence shown in SEQ ID NO:16, and recognizes the tumor antigen MART-1, or comprises a CDR1 having the amino acid sequence shown in SEQ ID NO:22, a CDR2 having the amino acid sequence shown in SEQ ID NO:23, and a CDR3 having the amino acid sequence shown in 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), wherein the TCR β chain comprises a TCR β chain constant region, the TCR β chain constant region comprising, in order, a TCR β chain extracellular constant region, a TCR β chain transmembrane region, and a TCR β chain intracellular constant region. Optionally, the 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, a mouse, or another 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 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, for example, a mutant TCR β chain intracellular constant region in which at least one lysine in a wild-type TCR β chain intracellular constant region derived from a human, mouse, or other mammalian species has been mutated to arginine or alanine.

[0051] In some embodiments, the mutant TCR β chain intracellular constant region is a wild-type TCR β chain intracellular constant region in which at least one lysine has been mutated to arginine or alanine, and the wild-type TCR β chain intracellular constant region comprises or consists of the amino acid sequence set forth 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 set forth 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 mutant 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 antigens (e.g., NYESO-1, AFP, and MART-1), lipid antigens (e.g., β-GlcCer, eLPA, and LPE), and polysaccharide 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: 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, or CDR3, which is contained 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 the amino acid sequence shown in SEQ ID NO:18, a CDR2 having the amino acid sequence shown in SEQ ID NO:19, and a CDR3 having the amino acid sequence shown in SEQ ID NO:20, and recognizes the tumor antigen MART-1, or comprises a CDR1 having the amino acid sequence shown in SEQ ID NO:26, a CDR2 having the amino acid sequence shown in SEQ ID NO:27, and a CDR3 having the amino acid sequence shown in 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, a mouse, or another mammalian species.

[0060] In some embodiments, the TCR beta 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 the amino acid sequence shown in 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 aforementioned TCR α chain and TCR β chain, and the TCR α chain intracellular constant region and the TCR β chain intracellular constant region cannot simultaneously be wild-type. For example, when the TCR α chain intracellular constant region is any of the aforementioned wild-type TCR α chain intracellular constant regions, the TCR β chain intracellular constant region is any of the aforementioned mutant TCR β chain intracellular constant regions; and when the TCR α chain intracellular constant region is any of the aforementioned mutant TCR α chain intracellular constant regions, the TCR β chain intracellular constant region is any of the aforementioned 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, wherein the T cell receptor comprises 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 order, a TCR β chain extracellular constant region, a TCR β chain transmembrane region, and a TCR β chain intracellular constant region, 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 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 may be 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 comprising or consisting of the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:47, or SEQ ID NO:48.

[0070] Furthermore, the TCR α chain comprises a TCR α chain constant region, which comprises a TCR α chain extracellular constant region, a TCR α chain transmembrane region, and a TCR α chain intracellular constant region 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 set forth in SEQ ID NO:4; Furthermore, 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 order, a TCR α chain extracellular constant region, a TCR α chain transmembrane region, and a TCR α chain intracellular constant region, 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; (2) Provided is 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 or consisting of the amino acid sequence shown in SEQ ID NO:4.

[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] Furthermore, the TCR β chain comprises a TCR β chain constant region, and the TCR β chain constant region comprises 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 the amino acid sequence shown in 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 mutant 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 α chain variable regions and / or TCR β chain variable regions that are capable of binding to and recognizing one or more antigens, including, but not limited to, polypeptide antigens (e.g., NYESO-1, AFP, and MART-1), lipid antigens (e.g., β-GlcCer, eLPA, and LPE), and polysaccharide 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: G12V, TP53 R175H, β-GlcCer, eLPA, LPE, CA199, CA72-4, or CA125, and preferably 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.

