WO2023131053A1 - 一种t细胞受体及其制备方法和用途 - Google Patents

一种t细胞受体及其制备方法和用途 Download PDF

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WO2023131053A1
WO2023131053A1 PCT/CN2022/143567 CN2022143567W WO2023131053A1 WO 2023131053 A1 WO2023131053 A1 WO 2023131053A1 CN 2022143567 W CN2022143567 W CN 2022143567W WO 2023131053 A1 WO2023131053 A1 WO 2023131053A1
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tcr
chain
amino acid
acid sequence
constant region
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PCT/CN2022/143567
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French (fr)
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李贵登
程洪成
邱雅静
许悦
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苏州系统医学研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • This application relates to the field of T cell receptors, in particular to the construction and application of modified T cell receptors.
  • T cells Malignant tumors seriously threaten human health and life, and are one of the main causes of human death, and its incidence is increasing year by year.
  • Traditional tumor treatment methods are mainly based on surgery, supplemented by radiotherapy and chemotherapy.
  • tumor immunotherapy targets the immune system to stimulate systemic responses to tumors.
  • tumor immunotherapy can significantly improve the prognosis of patients with specific tumor types.
  • the adoptive transfer of antigen-specific T cells is one of the main means of tumor immunotherapy, and has been studied for the treatment of hematological tumors and solid tumors. It is mainly based on the fact that T cell receptors on the surface of T cells can recognize different tumor antigens, so as to kill and eliminate tumor cells.
  • the T cell adoptive therapy that has been studied more at this stage mainly includes CAR-T cell therapy and TCR-T therapy, in which CAR-T is to couple the antigen-binding part of an antibody that can recognize a certain tumor antigen to the CD3- ⁇ chain in vitro as A chimeric protein is used to transfect the patient's T cells through gene transduction to express the chimeric antigen receptor.
  • TCR-T therapy T cells recognize the antigen presented by the Major Histocompatibility Complex (MHC) on the surface of the target cell through the TCR on its surface, so as to achieve direct attack and killing of the target cell.
  • MHC Major Histocompatibility Complex
  • It is a T cell therapy technology developed based on natural TCR or slight modification of TCR. It has broader applicability due to its ability to recognize tumor epitopes presented by major histocompatibility complex molecules on the surface of tumor cells.
  • TCR-T cell therapy depends on the antigen TCR expressed on the surface of T cells, and TCR is a receptor on the surface of T cells, which binds to CD3 in a non-covalent bond to form a TCR-CD3 complex.
  • the antigen presented by MHC activates T cells and promotes the division and differentiation of T cells.
  • most TCRs are heterodimers composed of ⁇ and ⁇ chains, both of which contain an antigen-binding region, a constant region, and a transmembrane domain. Instead, these ⁇ and ⁇ chains are covalently linked by disulfide bonds between conserved cysteine residues located within the anchorage regions of each chain.
  • TCR-T can target most intracellular antigens, not limited to surface antigens, that is, TCR-T can target most tumor antigens, especially the ability to recognize tumor cell intracellular antigens. Therefore, T cell receptors have a wider range of tumor recognition than antibody drugs and CAR-T, which relies on antibodies to recognize tumors.
  • CAR-T has shortcomings in the treatment of solid tumors, and TCR-T technology is expected to solve this problem.
  • TCR-T optimization and transformation methods to improve or enhance the therapeutic effect of TCR-T tumors, which mainly through the modification of the "probe"-TCR that T cells bind to tumor antigens, to strengthen the recognition of T cells against tumor cells.
  • the process improves the affinity of T lymphocytes to tumor cells, so that T cells that have no tumor recognition ability can effectively recognize and kill tumor cells.
  • TCR-T Increase the expression of exogenous TCR to enhance the anti-tumor effect of TCR-T.
  • Murinization of TCR constant region mouseization of TCR constant region by means of genetic engineering to reduce or prevent the mismatch between endogenous and exogenous TCR, promote the preferential pairing of exogenous TCR, and increase the surface antigen of T cells TCR expression level, thereby enhancing the anti-tumor effect of TCR-T; b.
  • TCR constant region cysteine insertion method the constant region of TCR ⁇ chain and ⁇ chain introduces cysteine to promote the preferential pairing of exogenous TCR , increase the surface expression level of exogenous TCR, and reduce the mismatch with the endogenous TCR chain, this method can improve the effectiveness and safety of tumor-reactive T cells;
  • TCR transmembrane region inserts hydrophobic mutations By increasing the hydrophobicity of the transmembrane region of the TCR ⁇ chain and improving TCR stability, the expression of TCR on the surface of T cells is enhanced, thereby improving cell affinity and anti-tumor TCR activity.
  • Synthetic T cell receptor and antigen receptor method (STAR: synthetic T cell receptor and antigen receptor): Insert the VH and VL of the antibody antigen-binding region into the TCR ⁇ constant region, which has both the high affinity of CAR and the TCR The complex has high signaling ability, thereby enhancing the anti-tumor effect of TCR-T.
  • T cell dysfunction (1) T cell dysfunction. a. Continuous antigenic stimulation inside the tumor microenvironment leads to exhaustion of CD8 + T cells, such as increased expression of inhibitory receptors such as PD-1, Lag-3, CD39, and TIM-3; b. The tumor microenvironment with low oxygen and low pH makes The progressive loss of T cell effector function, such as decreased secretion of pro-inflammatory cytokines such as IFN- ⁇ and TNF- ⁇ , leads to poor proliferation and self-renewal ability of T cells, and metabolic activity disorders.
  • CD8 + T cells such as increased expression of inhibitory receptors such as PD-1, Lag-3, CD39, and TIM-3
  • the tumor microenvironment with low oxygen and low pH makes The progressive loss of T cell effector function, such as decreased secretion of pro-inflammatory cytokines such as IFN- ⁇ and TNF- ⁇ , leads to poor proliferation and self-renewal ability of T cells, and metabolic activity disorders.
  • TCR-T cells The expression level of TCR on the surface of T cells decreased. Antigen stimulation in the tumor microenvironment leads to a decrease in the expression of exogenous TCR on the surface of CD8 + T cells or accelerated degradation, so that there are not enough TCR-T cells to attack tumor cells.
  • the purpose of this application is to provide a T cell receptor that can inhibit degradation and enhance the anti-tumor effect of TCR-T cells.
  • the present application provides a TCR ⁇ chain isolated from a T cell receptor (TCR) or a fragment thereof, the TCR ⁇ chain includes a TCR ⁇ chain constant region, and the TCR ⁇ chain constant region includes sequentially connected TCR ⁇ chain cells External constant region, TCR ⁇ chain transmembrane region and TCR ⁇ chain intracellular constant region, the TCR ⁇ chain intracellular constant region is a mutated TCR ⁇ chain intracellular constant region, and the mutated TCR ⁇ chain intracellular constant region It is formed by mutation of at least one serine in the intracellular constant region of the wild-type TCR ⁇ chain to alanine.
  • TCR T cell receptor
  • the application provides a TCR beta chain isolated from a T cell receptor (TCR) or a fragment thereof, the TCR beta chain comprising a TCR beta chain constant region, the TCR beta chain constant region comprising sequentially connected TCR beta chains Extracellular constant region, TCR ⁇ chain transmembrane region and TCR ⁇ chain intracellular constant region, the TCR ⁇ chain intracellular constant region is a mutated TCR ⁇ chain intracellular constant region, and the mutated TCR ⁇ chain intracellular constant region The region is formed by mutation of at least one lysine to arginine or alanine in the intracellular constant region of the wild-type TCR ⁇ chain.
  • TCR T cell receptor
  • the application provides an isolated T cell receptor or a fragment thereof, the T cell receptor comprising any one of the TCR ⁇ chain and/or TCR ⁇ chain.
  • the application provides one or more T cell receptors, such as F5-WT TCR, F5-SA TCR, F5-KR TCR or F5-dMUT TCR, such as 1G4-WT TCR, 1G4-SA TCR, 1G4 -KR TCR or 1G4-dMUT TCR.
  • T cell receptors such as F5-WT TCR, F5-SA TCR, F5-KR TCR or F5-dMUT TCR.
  • the application provides an isolated nucleic acid or a fragment thereof, which encodes any one of the aforementioned isolated TCR ⁇ chains or fragments thereof, TCR ⁇ chains or fragments thereof, or T cell receptors or fragments thereof.
  • the present application provides a nucleic acid construct comprising any one of the aforementioned isolated nucleic acids or fragments thereof.
  • the present application provides a vector comprising any one of the aforementioned nucleic acid constructs.
  • the present application provides an engineered cell comprising any one of the aforementioned nucleic acid constructs or vectors.
  • the present application also provides a TCR complex, which is produced in T cells by any one of the aforementioned TCR nucleic acid constructs of the present application.
  • the present application provides functional testing of TCR-T cells during in vitro culture.
  • the present application provides the isolated TCR ⁇ chain or fragment thereof, TCR ⁇ chain or fragment thereof, T cell receptor or fragment thereof, nucleic acid or fragment thereof, nucleic acid construct, vector or engineering described in any one of the foregoing
  • mutant TCR-T cell therapy provided by this application can be used alone or in combination with other therapies, or in combination with PD-1/PD-L1 antibodies, or in combination with cytokine therapy, or in combination with radiotherapy and chemotherapy.
  • the present application provides a method for transforming T cell receptors, including:
  • This application uses the mutation of the ubiquitination modification site in the intracellular constant region of the T cell receptor ⁇ chain and ⁇ chain to inhibit the degradation of the T cell receptor after the activation of the TCR antigen signal and maintain the level of TCR on the cell surface. This method is applicable Different TCR-T cell therapy.
  • SA TCR, KR TCR and dMUT TCR cells after antigen stimulation, compared with WT TCR cells (wild-type TCR cells), SA TCR, KR TCR and dMUT TCR cells have more cell surface TCR expression, and in the process of in vitro culture, SA TCR, KR TCR and dMUT TCR cells exhibited a phenotype that differentiated toward central memory T cells, and in vitro depletion models showed lower levels of depletion in KR TCR and dMUT TCR cells compared to WT TCR, and this phenotypic shift benefited SA TCR, KR TCR and a healthier mitochondrial state in dMUT TCR cells, which is manifested in the mutant TCR-T cells cultured in vitro exhibiting lower mitochondrial membrane potential and ROS levels.
  • the present application found that compared with WT TCR, KR TCR and dMUT TCR cells had stronger anti-tumor effects through the mouse xenograft tumor-T cell adoptive model, and found that the dMUT TCR mutant group was more effective in mouse spleen and There are more TCR-T cells in the blood, while in tumor tissue, the proportion of KR TCR and dMUT TCR cells is significantly increased.
  • SA TCR, KR TCR and dMUT TCR groups accumulated more central memory T cells in mouse spleen, while dMUT-mutated TCR-T cells exhibited lower T cell Depleted phenotype and proliferative capacity. Therefore, KR TCR and dMUT TCR cells showed a more advantageous potential to inhibit tumor growth during the adoptive treatment of equivalent TCR-T cells.
  • this application not only inhibits the degradation of TCR, but also improves the efficacy of TCR-T cell therapy by mutating the amino acids of the constant region of TCR cells, which can effectively curb the growth of tumors, and the tumors are not limited to solid tumors. , can be hematoma or lymphoma.
  • FIG. 1 Antigen stimulation promotes the degradation of cell surface TCRs.
  • A In K562-NYESO-1-inoculated tumor-bearing mice, 12 days after 1G4-TCR-T cells were adopted, the expression of TCR on the surface of TCR-T cells in the spleen and tumor was detected.
  • B In vitro functional experiments were performed on primary human T cells. 1G4-TCR-T cells were cultured in vitro and incubated with K562-NYESO-1 and K562-MART-1 cells for 12 hours to detect the degradation of TCR.
  • C Activated F5-TCR-T cells were cultured in vitro, and after CD3 antibody stimulation for 12 hours, the degradation of TCR was detected.
  • D Activated 1G4-TCR-T cells were cultured in vitro, and after being stimulated with CD3 antibody for 12 hours, the degradation of TCR was detected.
  • FIG. 1G4-TCR-T cells Result plot of mutations inhibiting TCR downregulation and degradation. All in vitro functional experiments were performed on primary human T cells.
  • A Schematic diagram of degradation-inhibited TCR mutations.
  • B Expression of mutated 1G4-TCR on primary human T cells.
  • C Different mutant F5-TCR-T cells activated in vitro, after CD3 antibody stimulation for 12 hours, the degradation of different TCRs was detected ("R” means no CD3 antibody stimulation, "S” means CD3 antibody stimulation).
  • R means no CD3 antibody stimulation
  • S means CD3 antibody stimulation
  • D Different mutant 1G4-TCR-T cells activated in vitro, after CD3 antibody stimulation for 12 hours, the degradation of different TCRs was detected ("R” means no CD3 antibody stimulation, "S” means CD3 antibody stimulation).
  • E 1G4-TCR-T cells with different mutations activated in vitro, stimulated with CD3 antibody for different times, and the expression of different TCRs were detected.
  • FIG. 3 Diagram of the effect of mutations on the function of 1G4-TCR-T cells in vitro. All in vitro functional experiments were performed on primary human T cells.
  • B Activated 1G4-TCR-T cells, detecting memory T cell differentiation indicator CD62L.
  • C 1G4-TCR-T cells activated in vitro, and memory T cell differentiation index TCF-1 was detected.
  • D Using the in vitro depletion model, the expression of LAG-3, a T cell depletion molecule, was detected in different mutant TCR-T cells.
  • FIG. 4 Comparison of mitochondrial functional status in 1G4-TCR-T cells with different mutations. All in vitro experiments were performed on primary human T cells.
  • A The number of intracellular mitochondria was detected after different TCR-T cells were activated in vitro for 12 days.
  • B Detection of changes in mitochondrial membrane potential in mutated 1G4-TCR-T cells.
  • C 1G4-TCR-T cells activated in vitro, and the production level of mitochondrial ROS in the cells was detected.
  • D After different TCR-T cells were activated in vitro for 12 days and treated with 2-NBDG, FACS flow analysis was performed to detect the uptake of glucose by TCR-T cells.
  • E Bodipy FL C16 uptake assay to detect the utilization efficiency of extracellular fatty acids by TCR-T cells in different mutant groups.
  • FIG. 1G4-TCR-T cells Comparison of anti-tumor effects of 1G4-TCR-T cells with different mutations.
  • A K562-NYESO-1NSG tumor-bearing mice inoculated with 1G4-TCR-T cells, statistical diagrams of tumor size at different time points. After K562-NYESO-1NSG tumor-bearing mice adopted T cells, the weight of tumor tissue in different mutation groups was detected on the twelfth day (B), and the blood (C), spleen (D) and tumor tissue (E) of tumor-bearing mice were detected at the same time ) in the proportion of adoptive TCR-T cells.
  • FIG. 6 Diagram of the effect of mutations on the function of 1G4-TCR-T cells in mice.
  • A In the spleen of NSG tumor-bearing mice, CD45RO and CD27 were used as memory T cell differentiation indicators, and different mutation groups were detected by flow cytometry.
  • B In the tumor tissue of NSG tumor-bearing mice, the expression of CD62L, a memory T cell differentiation indicator in different mutation groups, was detected.
  • C In tumor tissues of NSG tumor-bearing mice, the expression of memory T cell differentiation index CCR7 in different mutation groups was detected.
  • D In the tumor tissue of NSG tumor-bearing mice, the proportion and statistical chart of PD-1 + TIM-3 + double-positive cells of exhausted T cells in different mutation groups were detected.
  • TCR-T cell immunosuppressive molecules PD-1(E), TIM-3(F), LAG-3(G) and CD39(H) in different mutation groups in tumor tissues of NSG tumor-bearing mice were detected.
  • I In the tumor tissues of NSG tumor-bearing mice, the expression of transcription factor TOX in different mutation groups was detected.
  • J Detection of the proportion of IFN- ⁇ + positive CD8 + T cells in the tumor tissues of NSG tumor-bearing mice.
  • K Detection of the proportion of IFN- ⁇ + TNF- ⁇ + double-positive TCR-T cells in the tumor tissues of NSG tumor-bearing mice.
  • the present application provides a TCR ⁇ chain isolated from a T cell receptor (TCR) or a fragment thereof, the TCR ⁇ chain includes a TCR ⁇ chain constant region, and the TCR ⁇ chain constant region includes sequentially connected TCR ⁇ chain cells External constant region, TCR ⁇ chain transmembrane region and TCR ⁇ chain intracellular constant region.
  • the TCR ⁇ chain further includes a TCR ⁇ chain variable region.
  • the TCR ⁇ chain constant region and its corresponding TCR ⁇ chain extracellular constant region, TCR ⁇ chain transmembrane region and TCR ⁇ chain intracellular constant region are respectively derived from human, mouse or other Wild-type T cell receptor (TCR) constant region of a mammalian species.
  • TCR Wild-type T cell receptor
  • the intracellular constant region of the TCR ⁇ chain is a wild-type TCR ⁇ chain intracellular constant region of human origin, mouse origin or other mammalian species, for example comprising the amino acid sequence shown in SEQ ID NO: 3 or composed of The wild-type TCR ⁇ chain intracellular constant region composed of the amino acid sequence.
  • the TCR ⁇ chain intracellular constant region is a mutated TCR ⁇ chain intracellular constant region
  • the mutated TCR ⁇ chain intracellular constant region is at least one of the wild-type TCR ⁇ chain intracellular constant regions
  • Serine is mutated to alanine, for example, at least one serine in the intracellular constant region of the wild-type TCR ⁇ chain of human, mouse or other mammalian species is mutated to alanine to form a mutated TCR ⁇ chain intracellular constant region.
  • the intracellular constant region of the mutated TCR ⁇ chain is formed by mutation of at least one serine in the intracellular constant region of the wild-type TCR ⁇ chain to alanine, and the intracellular constant region of the wild-type TCR ⁇ chain comprises The amino acid sequence shown in SEQ ID NO: 3 or composed of said amino acid sequence, for example, in the amino acid sequence shown in SEQ ID NO: 3, one or two (all) serines are mutated to alanine to form a mutated TCR ⁇ chain intracellular constant region.
  • the intracellular constant region of the mutated TCR ⁇ chain comprises or consists of the amino acid sequence shown in SEQ ID NO:4.
  • the TCR ⁇ chain includes a TCR ⁇ chain variable region, and the TCR ⁇ chain variable region can bind and recognize one or more antigens, including but not limited to polypeptide antigens (such as NYESO-1 , AFP and MART-1), lipid antigens (such as ⁇ -GlcCer, eLPA and LPE) and polysaccharide antigens (such as CA199, CA72-4 and CA125).
  • polypeptide antigens such as NYESO-1 , AFP and MART-1
  • lipid antigens such as ⁇ -GlcCer, eLPA and LPE
  • polysaccharide antigens such as CA199, CA72-4 and CA125.
  • 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.
  • 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, ROR
  • MAGE-A1/A3/A4/A6/A10/C2 gp100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1/2, BRLF-1, BMLF-1, HPV- One or more antigens among 16E6/E7, KRAS G12D, KRAS G12V, TP53R175H, ⁇ -GlcCer, eLPA, LPE, CA199, CA72-4 or CA125.
  • the antigen is a tumor antigen, such as MAGE-A1/A3/A4/A6/A10/C2, gp100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1/ 2.
  • a tumor antigen such as MAGE-A1/A3/A4/A6/A10/C2, gp100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1/ 2.
  • the antigen may be a cell surface antigen, or an intracellular antigen.
  • the TCR ⁇ chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, which recognizes the tumor antigen MART-1; or comprises the amino acid sequence shown in SEQ ID NO: 21 or consists of Its composition, which recognizes the tumor antigen NYESO-1.
  • the TCR ⁇ chain variable region comprises a complementarity determining region (CDR), such as CDR1, CDR2, CDR3.
  • CDR complementarity determining region
  • the complementarity determining region is included in the amino acid sequence shown in SEQ ID NO: 13, or in the amino acid sequence shown in SEQ ID NO: 21.
  • the TCR ⁇ chain variable region comprises 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 CDR3 of the amino acid sequence shown in SEQ ID NO:16 , which recognizes the tumor antigen MART-1; or comprises 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 CDR3 of the amino acid sequence shown in SEQ ID NO:24, which recognizes the tumor Antigen NYESO-1.
