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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French (fr)
Chinese (zh)
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李贵登
程洪成
邱雅静
许悦
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Suzhou Institute of Systems Medicine
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Suzhou Institute of Systems Medicine
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Priority to KR1020247025309A priority Critical patent/KR20240130120A/ko
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Priority to JP2024540916A priority patent/JP2025501358A/ja
Priority to CN202280079730.8A priority patent/CN118475601A/zh
Publication of WO2023131053A1 publication Critical patent/WO2023131053A1/zh
Priority to US18/763,813 priority patent/US20240358832A1/en
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    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
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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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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110818802A (zh) * 2018-08-08 2020-02-21 华夏英泰（北京）生物技术有限公司 一种嵌合t细胞受体star及其应用
CN110857319A (zh) * 2018-08-24 2020-03-03 杭州康万达医药科技有限公司 一种分离的t细胞受体、其修饰的细胞、编码核酸及其应用
CN113396216A (zh) * 2019-02-04 2021-09-14 Ksq治疗公司 用于改进的免疫疗法的组合基因靶标
WO2021238903A1 (zh) * 2020-05-25 2021-12-02 华夏英泰（北京）生物技术有限公司 增强型合成t细胞受体抗原受体

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2651174A1 (en) * 2006-05-03 2007-11-15 Government Of The United States Of America, Represented By The Secretary , Department Of Health And Human Services Chimeric t cell receptors and related materials and methods of use
EA202091977A1 (ru) * 2018-05-28 2021-02-09 Драгонфлай Терапьютикс, Инк. Мультиспецифические связывающие белки, которые связывают cd33, nkg2d и cd16, и способы применения
CN115776991A (zh) * 2020-05-07 2023-03-10 华夏英泰（北京）生物技术有限公司 改进的t细胞受体-共刺激分子嵌合物

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110818802A (zh) * 2018-08-08 2020-02-21 华夏英泰（北京）生物技术有限公司 一种嵌合t细胞受体star及其应用
CN110857319A (zh) * 2018-08-24 2020-03-03 杭州康万达医药科技有限公司 一种分离的t细胞受体、其修饰的细胞、编码核酸及其应用
CN113396216A (zh) * 2019-02-04 2021-09-14 Ksq治疗公司 用于改进的免疫疗法的组合基因靶标
WO2021238903A1 (zh) * 2020-05-25 2021-12-02 华夏英泰（北京）生物技术有限公司 增强型合成t细胞受体抗原受体

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
DANIEL SOMMERMEYER ET AL., J IMMUNOL, vol. 184, no. 11, 1 June 2010 (2010-06-01), pages 6223 - 31
E. MEYERSW. MILLER, CABIOS, vol. 4, 1989, pages 11 - 17
FEIGE, M. J. ET AL.: "Dimerization-dependent Folding Underlies Assembly Control of the Clonotypic αβT Cell Receptor Chains", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 290, no. 44, 30 October 2015 (2015-10-30), pages 26821 - 26831, XP055546197, DOI: 10.1074/jbc.M115.689471 *
KAREN FRONING, MAGUIRE JACK, SERENO ARLENE, HUANG FLORA, CHANG SHAWN, WEICHERT KENNETH, FROMMELT ANTON J., DONG JESSICA, WU XIUFEN: "Computational stabilization of T cell receptors allows pairing with antibodies to form bispecifics", NATURE COMMUNICATIONS, vol. 11, no. 1, XP055747026, DOI: 10.1038/s41467-020-16231-7 *
LONGCHANG XU, WEI WEI: "Research Challenges and Examination Considerations for TCR T Cell Therapy", CHINESE JOURNAL OF BIOLOGICALS, vol. 33, no. 3, 19 March 2020 (2020-03-19), pages 355 - 360, XP093078262, DOI: 10.13200/j.cnki.cjb.003022 *
NEEDLEMAWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 444 - 453, Retrieved from the Internet <URL:http://www.gcg.com>
SANTOSHA A. VARDHANA ET AL., NAT IMMUNOL., vol. 21, no. 9, September 2020 (2020-09-01), pages 1022 - 1033
See also references of EP4461366A4

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