[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 containing three complementarity-determining regions (CDRs) contained in the amino acid sequence shown in SEQ ID NO: 17; (2) a TCR β chain variable region containing three complementarity-determining regions (CDRs) contained in the amino acid sequence shown in 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 shown in SEQ ID NO: 26, CDR2 of the amino acid sequence shown in SEQ ID NO: 27, and / or CDR3 of 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 contains a TCR β chain variable region that contains the amino acid sequence shown in SEQ ID NO:25 or consists 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 containing three complementarity-determining regions (CDRs) contained in the amino acid sequence shown in SEQ ID NO: 13; (2) a TCR α chain variable region containing 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 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) It contains the 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 α chain variable region and the TCR β 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 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 CDR3 having the amino acid sequence shown in SEQ ID NO: 16, and the TCR β chain variable region comprises CDR1 having the amino acid sequence shown in SEQ ID NO: 18, CDR2 having the amino acid sequence shown in SEQ ID NO: 19, and CDR3 having the amino acid sequence shown in SEQ ID NO: 20; (4) The TCR α chain variable region comprises CDR1 having the amino acid sequence shown in SEQ ID NO: 22, CDR2 having the amino acid sequence shown in SEQ ID NO: 23, and CDR3 having the amino acid sequence shown in SEQ ID NO: 24, and the TCR β chain variable region comprises 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 CDR3 having the 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 comprising or consisting of the amino acid sequence shown in SEQ ID NO:21, and those comprising or consisting of the amino acid sequence shown in SEQ ID NO:25.

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

[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 TCR alpha chain constant regions and / or TCR beta chain constant regions described above. For example, the TCR alpha chain constant region and 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 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 α chain variable region has the amino acid sequence of SEQ ID NO: 13, and the F5-TCR β chain variable region has the amino acid sequence of SEQ ID NO: 17; Furthermore, the F5-WT TCR α chain constant region has the amino acid sequence of SEQ ID NO:5, and the F5-WT TCR β chain constant region has the amino acid sequence of SEQ ID NO:11; Furthermore, the F5-SA TCR α chain constant region has the amino acid sequence of SEQ ID NO:6, and the F5-SA TCR β chain constant region has the amino acid sequence of SEQ ID NO:11; Furthermore, the F5-KR TCR α chain constant region has the amino acid sequence of SEQ ID NO:5, and the F5-KR TCR β chain constant region has the amino acid sequence of SEQ ID NO:12; Furthermore, the F5-dMUT TCR α chain constant region has the amino acid sequence of SEQ ID NO:6, and the F5-dMUT TCR β chain constant region has 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 has the amino acid sequence of SEQ ID NO:21, or the 1G4-TCR beta chain variable region has the amino acid sequence of SEQ ID NO:25; Furthermore, the 1G4-WT TCR α chain constant region has the amino acid sequence of SEQ ID NO:5, and the 1G4-WT TCR β chain constant region has the amino acid sequence of SEQ ID NO:11; Furthermore, the 1G4-SA TCR α chain constant region has the amino acid sequence of SEQ ID NO:6, and the 1G4-SA TCR β chain constant region has the amino acid sequence of SEQ ID NO:11; Furthermore, the 1G4-KR TCR α chain constant region has the amino acid sequence of SEQ ID NO:5, and the 1G4-KR TCR β chain constant region has the amino acid sequence of SEQ ID NO:12.

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

[0088] In another aspect, any of the above-mentioned 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 of 3 to 25 amino acid residues in length in which 1 to 5 adjacent repeats of motifs such as GGGS, GGGGS, GSGSA, and GGSGG are linked together.

[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 the 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 the 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 is transcriptionally active in a host cell, a transcription terminator sequence that is recognized in a host cell to terminate transcription and that is operably linked to the 3' end of the coding sequence, and a preamble sequence operably linked to the 5' end of the coding sequence that is important for host cell translation.

[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 CRISPR Gene editing vectors, for example, retroviral vectors, lentiviral vectors, baculoviral vectors, herpesvirus vectors, adenoviral vectors, and adeno-associated virus (AAV) vectors.

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

[0115] Typically, expression of a polynucleotide sequence encoding a 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, and animal viruses.

[0116] Suitable expression vectors contain one or more promoter sequences that function in the organism, 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), and 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 turn on when expression of the polynucleotide sequence is expected and turn off when expression is not expected.