  • the extracellular constant region of the TCR ⁇ chain is the extracellular constant region of the TCR ⁇ chain derived from human, mouse or other mammalian species.
  • the extracellular constant region of the TCR ⁇ chain comprises the amino acid sequence of SEQ ID NO: 1, or comprises the amino acid sequence of SEQ ID NO: 1 having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutated amino acid sequences, or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence of SEQ ID NO: 1 %, 98% or 99% of an amino acid sequence, or consists of said amino acid sequence.
  • the TCR ⁇ chain transmembrane region is a TCR ⁇ chain transmembrane region derived from human, mouse or other mammalian species.
  • the transmembrane region of the TCR ⁇ chain comprises the amino acid sequence of SEQ ID NO: 2, or comprises the amino acid sequence of SEQ ID NO: 2 having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutated amino acid sequences, or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of an amino acid sequence, or consists of said amino acid sequence.
  • the TCR alpha chain constant region comprises or consists of the amino acid sequence of SEQ ID NO:5 or SEQ ID NO:6.
  • the application provides a TCR beta chain isolated from a T cell receptor (TCR) or a fragment thereof, the TCR beta chain comprising a TCR beta chain constant region, the TCR beta chain constant region comprising sequentially connected TCR beta chains Extracellular constant region, TCR ⁇ chain transmembrane region and TCR ⁇ chain intracellular constant region.
  • TCR ⁇ chain further includes a TCR ⁇ chain variable region.
  • the TCR ⁇ chain constant region and its corresponding TCR ⁇ chain extracellular constant region, TCR ⁇ chain transmembrane region and TCR ⁇ chain intracellular constant region are respectively derived from human, mouse or other Wild-type T cell receptor (TCR) constant region of a mammalian species.
  • TCR Wild-type T cell receptor
  • the intracellular constant region of the TCR ⁇ chain is a wild-type TCR ⁇ chain intracellular constant region of human origin, mouse origin or other mammalian species, such as comprising the amino acid sequence shown in SEQ ID NO: 9 or composed of The wild-type TCR ⁇ chain intracellular constant region composed of said amino acid sequence; for example comprising the amino acid sequence shown in SEQ ID NO:47 or the wild-type TCR ⁇ chain intracellular constant region composed of said amino acid sequence; for example comprising SEQ ID NO The amino acid sequence shown in: 48 or the intracellular constant region of the wild-type TCR ⁇ chain composed of said amino acid sequence.
  • the TCR ⁇ chain intracellular constant region is a mutated TCR ⁇ chain intracellular constant region
  • the mutated TCR ⁇ chain intracellular constant region is at least one of the wild-type TCR ⁇ chain intracellular constant regions Mutation of lysine to arginine or alanine, for example, mutation of at least one lysine to arginine or alanine in the intracellular constant region of the wild-type TCR ⁇ chain of human, mouse or other mammalian species Acid-forming mutant TCR ⁇ chain intracellular constant region.
  • the intracellular constant region of the mutated TCR ⁇ chain is formed by mutation of at least one lysine in the intracellular constant region of the wild-type TCR ⁇ chain to arginine or alanine, and the wild-type TCR ⁇ chain
  • the chain intracellular constant region comprises or consists of the amino acid sequence shown in SEQ ID NO: 9, SEQ ID NO: 47 or SEQ ID NO: 48, for example, one of the amino acid sequences shown in SEQ ID NO: 9, 2 or 3 (all) lysines are mutated to arginine or alanine to form a mutated TCR ⁇ chain intracellular constant region; for example, 1 or 2 ( All) lysine is mutated to arginine or alanine to form a mutated TCR ⁇ chain intracellular constant region.
  • the intracellular constant region of the mutated TCR ⁇ chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 10.
  • the TCR ⁇ chain includes a TCR ⁇ chain variable region, and the TCR ⁇ chain variable region can bind and recognize one or more antigens, including but not limited to polypeptide antigens (such as NYESO-1 , AFP and MART-1), lipid antigens (such as ⁇ -GlcCer, eLPA and LPE) and polysaccharide antigens (such as CA199, CA72-4 and CA125).
  • polypeptide antigens such as NYESO-1 , AFP and MART-1
  • lipid antigens such as ⁇ -GlcCer, eLPA and LPE
  • polysaccharide antigens such as CA199, CA72-4 and CA125.
  • 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.
  • 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, ROR
  • MAGE-A1/A3/A4/A6/A10/C2 gp100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1/2, BRLF-1, BMLF-1, HPV- One or more antigens among 16E6/E7, KRAS G12D, KRAS G12V, TP53R175H, ⁇ -GlcCer, eLPA, LPE, CA199, CA72-4 or CA125.
  • the antigen is a tumor antigen, such as MAGE-A1/A3/A4/A6/A10/C2, gp100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1/ 2.
  • a tumor antigen such as MAGE-A1/A3/A4/A6/A10/C2, gp100, CEA, NYESO-1, AFP, MART-1, HERV-E, HER2, LMP1/ 2.
  • the antigen may be a cell surface antigen, or an intracellular antigen.
  • the TCR ⁇ chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 17, which recognizes the tumor antigen MART-1; or comprises the amino acid sequence shown in SEQ ID NO: 25 or consists of Its composition, which recognizes the tumor antigen NYESO-1.
  • the TCR beta chain variable region comprises a complementarity determining region (CDR), such as CDR1, CDR2, CDR3.
  • CDR complementarity determining region
  • the complementarity determining region is included in the amino acid sequence shown in SEQ ID NO: 17, or in the amino acid sequence shown in SEQ ID NO: 25.
  • the TCR beta chain variable region comprises 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 CDR3 of the amino acid sequence shown in SEQ ID NO:20 , which recognizes the tumor antigen MART-1; or comprises 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 CDR3 of the amino acid sequence shown in SEQ ID NO:28, which recognizes the tumor Antigen NYESO-1.
  • the extracellular constant region of the TCR ⁇ chain is the extracellular constant region of the TCR ⁇ chain derived from human, mouse or other mammalian species.
  • the extracellular constant region of the TCR ⁇ chain comprises the amino acid sequence of SEQ ID NO: 7, or comprises the amino acid sequence of SEQ ID NO: 7 having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutated amino acid sequences, or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the amino acid sequence of SEQ ID NO:7 %, 98% or 99% of an amino acid sequence, or consists of said amino acid sequence.
  • the TCR ⁇ chain transmembrane region is a TCR ⁇ chain transmembrane region derived from human, mouse or other mammalian species.
  • the transmembrane region of the TCR ⁇ chain comprises the amino acid sequence of SEQ ID NO: 8, or comprises an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 mutated amino acid sequences, or comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of an amino acid sequence, or consists of said amino acid sequence.
  • the TCR beta chain constant region comprises or consists of the amino acid sequence of SEQ ID NO: 11 or SEQ ID NO: 12.
  • the present application provides an isolated T cell receptor or a fragment thereof, the T cell receptor comprising any one of the aforementioned TCR ⁇ chain and/or TCR ⁇ chain.
  • the T cell receptor includes any of the aforementioned TCR ⁇ chains, and the intracellular constant region of the TCR ⁇ chain is selected from:
  • the mutated TCR ⁇ chain intracellular constant region is a serine mutation of at least one of the wild-type TCR ⁇ chain intracellular constant region described in (1) or (2) is formed for alanine; or
  • the T cell receptor includes any of the aforementioned TCR ⁇ chains, and the intracellular constant region of the TCR ⁇ chain is selected from:
  • the mutated TCR ⁇ chain intracellular constant region is lysine of at least one of the wild-type TCR ⁇ chain intracellular constant region described in (1) or (2) acid mutation to arginine or alanine formation; or
  • the T cell receptor includes any one of the aforementioned TCR ⁇ chain and TCR ⁇ chain, and wherein the intracellular constant region of the TCR ⁇ chain is different from the intracellular constant region of the TCR ⁇ chain.
  • Wild type when the intracellular constant region of the TCR ⁇ chain is any of the aforementioned wild-type TCR ⁇ chain intracellular constant regions, the intracellular constant region of the TCR ⁇ chain is the intracellular constant region of any of the aforementioned mutated TCR ⁇ chains; when the TCR When the intracellular constant region of the ⁇ chain is the intracellular constant region of any of the aforementioned mutated TCR ⁇ chains, the intracellular constant region of the TCR ⁇ chain is any of the aforementioned wild-type or mutated intracellular constant regions of the TCR ⁇ chain.
  • the application provides an isolated T cell receptor or a fragment thereof, the T cell receptor comprising a TCR ⁇ chain and a TCR ⁇ chain, wherein the TCR ⁇ chain comprises a TCR ⁇ chain variable region and the TCR ⁇ chain constant region, the TCR ⁇ chain constant region includes the TCR ⁇ chain extracellular constant region, the TCR ⁇ chain transmembrane region and the TCR ⁇ chain intracellular constant region connected in sequence, and the TCR ⁇ chain intracellular constant region selected from:
  • the intracellular constant region of the mutated TCR ⁇ chain wherein the mutated TCR ⁇ chain intracellular constant region is at least one lysine in the wild-type TCR ⁇ chain intracellular constant region mutated to arginine or alanine ;or
  • intracellular constant region of the wild-type TCR ⁇ chain is selected from any of the aforementioned wild-type TCR ⁇ chain intracellular constant regions of the present application, for example:
  • a wild-type TCR ⁇ 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.
  • the TCR ⁇ chain includes a TCR ⁇ chain constant region
  • the TCR ⁇ chain constant region includes a TCR ⁇ chain extracellular constant region, a TCR ⁇ chain transmembrane region and a TCR ⁇ chain intracellular constant region connected in sequence, so
  • the intracellular constant region of the TCR ⁇ chain is selected from any of the aforementioned wild-type TCR ⁇ chain intracellular constant regions or mutated TCR ⁇ chain intracellular constant regions of the present application, for example:
  • the mutated TCR ⁇ chain intracellular constant region is a serine mutation of at least one of the wild-type TCR ⁇ chain intracellular constant region described in (1) or (2) is alanine;
  • the TCR ⁇ chain includes a TCR ⁇ chain variable region.
  • the present application provides an isolated T cell receptor or a fragment thereof, the T cell receptor includes a TCR ⁇ chain and a TCR ⁇ chain, wherein the TCR ⁇ chain includes a TCR ⁇ chain that can be Variable region and TCR ⁇ chain constant region, the TCR ⁇ chain constant region includes TCR ⁇ chain extracellular constant region, TCR ⁇ chain transmembrane region and TCR ⁇ chain intracellular constant region connected in sequence, the TCR ⁇ chain intracellular constant region
  • the constant region is selected from:
  • intracellular constant region of the wild-type TCR ⁇ chain is selected from any of the aforementioned wild-type TCR ⁇ chain intracellular constant regions of the present application, for example:
  • a wild-type TCR ⁇ chain intracellular constant region comprising or consisting of the amino acid sequence shown in SEQ ID NO:3.
  • the TCR ⁇ chain includes a TCR ⁇ chain constant region
  • the TCR ⁇ chain constant region includes a TCR ⁇ chain extracellular constant region, a TCR ⁇ chain transmembrane region and a TCR ⁇ chain intracellular constant region connected in sequence, so
  • the intracellular constant region of the TCR ⁇ chain is selected from any of the aforementioned wild-type TCR ⁇ chain intracellular constant regions or mutated TCR ⁇ chain intracellular constant regions of the present application, for example:
  • the mutated TCR ⁇ chain intracellular constant region is lysine of at least one of the wild-type TCR ⁇ chain intracellular constant region described in (1) or (2) acid mutation to arginine or alanine formation;
  • the TCR ⁇ chain includes a variable region of the TCR ⁇ chain.
  • the T cell receptor comprises the amino acid sequence shown in SEQ ID NO: 3 or the wild-type TCR ⁇ chain intracellular constant region consisting of the amino acid sequence, and/or comprises the amino acid sequence shown in SEQ ID NO: 10 The amino acid sequence or the mutated TCR ⁇ chain intracellular constant region consisting of the amino acid sequence is shown.
  • the T cell receptor comprises the amino acid sequence shown in SEQ ID NO: 4 or a mutated TCR ⁇ chain intracellular constant region consisting of the amino acid sequence, and/or comprises SEQ ID NO: 9, The amino acid sequence shown in SEQ ID NO: 47 or SEQ ID NO: 48 or the intracellular constant region of the wild-type TCR ⁇ chain composed of the amino acid sequence.
  • the amino acid sequence shown in the T cell receptor SEQ ID NO: 4 or the mutated TCR ⁇ chain intracellular constant region consisting of the amino acid sequence and/or comprises the amino acid sequence shown in SEQ ID NO: 10 The amino acid sequence or the mutated TCR ⁇ chain intracellular constant region consisting of said amino acid sequence.
  • any of the aforementioned T cell receptors includes any of the aforementioned TCR ⁇ chain variable regions and/or TCR ⁇ chain variable regions.
  • the TCR ⁇ chain variable region and/or the TCR ⁇ chain variable region can bind and recognize one or more antigens, including but not limited to polypeptide antigens (such as NYESO-1, AFP and MART-1), lipids Antigens (such as ⁇ -GlcCer, eLPA and LPE) and polysaccharide antigens (such as CA199, CA72-4 and CA125).
  • polypeptide antigens such as NYESO-1, AFP and MART-1
  • lipids Antigens such as ⁇ -GlcCer, eLPA and LPE
  • polysaccharide antigens such as CA199, CA72-4 and CA125.
  • the antigen can be 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 One or more antigens from G12D, KRAS G12V, TP53R175H,
  • the T cell receptor of any of the foregoing includes the variable region of the TCR beta chain of any of the foregoing, for example:
  • variable region of the TCR ⁇ chain comprises three CDRs contained in the amino acid sequence shown in SEQ ID NO:17;
  • variable region of the TCR ⁇ chain comprises three CDRs contained in the amino acid sequence shown in SEQ ID NO:25;
  • the TCR ⁇ chain variable region comprises 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;
  • the TCR ⁇ chain variable region comprises 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;
  • variable region of the TCR ⁇ chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 17; or
  • variable region of the TCR ⁇ chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 25.
  • the T cell receptor of any of the foregoing includes the variable region of the TCR alpha chain of any of the foregoing, for example:
  • variable region of the TCR ⁇ chain comprises three CDRs contained in the amino acid sequence shown in SEQ ID NO:13;
  • variable region of the TCR ⁇ chain comprises three CDRs contained in the amino acid sequence shown in SEQ ID NO:21;
  • variable region of the TCR ⁇ chain comprises 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;
  • variable region of the TCR ⁇ chain comprises 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;
  • variable region of the TCR ⁇ chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 13; or
  • variable region of the TCR ⁇ chain comprises or consists of the amino acid sequence shown in SEQ ID NO: 21.
  • the TCR ⁇ chain variable region and the TCR ⁇ chain variable region of the T cell receptor of any one of the foregoing are selected from:
  • the TCR ⁇ chain variable region comprises three complementarity determining regions CDRs contained in the amino acid sequence shown in SEQ ID NO:13, and the TCR ⁇ chain variable region comprises the amino acid sequence shown in SEQ ID NO:17. Contains three complementarity determining regions CDR;
  • the TCR ⁇ chain variable region comprises three complementarity determining regions CDRs contained in the amino acid sequence shown in SEQ ID NO:21, and the TCR ⁇ chain variable region comprises the amino acid sequence shown in SEQ ID NO:25. Contains three complementarity determining regions CDR;
  • the TCR ⁇ chain variable region comprises 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 CDR3 of the amino acid sequence shown in SEQ ID NO:16, said The TCR ⁇ chain variable region comprises 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 CDR3 of the amino acid sequence shown in SEQ ID NO:20;
  • the TCR ⁇ chain variable region comprises 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 CDR3 of the amino acid sequence shown in SEQ ID NO:24, said The TCR ⁇ chain variable region comprises 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 CDR3 of the amino acid sequence shown in SEQ ID NO:28;
  • the TCR ⁇ chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO: 13, and the TCR ⁇ chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17 or consists of the amino acid sequence composed; or
  • the TCR ⁇ chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:21, and the TCR ⁇ chain variable region comprises the amino acid sequence shown in SEQ ID NO:25 or consists of the amino acid sequence composition.
  • the T cell receptor includes any one of the aforementioned TCR ⁇ chain extracellular constant region and/or TCR ⁇ chain extracellular constant region.
  • the T cell receptor includes any of the aforementioned TCR ⁇ chain transmembrane regions and/or TCR ⁇ chain transmembrane regions.
  • the T cell receptor includes any one of the aforementioned TCR ⁇ chain constant region and/or TCR ⁇ chain constant region.
  • the TCR ⁇ chain constant region and the TCR ⁇ chain constant region are selected from:
  • the TCR alpha chain constant region comprises the amino acid sequence of SEQ ID NO:5 or consists of the amino acid sequence
  • the TCR beta chain constant region comprises the amino acid sequence of SEQ ID NO:12 or consists of the amino acid sequence
  • the TCR ⁇ chain constant region comprises the amino acid sequence of SEQ ID NO: 6 or consists of the amino acid sequence
  • the TCR ⁇ chain constant region comprises the amino acid sequence of SEQ ID NO: 12 or consists of the amino acid sequence
  • the TCR alpha chain constant region comprises the amino acid sequence of SEQ ID NO:6 or consists of the amino acid sequence
  • the TCR beta chain constant region comprises the amino acid sequence of SEQ ID NO:11 or consists of the amino acid sequence .
  • 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.
  • F5-TCR variable region recognizes tumor antigen MART-1
  • the F5-TCR ⁇ chain variable region is the amino acid sequence of SEQ ID NO: 13
  • the F5-TCR ⁇ chain variable region is SEQ ID NO: 17 amino acid sequence
  • the F5-WT TCR ⁇ chain constant region is the amino acid sequence of SEQ ID NO:5
  • the F5-WT TCR ⁇ chain constant region is the amino acid sequence of SEQ ID NO:11;
  • the F5-SA TCR ⁇ chain constant region is the amino acid sequence of SEQ ID NO:6, and the F5-SA TCR ⁇ chain constant region is the amino acid sequence of SEQ ID NO:11;
  • the F5-KR TCR ⁇ chain constant region is the amino acid sequence of SEQ ID NO:5
  • the F5-KR TCR ⁇ chain constant region is the amino acid sequence of SEQ ID NO:12
  • F5-dMUT TCR alpha chain constant region is the amino acid sequence of SEQ ID NO:6
  • F5-dMUT TCR beta chain constant region is the amino acid sequence of SEQ ID NO:12.
  • the application provides one or more T cell receptors 1G4-TCR, including 1G4-WT TCR, 1G4-SA TCR, 1G4-KR TCR or 1G4-dMUT TCR.
  • the 1G4-TCR variable region recognizes tumor antigen NYESO-1
  • the 1G4-TCR ⁇ chain variable region is the amino acid sequence of SEQ ID NO: 21 or
  • the 1G4-TCR ⁇ chain variable region is SEQ ID NO : the amino acid sequence of 25;
  • 1G4-WT TCR ⁇ chain constant region is the amino acid sequence of SEQ ID NO:5
  • the 1G4-WT TCR ⁇ chain constant region is the amino acid sequence of SEQ ID NO:11;
  • 1G4-SA TCR alpha chain constant region is the amino acid sequence of SEQ ID NO:6
  • 1G4-SA TCR beta chain constant region is the amino acid sequence of SEQ ID NO:11;
  • 1G4-KR TCR alpha chain constant region is the amino acid sequence of SEQ ID NO:5
  • 1G4-KR TCR beta chain constant region is the amino acid sequence of SEQ ID NO:12.
  • 1G4-dMUT TCR alpha chain constant region is the amino acid sequence of SEQ ID NO:6
  • the 1G4-dMUT TCR beta chain constant region is the amino acid sequence of SEQ ID NO:12.
  • any of the aforementioned TCR ⁇ chains, TCR ⁇ chains or T cell receptors is further combined with a signal peptide, and the signal peptide forms a signal peptide with the TCR ⁇ chain variable region and/or the TCR ⁇ chain variable region- Variable region structure.
  • the signal peptide is selected from human growth hormone signal peptide, CD8 ⁇ signal peptide, and immunoglobulin signal peptide.
  • the signal peptide comprises or consists of the amino acid sequence of SEQ ID NO:29 and/or SEQ ID NO:30.
  • the TCR alpha chain signal peptide amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 29.
  • the TCR beta chain signal peptide amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO:30.
  • the above-mentioned parts that form any of the aforementioned TCR ⁇ chains, TCR ⁇ chains or T cell receptors of the present application can be directly connected to each other, or connected through a linker sequence.
  • the linker sequence may be a sequence in which motifs such as GGGS, GGGGS, GSGSA and GGSGG are adjacently connected, repeating 1 to 5 motifs, and having a length of 3 to 25 amino acid residues.
  • the application provides an isolated nucleic acid or a fragment thereof, which encodes any one of the aforementioned isolated TCR ⁇ chains or fragments thereof, TCR ⁇ chains or fragments thereof, or T cell receptors or fragments thereof.
  • the nucleic acid sequence encoding the wild-type TCR ⁇ chain constant region comprises the nucleic acid sequence of SEQ ID NO: 31 or consists of said nucleic acid sequence.
  • the nucleic acid sequence encoding the wild-type TCR beta chain constant region comprises the nucleic acid sequence of SEQ ID NO: 32 or consists of said nucleic acid sequence.
  • the nucleic acid sequence encoding the SA TCR alpha chain constant region comprises the nucleic acid sequence of SEQ ID NO: 33 or consists of said nucleic acid sequence.
  • the nucleic acid sequence encoding the KR TCR beta chain constant region comprises the nucleic acid sequence of SEQ ID NO: 34 or consists of said nucleic acid sequence.
  • the nucleic acid sequence encoding the F5-TCR alpha chain variable region comprises the nucleic acid sequence of SEQ ID NO: 35 or consists of said nucleic acid sequence.
  • the nucleic acid sequence encoding the F5-TCR beta chain variable region comprises the nucleic acid sequence of SEQ ID NO: 36 or consists of said nucleic acid sequence.
  • the nucleic acid sequence encoding the 1G4-TCR alpha chain variable region comprises the nucleic acid sequence of SEQ ID NO: 37 or consists of said nucleic acid sequence.
  • the nucleic acid sequence encoding the 1G4-TCR beta chain variable region comprises the nucleic acid sequence of SEQ ID NO: 38 or consists of said nucleic acid sequence.
  • the nucleic acid sequence encoding F5-WT-TCR comprises or consists of the nucleic acid sequence of SEQ ID NO:39.
  • the nucleic acid sequence encoding F5-SA-TCR comprises the nucleic acid sequence of SEQ ID NO: 40 or consists of said nucleic acid sequence.
  • the nucleic acid sequence encoding F5-KR-TCR comprises the nucleic acid sequence of SEQ ID NO: 41 or consists of said nucleic acid sequence.
  • the nucleic acid sequence encoding F5-dMUT-TCR comprises the nucleic acid sequence of SEQ ID NO: 42 or consists of said nucleic acid sequence.
  • the nucleic acid sequence encoding 1G4-WT-TCR comprises or consists of the nucleic acid sequence of SEQ ID NO:43.
  • the nucleic acid sequence encoding 1G4-SA-TCR comprises or consists of the nucleic acid sequence of SEQ ID NO:44.
  • the nucleic acid sequence encoding 1G4-KR-TCR comprises the nucleic acid sequence of SEQ ID NO: 45 or consists of said nucleic acid sequence.
  • the nucleic acid sequence encoding 1G4-dMUT-TCR comprises the nucleic acid sequence of SEQ ID NO: 46 or consists of said nucleic acid sequence.
  • the present application provides a nucleic acid construct comprising any one of the aforementioned isolated nucleic acids or fragments thereof.
  • the nucleic acid construct further includes one or more regulatory sequences operably linked to the aforementioned nucleic acid.
  • regulatory sequences operably linked to the aforementioned nucleic acid.
  • the present application provides a vector comprising any one of the aforementioned nucleic acid constructs.
  • the vector is an expression vector or a crisper gene editing vector, such as a retrovirus vector, a lentivirus vector, a baculovirus vector, a herpes virus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector.
  • a retrovirus vector such as a retrovirus vector, a lentivirus vector, a baculovirus vector, a herpes virus vector, an adenovirus vector, or an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • the construction of the retroviral vector mainly includes an origin of replication, a 5'-LTR, a 3'-LTR and any one of the aforementioned nucleic acid sequences or nucleic acid constructs.
  • the promoter is operably linked to the nucleic acid sequence encoding the T cell receptor, and the nucleic acid construct is incorporated into an expression vector to realize the expression of the polynucleotide sequence encoding the T cell receptor.
  • Nucleic acid sequences encoding T cell receptors of the present application can be cloned into many types of vectors. For example, including but not limited to plasmids, phages, animal viruses, etc.
  • Suitable expression vectors contain one or more promoter sequences functional in the organism, an origin of replication, suitable restriction sites and selectable markers.
  • Suitable promoters include, but are not limited to, immediate early cytomegalovirus (CMV) promoter, elongation growth factor-1a (EF-1a), simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immune Defective virus (HIV), long terminal repeat (LTR) promoter and other constitutive promoter sequences, which can drive high-level expression of any polynucleotide sequence connected to it; also include but not limited to metallothionein promoter, tetracycline promoter An inducible promoter, such as a promoter, is used to turn on when expression of a polynucleotide sequence is desired and to turn off when expression is not desired.
  • CMV immediate early cytomegalovirus
  • EF-1a elongation growth factor-1a
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HV human immune Defective virus
  • LTR long terminal repeat
  • An inducible promoter
  • Selectable markers include marker genes and reporter genes in order to facilitate the identification of cells that are selectively expressed by viral vector-infected cell populations.
  • Suitable marker genes include, but are not limited to, antibiotic resistance genes, neo genes, and the like.
  • Suitable reporter genes include, but are not limited to, ⁇ -galactosidase, green fluorescent protein gene, luciferase, chloramphenicol acetyltransferase, and the like.
  • the application provides an engineered cell comprising any one of the aforementioned nucleic acid constructs or vectors.
  • the engineered cells are primary cells obtained from a subject.
  • the subject is a mammalian subject, eg, the subject is a human.
  • the engineered cells are T cells, preferably human T cells.
  • the engineered cells are NK cells, NKT cells, macrophages, ⁇ T cells, human CD4 + T cells, CD8 + T cells, or a mixed cell population of CD4 + T/CD8 + T cells.
  • the TCR nucleic acid construct of the present application or the vector comprising the nucleic acid construct can be used to express or produce TCR or engineered cells comprising the TCR.
  • the methods include:
  • the present application uses retroviruses to introduce target sequences into host cells. Retrovirus infection is currently a very widely used method for infecting primary cells of human or mouse origin.
  • virus particles are produced by packaging with preferred viral envelope proteins or capsid proteins. Then the recombined virus particles are delivered into the host or cells cultured in vitro to complete the expression of the target gene in the host cells.
  • virus particles include TCR nucleic acid sequences, retroviral vectors and nucleic acid sequences of packaging proteins.
  • the present application also provides a TCR complex, which is produced in T cells by any one of the aforementioned TCR nucleic acid constructs of the present application.
  • the present application provides functional testing of TCR-T cells during in vitro culture. Specifically, it includes TCR-T cell memory phenotype detection, TCR-T cell exhaustion phenotype detection in vitro exhaustion model and TCR-T cell mitochondrial phenotype detection.
  • TCR-T cell memory phenotype and mitochondrial phenotype detection were detected by cell flow cytometry after the activation of primary human T cells.
  • the exhaustion model constructed in vitro was used to detect the exhaustion of mutant TCR-T cells.
  • the CD3 antibody was used to stimulate TCR-T cells with different mutations in vitro for multiple rounds, and then flow cytometry was used to detect the expression of exhaustion-related molecules.
  • the present application provides the isolated TCR ⁇ chain or fragment thereof, TCR ⁇ chain or fragment thereof, T cell receptor or fragment thereof, nucleic acid or fragment thereof, nucleic acid construct, vector or engineered
  • TCR ⁇ chain or fragment thereof TCR ⁇ chain or fragment thereof, T cell receptor or fragment thereof, nucleic acid or fragment thereof, nucleic acid construct, vector or engineered
  • the mutant TCR-T cell therapy provided by this application can be used alone or in combination with other therapies, or in combination with PD-1/PD-L1 antibodies, or in combination with cytokine therapy, or in combination with radiotherapy and chemotherapy.
  • the amount and frequency of use are determined by various factors such as the characteristics of the disease and the severity of the disease.
  • the TCR-T cell therapy provided by the present application can be used multiple times at a low dose, and the above-mentioned dosage range is 10 4 -10 9 cells/kg.
  • the adoptive therapy of TCR-T cells provided by this application follows the internationally recognized adoptive technology. In one example herein, 5 ⁇ 10 6 TCR-T cells were implemented by tail vein injection. In principle, T cell therapy combinations could be injected directly into tumor tissue or the site of infection.
  • the present application provides a method for transforming T cell receptors, including:
  • T cell receptors are molecules present on the surface of T cells that are responsible for recognizing peptide-MHC complexes.
  • the TCR is an intact or full-length TCR.
  • the present application provides a TCR fragment that is smaller than a full-length TCR but still retains binding to a specific antigenic peptide of an MHC molecule, ie, the MHC-peptide complex.
  • the TCR is a heterodimer composed of alpha and beta chains.
  • the TCR can also be a single chain TCR (scTCR).
  • variable region refers to the domain of the TCR alpha or beta chain that is involved in the binding of the TCR to the antigen-MHC complex.
  • the variable regions of the ⁇ and ⁇ chains of natural TCRs generally have a similar structure, each containing four conserved framework regions (FRs) and three hypervariable regions or complementarity determining regions (CDRs). Among them, CDR3 in each variable region is the main CDR responsible for recognizing the processed antigen.
  • a single TCR alpha chain variable region or TCR beta chain variable region may be sufficient to confer binding to the peptide-MHC complex.
  • the TCR ⁇ chain of the present application may be a human TCR ⁇ chain, a humanized TCR ⁇ chain, a chimeric TCR ⁇ chain or a murine TCR ⁇ chain.
  • the TCR alpha chain is a chimeric TCR alpha chain comprising sequences derived from more than one species, such as sequences derived from human and mouse. For example, exchanging human TCR constant regions with murine counterparts can improve human T cell function and expression levels (see, e.g., Daniel Sommermeyer et al., J Immunol. 2010 Jun 1;184(11):6223-31 ., which is incorporated herein by reference).
  • a TCR may comprise human-derived variable regions and murine-derived constant regions.
  • the TCR ⁇ chain of the present application can also be human TCR ⁇ chain, humanized TCR ⁇ chain, chimeric TCR ⁇ chain or murine TCR ⁇ chain.
  • a chimeric TCR beta chain comprising sequences derived from more than one species, for example sequences derived from human and mouse.
  • the TCR ⁇ chain constant region and/or TCR ⁇ chain constant region described in this application includes an extracellular constant region, a transmembrane region and an intracellular constant region connected in sequence, wherein the extracellular constant region may include a TCR ⁇ chain and a TCR ⁇
  • the hinge region of the chain is involved in the formation of the disulfide bond of the TCR ⁇ chain and the TCR ⁇ chain;
  • the transmembrane region is also a constant region, and its functions include participating in the cell membrane anchoring of the TCR ⁇ chain and the TCR ⁇ chain and the interaction with the CD3 subunit to form a TCR - CD3 complex;
  • the possible role of the intracellular constant region includes participating in TCR signal transduction, TCR-CD3 complex conformational transition and signal transduction.
  • antigen refers to molecules on the cell surface or intracellularly presented by MHC molecules or MHC-like molecules, which can be bound by antibodies or T cell receptors (TCR), including but not limited to polypeptide antigens (such as NYESO-1, AFP and MART-1), lipid antigens (such as ⁇ -GlcCer, eLPA and LPE) or polysaccharide antigens (such as CA199, CA72-4 and CA125).
  • TCR T cell receptors
  • the antigen may be a tumor antigen, such as tumor cell-associated antigen (Tumor-associated antigen, TAA), or tumor-specific antigen (Tumor specific antigen, TSA).
  • isolated refers to material that has been removed from its natural state or otherwise manipulated, such as alpha chains, beta chains, T cell receptors and nucleic acids as described herein.
  • An isolated material can be substantially or essentially free of components that normally accompany it in its natural state, or it can be manipulated to be in 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 may also or alternatively be in enriched, partially purified or purified form.
  • wild type in the present invention refers to a type of naturally occurring amino acid sequence or encoding nucleic acid sequence, or has at least 90%-100% identity with the naturally occurring amino acid sequence or encoding nucleic acid sequence, or has at least 90%-100% identity with the naturally occurring Compared with the amino acid sequence or coding nucleic acid sequence, there are no more than 1-10 or 1-5 amino acid mutations (especially conservative amino acid substitutions), and still have the same or similar activity and/or function as this region.
  • Wild type is introduced as a template for the corresponding mutation or mutation combination in the present application, for example, "wild type derived from human, mouse or other mammalian species” means that the region has natural human, natural mouse or The amino acid sequence or coding nucleic acid sequence of the region of rat or other mammalian species, or consists of the amino acid sequence or coding nucleic acid sequence.
  • a region "derived from" a human, murine or other mammalian species also encompasses a region that is substantially identical to the amino acid sequence or nucleic acid encoding the region of a native human, native mouse or rat, or other mammalian species Sequence identity, for example at least 90%-100% identity compared to a naturally occurring amino acid sequence or encoding nucleic acid sequence, or no more than 1-10 or 1-5 compared to a naturally occurring amino acid sequence or encoding nucleic acid sequence Amino acid mutations (especially conservative amino acid substitutions), and still have the same or similar activity and/or function as this region.
  • mutation is understood as a substitution, deletion or addition of one or more amino acids or nucleic acids. For example, it may be a conservative substitution of an amino acid, which is known in the art and includes amino acid substitutions in which one amino acid with certain physical and/or chemical properties is exchanged for another amino acid with the same chemical or physical properties .
  • the conservative amino acid substitution can be an acidic amino acid for another acidic amino acid (e.g., Asp or Glu), an amino acid with a non-polar side chain for another amino acid with a non-polar side chain (e.g., Ala, Gly, Val, He, Leu, Met, Phe, Pro, Trp, Val, etc.), a basic amino acid replaces another basic amino acid (Lys, Arg, etc.), an amino acid with a polar side chain replaces another one with a polar side chain Amino acids (Asn, Cys, Gin, Ser, Thr, Tyr, etc.), etc., said conservative substitutions may be based, for example, on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. sex to proceed.
  • an amino acid with a non-polar side chain for another amino acid with a non-polar side chain e.g., Ala, Gly, Val, He
  • the intracellular constant region of the mutated TCR ⁇ chain refers to the replacement of at least one serine in the intracellular constant region of the wild-type TCR ⁇ chain with alanine; the mutated TCR ⁇ chain intracellular constant region refers to the intracellular constant region of the wild-type TCR ⁇ chain. At least one lysine in the constant region is substituted with arginine or alanine.
  • the sequences are aligned for optimal comparison purposes (e.g., a first and second amino acid sequence or nucleic acid sequence may be placed between a first and a second amino acid sequence or nucleic acid sequence for optimal alignment). Gaps may be introduced in one or both or non-homologous sequences may be discarded for comparison purposes).
  • the length of the aligned reference sequence is at least 30%, preferably at least 40%, more preferably at least 50%, 60% and even more preferably at least 70%, 80% , 90%, 100% of the reference sequence length.
  • the amino acid residues or nucleic acids at corresponding amino acid positions or nucleic acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleic acid as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the comparison of sequences and the calculation of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the Needlema and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm (available at http://www.gcg.com available), use the Blossum 62 matrix or the PAM250 matrix with gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 to determine the distance between two amino acid sequences. percent identity.
  • using the GAP program in the GCG software package (available at http://www.gcg.com), using the NWSgapdna.CMP matrix and gap weights of 40, 50, 60, 70 or 80 and Length weights of 1, 2, 3, 4, 5 or 6 determine the percent identity between two nucleic acid sequences.
  • a particularly preferred parameter set (and one that should be used unless otherwise stated) is the Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.
  • nucleic acid sequences and protein sequences described herein can further be used as "query sequences" to perform searches against public databases, eg, to identify other family member sequences or related sequences.
  • nucleotide or “nucleic acid” are used interchangeably herein to refer to nucleic acid strands of any length, and include DNA and RNA.
  • Nucleic acids may be deoxyribonucleic acids, ribonucleic acids, modified nucleic acids or bases, and/or their analogs, or any substrate capable of incorporation into a strand by DNA or RNA polymerases.
  • the nucleic acid sequence described herein can be obtained by designing primers based on the nucleotide sequence disclosed herein by PCR amplification, or by preparing a cDNA library for PCR amplification by skilled personnel.
  • SA refers to the mutation of at least one serine to alanine in the amino acid sequence of the intracellular constant region of the ⁇ chain
  • KR refers to At least one lysine in the amino acid sequence of the internal constant region is mutated to arginine
  • dMUT refers to the simultaneous presence of the "SA” mutation in the intracellular constant region of the ⁇ chain and the "KR” mutation in the amino acid sequence of the intracellular constant region of the ⁇ chain .
  • F5-TCR refers to a type of TCR that can recognize the tumor antigen MART-1
  • the F5-WT TCR, F5-SA TCR, F5-KR TCR and F5-dMUT TCR included in it have the same TCR ⁇ -chain variable region and TCR ⁇ -chain variable region
  • the F5-WT TCR, F5-SA TCR, F5-KR TCR and F5-dMUT TCR are based on mutations in the ⁇ -chain intracellular constant region or ⁇ -chain intracellular constant region Divide differently.
  • 1G4-TCR in this application refers to a type of TCR that can recognize the tumor antigen NYESO-1
  • the 1G4-WT TCR, 1G4-SA TCR, 1G4-KR TCR or 1G4-dMUT TCR included in it have the same TCR ⁇ Chain variable region and TCR ⁇ chain variable region
  • the 1G4-WT TCR, 1G4-SA TCR, 1G4-KR TCR or 1G4-dMUT TCR is different according to the mutation in the intracellular constant region of the ⁇ chain or the intracellular constant region of the ⁇ chain And divide.
  • Vector means that it is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing said genes or sequences in the host cell.
  • vectors include, but are not limited to, viral vectors, plasmids, cosmids, or phage vectors.
  • host cell refers to a cell into which exogenous nucleic acid has been introduced, including the progeny of these cells.
  • restriction sites will introduce one or more irrelevant residues at both ends of the expressed amino acid sequence, and will not affect the activity of the target sequence. For example, including but not limited to NotI, BamHI, XhoI and other enzyme cutting sites.
  • the amino-terminal or carboxy-terminal of the constructed protein contains one or more protein tags.
  • protein tags including but not limited to FLAG, HA, c-Myc, Poly-His, etc.
  • F5-TCR and 1G4-TCR respectively, wherein F5-TCR includes F5-WT TCR, F5-SA TCR, F5-KR TCR and F5-dMUT TCR, wherein 1G4-TCR includes 1G4-WT TCR, 1G4-SA TCR, 1G4-KR TCR and 1G4-dMUT TCR.
  • each F5-TCR or 1G4-TCR includes a TCR ⁇ chain and a TCR ⁇ chain respectively, wherein the TCR ⁇ chain is composed of a human growth hormone signal peptide, a TCR ⁇ chain variable region, a TCR ⁇ chain extracellular constant region, and a TCR ⁇ chain transmembrane region.
  • the intracellular region of the TCR ⁇ chain is sequentially connected; the TCR ⁇ chain is composed of the human growth hormone signal peptide, the variable region of the TCR ⁇ chain, the extracellular constant region of the TCR ⁇ chain, the transmembrane region of the TCR ⁇ chain, and the intracellular region of the TCR ⁇ chain.
  • the TCR ⁇ chain is connected in series with the TCR ⁇ chain through the F2A peptide.
  • the full-length nucleotide sequences of the F5-WT TCR, F5-SA TCR, F5-KR TCR and F5-dMUT TCR, and 1G4-WT TCR, 1G4-SA TCR, 1G4-KR TCR and 1G4-dMUT TCR were respectively as described in SEQ ID NO:39-46.