[0118] Selectable markers include marker genes and reporter genes that facilitate the identification of selectively expressing cells in a cell population infected with 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 genes, luciferase, chloramphenicol acetyltransferase, etc.

[0119] In another aspect, the present application provides an engineered cell comprising any of the foregoing 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, e.g., 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 containing said nucleic acid constructs can be used by conventional recombinant DNA techniques to express or produce TCRs or engineered cells containing said TCRs. Typically, the method comprises: (1) transforming or transducing a suitable host cell with a TCR nucleic acid construct of the present application or a vector containing said nucleic acid construct; (2) culturing host cells in an appropriate medium; Culture medium or host The cells are then transformed into the engineered cells of the present invention. Cells and (3) isolating and purifying the product.

[0124] Methods for introducing various nucleic acid sequences or vectors into engineered cells are now well known in the art, and 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 host cells. Retroviral infection is a method currently widely used to infect primary cells of human or mouse origin. In some embodiments, various mutant TCR nucleic acid sequences are constructed into retroviral vectors, which are then packaged with a preferred viral envelope or capsid protein to produce viral particles. The recombinant viral particles are then delivered to a host or in vitro cultured cells, where expression of the gene of interest is achieved in the host cells.

[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, this includes detection of the memory phenotype of TCR-T cells, detection of the exhaustion phenotype of TCR-T cells in an in vitro exhaustion model, and detection of the mitochondrial phenotype of TCR-T cells. The above-mentioned detection of the TCR-T cell memory phenotype and mitochondrial phenotype was detected using cell flow cytometry after activation of human primary T cells. The above-mentioned detection of mutant TCR-T cell exhaustion uses an in vitro constructed exhaustion model. Specifically, TCR-T cells with various mutations are stimulated multiple times in vitro using a CD3 antibody, and then the expression of exhaustion-associated molecules is detected by flow cytometry.

[0129] In another aspect, the present application provides a method for producing a method of manufacturing a pharmaceutical composition comprising: (1) Inhibition of T cell receptor degradation 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.

[0130] Meanwhile, the mutant TCR-T cell therapy of 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 used alone or in combination, the dosage and frequency of the above therapies are determined by various factors, such as the characteristics and severity of the disease. Theoretically, the TCR-T cell therapy of the present application can be used multiple times at a low dose, and the above dose range is 10 4 ~10 9The adoptive therapy method of TCR-T cells according to the present application is carried out in accordance with internationally recognized adoptive techniques, and in one example of the present application, 5×10 6 This is achieved by injecting 100 TCR-T cells into the tail vein. In principle, those used in combination 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 of manufacturing a pharmaceutical composition 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, several terms are defined below.

[0133] T cell receptors (TCRs) are molecules present on the surface of T cells that recognize peptide-MHC complexes. 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 the 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 a domain of the TCR α or β chain that is involved in binding of the TCR to an antigen-MHC complex. The variable regions of the α and β chains of native TCRs generally have similar structures, with each structural region containing four conserved framework regions (FRs) and three hypervariable regions or complementarity-determining regions (CDRs). Of these, CDR3 of each variable region is the primary CDR responsible for recognizing 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 α chain of the present application can be a human TCR α chain, a humanized TCR α chain, a chimeric TCR α chain, or a murine-derived TCR α chain. In some embodiments of the present application, the TCR α chain is a chimeric TCR α chain, which includes sequences from multiple species, such as sequences from human and mouse. For example, replacing a human TCR constant region with a murine counterpart can 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 murine 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 a human and a mouse.