  • Embodiment 2 Retroviral packaging preparation method
  • the TCR expression vector (pMSGV-TCR) constructed in Example 1 was co-transfected with the envelope plasmid pHIT60/RD114 into HEK293T cells to package the retrovirus.
  • the above-mentioned mixed liposome liquid was added to HEK293T cell culture dishes, and placed in a 37° C. incubator for 48 hours. Then the virus liquid was collected, filtered through a 0.22 ⁇ m filter membrane, and placed in a -80°C refrigerator for later use.
  • Human primary T cell culture Human primary T cells were purchased from a commercial company (ALLCELLS). The complete medium for culturing primary T cells contains 5% human serum (Gemini#100-512), RPMI-1640 medium (Hyclone#SH30809.01) containing double antibody (Gibco#15140-122) and 1xGlutMAX (Gibco #35050-061), and at the same time, 100 U/ml rhIL-2 (PeproTech#200-02) should be added to the culture medium.
  • Virus infection experiments were performed after culturing in a 37°C incubator for 48 hours.
  • Infect human primary T cells with retrovirus Resuspend human primary T cells in a 24-well plate, pipette 750 ⁇ l into EP tube (Axygen#MCT-105C), centrifuge at 2500 rpm for 5 minutes, discard the supernatant, and use 500 ⁇ l phase Resuspend T cells in the corresponding virus solution (i.e. mutant TCR retrovirus), add 0.75 ⁇ l of Polybrene (SANTA CRUZ #SC-134220), pipette evenly and add to the corresponding 24-well plate, and the infection system is 750 ⁇ l at this time .
  • Flow cytometric analysis was performed using a BD LSR Fortessa instrument (BD Bioscience).
  • BD Bioscience For the detection of cell surface molecules: pipette 2x105 cells into a 96-well U-bottom plate, centrifuge at 1800rpm for 5 minutes, discard the supernatant, add the loss antibody prepared in FACS buffer (1xPBS containing 2% serum), and place on ice Stain in the dark for 25 minutes, wash once with FACS buffer, and perform flow cytometry detection.
  • cytokine staining For cytokine staining: pipette 2x105 cells into a 96-well U-bottom plate, centrifuge at 1800rpm for 5 minutes, discard the supernatant, add the loss antibody prepared in FACS buffer (1xPBS containing 2% serum), and keep on ice Light staining for 25 minutes, washed once with FACS buffer, fixed cells with paraformaldehyde fixative (Biolegend #420801) for 20 minutes, washed once with FACS buffer, added cells prepared with permeabilization solution (Invitrogen #00-8333-56) Antibody for factor flow cytometry, stained on ice for 25 minutes in the dark, washed once with FACS buffer, and detected by flow cytometry.
  • FACS buffer 1xPBS containing 2% serum
  • TCR expression down-regulation experiment caused by tumor antigen stimulation in vivo K562-NYESO-1 inoculated tumor-bearing mice, 12 days after the adoption of 1G4-TCR-T cells, detected the expression of TCR on the surface of TCR-T cells in the spleen and tumor.
  • Figure 1 (A) tumor antigen stimulation in tumor tissue caused down-regulation of TCR expression.
  • 1G4-TCR-T cells were mixed with K562-NYESO-1 target cells or K562-MART-1 non-target cells at a ratio of 1:1 in a 24-well plate and placed in a 37°C incubator Incubate for 12 hours (it should be understood that the specific time is determined by different experiments), aspirate cells for flow cytometric FACS detection, detect TCR down-regulation by staining TCR on the surface of T cells, and analyze TCR degradation level by staining after cell fixation and membrane rupture Intracellular TCR was detected, and the results are shown in Figure 1(B).
  • Target cell antigen stimulation promoted TCR downregulation and degradation;
  • hCD3 antibody stimulation caused TCR downregulation and degradation
  • 1G4-TCR-T cells and F5-TCR after activation After T cells were resuspended, they were added to a 24-well plate coated with hCD3 antibody and incubated in a 37°C incubator for 12 hours (it should be understood that the specific time is determined by different experiments), and the cells were drawn for flow cytometric FACS detection. TCR down-regulation was measured by staining TCR on the surface of T cells, and the level of TCR degradation was detected by staining intracellular TCR after cell fixation and permeabilization.
  • T cell function test in vitro after in vitro activated human primary T cells were infected with different mutant 1G4-TCR retroviruses, T cells were cultured in RPMI-1640 complete medium for 12 days, and 2x105 cells were drawn into 96-well plates , for flow cytometry FACS detection, the detection index is the expression of T cell memory molecules such as: CD62L, TCF1, CD27 and CD45RO, the results are shown in Figure 3 (AC), compared with the WT TCR group, SA TCR, KR TCR and The proportion of CD27 + CD45RO + (central memory T cells) in dMUT TCR-T cells was significantly increased, and the mutation group was more inclined to differentiate into memory T cells. At the same time, the expression of T cell memory molecules CD62L and TCF1 increased, especially in dMUT TCR -T cells are most pronounced.
  • T cell memory molecules such as: CD62L, TCF1, CD27 and CD45RO
  • T cell exhaustion model in vitro Human primary T cells activated in vitro were infected with 1G4-TCR retrovirus with different mutations for 48 hours, then the T cells were taken out, centrifuged at 2500 rpm for 5 minutes, and TCR-T cells were resuspended in 2 ⁇ g of complete medium /ml hCD3 antibody pre-coated 24-well plate (10 6 cells per well), stimulate TCR-T cells again for 48 hours, then repeat the above steps 2 times, end the culture, collect TCR-T cells for flow detection .
  • the construction of this in vitro depletion model refers to relevant articles recognized in the field (Santosha A.Vardhana et al., Nat Immunol.
  • Mitochondrial index detection After in vitro activated human primary T cells were infected with different mutant 1G4-TCR retroviruses, T cells were cultured in RPMI-1640 complete medium for 12 days, 2x105 cells were drawn into EP tubes, and added to MitoTracker Green (Invitrogen#M7514) staining, the final concentration was 50nM, FACS detection was performed after staining at 37°C for 1 hour in the dark. The results are shown in Figure 4(A). Compared with WT TCR cells, TCR-T cells with different mutations , there was no significant change in the number of mitochondria.
  • the detection method for the mitochondrial membrane potential is to pipette 2x105 cells into the EP tube, add TMRE (Invitrogen#T669) for staining, the final concentration is 200nM, and perform flow cytometric FACS detection after staining at 37°C for 1 hour in the dark, and the results are shown in Figure 4 (B) As shown, compared with WT TCR cells, mitochondrial membrane potential was significantly reduced in SA TCR, KR TCR and dMUT TCR-T cells, especially in dMUT TCR-T cells.
  • the detection method for mitochondrial reactive oxygen species is to draw 2x105 cells into EP tubes, add MitoSOX (Invitrogen#M36008) for staining, the final concentration is 5 ⁇ M, and perform flow FACS detection after 1 hour at 37°C in the dark, as shown in Figure 4 ( C) As shown, mitochondrial ROS production was significantly reduced in SA TCR, KR TCR and mutant TCR-T cells compared to WT TCR cells.
  • Metabolism-related index detection Human primary T cells infected with mutant 1G4-TCR retrovirus were cultured in RPMI-1640 complete medium in a 37°C incubator for 12 days in vitro, 2x105 cells were drawn into EP tubes, and centrifuged at 2500rpm for 5 minutes , Discard the supernatant, then resuspend T cells in a 24-well plate using sugar-free and serum-free medium, treat the cells with 2-NBDG (Invitrogen #N13195) or 1 ⁇ M Bodipy FL C16 (Invitrogen #D3821) at a final concentration of 50 ⁇ M, After staining at 37°C in the dark for 30 minutes, flow cytometric FACS detection was performed.
  • 2-NBDG Invitrogen #N13195
  • Bodipy FL C16 Invitrogen #D3821
  • mutant TCR-T cells were carried out in immunodeficient NSG mice aged 4-8 weeks.
  • 8x105 K562-NYESO-1 cells were inoculated subcutaneously in the right hindlimb axilla of NSG mice, and the target cells were The mice were grown subcutaneously for about 8-12 days, and the tumor volume was detected using a vernier caliper. Specifically, when the tumor volume was 80mm 3 -120mm 3 , NSG mice were injected with 5x106 human primary TCRs infected with different mutations 1G4-TCR into the tail vein.
  • the blood, spleen and tumor tissue of NSG mice are collected, and the weight of the tumor tissue is first weighed. Then use erythrocyte lysate (Biolegend #420301) to lyse the red blood cells of the mouse blood and spleen respectively. The mouse tumor tissue was ground and then subjected to Percoll density gradient centrifugation (Cytiva #17089110), and the mouse blood, spleen and tumor tissue were collected. Human primary T cells adopted from the tail vein were analyzed by flow cytometry.
  • Figure 5(A) found through statistics on tumor growth volume that, compared with the WT TCR group, the experimental group adopting KR TCR-T cells and dMUT TCR-T cells, Among them, the tumor growth rate was significantly slowed down. After weighing the tumor tissue, it was found that in the experimental group adopting KR TCR-T cells and dMUT TCR-T cells, the tumor weight was significantly reduced, as shown in Figure 5(B).
  • the proportions of adopted CD8 + TCR-T cells in the spleen, blood and tumor tissues of tumor-bearing NSG mice were counted, and the results are shown in Figure 5 (CE).
  • TCR-T cell cytokines in tumor tissues was detected, as shown in Figure 6 (J, K), the ratio of IFN- ⁇ + CD8 + cells in the adoptive KR TCR-T cell group and dMUT TCR-T cell group Significantly increased, showing that KR TCR and dMUT TCR-T cells have stronger anti-tumor effects, and found TNF- ⁇ + IFN- ⁇ + double positive cells in CD8 + KR TCR-T cells and CD8 + dMUT TCR-T cells The ratio was significantly increased, indicating that KR TCR-T cells produced more cytokines than WT TCR cells, but less than dMUT TCR-T cells, which indicated that dMUT TCR-T cells had stronger effector functions in tumor tissues.
  • T cell adoptive immunotherapy whether the limited T cells in the patient's body are collected to expand TCR-T cells in large quantities, or the persistence of T cell function and the occurrence of T cell exhaustion in tumor patients after adoption are the current TCR-T cells. - Difficulties faced by T cell therapy.
  • the mutant TCR-T cell transformation method provided in this application especially the dMUT TCR-T transformation method, enables TCR-T cells to exhibit more persistent anti-tumor efficacy after in vitro expansion and reinfusion into tumor models, and Can resist the occurrence of T cell exhaustion. This suggests that TCR-T cells engineered with dMUT TCR mutations are expected to achieve sustained and efficient anti-tumor functions in clinical production and treatment.

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Abstract

提供一种T细胞受体的构建及其用途。利用T细胞受体α链和β链胞内恒定区位点的突变，抑制了TCR抗原信号激活后T细胞受体的降解，维持了细胞表面TCR的水平，提高TCR-T细胞疗法的功效，该方法适用于不同的TCR-T细胞疗法。

Description

一种T细胞受体及其制备方法和用途 技术领域
本申请涉及T细胞受体领域，具体涉及经改造的T细胞受体的构建及其用途。
背景技术
恶性肿瘤严重威胁人类的健康和生命,是人类死亡的主要病因之一,其发病率逐年上升。传统的肿瘤治疗方法主要是以手术治疗为主，放疗，化疗为辅，而与传统疗法相比，肿瘤免疫疗法是以免疫系统为靶点，激发对肿瘤的全身性反应，此外，据临床数据显示肿瘤免疫治疗能够显著改善特定肿瘤类型患者的预后。而抗原特异T细胞的过继转移是肿瘤免疫治疗的主要手段之一，现已经被研究用于血液瘤以及实体瘤治疗。它主要是基于T细胞表面的T细胞受体可以识别不同的肿瘤抗原，从而实现对肿瘤细胞的杀伤和清除。
现阶段研究较多的T细胞过继疗法主要包括CAR-T细胞疗法和TCR-T疗法，其中CAR-T是将能识别某种肿瘤抗原的抗体的抗原结合部分与CD3-δ链体外偶联为一个嵌合蛋白，通过基因转导的方法转染患者的T细胞，使其表达嵌合抗原受体。当患者的T细胞被“重新编码”后，生成大量的肿瘤T细胞，从而达到发挥杀伤肿瘤作用。而TCR-T疗法是T细胞通过其表面的TCR识别靶细胞表面的主要组织相融性复合物(Major Histocompatibility Complex，MHC)所呈递的抗原，从而实现对靶细胞的直接攻击和杀伤。它是基于天然的TCR或者对TCR进行微弱的改造所开发的T细胞治疗技术。由于其能够识别肿瘤细胞表面主要组织相容性复合物分子所呈递的肿瘤表位，因而具有更广泛的适用性。
TCR-T细胞疗法的靶向性取决于T细胞表面表达的抗原TCR，而TCR是T细胞表面的受体，其以非共价键与CD3结合，形成TCR-CD3复合物，通过识 别并结合MHC呈递的抗原从而激活T细胞，促进T细胞的分裂与分化。与CAR不同的是，大多数TCR是由α链和β链组成的异源二聚体，其α链和β链均包含抗原结合区、恒定区和跨膜结构域。而这些α链和β链通过位于每条链固定区域内的保守半胱氨酸残基之间的二硫键共价连接。与CAR-T疗法相比较，TCR-T可针对大多数胞内抗原，而不局限在表面抗原，即TCR-T可以靶向大部分的肿瘤抗原，特别是能够识别肿瘤细胞胞内抗原。因此，T细胞受体对肿瘤的识别范围比抗体类药物以及依赖于抗体识别肿瘤的CAR-T的应用范围更广。此外，CAR-T在实体瘤治疗方面存在不足，而TCR-T技术有望解决这一难题。
目前，已经有多种TCR-T优化改造的方法用于改善或增强TCR-T肿瘤治疗效果，其主要通过改造T细胞结合肿瘤抗原的“探头”-TCR，以加强T细胞针对肿瘤细胞的识别过程，提高T淋巴细胞对于肿瘤细胞的亲和力，使得原来无肿瘤识别能力的T细胞能够有效地识别并杀伤肿瘤细胞。具体的包括：
(1)增加外源性TCR的表达，以增强TCR-T抗肿瘤效果。a.TCR恒定区鼠源化:借助基因工程手段将TCR恒定区鼠源化，以降低或阻止内源性和外源性TCR错配，促进外源性TCR的优先配对，提高T细胞表面抗原TCR表达水平，从而增强TCR-T的抗肿瘤效果；b.TCR恒定区半胱氨酸插入法：TCR α链和β链的恒定区引入半胱氨酸，以促进外源性TCR的优先配对，增加外源性的TCR表面表达水平，并减少与内源性TCR链的错配，此方法可以提高肿瘤反应性T细胞的有效性和安全性；c.TCR跨膜区插入疏水性突变：通过增加TCR α链跨膜区的疏水性，提高TCR稳定性，使得T细胞表面TCR表达增强，从而提高细胞亲和力和抗肿瘤TCR活性。
(2)增加外源性TCR与肿瘤抗原的亲和力。合成T细胞受体和抗原受体法(STAR：synthetic T cell receptor and antigen receptor)：将抗体抗原结合区域 的VH和VL分别插入至到TCR αβ恒定区，既具有CAR的高亲和力，又具有TCR复合体高信号传递能力，从而增强TCR-T的抗肿瘤效果。
虽然修饰的TCR-T细胞过继疗法已经在转移性黑色素瘤和其他恶性肿瘤的治疗过程中取得很好的效果，但是在肿瘤治疗过程依然存在诸多局限性：
(1)T细胞功能紊乱。a.肿瘤微环境内部持续的抗原刺激导致CD8 +T细胞耗竭，如PD-1、Lag-3、CD39以及TIM-3等抑制性受体表达增加；b.低氧低pH的肿瘤微环境使得T细胞效应功能进行性丧失，如IFN-γ，TNF-α等促炎性细胞因子分泌水平降低以及导致T细胞增殖和自我更新能力变差，代谢活性失调等。
(2)T细胞表面TCR表达水平降低。肿瘤微环境内抗原刺激导致CD8 +T细胞表面外源性TCR表达降低或是降解加快，使得没有足够的TCR-T细胞攻击肿瘤细胞。
(3)临床治疗过程中存在治疗毒性。目前，TCR-T靶向的抗原很少具有肿瘤组织，因此，在治疗过程中会产生肿瘤靶向的瘤外毒性。
发明内容
发明要解决的问题
鉴于上述所述的TCR-T细胞疗法的一些技术缺点，本申请的目的在于提供一种能够抑制降解的T细胞受体，并增强TCR-T细胞的抗肿瘤效果。
用于解决问题的方案
一方面，本申请提供一种T细胞受体(TCR)分离的TCR α链或其片段，所述TCR α链包括TCR α链恒定区，所述TCR α链恒定区包括依次连接的TCRα链胞外恒定区、TCR α链跨膜区和TCR α链胞内恒定区，所述TCR α链胞内恒定区为突变的TCR α链胞内恒定区，所述突变的TCR α链胞内恒定区是野生 型TCR α链胞内恒定区中至少一个丝氨酸突变为丙氨酸形成。
另一方面，本申请提供一种T细胞受体(TCR)分离的TCR β链或其片段，所述TCR β链包括TCR β链恒定区，所述TCR β链恒定区包括依次连接的TCRβ链胞外恒定区、TCR β链跨膜区和TCR β链胞内恒定区，所述TCR β链胞内恒定区为突变的TCR β链胞内恒定区，所述突变的TCR β链胞内恒定区是野生型TCR β链胞内恒定区中至少一个赖氨酸突变为精氨酸或丙氨酸形成。
另一方面，本申请提供一种分离的T细胞受体或其片段，所述T细胞受体包括前述任一项的TCR α链和/或TCR β链。
另一方面，本申请提供一种或多种T细胞受体，例如F5-WT TCR、F5-SA TCR、F5-KR TCR或F5-dMUT TCR，例如1G4-WT TCR、1G4-SA TCR、1G4-KR TCR或1G4-dMUT TCR。
另一方面，本申请提供一种分离的核酸或其片段，其编码前述任意一项分离的TCR α链或其片段、TCR β链或其片段、或T细胞受体或其片段。
另一方面，本申请提供一种核酸构建物，其包含前述任意一项分离的核酸或其片段。
另一方面，本申请提供一种载体，其包含前述任意一项的核酸构建物。
另一方面，本申请提供一种工程化细胞，其包含前述任意一项的核酸构建物或载体。
另一方面，本申请还提供一种TCR复合物，其由本申请的前述任意一项TCR核酸构建物在T细胞中产生。
另一方面，本申请提供了TCR-T细胞在体外培养过程中进行了功能性检测。
另一方面，本申请提供了前述任一项所述分离的TCR α链或其片段、TCR β链或其片段、T细胞受体或其片段、核酸或其片段、核酸构建物、载体或工程化 细胞在下述任意一种或多种用途中的应用：
(1)在抗原刺激过程中的抑制T细胞受体降解中的应用；
(2)提供对肿瘤的杀伤效果，或抑制肿瘤生长；
(3)维持T细胞的增殖能力；
(4)抗肿瘤的免疫药物及细胞的制备。
另一方面，本申请提供的突变TCR-T细胞疗法可以单独使用或联合其他疗法同时使用，或联合PD-1/PD-L1抗体，或联合细胞因子疗法，或联合放化疗，共同结合使用。
另一方面，本申请提供一种T细胞受体的改造方法，包括：
(1)将野生型TCR β链胞内恒定区中至少一个的赖氨酸突变为精氨酸或丙氨酸；和/或
(2)将野生型TCR α链胞内恒定区中至少一个的丝氨酸突变为丙氨酸。
发明的效果
本申请利用T细胞受体α链和β链胞内恒定区泛素化修饰位点的突变，抑制了TCR抗原信号激活后T细胞受体的降解，维持了细胞表面TCR的水平，该方法适用于不同的TCR-T细胞疗法。
一方面，抗原刺激之后，相比于WT TCR细胞(野生型TCR细胞)，SA TCR、KR TCR和dMUT TCR细胞有更多的细胞表面TCR表达，并且体外培养的过程中，SA TCR、KR TCR和dMUT TCR细胞展现出向中央记忆T细胞分化的表型，体外耗竭模型显示相较于WT TCR，KR TCR和dMUT TCR细胞耗竭水平更低，这种表型的转变得益于SA TCR、KR TCR和dMUT TCR细胞内更健康的线粒体状态，其表现为体外培养的突变体TCR-T细胞展现出更低的线粒体膜电势及ROS水平。另一方面，本申请通过小鼠移植瘤-T细胞过继模型，发现相比较于WT TCR，KR TCR和dMUT TCR细胞有更强的抗肿瘤效果，并发现dMUT TCR 突变体组在小鼠脾脏和血液中有更多的TCR-T细胞存在，而在肿瘤组织中，KR TCR和dMUT TCR细胞数量比例明显增多。从细胞表型来说，在小鼠脾脏中SA TCR，KR TCR和dMUT TCR组积累了更多的中央记忆T细胞，而在肿瘤组织中dMUT突变的TCR-T细胞展现出更低的T细胞耗竭表型及增殖能力。因此，在过继等量的TCR-T细胞治疗的过程中，KR TCR和dMUT TCR细胞表现出更具优势的抑制肿瘤生长的潜力。
可以理解的是，本申请通过对TCR胞内恒定区氨基酸的突变，不仅抑制TCR降解，也提高了TCR-T细胞疗法的功效，可以有效的遏制肿瘤的生长，所述肿瘤不局限于实体瘤，可以是血液瘤或者淋巴瘤。
附图说明
图1.抗原刺激促进了细胞表面TCR的降解。(A)K562-NYESO-1接种的荷瘤小鼠，1G4-TCR-T细胞过继第十二天后，检测脾脏及肿瘤中TCR-T细胞表面TCR的表达情况。(B)体外功能实验均在人原代T细胞上进行，体外培养1G4-TCR-T细胞，分别跟K562-NYESO-1和K562-MART-1细胞共孵育12小时，检测TCR的降解情况。(C)体外培养活化的F5-TCR-T细胞，CD3抗体刺激12小时后，检测TCR的降解情况。(D)体外培养活化的1G4-TCR-T细胞，CD3抗体刺激12小时后，检测TCR的降解情况。
图2.突变抑制TCR下调和降解的结果图。体外功能实验均在人原代T细胞上进行。(A)降解受抑制的TCR突变模式图。(B)突变后的1G4-TCR在人原代T细胞上的表达。(C)体外活化的不同突变F5-TCR-T细胞，CD3抗体刺激12小时后，检测不同TCR的降解情况(“R”代表无CD3抗体刺激，“S”代表有CD3抗体刺激)。(D)体外活化的不同突变1G4-TCR-T细胞，CD3抗体刺激12小时后，检测不同TCR的降解情 况(“R”代表无CD3抗体刺激，“S”代表有CD3抗体刺激)。(E)体外活化的不同突变1G4-TCR-T细胞，CD3抗体刺激不同时间，检测不同TCR的表达情况。
图3.突变对1G4-TCR-T细胞体外功能的影响图。体外功能实验均在人原代T细胞上进行。(A)体外活化的TCR-T细胞，以CD45RO和CD27为记忆T细胞分化指标，进行流式检测。(B)活化的1G4-TCR-T细胞，检测记忆T细胞分化指标CD62L。(C)体外活化的1G4-TCR-T细胞，检测记忆T细胞分化指标TCF-1。(D)利用体外耗竭模型，检测不同突变体TCR-T细胞耗竭分子LAG-3的表达。(E)利用体外耗竭模型，检测TCR-T细胞耗竭关键转录因子TOX的表达。(F)利用体外耗竭模型，检测突变体TCR-T细胞因子的产生。
图4.不同突变1G4-TCR-T细胞线粒体功能状态的对比图。体外实验均在人原代T细胞上进行。(A)不同TCR-T细胞体外激活12天后检测胞内线粒体数量。(B)检测突变后的1G4-TCR-T细胞内线粒体膜电势的变化。(C)体外活化的1G4-TCR-T细胞，检测其胞内线粒体ROS的产生水平。(D)不同TCR-T细胞体外激活12天后，2-NBDG处理后，FACS流式分析，检测TCR-T细胞对葡萄糖的摄取。(E)Bodipy FL C16摄取实验检测不同突变组TCR-T细胞对胞外脂肪酸的利用效率。
图5.不同突变1G4-TCR-T细胞的抗肿瘤效果对比图。(A)1G4-TCR-T细胞接种的K562-NYESO-1NSG荷瘤小鼠，不同时间点肿瘤大小的统计图。K562-NYESO-1NSG荷瘤小鼠过继T细胞后，第十二天检测不同突变组肿瘤组织的重量(B)，同时检测荷瘤小鼠血液(C)，脾脏(D)及肿瘤组织(E)中过继TCR-T细胞的比例。
图6.突变对1G4-TCR-T细胞在小鼠体内功能的影响图。(A)NSG荷瘤小鼠脾脏内，以CD45RO和CD27为记忆T细胞分化指标，对不同突变组进行流式检测。(B)NSG荷瘤小鼠肿瘤组织内，检测不同突变组记忆T细胞分化指标CD62L的表达。(C)NSG荷瘤小鼠肿瘤组织内，检测不同突变组记忆T细胞分化指标CCR7的表达。(D)NSG荷瘤小 鼠肿瘤组织内，检测不同突变组耗竭T细胞PD-1 +TIM-3 +双阳性细胞比例及统计图。检测NSG荷瘤小鼠肿瘤组织内不同突变组TCR-T细胞免疫抑制性分子PD-1(E)，TIM-3(F)，LAG-3(G)和CD39(H)的表达。(I)NSG荷瘤小鼠肿瘤组织内，检测不同突变组转录因子TOX的表达。(J)检测NSG荷瘤小鼠肿瘤组织内，IFN-γ +阳性的CD8 +T细胞比例。(K)检测NSG荷瘤小鼠肿瘤组织内，IFN-γ +TNF-α +双阳性的TCR-T细胞比例。
具体实施方式
一方面，本申请提供一种T细胞受体(TCR)分离的TCR α链或其片段，所述TCR α链包括TCR α链恒定区，所述TCR α链恒定区包括依次连接的TCRα链胞外恒定区、TCR α链跨膜区和TCR α链胞内恒定区。可选的，所述TCR α链进一步包括TCR α链可变区。
在一些实施方式中，所述TCR α链恒定区及其相对应的TCR α链胞外恒定区、TCR α链跨膜区和TCR α链胞内恒定区分别来源于人源、鼠源或其他哺乳动物种属的野生型T细胞受体(TCR)恒定区。
在一些实施方式中，所述TCR α链胞内恒定区为人源、鼠源或其他哺乳动物种属的野生型TCR α链胞内恒定区，例如包含SEQ ID NO:3所示氨基酸序列或由所述氨基酸序列组成的野生型TCR α链胞内恒定区。
在一些实施方式中，所述TCR α链胞内恒定区为突变的TCR α链胞内恒定区，所述突变的TCR α链胞内恒定区是野生型TCR α链胞内恒定区中至少一个丝氨酸突变为丙氨酸形成，例如人源、鼠源或其他哺乳动物种属的野生型TCR α链胞内恒定区中至少一个丝氨酸突变为丙氨酸形成突变的TCR α链胞内恒定区。
在一些实施方式中，所述突变的TCR α链胞内恒定区是野生型TCR α链胞内恒定区中至少一个丝氨酸突变为丙氨酸形成，所述野生型TCR α链胞内恒定区包含SEQ ID NO:3所示氨基酸序列或由所述氨基酸序列组成的，例如SEQ ID NO:3所示氨基酸序列中1个或2个(全部)丝氨酸突变为丙氨酸形成突变的TCRα链胞内恒定区。
在一些实施方式中，所述突变的TCR α链胞内恒定区包含SEQ ID NO:4所示氨基酸序列或由所述氨基酸序列组成的。
在一些实施方式中，所述TCR α链包括TCR α链可变区，所述TCR α链可变区可以结合并识别一种或多种抗原，包括但不限于多肽类抗原(例如NYESO-1、AFP和MART-1)、脂类抗原(例如β-GlcCer、eLPA和LPE)和多糖类抗原(例如CA199、CA72-4和CA125)。
在一些实施方式中，所述抗原为肿瘤抗原、微生物抗原或自身抗原，例如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 G12D、KRAS G12V、TP53R175H、β-GlcCer、eLPA、LPE、CA199、CA72-4或CA125中的一种或多种抗原。
在一些实施方式中，所述抗原为肿瘤抗原，例如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、TP53R175H、β-GlcCer、eLPA、LPE、CA199、CA72-4或CA125中的一种或 多种。所述抗原可以是细胞表面抗原，或细胞胞内抗原。
在一些实施方式中，所述TCR α链可变区包含SEQ ID NO:13所示氨基酸序列或由其组成，其识别肿瘤抗原MART-1；或包含SEQ ID NO:21所示氨基酸序列或由其组成，其识别肿瘤抗原NYESO-1。
在一些实施方式中，所述TCR α链可变区包含互补决定区(CDR)，如CDR1，CDR2，CDR3。所述互补决定区包含于SEQ ID NO:13所示氨基酸序列中，或包含SEQ ID NO:21所示氨基酸序列中。
在一些实施方式中，所述TCR α链可变区包含SEQ ID NO:14所示氨基酸序列的CDR1、SEQ ID NO:15所示氨基酸序列的CDR2和SEQ ID NO:16所示氨基酸序列的CDR3，其识别肿瘤抗原MART-1；或包含SEQ ID NO:22所示氨基酸序列的CDR1、SEQ ID NO:23所示氨基酸序列的CDR2和SEQ ID NO:24所示氨基酸序列的CDR3，其识别肿瘤抗原NYESO-1。
在一些实施方式中，所述TCR α链胞外恒定区为来源于人源、鼠源或其他哺乳动物种属的TCR α链胞外恒定区。
在一些实施方式中，所述TCR α链胞外恒定区为包含SEQ ID NO:1的氨基酸序列，或包含与SEQ ID NO:1的氨基酸序列相比具有1、2、3、4、5、6、7、8、9或10个突变的氨基酸序列，或包含与SEQ ID NO:1的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列，或由所述氨基酸序列组成。
在一些实施方式中，所述TCR α链跨膜区为来源于人源、鼠源或其他哺乳动物种属的TCR α链跨膜区。
在一些实施方式中，所述TCR α链跨膜区包含SEQ ID NO:2的氨基酸序列，或包含与SEQ ID NO:2的氨基酸序列相比具有1、2、3、4、5、6、7、8、9或 10个突变的氨基酸序列，或包含与SEQ ID NO:2的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列，或由所述氨基酸序列组成。
在一些实施方式中，所述TCR α链恒定区包含SEQ ID NO:5或SEQ ID NO:6的氨基酸序列或由所述氨基酸序列组成。
一方面，本申请提供一种T细胞受体(TCR)分离的TCR β链或其片段，所述TCR β链包括TCR β链恒定区，所述TCR β链恒定区包括依次连接的TCR β链胞外恒定区、TCR β链跨膜区和TCR β链胞内恒定区。可选的，所述TCR β链进一步包括TCR β链可变区。
在一些实施方式中，所述TCR β链恒定区及其相对应的TCR β链胞外恒定区、TCR β链跨膜区和TCR β链胞内恒定区分别来源于人源、鼠源或其他哺乳动物种属的野生型T细胞受体(TCR)恒定区。
在一些实施方式中，所述TCR β链胞内恒定区为人源、鼠源或其他哺乳动物种属的野生型TCR β链胞内恒定区，例如包含SEQ ID NO:9所示氨基酸序列或由所述氨基酸序列组成的野生型TCR β链胞内恒定区；例如包含SEQ ID NO:47所示氨基酸序列或由所述氨基酸序列组成的野生型TCR β链胞内恒定区；例如包含SEQ ID NO:48所示氨基酸序列或由所述氨基酸序列组成的野生型TCR β链胞内恒定区。