[0137] The TCR α chain constant region and / or TCR β chain constant region described herein comprises an extracellular constant region, a transmembrane region, and an intracellular constant region, which are linked in order. The extracellular constant region may include 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 anchors of the TCR α chain and the TCR β chain and the CD3 subunit to form the TCR-CD3 complex. Possible functions of the intracellular constant region include being involved in conformational changes in 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 is presented intracellularly by an MHC molecule or an MHC-like molecule and can be bound by an antibody or a T cell receptor (TCR), including, but not limited to, polypeptide antigens (e.g., NYESO-1, AFP, and MART-1), lipid antigens (e.g., β-GlcCer, eLPA, and LPE), or polysaccharide antigens (e.g., CA199, CA72-4, and CA125). The antigen may also 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 human beings, such as the α chain, β chain, T cell receptor, and nucleic acids described herein. Isolated material can be largely or substantially free from components that normally accompany it in its natural state, or it can be manipulated to an artificial state with components that normally accompany it in its natural state. Isolated material can be in natural, chemically synthesized, or recombinant form. Isolated material can alternatively be in enriched, partially purified, or purified form.

[0140] It should be understood that "wild type" in the present invention means a region that has at least 90% to 100% identity to a naturally occurring amino acid sequence or encoding nucleic acid sequence, or a region that has 1 to 10 or 1 to 5 amino acid mutations (particularly conservative amino acid substitutions) to a naturally occurring amino acid sequence or encoding nucleic acid sequence, but still has the same or similar activity and / or function as the region.

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

[0142] The term "mutation" can be understood to mean the substitution, deletion, or addition of one or more amino acids or nucleic acids. For example, conservative amino acid substitutions can be made. Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid with particular physical and / or chemical properties is replaced with another amino acid with the same chemical or physical properties. For example, conservative amino acid substitutions can be made by substituting an acidic amino acid for another acidic amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid for another basic amino acid (e.g., Lys, Arg), or an amino acid with a polar side chain for another amino acid with a polar side chain (e.g., Asn, Cys, Gin, Ser, Thr, Tyr, etc.). Conservative substitutions can be made, for example, based on similarities 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 a region in which at least one serine in the wild-type TCR α chain intracellular constant region is substituted with alanine. A mutated TCR β chain intracellular constant region refers to a region in which at least one lysine in the wild-type TCR β chain intracellular constant region is substituted with arginine or alanine.

[0144] The identity between sequences is calculated 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 into 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%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. The amino acid residues or nucleic acids at 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 as the corresponding position in the second sequence, the molecules are identical at this position.

[0146] Sequence comparison and calculation of the percentage identity between two sequences may be performed 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 into the GAP program in the GCG software package (available at http: / / www.gcg.com) using a Blossum 62 matrix or a PAM250 matrix, a gap weight of 16, 14, 12, 10, 8, 6, or 4, and a length weight 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, a gap weight of 40, 50, 60, 70, or 80, and a length weight 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 "query sequences" to perform searches against common databases, e.g., to identify other family member or related sequences.

[0149] As known in the art, the terms "nucleotide" and "nucleic acid," which may be used interchangeably herein, refer to a nucleic acid chain of any length, including DNA and RNA. A nucleic acid may be deoxyribonucleic acid, ribonucleic acid, modified nucleic acids or bases, 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 a skilled artisan preparing a cDNA library for PCR amplification.

[0151] In this application, the terms "wild type" and "WT" can be used interchangeably. "SA" refers to a mutation of at least one serine in the amino acid sequence of the α chain intracellular constant region to alanine. "KR" refers to a mutation of at least one lysine in the amino acid sequence of the β chain intracellular constant region to arginine. It should be understood that "dMUT" refers to the simultaneous presence of the "SA" mutation in the α chain intracellular constant region and the "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 TCRs 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 this application, 1G4-TCR refers to a TCR capable of recognizing the tumor antigen NYESO-1, and the 1G4-WT TCR, 1G4-SA TCR, 1G4-KR TCR, and 1G4-dMUT TCR included therein 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 procedures, it should be understood that the designed appropriate enzyme cleavage sites will introduce one or more irrelevant residues at both ends of the expressed amino acid sequence without affecting the activity of the target sequence, including, but 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.

[0157] It should be understood that for purposes of purifying 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, etc.