在一些实施方式中，所述TCR β链胞内恒定区为突变的TCR β链胞内恒定区，所述突变的TCR β链胞内恒定区是野生型TCR β链胞内恒定区中至少一个赖氨酸突变为精氨酸或丙氨酸形成，例如人源、鼠源或其他哺乳动物种属的野生型TCR β链胞内恒定区中至少一个赖氨酸突变为精氨酸或丙氨酸形成突变的TCR β链胞内恒定区。
在一些实施方式中，所述突变的TCR β链胞内恒定区是野生型TCR β链胞内恒定区中至少一个赖氨酸突变为精氨酸或丙氨酸形成，所述野生型TCR β链胞内恒定区包含SEQ ID NO:9、SEQ ID NO:47或SEQ ID NO:48所示氨基酸序列或由所述氨基酸序列组成的，例如SEQ ID NO:9所示氨基酸序列中1个、2个或3个(全部)赖氨酸突变为精氨酸或丙氨酸形成突变的TCR β链胞内恒定区；例如SEQ ID NO:47或SEQ ID NO:48中1个或2个(全部)赖氨酸突变为精氨酸或丙氨酸形成突变的TCR β链胞内恒定区。
在一些实施方式中，所述突变的TCR β链胞内恒定区包含SEQ ID NO:10所示氨基酸序列或由所述氨基酸序列组成的。
在一些实施方式中，所述TCR β链包括TCR β链可变区，所述TCR β链可变区可以结合并识别一种或多种抗原，包括但不限于多肽类抗原(例如NYESO-1、AFP和MART-1)、脂类抗原(例如β-GlcCer、eLPA和LPE)和多糖类抗原(例如CA199、CA72-4和CA125)。
在一些实施方式中，所述抗原为肿瘤抗原、微生物抗原或自身抗原，例如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 G12D、KRAS G12V、TP53R175H、β-GlcCer、eLPA、LPE、CA199、CA72-4或CA125中的一种或多种抗原。
在一些实施方式中，所述抗原为肿瘤抗原，例如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、TP53R175H、β-GlcCer、eLPA、LPE、CA199、CA72-4或CA125中的一种或多种。所述抗原可以是细胞表面抗原，或细胞胞内抗原。
在一些实施方式中，所述TCR β链可变区包含SEQ ID NO:17所示氨基酸序列或由其组成，其识别肿瘤抗原MART-1；或包含SEQ ID NO:25所示氨基酸序列或由其组成，其识别肿瘤抗原NYESO-1。
在一些实施方式中，所述TCR β链可变区包含互补决定区(CDR)，如CDR1，CDR2，CDR3。所述互补决定区包含于SEQ ID NO:17所示氨基酸序列中，或包含SEQ ID NO:25所示氨基酸序列中。
在一些实施方式中，所述TCR β链可变区包含SEQ ID NO:18所示氨基酸序列的CDR1、SEQ ID NO:19所示氨基酸序列的CDR2和SEQ ID NO:20所示氨基酸序列的CDR3，其识别肿瘤抗原MART-1；或包含SEQ ID NO:26所示氨基酸序列的CDR1、SEQ ID NO:27所示氨基酸序列的CDR2和SEQ ID NO:28所示氨基酸序列的CDR3，其识别肿瘤抗原NYESO-1。
在一些实施方式中，所述TCR β链胞外恒定区为来源于人源、鼠源或其他哺乳动物种属的TCR β链胞外恒定区。
在一些实施方式中，所述TCR β链胞外恒定区为包含SEQ ID NO:7的氨基酸序列，或包含与SEQ ID NO:7的氨基酸序列相比具有1、2、3、4、5、6、7、8、9或10个突变的氨基酸序列，或包含与SEQ ID NO:7的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列，或由所述氨基酸序列组成。
在一些实施方式中，所述TCR β链跨膜区为来源于人源、鼠源或其他哺乳动物种属的TCR β链跨膜区。
在一些实施方式中，所述TCR β链跨膜区包含SEQ ID NO:8的氨基酸序列，或包含与SEQ ID NO:8的氨基酸序列相比具有1、2、3、4、5、6、7、8、9或10个突变的氨基酸序列，或包含与SEQ ID NO:8的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列，或由所述氨基酸序列组成。
在一些实施方式中，所述TCR β链恒定区包含SEQ ID NO:11或SEQ ID NO:12的氨基酸序列或由所述氨基酸序列组成。
另一方面，本申请提供一种分离的T细胞受体或其片段，所述T细胞受体包括前述任意一种TCR α链和/或TCR β链。
在一些实施方式中，所述T细胞受体包括前述任意一种TCR α链，所述TCR α链胞内恒定区选自：
(1)来源于人源、鼠源或其他哺乳动物种属的野生型TCR α链胞内恒定区；
(2)包含SEQ ID NO:3所示氨基酸序列或由所述氨基酸序列组成的野生型TCR α链胞内恒定区；
(3)突变的TCR α链胞内恒定区，所述突变的TCR α链胞内恒定区为(1)或(2)所述的野生型TCR α链胞内恒定区中至少一个的丝氨酸突变为丙氨酸形成；或
(4)包含SEQ ID NO:4所示氨基酸序列或由所述氨基酸序列组成的突变的TCR α链胞内恒定区。
在一些实施方式中，所述T细胞受体包括前述任意一种TCR β链，所述TCRβ链胞内恒定区选自：
(1)来源于人源、鼠源或其他哺乳动物种属的野生型TCR β链胞内恒定区；
(2)包含SEQ ID NO:9、SEQ ID NO:47或SEQ ID NO:48所示氨基酸序列 或由所述氨基酸序列组成的野生型TCR β链胞内恒定区；
(3)突变的TCR β链胞内恒定区，所述突变的TCR β链胞内恒定区为(1)或(2)所述的野生型TCR β链胞内恒定区中至少一个的赖氨酸突变为精氨酸或丙氨酸形成；或
(4)包含SEQ ID NO:10所示氨基酸序列或由所述氨基酸序列组成的突变的TCR β链胞内恒定区。
在一些实施方式中，所述T细胞受体包括前述任意一种TCR α链和TCR β链，并且，其中所述TCR α链胞内恒定区与所述TCR β链胞内恒定区不同时为野生型。例如，当TCR α链胞内恒定区为前述任意一种野生型TCR α链胞内恒定区时，TCR β链胞内恒定区为前述任意一种突变的TCR β链胞内恒定区；当TCR α链胞内恒定区为前述任意一种突变的TCR α链胞内恒定区时，TCR β链胞内恒定区为前述任意一种野生型或突变的TCR β链胞内恒定区。
在一些实施方式中，本申请提供一种分离的T细胞受体或其片段，所述T细胞受体包括TCR α链和TCR β链，其中，所述TCR β链包括TCR β链可变区和TCR β链恒定区，所述TCR β链恒定区包括依次连接的TCR β链胞外恒定区、TCR β链跨膜区和TCR β链胞内恒定区，所述TCR β链胞内恒定区选自：
(1)突变的TCR β链胞内恒定区，所述突变的TCR β链胞内恒定区为野生型TCR β链胞内恒定区中至少一个的赖氨酸突变为精氨酸或丙氨酸；或
(2)包含SEQ ID NO:10所示氨基酸序列或由所述氨基酸序列组成的突变的TCR β链胞内恒定区。
进一步的，所述野生型TCR β链胞内恒定区选自本申请前述任意一种野生型TCR β链胞内恒定区，例如：
(1)来源于人源、鼠源或其他哺乳动物种属的野生型TCR β链胞内恒定区；
(2)包含SEQ ID NO:9、SEQ ID NO:47或SEQ ID NO:48所示氨基酸序列或由所述氨基酸序列组成的野生型TCR β链胞内恒定区。
进一步的，所述TCR α链包括TCR α链恒定区，所述TCR α链恒定区包括依次连接的TCR α链胞外恒定区、TCR α链跨膜区和TCR α链胞内恒定区，所述TCR α链胞内恒定区选自本申请前述任意一种野生型TCR α链胞内恒定区或突变的TCR α链胞内恒定区，例如：
(1)来源于人源、鼠源或其他哺乳动物种属的野生型TCR α链胞内恒定区；
(2)包含SEQ ID NO:3所示氨基酸序列或由所述氨基酸序列组成的野生型TCR α链胞内恒定区；
(3)突变的TCR α链胞内恒定区，所述突变的TCR α链胞内恒定区为(1)或(2)所述的野生型TCR α链胞内恒定区中至少一个的丝氨酸突变为丙氨酸；
(4)包含SEQ ID NO:4所示氨基酸序列或由所述氨基酸序列组成的突变的TCR α链胞内恒定区；
进一步的，所述TCR α链包括TCR α链可变区。
在一些实施方式中，本申请提供一种分离的T细胞受体或其片段，所述T细胞受体包括TCR α链和TCR β链，其特征在于，所述TCR α链包括TCR α链可变区和TCR α链恒定区，所述TCR α链恒定区包括依次连接的TCR α链胞外恒定区、TCR α链跨膜区和TCR α链胞内恒定区，所述TCR α链胞内恒定区选自：
(1)突变的TCR α链胞内恒定区，所述突变的TCR α链胞内恒定区为野生型TCR α链胞内恒定区中至少一个丝氨酸突变为丙氨酸；
(2)包含SEQ ID NO:4所示氨基酸序列或由所述氨基酸序列组成的突变的TCR α链胞内恒定区。
进一步的，所述野生型TCR α链胞内恒定区选自本申请前述任意一种野生型TCRα链胞内恒定区，例如：
(1)来源于人源、鼠源或其他哺乳动物种属的野生型TCR α链胞内恒定区；
(2)包含SEQ ID NO:3所示氨基酸序列或由所述氨基酸序列组成的野生型TCR α链胞内恒定区。
进一步的，所述TCR β链包括TCR β链恒定区，所述TCR β链恒定区包括依次连接的TCR β链胞外恒定区、TCR β链跨膜区和TCR β链胞内恒定区，所述TCR β链胞内恒定区选自本申请前述任意一种野生型TCR β链胞内恒定区或突变的TCR β链胞内恒定区，例如：
(1)来源于人源、鼠源或其他哺乳动物种属的野生型TCR β链胞内恒定区；
(2)包含SEQ ID NO:9、SEQ ID NO:47或SEQ ID NO:48所示氨基酸序列或由所述氨基酸序列组成的野生型TCR β链胞内恒定区；
(3)突变的TCR β链胞内恒定区，所述突变的TCR β链胞内恒定区为(1)或(2)所述的野生型TCR β链胞内恒定区中至少一个的赖氨酸突变为精氨酸或丙氨酸形成；
(4)包含SEQ ID NO:10所示氨基酸序列或由所述氨基酸序列组成的突变的TCR β链胞内恒定区。
进一步的，所述TCR β链包括TCR β链可变区。
在一些实施方式中，所述T细胞受体包含SEQ ID NO:3所示氨基酸序列或由所述氨基酸序列组成的野生型TCR α链胞内恒定区，和/或包含SEQ ID NO:10所示氨基酸序列或由所述氨基酸序列组成的突变的TCR β链胞内恒定区。
在一些实施方式中，所述T细胞受体包含SEQ ID NO:4所示氨基酸序列或由所述氨基酸序列组成的突变的TCR α链胞内恒定区，和/或包含SEQ ID NO:9、 SEQ ID NO:47或SEQ ID NO:48所示氨基酸序列或由所述氨基酸序列组成的野生型TCR β链胞内恒定区。
在一些实施方式中，所述T细胞受体SEQ ID NO:4所示氨基酸序列或由所述氨基酸序列组成的突变的TCR α链胞内恒定区，和/或包含SEQ ID NO:10所示氨基酸序列或由所述氨基酸序列组成的突变的TCR β链胞内恒定区。
在一些实施方式中，前述任意一种T细胞受体包括前述任意一种TCR α链可变区和/或TCR β链可变区。所述TCR α链可变区和/或TCR β链可变区可以结合并识别一种或多种抗原，包括但不限于多肽类抗原(例如NYESO-1、AFP和MART-1)、脂类抗原(例如β-GlcCer、eLPA和LPE)和多糖类抗原(例如CA199、CA72-4和CA125)。所述抗原可以是肿瘤抗原、微生物抗原或自身抗原，例如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 G12D、KRAS G12V、TP53R175H、β-GlcCer、eLPA、LPE、CA199、CA72-4或CA125中的一种或多种抗原；优选为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、TP53R175H、β-GlcCer、eLPA、LPE、CA199、CA72-4或CA125中的一种或多种。所述抗原可以是细胞表面抗原，或细胞胞内抗原。
在一些实施方式中，前述任一项的T细胞受体包括前述任一项的TCR β链可变区，例如：
(1)TCR β链可变区包含SEQ ID NO:17所示氨基酸序列所含的三个互补决定区CDR；
(2)TCR β链可变区包含SEQ ID NO:25所示氨基酸序列所含的三个互补决定区CDR；
(3)TCR β链可变区包含SEQ ID NO:18所示氨基酸序列的CDR1、SEQ ID NO:19所示氨基酸序列的CDR2和/或SEQ ID NO:20所示氨基酸序列的CDR3；
(4)TCR β链可变区包含SEQ ID NO:26所示氨基酸序列的CDR1、SEQ ID NO:27所示氨基酸序列的CDR2和/或SEQ ID NO:28所示氨基酸序列的CDR3；
(5)TCR β链可变区包含SEQ ID NO:17所示氨基酸序列或由其组成；或
(6)TCR β链可变区包含SEQ ID NO:25所示氨基酸序列或由其组成。
在一些实施方式中，前述任一项的T细胞受体包括前述任一项的TCR α链可变区，例如：
(1)TCR α链可变区包含SEQ ID NO:13所示氨基酸序列所含的三个互补决定区CDR；
(2)TCR α链可变区包含SEQ ID NO:21所示氨基酸序列所含的三个互补决定区CDR；
(3)TCR α链可变区包含SEQ ID NO:14所示氨基酸序列的CDR1、SEQ ID NO:15所示氨基酸序列的CDR2和/或SEQ ID NO:16所示氨基酸序列的CDR3；
(4)TCR α链可变区包含SEQ ID NO:22所示氨基酸序列的CDR1、SEQ ID NO:23所示氨基酸序列的CDR2和/或SEQ ID NO:24所示氨基酸序列的CDR3；
(5)TCR α链可变区包含SEQ ID NO:13所示氨基酸序列或由其组成；或
(6)TCR α链可变区包含SEQ ID NO:21所示氨基酸序列或由其组成。
在一些实施方式中，前述任一项的T细胞受体的TCR α链可变区和TCR β 链可变区选自：
(1)所述TCR α链可变区包含SEQ ID NO:13所示氨基酸序列所含的三个互补决定区CDR，所述TCR β链可变区包含SEQ ID NO:17所示氨基酸序列所含的三个互补决定区CDR；
(2)所述TCR α链可变区包含SEQ ID NO:21所示氨基酸序列所含的三个互补决定区CDR，所述TCR β链可变区包含SEQ ID NO:25所示氨基酸序列所含的三个互补决定区CDR；
(3)所述TCR α链可变区包含SEQ ID NO:14所示氨基酸序列的CDR1、SEQ ID NO:15所示氨基酸序列的CDR2和SEQ ID NO:16所示氨基酸序列的CDR3，所述TCR β链可变区包含SEQ ID NO:18所示氨基酸序列的CDR1、SEQ ID NO:19所示氨基酸序列的CDR2和SEQ ID NO:20所示氨基酸序列的CDR3；
(4)所述TCR α链可变区包含SEQ ID NO:22所示氨基酸序列的CDR1、SEQ ID NO:23所示氨基酸序列的CDR2和SEQ ID NO:24所示氨基酸序列的CDR3，所述TCR β链可变区包含SEQ ID NO:26所示氨基酸序列的CDR1、SEQ ID NO:27所示氨基酸序列的CDR2和SEQ ID NO:28所示氨基酸序列的CDR3；
(5)所述TCR α链可变区包含SEQ ID NO:13所示氨基酸序列或由其组成，所述TCR β链可变区包含SEQ ID NO:17所示氨基酸序列或由所述氨基酸序列组成；或
(6)所述TCR α链可变区包含SEQ ID NO:21所示氨基酸序列或由其组成，所述TCR β链可变区包含SEQ ID NO:25所示氨基酸序列或由所述氨基酸序列组成。
在一些实施方式中，所述T细胞受体包括前述任意一种TCR α链胞外恒定区和/或TCR β链胞外恒定区。
在一些实施方式中，所述T细胞受体包括前述任意一种TCR α链跨膜区和/或TCR β链跨膜区。
在一些实施方式中，所述T细胞受体包括前述任意一种TCR α链恒定区和/或TCR β链恒定区。例如，所述TCR α链恒定区和TCR β链恒定区选自：
(1)所述TCR α链恒定区包含SEQ ID NO:5的氨基酸序列或由所述氨基酸序列组成，所述TCR β链恒定区包含SEQ ID NO:12的氨基酸序列或由所述氨基酸序列组成；
(2)所述TCR α链恒定区包含SEQ ID NO:6的氨基酸序列或由所述氨基酸序列组成，所述TCR β链恒定区包含SEQ ID NO:12的氨基酸序列或由所述氨基酸序列组成；或
(3)所述TCR α链恒定区包含SEQ ID NO:6的氨基酸序列或由所述氨基酸序列组成，所述TCR β链恒定区包含SEQ ID NO:11的氨基酸序列或由所述氨基酸序列组成。
另一方面，本申请提供一种或多种T细胞受体F5-TCR，包括F5-WT TCR、F5-SA TCR、F5-KR TCR或F5-dMUT TCR。所述F5-TCR可变区识别肿瘤抗原MART-1，所述F5-TCR α链可变区为SEQ ID NO:13的氨基酸序列，所述F5-TCRβ链可变区为SEQ ID NO:17的氨基酸序列；
进一步，所述F5-WT TCR α链恒定区为SEQ ID NO:5的氨基酸序列，F5-WT TCR β链恒定区为SEQ ID NO:11的氨基酸序列；
进一步，所述F5-SA TCR α链恒定区为SEQ ID NO:6的氨基酸序列，F5-SA TCR β链恒定区为SEQ ID NO:11的氨基酸序列；
进一步，所述F5-KR TCR α链恒定区为SEQ ID NO:5的氨基酸序列，F5-KR TCR β链恒定区为SEQ ID NO:12的氨基酸序列；
进一步，所述F5-dMUT TCR α链恒定区为SEQ ID NO:6的氨基酸序列，F5-dMUT TCR β链恒定区为SEQ ID NO:12的氨基酸序列。
另一方面，本申请提供一种或多种T细胞受体1G4-TCR，包括1G4-WT TCR、1G4-SA TCR、1G4-KR TCR或1G4-dMUT TCR。所述1G4-TCR可变区识别肿瘤抗原NYESO-1，所述1G4-TCR α链可变区为SEQ ID NO:21的氨基酸序列或，所述1G4-TCR β链可变区为SEQ ID NO:25的氨基酸序列；
进一步，所述1G4-WT TCR α链恒定区为SEQ ID NO:5的氨基酸序列，1G4-WT TCR β链恒定区为SEQ ID NO:11的氨基酸序列；
进一步，所述1G4-SA TCR α链恒定区为SEQ ID NO:6的氨基酸序列，1G4-SA TCR β链恒定区为SEQ ID NO:11的氨基酸序列；
进一步，所述1G4-KR TCR α链恒定区为SEQ ID NO:5的氨基酸序列，1G4-KR TCR β链恒定区为SEQ ID NO:12的氨基酸序列。
进一步，所述1G4-dMUT TCR α链恒定区为SEQ ID NO:6的氨基酸序列，1G4-dMUT TCR β链恒定区为SEQ ID NO:12的氨基酸序列。
另一方面，前述任意一种TCR α链、TCR β链或T细胞受体进一步与信号肽结合，所述信号肽与TCR α链可变区和/或TCR β链可变区形成信号肽-可变区结构。
在一些实施方式中，所述信号肽选自人生长激素信号肽、CD8α信号肽、免疫球蛋白信号肽。
在一些实施方式中，所述信号肽包含SEQ ID NO:29和/或SEQ ID NO:30的氨基酸序列或由所述氨基酸序列组成。
在一些实施方式中，TCR α链信号肽氨基酸序列包含SEQ ID NO:29的氨基酸序列或由所述氨基酸序列组成。
在一些实施方式中，TCR β链信号肽氨基酸序列包含SEQ ID NO:30的氨基酸序列或由所述氨基酸序列组成。
形成本申请的前述任意一种TCR α链、TCR β链或T细胞受体的上述各个部分，相互之间可直接相连，或通过接头序列连接。接头序列可以是GGGS、GGGGS、GSGSA和GGSGG等基序相邻连接的，重复1～5个基序，长度3～25个氨基酸残基的序列。
另一方面，本申请提供一种分离的核酸或其片段，其编码前述任意一项分离的TCR α链或其片段、TCR β链或其片段、或T细胞受体或其片段。
在一些实施方式中，编码野生型TCR α链恒定区核酸序列包含SEQ ID NO:31的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码野生型TCR β链恒定区核酸序列包含SEQ ID NO:32的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码SA TCR α链恒定区核酸序列包含SEQ ID NO:33的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码KR TCR β链恒定区核酸序列包含SEQ ID NO:34的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码F5-TCR α链可变区核酸序列包含SEQ ID NO:35的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码F5-TCR β链可变区核酸序列包含SEQ ID NO:36的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码1G4-TCR α链可变区核酸序列包含SEQ ID NO:37的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码1G4-TCR β链可变区核酸序列包含SEQ ID NO:38 的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码F5-WT-TCR核酸序列包含SEQ ID NO:39的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码F5-SA-TCR核酸序列包含SEQ ID NO:40的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码F5-KR-TCR核酸序列包含SEQ ID NO:41的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码F5-dMUT-TCR核酸序列包含SEQ ID NO:42的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码1G4-WT-TCR核酸序列包含SEQ ID NO:43的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码1G4-SA-TCR核酸序列包含SEQ ID NO:44的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码1G4-KR-TCR核酸序列包含SEQ ID NO:45的核酸序列或由所述核酸序列组成。