[0158] It should be understood that the above examples (embodiments) are all illustrative and are not intended to encompass all possible embodiments within the scope of the claims. Various modifications and variations 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 other embodiments of the present application that may not be explicitly described. Therefore, the above examples merely 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] JPEG2023131053000002.jpg194147JPEG2023131053000003.jpg193147JPEG2023131053000004.jpg192146JPEG2023131053000005.jpg191144JPEG2023131053000006.jpg191145JPEG2023131053000007.jpg190146JPEG2023131053000008.jpg172148Example 1. Construction of TCR expression vector

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

[0161] As described above, each F5-TCR or 1G4-TCR comprises a TCR α chain and a TCR β chain, respectively. The TCR α chain is composed 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, which are linked in this order. The TCR β chain is composed 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, which are linked in this order. The TCR α chain and the TCR β chain are linked in tandem 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 was mutated to alanine in the TCR α chain intracellular constant region and lysine was 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 set forth in SEQ ID NOs: 39 to 46, respectively.

[0164] All TCR nucleotide 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-transfected with the envelope plasmid pHIT60 / RD114 into HEK293T cells to package retrovirus. Specifically, the ratio of the packaging system was MSGV-TCR:pHIT60:RD114 = 2:2:1.3. The mixture was homogenized in OPTI-DMEM medium, and 15.9 μl of TransIT293-Mirus transfection reagent (Mirusbio #MIR 2700) was added. After homogenization by pipetting, the mixture was left at room temperature for 25 minutes. The liposome mixture was then added to an HEK293T cell culture dish and cultured in a 37°C incubator for 48 hours. The virus solution was then collected, filtered through a 0.22 μm filter, and stored in a -80°C refrigerator. Example 3. Method for preparing and culturing human primary mutant TCR-T cells

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

[0167] Infection of human primary T cells with retrovirus: 750 μl of resuspended human primary T cells in a 24-well plate was aspirated into an EP tube (Axygen #MCT-105C) and centrifuged at 2500 rpm for 5 minutes. The supernatant was discarded. The T cells were resuspended in 500 μl of the corresponding virus solution (i.e., mutant TCR retrovirus), 0.75 μl of Polybrene (SANTA CRUZ #SC-134220), and the mixture was pipetted uniformly and added to the corresponding 24-well plate. The infection volume was 750 μl. After centrifugation at 2500 rpm for 90 minutes at 30°C, 500 μl of the supernatant from the 24-well plate was aspirated into an EP tube, centrifuged at 2500 rpm for 5 minutes, the supernatant was discarded, and 750 μl of complete T cell medium (1.25x rhIL-2) was added to the 24-well plate. The plate was then placed in a 37°C incubator and cultured for 24 hours. The next day, a second viral infection was performed, and the specific steps were the same as those described above. After infection, the human primary T cells were replenished with fresh RPMI-1640 complete medium every 1–2 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 minutes. The supernatant was discarded, and flow cytometry antibodies prepared in FACS buffer (containing 2% serum in 1x PBS) were added. The cells were stained on ice for 25 minutes away from light, washed once with FACS buffer, and detected by a flow cytometer.