在一些实施方式中，编码1G4-dMUT-TCR核酸序列包含SEQ ID NO:46的核酸序列或由所述核酸序列组成。另一方面，本申请提供一种核酸构建物，其包含前述任意一项分离的核酸或其片段。
在一些实施方式中，所述核酸构建物还包括与前述核酸操作性连接的一个或多个调控序列。例如，包括但不限于在宿主细胞中显示转录活性的启动子序列、在宿主细胞中被识别并终止转录的与编码序列的3’末端操作性连接的转录终止子序列、对宿主细胞翻译重要的与编码序列的5’末端操作性连接的前导序列。
另一方面，本申请提供一种载体，其包含前述任意一项的核酸构建物。
在一些实施方式中，所述载体为表达载体或crisper基因编辑载体，例如逆转录病毒载体、慢病毒载体、杆状病毒载体、疱疹病毒载体、腺病毒载体、腺相关病毒(AAV)载体。
在一些实施方式中，所述逆转录病毒载体的构建主要包括复制起始位点、5’-LTR、3’-LTR及前述任意一项的核酸序列或核酸构建物。
通常可操作性的将启动子与编码T细胞受体的核酸序列相连，并将核酸构建物并入表达载体，实现编码T细胞受体的多核苷酸序列的表达。编码本申请T细胞受体的核酸序列可被克隆入许多类型载体。例如，包括但不限于质粒、噬菌体、动物病毒等。
合适的表达载体包含一种或多种在有机体中起作用的启动子序列、复制起点、合适的酶切位点和可选择的标记。
合适的启动子包括但不限于即时早期巨细胞病毒(CMV)启动子、延伸生长因子-1a(EF-1a)、人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)、长末端重复(LTR)启动子等组成型启动子序列，能够驱动任何连接其上的任何多核苷酸序列高水平表达；也包括但不限于金属硫蛋白启动子、四环素启动子等诱导型启动子，用于在期望多核苷酸序列表达时打开，不期望其表达时关闭。
可选择的标记包括标记基因和报告基因，为了便于鉴定通过病毒载体感染的细胞群中的选择表达的细胞。合适的标记基因包括但不限于抗生素抗性基因、neo基因等。合适的报告基因包括但不限于β-半乳糖苷酶、绿色荧光蛋白基因、荧光素酶、氯霉素乙酰转移酶等。
另一方面，本申请提供一种工程化细胞，其包含前述任意一项的核酸构建 物或载体。
在一些实施方式中，所述工程化细胞为获自受试者的原代细胞。在一些实施方式中，受试者为哺乳动物受试者，例如受试者为人类。
在一些实施方式中，工程化细胞为T细胞，优选地人T细胞。
在一些实施方式中，工程化细胞为NK细胞，NKT细胞，巨噬细胞，γδT细胞，人CD4 +T细胞，CD8 +T细胞，或CD4 +T/CD8 +T细胞的混合细胞群。
通过常规的重组DNA技术，可利用本申请的TCR核酸构建物或包含所述核酸构建物的载体来表达或生产TCR或包含所述TCR的工程化细胞。通常所述方法包括：
(1)用编码本申请的TCR核酸构建物或包含所述核酸构建物的载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养宿主细胞；
(3)从培养基或细胞中分离、纯化出本申请工程化细胞或TCR。
将不同核酸序列或载体导入工程化细胞的方法目前是领域内已经熟知的，通过物理方法，化学方法及生物学方法可以将目的基因导入宿主细胞。
在一些实施方式中，本申请使用逆转录病毒来完成目的序列导入宿主细胞，逆转录病毒感染法是目前使用非常广泛的感染人或鼠源原代细胞的方法。本申请一些实施方式中，将不同突变TCR核酸序列构建到逆转录病毒载体之后，通过使用优选的病毒包膜蛋白或衣壳蛋白包装产生病毒颗粒。随后将重组的病毒颗粒传递进宿主或离体培养的细胞中，完成目的基因在宿主细胞的表达。
上述的病毒颗粒中包含TCR核酸序列、逆转录病毒载体及包装蛋白的核酸序列。
另一方面，本申请还提供一种TCR复合物，其由本申请的前述任意一项TCR 核酸构建物在T细胞中产生。
另一方面，本申请提供了TCR-T细胞在体外培养过程中进行了功能性检测。具体的，包括TCR-T细胞记忆性表型检测，体外耗竭模型中检测TCR-T细胞耗竭表型以及TCR-T细胞线粒体表型检测。上述TCR-T细胞记忆性表型和线粒体表型检测在人原代T细胞激活后，利用细胞流式进行检测。上述突变TCR-T细胞耗竭检测使用了体外构建好的耗竭模型，具体的，利用CD3抗体体外多轮刺激不同突变的TCR-T细胞，然后利用流式细胞技术检测耗竭相关分子表达。
另一方面，本申请提供了前述任一项所述分离的TCR α链或其片段、TCRβ链或其片段、T细胞受体或其片段、核酸或其片段、核酸构建物、载体或工程化细胞在下述任意一种或多种用途中的应用：
(1)在抗原刺激过程中的抑制T细胞受体降解中的应用；
(2)提供对肿瘤的杀伤效果，或抑制肿瘤生长；
(3)维持T细胞的增殖能力；
(4)抗肿瘤的免疫药物及细胞的制备。
另一方面，本申请提供的突变TCR-T细胞疗法可以单独使用或联合其他疗法同时使用，或联合PD-1/PD-L1抗体，或联合细胞因子疗法，或联合放化疗，共同结合使用。上述疗法单独使用或联合使用时，其使用数量和频率由病症特性及病症的严重程度等多种因素共同确定。理论上，本申请所提供的TCR-T细胞疗法可以低剂量多次使用，上述的使用剂量范围为10 4-10 9个细胞/千克。而本申请所提供的TCR-T细胞的过继治疗方式遵循国际上公认的过继技术实施。在本文的一个实施例中5x10 6个TCR-T细胞通过尾静脉注射进行实施。原则上，T细胞疗法联用物可以直接注入肿瘤组织或感染部位。
另一方面，本申请提供一种T细胞受体的改造方法，包括：
(1)将前述任一项野生型TCR β链胞内恒定区中至少一个的赖氨酸突变为精氨酸或丙氨酸；和/或
(2)将前述任一项野生型TCRα链胞内恒定区中至少一个的丝氨酸突变为丙氨酸。
为了更容易理解本申请公开内容，定义如下某些术语。
T细胞受体(TCR)是存在于T细胞表面的分子，其负责识别肽-MHC复合物。在一些实施方案中，所述TCR是完整或全长TCR。在一些实施方案中，本申请提供一种TCR片段，其小于全长TCR，但仍保留与MHC分子的特定抗原肽即MHC-肽复合物的结合。在一些实施方案中，TCR是由α和β链组成的异二聚体。在一些实施方案中，TCR也可以是单链TCR(scTCR)。
术语“可变区”或“可变域”是指TCRα或β链的结构域，其参与TCR与抗原-MHC复合物的结合。天然TCR的α链和β链的可变区通常具有类似的结构，每个结构区均包含四个保守框架区(FR)和三个高变区或互补决定区(CDR)。其中各可变区中的CDR3是负责识别经加工的抗原的主要CDR。单个TCRα链可变区或TCRβ链可变区可能足以赋予对肽-MHC复合物的结合。
应当理解，在一些实施方案中，本申请的TCRα链可以是人TCRα链、人源化TCRα链、嵌合TCRα链或鼠源TCRα链。在本申请的一些实施方案中，TCRα链是嵌合TCRα链，其包含衍生自多于一种物种的序列，例如衍生自人和小鼠的序列。举例来说，将人TCR恒定区用鼠对应物交换可以改善人T细胞的功能以及表达水平(参见，例如，Daniel Sommermeyer et al.,J Immunol.2010 Jun 1；184(11):6223-31.,其通过引用并入本文作为参考)。因此，TCR可以包含人衍生的可变区和鼠衍生的恒定区。
同样，在一些实施方案中，本申请的TCRβ链也可以是人TCRβ链、人源化 TCRβ链、嵌合TCRβ链或鼠源TCRβ链。嵌合TCRβ链，其包含衍生自多于一种物种的序列，例如衍生自人和小鼠的序列。
依据本申请所描述的TCR α链恒定区和/或TCRβ链恒定区，其包括依次连接的胞外恒定区、跨膜区和胞内恒定区，其中胞外恒定区可以包括TCR α链和TCRβ链铰链区，参与TCR α链TCR β链二硫键的形成；跨膜区亦属于恒定区，其作用包括参与TCR α链、TCRβ链的细胞膜锚定以及与CD3亚基的相互作用，形成TCR-CD3复合物；胞内恒定区可能的作用包括参与TCR信号转导后，TCR-CD3复合物构象的转变及信号传导。
术语“抗原”是指细胞表面分子或胞内由MHC分子或MHC-like分子呈递，可被抗体或T细胞受体(TCR)结合的分子，包括但不限于多肽类抗原(例如NYESO-1、AFP和MART-1)、脂类抗原(例如β-GlcCer、eLPA和LPE)或多糖类抗原(例如CA199、CA72-4和CA125)。所述抗原可以是肿瘤抗原，例如肿瘤细胞相关抗原(Tumor-associated antigen，TAA)，或肿瘤特异性抗原(Tumor specific antigen，TSA)。
术语“分离的”是指已经从其天然状态移出或以其他方式进行人为操作的材料，例如本文所述的α链、β链、T细胞受体和核酸。分离的材料可以基本上或实质上没有通常在其天然状态伴随其的组分，或者可以经操作而处于人造状态，其与通常在其天然状态伴随其的组分在一起。分离的材料可以是天然的、化学合成的或重组的形式。分离的材料也可以或备选地是富集的、部分纯化的或纯化的形式。
应当理解，本发明“野生型”是指一类天然存在的氨基酸序列或编码核酸序列，或与天然存在氨基酸序列或编码核酸序列相比具有至少90％-100％的同一性，或与天然存在氨基酸序列或编码核酸序列相比具有不超过1-10个或1-5个 氨基酸突变(尤其是保守氨基酸取代)，并且仍然具有与该区域相同或相似的活性和/或功能。
“野生型”作为本申请相应突变或突变组合的模板而引入，例如“来源于人源、鼠源或其他哺乳动物种属的野生型”，是指所述区域具有天然人、天然小鼠或大鼠、或其他哺乳动物种属的该区域的氨基酸序列或编码核酸序列，或由该氨基酸序列或编码核酸序列组成。“来源于”人、鼠或其他哺乳动物种属的区域还涵盖这样的区域，其基本上与天然人、天然小鼠或大鼠、或其他哺乳动物种属的该区域的氨基酸序列或编码核酸序列相同，例如与天然存在氨基酸序列或编码核酸序列相比具有至少90％-100％的同一性，或与天然存在氨基酸序列或编码核酸序列相比具有不超过1-10个或1-5个氨基酸突变(尤其是保守氨基酸取代)，并且仍然具有与该区域相同或相似的活性和/或功能。
术语“突变”可以理解为一个或多个氨基酸或核酸的取代、缺失或添加。例如可以是氨基酸的保守取代，保守氨基酸取代是本领域已知的，并且包括这样的氨基酸取代，其中一个具有一定物理和/或化学性质的氨基酸被交换为另一个具有相同化学或物理性质的氨基酸。例如，所述保守氨基酸取代可以是酸性氨基酸取代另一个酸性氨基酸(如，Asp或Glu)，具有非极性侧链的氨基酸取代另一个具有非极性侧链的氨基酸(如，Ala、Gly、Val、He、Leu、Met、Phe、Pro、Trp、Val等)，碱性氨基酸取代另一个碱性氨基酸(Lys、Arg等)，具有极性侧链的氨基酸取代另一个具有极性侧链的氨基酸(Asn、Cys、Gin、Ser，Thr，Tyr等)等，所述保守取代可以例如基于极性、电荷、溶解度、疏水性、亲水性和/或所涉及残基的两亲性质的相似性来进行。
在涉及本申请TCR α链胞内恒定区和/或TCR β链胞内恒定区的“突变”时，其优选为氨基酸的取代。例如，突变的TCR α链胞内恒定区是指野生型TCR α 链胞内恒定区中至少一个丝氨酸取代为丙氨酸；突变的TCR β链胞内恒定区是指野生型TCR β链胞内恒定区中至少一个赖氨酸取代为精氨酸或丙氨酸。
如下进行序列之间序列同一性的计算。
为确定两个氨基酸序列或两个核酸序列的同一性百分数，将所述序列出于最佳比较目的比对(例如，可以为了最佳比对而在第一和第二氨基酸序列或核酸序列之一或二者中引入空位或可以为比较目的而抛弃非同源序列)。在一个优选实施方案中，为比较目的，所比对的参考序列的长度是至少30％、优选地至少40％、更优选地至少50％、60％和甚至更优选地至少70％、80％、90％、100％的参考序列长度。随后比较在对应氨基酸位置或核酸位置处的氨基酸残基或核酸。当第一序列中的位置由第二序列中对应位置处的相同氨基酸残基或核酸占据时，则所述分子在这个位置处是相同的。
可以利用数学算法实现两个序列间的序列比较和同一性百分数的计算。在一个优选实施方案中，使用已经集成至GCG软件包的GAP程序中的Needlema和Wunsch((1970)J.Mol.Biol.48:444-453)算法(在http://www.gcg.com可获得)，使用Blossum 62矩阵或PAM250矩阵和空位权重16、14、12、10、8、6或4和长度权重1、2、3、4、5或6，确定两个氨基酸序列之间的同一性百分数。在又一个优选的实施方案中，使用GCG软件包中的GAP程序(在http://www.gcg.com可获得)，使用NWSgapdna.CMP矩阵和空位权重40、50、60、70或80和长度权重1、2、3、4、5或6，确定两个核酸序列之间的同一性百分数。特别优选的参数集合(和除非另外说明否则应当使用的一个参数集合)是采用空位罚分12、空位延伸罚分4和移码空位罚分5的Blossum 62评分矩阵。
还可以使用PAM120加权余数表、空位长度罚分12，空位罚分4，利用已经并入ALIGN程序(2.0版)的E.Meyers和W.Miller算法,((1989)CABIOS, 4:11-17)确定两个氨基酸序列或核酸序列之间的同一性百分数。
额外地或备选地，可以进一步使用本文所述的核酸序列和蛋白质序列作为“查询序列”以针对公共数据库执行检索，以例如鉴定其他家族成员序列或相关序列。
如本领域已知，在本文中可交换使用的“核苷酸”或“核酸”是指任何长度的核酸链，并且包括DNA和RNA。核酸可以是脱氧核糖核酸、核糖核酸、修饰的核酸或碱基、和/或它们的类似物、或者能够通过DNA或RNA聚合酶掺入链的任何底物。
本文所述的核酸序列可根据本文公开的核苷酸序列设计引物，通过PCR扩增法获得，或通过技术人员制备cDNA库进行PCR扩增获得。
应当理解，本申请中“野生型”和“WT”可以互换地使用；“SA”指α链胞内恒定区的氨基酸序列中至少一个丝氨酸突变为丙氨酸；“KR”指β链胞内恒定区的氨基酸序列中至少一个赖氨酸突变为精氨酸；“dMUT”指α链胞内恒定区中“SA”突变和β链胞内恒定区的氨基酸序列中“KR”突变同时存在。
应当理解，本申请中F5-TCR是指一类可以识别肿瘤抗原MART-1的TCR，其所包括的F5-WT TCR、F5-SA TCR、F5-KR TCR和F5-dMUT TCR具有相同的TCR α链可变区和TCR β链可变区，所述F5-WT TCR、F5-SA TCR、F5-KR TCR和F5-dMUT TCR依据α链胞内恒定区或β链胞内恒定区中突变不同而划分。
同样，本申请中1G4-TCR是指一类可以识别肿瘤抗原NYESO-1的TCR，其所包括的1G4-WT TCR、1G4-SA TCR、1G4-KR TCR或1G4-dMUT TCR具有相同的TCR α链可变区和TCR β链可变区，所述1G4-WT TCR、1G4-SA TCR、1G4-KR TCR或1G4-dMUT TCR依据α链胞内恒定区或β链胞内恒定区中突变不同而划分。
“载体”表示其能够将一种或多种所关注的基因或序列递送入宿主细胞并且优选在宿主细胞中表达所述基因或序列。载体的实例包括但不限于病毒载体、质粒、粘粒或噬菌体载体。
术语“宿主细胞”是指已经引入外源性核酸的细胞，包括这些细胞的子代。
应当理解，在基因克隆操作中，所设计的合适的酶切位点会在所表达的氨基酸序列的两端引入一个或多个不相干的残基，且并不影响目的序列的活性。例如，包括但不限于NotI、BamHI、XhoI等酶切位点。
应当理解，为了构建融合蛋白或获得自动定位到宿主细胞膜上的重组蛋白或促进重组蛋白的表达，需要将氨基酸片段添加至重组蛋白的N-末端、C-末端或蛋白内合适的区域，例如，包括但不限于信号肽、前导肽、2A肽、末端延伸等。
应当理解，为了对所构建重组蛋白进行纯化，所构建的蛋白的氨基端或羧基端含有一个或多个蛋白标签。例如，包括但不限于FLAG、HA、c-Myc、Poly-His等。
应当理解，以上实施例(实施方式)均为示例性的，不用于包含权利要求所包含的所有可能的实施方式。在不脱离本公开的范围的情况下，还可以在以上实施例的基础上做出各种变形和改变。同样的，也可以对以上实施例的各个技术特征进行任意组合，以形成可能没有被明确描述的本申请的另外的实施例。因此，上述实施例仅表达了本申请的几种实施方式，不对本申请专利的保护范围进行限制。
本申请序列表及注释
Figure PCTCN2022143567-appb-000001
Figure PCTCN2022143567-appb-000002
Figure PCTCN2022143567-appb-000003
Figure PCTCN2022143567-appb-000004
Figure PCTCN2022143567-appb-000005
Figure PCTCN2022143567-appb-000006
Figure PCTCN2022143567-appb-000007
Figure PCTCN2022143567-appb-000008
Figure PCTCN2022143567-appb-000009
实施例1.TCR表达载体构建
分别构建F5-TCR、1G4-TCR，其中F5-TCR包括F5-WT TCR、F5-SA TCR、F5-KR TCR和F5-dMUT TCR，其中1G4-TCR包括1G4-WT TCR、1G4-SA TCR、1G4-KR TCR和1G4-dMUT TCR。
依照前文所述，各F5-TCR或1G4-TCR分别包括TCRα链与TCRβ链，其中TCRα链分别由人生长激素信号肽、TCRα链可变区、TCRα链胞外恒定区、TCRα链跨膜区、TCRα链胞内区依次连接构成；TCRβ链分别由人生长激素信号肽、TCRβ链可变区、TCRβ链胞外恒定区、TCRβ链跨膜区、TCRβ链胞内区依次连接构成。TCRα链与TCRβ链通过F2A肽串联起来。以上各TCRα链与TCRβ链氨基酸或核苷酸序列如本申请前述实施方式所记载。
以上氨基酸序列以及TCRα链胞内恒定区丝氨酸突变为丙氨酸、TCRβ链恒定区赖氨酸突变为精氨酸的氨基酸序列均经过密码子优化后转换为碱基序列，并由公司合成(GenScript)。
所述F5-WT TCR、F5-SA TCR、F5-KR TCR和F5-dMUT TCR，以及1G4-WT TCR、1G4-SA TCR、1G4-KR TCR和1G4-dMUT TCR的全长核苷酸序列分别如 SEQ ID NO:39-46所记载。
本申请中所有TCR的碱基序列最终通过酶切，T4连接酶连接的方式克隆至pMSGV-LNGFR-P2A载体中。
实施例2.逆转录病毒包装制备方法
将从实施例1中所构建的TCR表达载体(pMSGV-TCR)，同包膜质粒pHIT60/RD114共转染到HEK293T细胞中，包装逆转录病毒。具体的，包装比例体系为MSGV-TCR：pHIT60：RD114＝2：2：1.3，OPTI-DMEM培养基中混匀，加入15.9μl的TransIT293-Mirus转染试剂(Mirusbio#MIR 2700)，吹打混匀后，室温下静置25分钟之后，将上述混合脂质体液体加入HEK293T细胞培养皿中，放入37℃培养箱培养48h。然后收取病毒液，经过0.22μm滤膜过滤，放置于-80℃冰箱备用。
实施例3.人原代突变TCR-T细胞的制备及培养方法
人原代T细胞培养：人原代T细胞购买于商业化公司(ALLCELLS)。培养原代T细胞的完全培养基包含5％的人血清(Gemini#100-512)，含有双抗(Gibco#15140-122)的RPMI-1640培养基(Hyclone#SH30809.01)及1xGlutMAX(Gibco#35050-061)，同时培养基中要加入100U/ml的rhIL-2(PeproTech#200-02)。用1ml上述完全培养基重悬人原代T细胞于经1μg/ml hCD3(Biolegend#317347)和1μg/ml hCD28抗体(Biolegend#102121)所包被的24孔板(Nest#702001)中，置于37℃培养箱培养48小时后进行病毒感染实验。
逆转录病毒感染人原代T细胞：重悬24孔板中的人原代T细胞，吸取750μl与EP管中(Axygen#MCT-105C)，2500rpm离心5分钟，弃掉上清，使用500μl相对应的病毒液(即突变TCR逆转录病毒)重悬T细胞，并加入0.75μl的 Polybrene(SANTA CRUZ#SC-134220)，吹打均匀后加入相对应的24孔板中，此时感染体系为750μl。30℃，2500rpm离心90分钟后，从24孔板中吸取500μl上清与EP管中，2500rpm离心5分钟，弃掉上清，加入750μl的T细胞完全培养基(1.25倍的rhIL-2)于24孔板中，放置37℃培养箱培养24小时。转天进行第二次病毒感染操作，具体步骤与前述操作相同，完成感染后的人原代T细胞每个1-2天补充新鲜的RPMI-1640完全培养基。
实施例4.细胞流式技术分析方法
流式细胞分析技术使用BD LSR Fortessa仪器(BD Bioscience)进行检测。对于细胞表面分子的检测：吸取2x10 5个细胞置于96孔U底板中，1800rpm离心5分钟后，弃掉上清，加入FACS缓冲液(含2％血清的1xPBS)配制的流失抗体，冰上避光染色25分钟，FACS缓冲液洗一次，流式上机检测。
对于细胞因子的染色：吸取2x10 5个细胞置于96孔U底板中，1800rpm离心5分钟后，弃掉上清，加入FACS缓冲液(含2％血清的1xPBS)配制的流失抗体，冰上避光染色25分钟，FACS缓冲液洗一次，使用多聚甲醛固定液(Biolegend#420801)固定细胞20分钟，FACS缓冲液洗一次，加入使用破膜液(Invitrogen#00-8333-56)配制的细胞因子流式抗体，冰上避光染色25分钟，FACS缓冲液洗一次，流式上机检测。
对于转录因子的染色：吸取2x10 5个细胞置于96孔U底板中，1800rpm离心5分钟后，弃掉上清，加入FACS缓冲液(含2％血清的1xPBS)配制的流失抗体，冰上避光染色25分钟，FACS缓冲液洗一次，使用FOXP3转录因子固定液(Invitrogen#00-5523-00)固定细胞20分钟，FACS缓冲液洗一次，加入使用破膜液配制的细胞因子流式抗体，冰上避光染色25分钟，FACS缓冲液洗一次，流式上机检测。流式数据使用FlowJo software软件(Tree Star)进行数据分析。