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

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

[0171] In vivo, we performed an experiment to detect downregulation of TCR expression by tumor antigen stimulation. Twelve days after the introduction of 1G4-TCR-T cells, we detected TCR expression on the surface of TCR-T cells in the spleen and tumors of tumor-bearing mice inoculated with K562-NYESO-1. As shown in Figure 1(A), tumor antigen stimulation downregulated TCR expression in tumor tissues. In vitro, we performed an experiment to detect downregulation and degradation of TCR expression by target cell antigen stimulation. 1G4-TCR-T cells were mixed with K562-NYESO-1 target cells or K562-MART-1 non-target cells at a 1:1 ratio in a 24-well plate and co-incubated at 37°C for 12 hours (the specific time was determined experimentally). Cells were then aspirated and subjected to flow FACS analysis. TCR downregulation was measured by staining the TCR on the T cell surface, while TCR degradation was detected by staining intracellular TCR after cell fixation and permeabilization. As shown in Figure 1(B), target cell antigen stimulation promoted TCR downregulation and degradation. Experiments on TCR downregulation and degradation by hCD3 antibody stimulation: After activation, 1G4-TCR T cells and F5-TCR T cells were resuspended and added to a 24-well plate coated with hCD3 antibody. They were then co-incubated in a 37°C incubator for 12 hours (the specific time was determined experimentally). Cells were then aspirated and subjected to flow FACS analysis. TCR downregulation was measured by staining the TCR on the T cell surface. Analysis of TCR degradation levels was performed by staining intracellular TCR after cell fixation and permeabilization. As shown in Figure 1(C) and (D), in vitro CD3 antibody stimulation promoted TCR downregulation and degradation in humanized T cells. Experiments comparing the expression of each mutant TCR: After activation, each mutant 1G4-TCR T cells were resuspended and subjected to FACS analysis to detect the expression of different TCRs. As shown in FIG. 2(B), there was no obvious difference in the expression of each mutant TCR compared to the wild-type TCR.Comparative experiments on downregulation and degradation of each mutant TCR following hCD3 antibody stimulation: 1G4-TCR and F5-TCR T cells were resuspended after activation and added to a 24-well plate coated with hCD3 antibody. They were then co-incubated at 37°C for the required time. TCR downregulation was measured by TCR surface staining, and TCR degradation was measured by intracellular TCR staining after cell fixation and permeabilization. As shown in Figure 2 (C and D), compared with the WT TCR, the SA TCR, KR TCR, and dMUT TCR in the mutant groups exhibited greater T cell membrane retention and significantly resisted downregulation and degradation upon antibody stimulation. This was particularly evident in the dMUT TCR group. As shown in Figure 2 (E), surface TCR expression at various time points following hCD3 antibody stimulation demonstrated that the dMUT TCR had the strongest resistance to degradation compared to the 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 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), SA TCR, KR TCR, and dMUT TCR T cells showed significantly higher expression of CD27 compared to the WT TCR group. + CD45RO + The proportion of central memory T cells (CMT 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 T cell memory molecules CD62L and TCF1 was also elevated, with dMUT TCR-T cells being the most prominent.

[0173] Construction of an in vitro T cell depletion model: 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 minutes. The TCR-T cells were resuspended in complete medium and plated onto 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, after which the above steps were repeated twice. After the incubation, the TCR-T cells were harvested and analyzed by flow cytometry. This in vitro depletion model was constructed based on a related publication known in the art (Santosha A. Vardhana et al., Nat Immunol. 2020 Sep;21(9):1022-1033). The detection indicators were depletion-related molecules such as PD-1, TIM3, LAG-3, and TOX, as well as cytokines such as TNF-α and IFN-γ. As shown in Figure 3 (D–F), the expression of the exhaustion-related molecules LAG-3 and TOX was significantly reduced 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 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. T cells were then cultured in RPMI-1640 complete medium for 12 days, and 2 × 10 5 The cells were absorbed into an EP tube and stained with MitoTracker Green (Invitrogen #M7514) at a final concentration of 50 nM. After staining for 1 hour at 37°C, away from light, flow FACS detection was performed. As shown in Figure 4(A), there was no significant change in the number of mitochondria in each mutant TCR-T cell compared to WT TCR cells. For the detection of mitochondrial membrane potential, 2 × 10 5Cells were aspirated into EP tubes and stained with TMRE (Invitrogen #T669) at a final concentration of 200 nM. After staining for 1 hour at 37°C, away from light, flow FACS detection was performed. As shown in Figure 4(B), compared to WT TCR cells, SA TCR, KR TCR, and dMUT TCR-T cells, especially dMUT TCR-T cells, showed significantly reduced mitochondrial membrane potential. For the detection of mitochondrial reactive oxygen species, 2 × 10 5 Cells were aspirated into EP tubes and stained with MitoSOX (Invitrogen #M36008) at a final concentration of 5 μM for 1 hour at 37°C, protected 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. Summarizing the results of the above examples, although each TCR mutation did not affect the number of mitochondria in T cells, mutant TCR cells, particularly dMUT TCR T cells, exhibited healthier mitochondria, which correlates with the memory phenotype and resistance to T cell exhaustion during in vitro culture of dMUT TCR T cells.