实施例5.抗原刺激引起的TCR降解
体内肿瘤抗原刺激引起TCR表达下调实验：K562-NYESO-1接种的荷瘤小鼠，1G4-TCR-T细胞过继第十二天后，检测脾脏及肿瘤中TCR-T细胞表面TCR的表达情况。如图1(A)所示肿瘤组织中肿瘤抗原刺激引起了TCR表达的下调。体外靶细胞抗原刺激引起TCR下调和降解实验：1G4-TCR-T细胞与K562-NYESO-1靶细胞或K562-MART-1非靶细胞按1:1比例混合24孔板中，37℃培养箱共孵育12小时(应理解的，具体时间由不同实验决定)，吸取细胞进行流式FACS检测，检测TCR下调通过染色T细胞表面TCR来测定，而分析TCR降解水平则通过细胞固定破膜之后染色胞内TCR进行检测，结果如图1(B)所示靶细胞抗原刺激促进了TCR的下调和降解；hCD3抗体刺激引起TCR下调和降解实验：活化之后的1G4-TCR-T细胞和F5-TCR-T细胞重悬后，加入由hCD3抗体包被过的24孔板中，37℃培养箱共孵育12小时(应理解的，具体时间由不同实验决定)，吸取细胞进行流式FACS检测，检测TCR下调通过染色T细胞表面TCR来测定，而分析TCR降解水平则通过细胞固定破膜之后染色胞内TCR进行检测，如图1(C、D)所示体外CD3抗体刺激促进了TCR在人源T细胞中的下调和降解。不同突变TCR的表达对比实验：活化之后的不同突变1G4-TCR-T细胞重悬后，FACS染色检测不同TCR的表达，如图2(B)所示不同突变TCR相较于野生型TCR其表达无明显差别。hCD3抗体刺激引起不同突变TCR下调和降解的对比实验：活化之后的1G4-TCR-T细胞和F5-TCR-T细胞重悬后，加入由hCD3抗体包被过的24孔板中，37℃培养箱共孵育实验所需时间，检测TCR下调通过染色T细胞表面TCR来测定，而分析TCR降解水平则通过细胞固定破膜之后染色胞内TCR进行检测，结果如图2(C、D)所示相比于WT TCR，突变组SA TCR，KR TCR和dMUT TCR展现出更多T细胞 膜驻留，并且能够显著抵抗由抗体刺激引起的下调和降解，而这一现象在dMUT TCR组显得尤其明显。而图(E)所示，由hCD3抗体刺激不同时间点检测表面TCR表达的实验显示，相比于其他组，dMUT TCR具有最强的抵抗降解的能力。
实施例6.T细胞体外功能检测及耗竭模型构建
T细胞体外功能检测：体外活化的人原代T细胞经不同突变1G4-TCR逆转录病毒感染后，T细胞在RPMI-1640完全培养基中培养12天，吸取2x10 5个细胞至96孔板中，进行流式FACS检测，检测指标为T细胞记忆性分子的表达如：CD62L、TCF1、CD27和CD45RO，结果如图3(A-C)所示，相比于WT TCR组，SA TCR、KR TCR和dMUT TCR-T细胞中CD27 +CD45RO +(中央记忆T细胞)比例显著增加，突变组更倾向于分化为记忆性T细胞，同时，T细胞记忆性分子CD62L和TCF1的表达升高，尤其dMUT TCR-T细胞最为明显。
T细胞体外耗竭模型构建：体外活化的人原代T细胞经不同突变1G4-TCR逆转录病毒感染48小时后，取出T细胞，2500rpm离心5分钟，用完全培养基重悬TCR-T细胞于2μg/ml hCD3抗体预先包被过的24孔板中(每孔10 6个细胞)，再次刺激TCR-T细胞48小时，之后重复上述步骤2次，结束培养，收取TCR-T细胞进行流式检测。此体外耗竭模型构建参考了本领域内公认的相关文章(Santosha A.Vardhana et al.,Nat Immunol.2020 Sep；21(9):1022-1033.)，检测指标为耗竭相关分子PD-1、TIM3、LAG-3、TOX等和细胞因子TNF-α以及IFN-γ，结果如图3(D-F)所示，耗竭相关分子LAG-3和TOX的表达在KR TCR-T细胞和dMUT TCR-T细胞中明显减少，同时有更多dMUT TCR-T细胞产生细胞因子TNF-α和IFN-γ，总体说明KR TCR-T细胞和dMUT TCR-T细胞能更好的抵抗T细胞耗竭的发生。
实施例7.T细胞线粒体功能检测及代谢分析
线粒体指标检测：体外活化的人原代T细胞经不同突变1G4-TCR逆转录病毒感染后，T细胞在RPMI-1640完全培养基中培养12天，吸取2x10 5个细胞至EP管中，加入MitoTracker Green(Invitrogen#M7514)染色，终浓度为50nM，37℃避光染色1小时后进行流式FACS检测，结果如图4(A)所示，相比于WT TCR细胞，不同突变TCR-T细胞中，线粒体数量无明显变化。对于线粒体膜电势的检测方法为吸取2x10 5个细胞至EP管中，加入TMRE(Invitrogen#T669)染色，终浓度为200nM，37℃避光染色1小时后进行流式FACS检测，结果如图4(B)所示，相比于WT TCR细胞，SA TCR，KR TCR和dMUT TCR-T细胞中线粒体膜电势明显降低，而dMUT TCR-T细胞尤为明显。对于线粒体活性氧的检测方法为吸取2x10 5个细胞至EP管中，加入MitoSOX(Invitrogen#M36008)染色，终浓度为5μM，37℃避光染色1小时后进行流式FACS检测，如图4(C)所示，相比于WT TCR细胞，SA TCR，KR TCR和突变TCR-T细胞中线粒体ROS的产生明显减少。总结上述实施例结果说明，不同的TCR突变并不影响T细胞内的线粒体数量，但突变TCR细胞尤其dMUT TCR-T细胞中线粒体展现出更加健康的状态，这与dMUT TCR-T细胞体外培养过程中表现出记忆性表型并抵抗T细胞耗竭有关。
代谢相关指标检测：感染突变1G4-TCR逆转录病毒的人原代T细胞，体外37℃培养箱RPMI-1640完全培养基中培养12天，吸取2x10 5个细胞于EP管中，2500rpm离心5分钟，弃掉上清，然后使用无糖无血清培养基重悬T细胞于24孔板中，终浓度50μM的2-NBDG(Invitrogen#N13195)或1μM的Bodipy FL C16(Invitrogen#D3821)处理细胞，37℃避光染色30分钟后进行流式FACS检测，流式上机之前可根据实验需求标记其他标记性分子，如图4(D、E)结果显示，相比于WT TCR细胞，SA TCR，KR TCR和dMUT TCR-T细胞对葡萄糖的摄入 量明显降低，而对于胞外脂肪酸的摄入则无明显变化。
实施例8.突变TCR-T细胞的抗肿瘤功能检测
突变TCR-T细胞的抗肿瘤实验在4-8周龄的免疫缺陷NSG小鼠体内进行。为比较突变TCR-T细胞相较于野生型TCR-T细胞具有更好的抗肿瘤效果，首先在NSG小鼠右侧后肢腋窝处皮下接种8x10 5个K562-NYESO-1细胞，待靶细胞在小鼠皮下生长8-12天左右，使用游标卡尺检测肿瘤体积，具体的，肿瘤体积在80mm 3-120mm 3时，NSG小鼠尾静脉注射5x10 6个感染了不同突变1G4-TCR的人原代T细胞，接下来每隔2天对肿瘤体积进行测量，大约14天左右时(具体操作时间由实验现象决定)，收取NSG小鼠的血液、脾脏和肿瘤组织，首先对肿瘤组织重量进行称量，然后使用红细胞裂解液(Biolegend#420301)分别对小鼠血液和脾脏进行裂解红细胞处理，小鼠肿瘤组织经研磨后进行Percoll密度梯度离心(Cytiva#17089110)，收取由小鼠血液、脾脏和肿瘤组织中所分离尾静脉过继的人原代T细胞，经流式染色后进行分析。
具体结果如图5和图6所示，首先，图5(A)通过对肿瘤生长体积的统计发现，相比较于WT TCR组，过继KR TCR-T细胞和dMUT TCR-T细胞的实验组，其中肿瘤生长速度明显减缓。对肿瘤组织进行称重后发现，过继KR TCR-T细胞和dMUT TCR-T细胞的实验组，肿瘤重量明显降低，如图5(B)所示。另外，本实施例中统计了荷瘤NSG小鼠脾脏、血液和肿瘤组织中过继的CD8 +TCR-T细胞的比例，结果如图5(C-E)所示，在小鼠脾脏和血液中，相比于WT TCR-T细胞，dMUT TCR-T细胞的比例明显升高。而在小鼠肿瘤组织中，相比于WT TCR-T细胞，KR TCR-T细胞和dMUT TCR-T细胞的比例明显升高。这说明KR TCR-T细胞和dMUT TCR-T细胞在NSG小鼠免疫过继模型中具有更强的抗肿瘤持久性。进一步通过细胞表型分析发现，相比于野生型TCR-T细胞， 脾脏中SA TCR、KR TCR和dMUT TCR-T细胞的CD27 +CD45RO +(中央记忆T细胞)比例明显增加，如图6(A)所示。同时通过对肿瘤组织中TCR-T细胞分析，发现相比于WT TCR-T细胞，dMUT TCR-T细胞表达更高的T细胞干性分子CD62L和CCR7，如图6(B、C)所示。图6(D)显示肿瘤组织之中，PD-1 +TIM-3 +双阳性的细胞比例在SA TCR、KR TCR和dMUT TCR组要明显少于WT TCR组，进一步，通过检测抑制性分子PD-1、TIM-3、LAG-3和CD39的荧光强度，发现与WT TCR-T细胞相比，突变组TCR-T细胞中抑制性分子的表达明显降低，如图6(E-H)所示。另外发现，耗竭关键转录因子TOX的表达在dMUT TCR-T细胞中明显减少如图6(I)。综上说明在体内抗肿瘤模型中，突变组TCR-T细胞能够抵抗T细胞耗竭的发生。同时检测了肿瘤组织中TCR-T细胞细胞因子的产生情况，如图6(J、K)所示，过继KR TCR-T细胞组和dMUT TCR-T细胞组，IFN-γ +CD8 +细胞比例明显增多，显示出KR TCR和dMUT TCR-T细胞有更强的抗肿瘤效果，同时发现CD8 +KR TCR-T细胞和CD8 +dMUT TCR-T细胞中TNF-α +IFN-γ +双阳性细胞比例明显增加，表明KR TCR-T细胞比WT TCR细胞产生更多的细胞因子，但少于dMUT TCR-T细胞，这说明dMUT TCR-T细胞在肿瘤组织中有更强的效应功能。
T细胞过继性免疫治疗中无论采集病人体内有限的T细胞去大量扩增TCR-T细胞，还是过继之后在肿瘤病人体内所面临的T细胞功能持久性降低及T细胞耗竭的发生都是目前TCR-T细胞治疗所面临的的困难。而本申请所提供的突变TCR-T细胞改造方法尤其是dMUT TCR-T改造，使得TCR-T细胞在经过体外扩增并回输到肿瘤模型中之后，展现出更加持久性抗肿瘤功效，并能抵抗T细胞耗竭的发生。这提示我们利用dMUT TCR突变改造的TCR-T细胞有望在临床生产及治疗上实现持续高效的抗肿瘤功能。

Claims (25)

  1. 一种分离的T细胞受体或其片段，所述T细胞受体包括TCRα链和TCRβ链，其特征在于，所述TCRβ链包括TCRβ链可变区和TCRβ链恒定区，所述TCRβ链恒定区包括依次连接的TCRβ链胞外恒定区、TCRβ链跨膜区和TCRβ链胞内恒定区，所述TCRβ链胞内恒定区选自：
    (1)突变的TCRβ链胞内恒定区，所述突变的TCRβ链胞内恒定区为野生型TCRβ链胞内恒定区中至少一个的赖氨酸突变为精氨酸或丙氨酸；或
    (2)包含SEQ ID NO:10所示氨基酸序列或由所述氨基酸序列组成的突变的TCRβ链胞内恒定区。
  2. 如权利要求1所述的分离的T细胞受体，其特征在于，所述野生型TCRβ链胞内恒定区选自：
    (1)来源于人源、鼠源或其他哺乳动物种属的野生型TCRβ链胞内恒定区；或
    (2)包含SEQ ID NO:9、SEQ ID NO:47或SEQ ID NO:48所示氨基酸序列或由所述氨基酸序列组成的野生型TCRβ链胞内恒定区。
  3. 如权利要求1-2任一项所述的分离的T细胞受体，其特征在于，所述TCRα链包括TCRα链恒定区，所述TCRα链恒定区包括依次连接的TCRα链胞外恒定区、TCRα链跨膜区和TCRα链胞内恒定区，所述TCRα链胞内恒定区选自：
    (1)来源于人源、鼠源或其他哺乳动物种属的野生型TCRα链胞内恒定区；
    (2)包含SEQ ID NO:3所示氨基酸序列或由所述氨基酸序列组成的野生型TCRα链胞内恒定区；
    (3)突变的TCRα链胞内恒定区，所述突变的TCRα链胞内恒定区为(1) 或(2)所述的野生型TCRα链胞内恒定区中至少一个的丝氨酸突变为丙氨酸；或
    (4)包含SEQ ID NO:4所示氨基酸序列或由所述氨基酸序列组成的突变的TCRα链胞内恒定区；
    可选的，所述TCRα链进一步包括TCRα链可变区。
  4. 一种分离的T细胞受体或其片段，所述T细胞受体包括TCRα链和TCRβ链，其特征在于，所述TCRα链包括TCRα链可变区和TCRα链恒定区，所述TCRα链恒定区包括依次连接的TCRα链胞外恒定区、TCRα链跨膜区和TCRα链胞内恒定区，所述TCRα链胞内恒定区选自：
    (1)突变的TCRα链胞内恒定区，所述突变的TCRα链胞内恒定区为野生型TCRα链胞内恒定区中至少一个丝氨酸突变为丙氨酸；或
    (2)包含SEQ ID NO:4所示氨基酸序列或由所述氨基酸序列组成的突变的TCRα链胞内恒定区。
  5. 如权利要求4所述的分离的T细胞受体，其特征在于，所述野生型TCRα链胞内恒定区选自：
    (1)来源于人源、鼠源或其他哺乳动物种属的野生型TCRα链胞内恒定区；或
    (2)包含SEQ ID NO:3所示氨基酸序列或由所述氨基酸序列组成的野生型TCRα链胞内恒定区。
  6. 如权利要求4-5任一项所述的分离的T细胞受体，其特征在于，所述TCRβ链包括TCRβ链恒定区，所述TCRβ链恒定区包括依次连接的TCRβ链胞外恒定区、TCRβ链跨膜区和TCRβ链胞内恒定区，所述TCRβ链胞内恒定区选自：
    (1)来源于人源、鼠源或其他哺乳动物种属的野生型TCRβ链胞内恒定区；
    (2)包含SEQ ID NO:9、SEQ ID NO:47或SEQ ID NO:48所示氨基酸序列或由所述氨基酸序列组成的野生型TCRβ链胞内恒定区；
    (3)突变的TCRβ链胞内恒定区，所述突变的TCRβ链胞内恒定区为(1)或(2)所述的野生型TCRβ链胞内恒定区中至少一个的赖氨酸突变为精氨酸或丙氨酸形成；或
    (4)包含SEQ ID NO:10所示氨基酸序列或由所述氨基酸序列组成的突变的TCRβ链胞内恒定区；
    可选的，所述TCRβ链进一步包括TCRβ链可变区。
  7. 前述权利要求任一项所述的分离的T细胞受体，其特征在于，所述分离的T细胞受体识别多肽类抗原、脂类抗原或多糖类抗原。
  8. 前述权利要求任一项所述的分离的T细胞受体，其特征在于，所述分离的T细胞受体识别肿瘤抗原、微生物抗原或自身抗原。
  9. 如权利要求8所述的分离的T细胞受体，其特征在于，所述分离的T细胞受体识别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 G12D、KRAS G12V、TP53 R175H、β-GlcCer、eLPA、LPE、CA199、CA72-4或CA125中的一种或多种抗原；优选为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或CA125中的一种或多种抗原。
  10. 前述权利要求任一项所述的分离的T细胞受体，其特征在于，所述TCRβ链可变区选自：
    (1)TCRβ链可变区包含SEQ ID NO:17所示氨基酸序列所含的三个互补决定区CDR；
    (2)TCRβ链可变区包含SEQ ID NO:25所示氨基酸序列所含的三个互补决定区CDR；
    (3)TCRβ链可变区包含SEQ ID NO:18所示氨基酸序列的CDR1、SEQ ID NO:19所示氨基酸序列的CDR2和/或SEQ ID NO:20所示氨基酸序列的CDR3；
    (4)TCRβ链可变区包含SEQ ID NO:26所示氨基酸序列的CDR1、SEQ ID NO:27所示氨基酸序列的CDR2和/或SEQ ID NO:28所示氨基酸序列的CDR3；
    (5)TCRβ链可变区包含SEQ ID NO:17所示氨基酸序列或由其组成；或
    (6)TCRβ链可变区包含SEQ ID NO:25所示氨基酸序列或由其组成。
  11. 前述权利要求任一项所述的分离的T细胞受体，其特征在于，所述TCRα链可变区选自：
    (1)TCRα链可变区包含SEQ ID NO:13所示氨基酸序列所含的三个互补决定区CDR；
    (2)TCRα链可变区包含SEQ ID NO:21所示氨基酸序列所含的三个互补决定区CDR；
    (3)TCRα链可变区包含SEQ ID NO:14所示氨基酸序列的CDR1、SEQ ID NO:15所示氨基酸序列的CDR2和/或SEQ ID NO:16所示氨基酸序列的CDR3；
    (4)TCRα链可变区包含SEQ ID NO:22所示氨基酸序列的CDR1、SEQ ID  NO:23所示氨基酸序列的CDR2和/或SEQ ID NO:24所示氨基酸序列的CDR3；
    (5)TCRα链可变区包含SEQ ID NO:13所示氨基酸序列或由其组成；或
    (6)TCRα链可变区包含SEQ ID NO:21所示氨基酸序列或由其组成。
  12. 前述权利要求任一项所述的分离的T细胞受体，还包括以下特征中的一项或多项：
    (1)所述TCRα链胞外恒定区来源于人源、鼠源或其他哺乳动物种属的TCRα链胞外恒定区；
    (2)所述TCRα链胞外恒定区包含SEQ ID NO:1的氨基酸序列，或包含与SEQ ID NO:1的氨基酸序列相比具有1、2、3、4、5、6、7、8、9或10个突变的氨基酸序列，或包含与SEQ ID NO:1的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列，或由所述氨基酸序列组成；
    (3)所述TCRα链跨膜区来源于人源、鼠源或其他哺乳动物种属的TCRα链跨膜区；
    (4)所述TCRα链跨膜区包含SEQ ID NO:2的氨基酸序列，或包含与SEQ ID NO:2的氨基酸序列相比具有1、2、3、4、5、6、7、8、9或10个突变的氨基酸序列，或包含与SEQ ID NO:2的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列，或由所述氨基酸序列组成；
    (5)所述TCRβ链胞外恒定区来源于人源、鼠源或其他哺乳动物种属的TCRβ链胞外恒定区；
    (6)所述TCRβ链胞外恒定区包含SEQ ID NO:7的氨基酸序列，或包含与SEQ ID NO:7的氨基酸序列相比具有1、2、3、4、5、6、7、8、9或10个突变的氨基酸序列，或包含与SEQ ID NO:7的氨基酸序列具有至少90％、91％、92％、 93％、94％、95％、96％、97％、98％或99％的氨基酸序列，或由所述氨基酸序列组成；
    (7)所述TCRβ链跨膜区来源于人源、鼠源或其他哺乳动物种属的TCRβ链跨膜区；
    (8)所述TCRβ链跨膜区包含SEQ ID NO:8的氨基酸序列，或包含与SEQ ID NO:8的氨基酸序列相比具有1、2、3、4、5、6、7、8、9或10个突变的氨基酸序列，或包含与SEQ ID NO:8的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的氨基酸序列，或由所述氨基酸序列组成。
  13. 前述权利要求任一项所述的分离的T细胞受体，其特征在于，所述TCRα链恒定区和TCRβ链恒定区选自：
    (1)所述TCRα链恒定区包含SEQ ID NO:5的氨基酸序列或由所述氨基酸序列组成，所述TCRβ链恒定区包含SEQ ID NO:12的氨基酸序列或由所述氨基酸序列组成；
    (2)所述TCRα链恒定区包含SEQ ID NO:6的氨基酸序列或由所述氨基酸序列组成，所述TCRβ链恒定区包含SEQ ID NO:12的氨基酸序列或由所述氨基酸序列组成；或
    (3)所述TCRα链恒定区包含SEQ ID NO:6的氨基酸序列或由所述氨基酸序列组成，所述TCRβ链恒定区包含SEQ ID NO:11的氨基酸序列或由所述氨基酸序列组成。
  14. 前述权利要求中任一项分离的T细胞受体，进一步包括信号肽，所述信号肽与TCRα链可变区和/或TCRβ链可变区形成信号肽-可变区结构。
  15. 如权利要求14所述的分离的T细胞受体，其特征在于，所述信号肽为人生长激素信号肽、CD8α信号肽或免疫球蛋白信号肽。
  16. 分离的核酸或其片段，其编码前述权利要求中任意一项分离的T细胞受体或其片段。
  17. 核酸构建物，其包含权利要求16所述分离的核酸或其片段。
  18. 载体，其包含权利要求17所述核酸构建物。
  19. 如权利要求18所述载体，其为表达载体或crisper基因编辑载体，优选为逆转录病毒载体。
  20. 工程化细胞，其包含权利要求17所述的核酸构建物，或权利要求18-19任一项所述的载体。
  21. 权利要求20所述工程化细胞为T细胞，例如人T细胞。
  22. 生产工程化细胞的方法，其包括：
    (1)用权利要求17所述的核酸构建物或权利要求18-19任一项所述的载体转化或转导宿主细胞；
    (2)在合适的培养基中培养宿主细胞；
    (3)从培养基或细胞中分离、纯化出工程化细胞。
  23. 前述任一项权利要求所述分离的T细胞受体或其片段、核酸或其片段、核酸构建物、载体或工程化细胞在下述任意一种或多种用途中的应用：
    (1)在抗原刺激过程中的抑制T细胞受体降解中的应用；
    (2)提供对肿瘤的杀伤效果，或抑制肿瘤生长；
    (3)维持T细胞的增殖能力；
    (4)抗肿瘤的免疫药物及细胞的制备。
  24. 一种T细胞受体的改造方法，包括：
    (1)将野生型TCRβ链胞内恒定区中至少一个的赖氨酸突变为精氨酸或丙氨酸；和/或
    (2)将野生型TCRα链胞内恒定区中至少一个的丝氨酸突变为丙氨酸。
  25. 如权利要求24所述的T细胞受体的改造方法，其特征在于：
    (1)所述野生型TCRβ链胞内恒定区选自权利要求2所述的野生型TCRβ链胞内恒定区；和/或
    (2)所述野生型TCRα链胞内恒定区选自权利要求5所述的野生型TCRα链胞内恒定区。
PCT/CN2022/143567 2022-01-05 2022-12-29 一种t细胞受体及其制备方法和用途 WO2023131053A1 (zh)

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