[0175] Detection of metabolic indicators: Human primary T cells infected with the mutant 1G4-TCR retrovirus were cultured in vitro for 12 days in RPMI-1640 complete medium at 37°C, and 2 × 10 5Cells were placed in EP tubes and centrifuged at 2500 rpm for 5 minutes. The supernatant was discarded. The T cells were then resuspended in glucose-free, serum-free medium and placed in a 24-well plate. The cells were treated with 50 μM 2-NBDG (Invitrogen #N13195) or 1 μM Bodipy FL C16 (Invitrogen #D3821) at a final concentration of 50 μM. The cells were then incubated at 37°C in the dark for 30 minutes, after which they were subjected to flow FACS detection. Depending on the experimental requirements, cells may be labeled with other markers before flow cytometry detection. Figure 4 (D and E) shows that compared to WT TCR cells, SA TCR, KR TCR, and dMUT TCR T cells showed significantly reduced glucose uptake and no significant changes in extracellular fatty acid uptake. Example 8. Detection of anti-tumor 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 superior antitumor effects to wild-type TCR-T cells, we first administered 8 × 10 5 K562-NYESO-1 cells were inoculated subcutaneously into the axilla of the right hind leg of NSG mice. After the target cells had grown subcutaneously in the mice for approximately 8–12 days, tumor volume was measured using a caliper. Specifically, tumors with a volume of 80 mm were measured. 3 ~120mm 3 In each case, 5 × 10 infected mice were infected with each mutant 1G4-TCR. 6 Human primary T cells were injected into the tail vein of NSG mice. Tumor volume was measured every two days. Approximately 14 days later (the specific duration of the procedure was determined based on the experimental outcome), blood, spleen, and tumor tissues were collected from the NSG mice. The tumor tissue was weighed, and then red blood cells in the mouse blood and spleen were lysed using an erythrocyte lysing kit (Biolegend #420301). The mouse tumor tissue was pulverized and then subjected to Percoll density gradient centrifugation (Cytiva #17089110). The human primary T cells introduced via the tail vein were collected from the mouse blood, spleen, and tumor tissue and analyzed by flow cytometry staining.

[0177] Specific results are shown in Figures 5 and 6. First, in Figure 5(A), tumor growth volume was statistically analyzed, revealing that the tumor growth rate was significantly slower in the experimental group transfected with KR TCR-T cells and dMUT TCR-T cells compared to the WT TCR group. Tumor tissue was weighed, and as shown in Figure 5(B), tumor weight was significantly reduced in the experimental group transfected with KR TCR-T cells and dMUT TCR-T cells. Furthermore, in this example, the transfected CD8 in the spleen, blood, and tumor tissue of tumor-bearing NSG mice was analyzed. + The percentage of TCR-T cells was calculated. 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 indicates that KR TCR-T cells and dMUT TCR-T cells have stronger antitumor persistence in the NSG mouse adoptive immunization model. Further cell phenotype analysis revealed that, as shown in Figure 6(A), the CD27 expression level of SA TCR, KR TCR, and dMUT TCR-T cells in the spleen was significantly higher than that of wild-type TCR-T cells. + CD45RO + The proportion of central memory T cells (CMCs) was significantly increased. Furthermore, analysis of TCR-T cells in tumor tissues revealed that dMUT TCR-T cells expressed higher levels of T cell memory molecules CD62L and CCR7 compared with WT TCR-T cells, as shown in Figure 6 (B and C). Figure 6 (D) shows the expression of PD-1 in SA TCR, KR TCR, and dMUT TCR groups in tumor tissues. + TIM-3 +The percentage of double-positive cells was significantly lower in the mutant TCR-T cells than in the WT TCR-T cells. Furthermore, by detecting the fluorescence intensity of the inhibitory molecules PD-1, TIM-3, LAG-3, and CD39, as shown in Figure 6 (E-H), we found that the expression of inhibitory molecules was significantly reduced in the mutant TCR-T cells compared to the WT TCR-T cells. Furthermore, as shown in Figure 6 (I), we found that the expression of TOX, a transcription factor important for exhaustion, was significantly reduced in the dMUT TCR-T cells. These results demonstrate that the mutant TCR-T cells are able to resist T cell exhaustion in an in vivo antitumor model. Furthermore, by detecting cytokine production by TCR-T cells in tumor tissues, we found that the adoptively transferred KR TCR-T cell group and the dMUT TCR-T cell group exhibited significantly reduced levels of IFN-γ. As shown in Figure 6 (J and K), these results suggest that the IFN-γ levels were significantly reduced in the adoptively transferred KR TCR-T cell group and the dMUT TCR-T cell group. + CD8 + The percentage of CD8 T cells was significantly increased. This suggests that KR TCR cells and dMUT TCR-T cells have stronger antitumor effects and CD8 + KR TCR-T cells and CD8 + TNF-α in dMUT TCR-T + IFN-γ + A high proportion of double-positive cells was observed. KR TCR-T cells produced more cytokines than WT TCR-T cells, but less than dMUT TCR-T cells, indicating that dMUT TCR-T cells have stronger effector functions in tumor tissues.

[0178] Current TCR-T cell therapies face challenges in collecting a patient's limited T cells to expand them into large numbers, and in tumor patients, the persistent decline in T cell function and the occurrence of T cell exhaustion after adoptive therapy. The present method for engineering mutant TCR-T cells, particularly the dMUT TCR-T engineering, enables TCR-T cells to demonstrate more sustained antitumor effects after in vitro expansion and reinfusion into tumor models, and also resists T cell exhaustion. This suggests that TCR-T cells engineered with the dMUT TCR mutation are expected to achieve sustained and efficient antitumor function in clinical production and therapy.

Claims

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, and the TCR β chain constant region comprises 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 or consisting of the amino acid sequence shown in SEQ ID NO:

10.

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 the isolated T cell receptor is selected from wild-type TCR β chain intracellular constant regions comprising or consisting of the amino acid sequence shown in 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, and the TCR α chain constant region comprises 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 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 the TCR alpha chain optionally further comprises a TCR alpha chain variable region.

4. 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, and the TCR α chain constant region comprises 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 according to claim 4, characterized in that it is selected from wild-type TCR α chain intracellular constant regions comprising or consisting of the amino acid sequence shown in SEQ ID NO:

3.

6. The TCR β chain comprises a TCR β chain constant region, and the TCR β chain constant region comprises 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 the amino acid sequence shown in 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. 10. 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. 10. 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, and 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 containing three complementarity-determining regions (CDRs) contained in the amino acid sequence represented by SEQ ID NO: 17; (2) a TCR β chain variable region containing 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 shown in SEQ ID NO: 26, CDR2 of the amino acid sequence shown in SEQ ID NO: 27, and / or CDR3 of 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) An isolated T cell receptor according to any one of the preceding claims, characterized in that it is selected from TCR β chain variable regions 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 containing three complementarity-determining regions (CDRs) contained in the amino acid sequence represented by SEQ ID NO: 13; (2) a TCR α chain variable region containing 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 TCR α chain variable regions comprising or consisting of the amino acid sequence shown in SEQ ID NO:

21.

12. 10. An isolated T cell receptor according to 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% identity 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 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. (5) The TCR β chain extracellular constant region is derived from the 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% identity 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. 10. The isolated T cell receptor of claim 1, 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 fragment thereof encoding the isolated T cell receptor or fragment thereof according to any one of the preceding claims.

17. A nucleic acid construct comprising the isolated nucleic acid or 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 CRISPR gene editing vector, preferably a retroviral vector.

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

21. 21. The engineered cell of claim 20, which is a T cell, for example a human T cell.

22. 1. A method for producing an engineered cell, comprising: (1) transforming or transducing a host cell with the nucleic acid construct according to claim 17 or the vector according to any one of claims 18 to 19; (2) culturing host cells in an appropriate medium; and (3) isolating and purifying the engineered cells from the medium or the host 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 capacity, 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 drugs 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, wherein the wild-type TCR α-chain intracellular constant region is selected from the wild-type TCR α-chain intracellular constant regions according to claim 5.

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