T cell antigen receptor as well as preparation method and application thereof

Abstract

The invention relates to a T cell antigen receptor, the T cell antigen receptor comprises an alpha chain and / or a beta chain, the alpha chain comprises any one or a combination of (a)-(b), (a) a constant region contains an amino acid segment as shown in SEQ ID NO: 1 (LVIVL); (b) the constant region comprises one or more cysteine residues capable of forming a non-natural disulfide bond with the beta chain; the constant region of the beta chain comprises one or more cysteine residues capable of forming a non-natural disulfide bond with the alpha chain. The T cell antigen receptor can significantly reduce the mismatch rate of TCR molecules and ensure correct expression of TCR, so that the specific recognition ability of T cells to tumor cells or pathogens is improved, the immunogenicity is significantly reduced, and the biological activity is improved.

Description

[0001] This application is a divisional application of the invention patent application with application number 2024108934427 and filing date 2024-07-04, entitled "T cell antigen receptor and preparation method and application thereof". TECHNICAL FIELD

[0002] The present application belongs to the field of biological medicine, and specifically relates to a T cell antigen receptor and a preparation method and application thereof. BACKGROUND

[0003] T cell receptor (TCR) is a molecule that specifically recognizes antigens and initiates immune responses in T lymphocytes, and is a heterodimeric cell surface protein of the immunoglobulin superfamily, which is associated with a non-variable protein of the CD3 complex involved in regulating signal transduction. TCR is the only receptor for specific antigen peptides presented on the major histocompatibility complex (MHC), and is essential for the cellular immune function of the immune system. The binding of antigen-specific TCR to the MHC complex initiates direct physical contact and interaction between T cells and antigen-presenting cells, leading to subsequent cell signaling and other physiological responses, allowing T cells of different antigen specificity to exert immune effects on their target cells.

[0004] The correct assembly of TCR is the key to the generation of TCR-T cells. TCR mispairing can cause cross-reactivity toxicity and other problems and become a technical difficulty in TCR-T cell therapy. The introduction of murine amino acids to reduce the mispairing rate may lead to excessive immunogenicity and affect the drugability. Therefore, it is necessary to develop a TCR modification scheme with good biological activity function to effectively reduce the TCR mispairing rate and immunogenicity. SUMMARY

[0005] The purpose of the present application is to provide a T cell antigen receptor, which comprises an alpha chain and / or a beta chain, wherein the alpha chain comprises any one or a combination of (a)-(b),

[0006] (a) the constant region contains an amino acid segment as shown in SEQ ID NO: 1 (LVIVL);

[0007] (b) the constant region contains one or more cysteine residues capable of forming a non-native disulfide bond with the beta chain;

[0008] the constant region of the beta chain contains one or more cysteine residues capable of forming a non-native disulfide bond with the alpha chain;

[0009] Preferably, the constant region amino acid sequence of the T cell antigen receptor is completely replaced by murine amino acids;

[0010] Preferably, the constant region amino acid sequence of the T cell antigen receptor is substituted with murine amino acids at least at one site, more preferably the constant region amino acid sequence of the T cell antigen receptor is substituted with 1 or 9 murine amino acids.

[0011] In a preferred technical solution of the present application, the transmembrane hydrophobic region of the constant region contains an amino acid segment as shown in SEQ ID NO: 1 (LVIVL).

[0012] In a preferred technical solution of the present application, the constant region of the alpha chain and the beta chain further comprises at least one leucine zipper.

[0013] In a preferred technical solution of the present application, the leucine zipper is selected from any one of SEQ ID NO: 2 (PGGRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNY), SEQ ID NO: 3 (PGGLTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAY), SEQ ID NO: 4 (AQCEKELQALEKENAQLEWELQALEKELAQ), SEQ ID NO: 5 (AQCEKELQALEKENAQLEWELQALEKELAQ).

[0014] In a preferred technical solution of the present application, the leucine zipper containing the sequence of SEQ ID NO: 2 and the leucine zipper containing the sequence of SEQ ID NO: 3 are used in pairs, and the leucine zipper containing the sequence of SEQ ID NO: 4 and the leucine zipper containing the sequence of SEQ ID NO: 5 are used in pairs.

[0015] In a preferred technical solution of the present application, the amino acid sequence of the constant region of the alpha chain comprises the following sequence: , wherein X1 is selected from any one of S, P, X2 is selected from any one of D, E, X3 is selected from any one of V, S, X4 is selected from any one of P, S, or an amino acid sequence having at least 85% homology with the above sequence;

[0016] The amino acid sequence of the constant region of the beta chain is as follows:

[0017] DLNKVFPPEVAVFEPS X5 AEI X6 HTQKATLVCLATGFFPDHVELSWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCG X7 TS X8SY X9 QGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF, wherein X5 is selected from any one of E, K, X6 is selected from any one of A, S, X7 is selected from any one of I, F, X8 is selected from any one of A, V, X9 is selected from any one of H, Q; or an amino acid sequence having at least 85% homology with the above sequence.

[0018] In the preferred technical scheme of the present application, the T cell antigen receptor comprises the amino acid sequence selected from any one of (1)-(7):

[0019] (1) the amino acid sequence of the constant region of the alpha chain thereof comprises SEQ ID NO: 6, or comprises an amino acid sequence having at least 85% homology with SEQ ID NO: 6, wherein the SEQ ID NO: 6 is:

[0020]

[0021] the amino acid sequence of the constant region of the beta chain thereof comprises SEQ ID NO: 7, or comprises an amino acid sequence having at least 85% homology with SEQ ID NO: 7, wherein the SEQ ID NO: 7 is:

[0022]

[0023] (2) the amino acid sequence of the constant region of the alpha chain thereof comprises SEQ ID NO: 8, or comprises an amino acid sequence having at least 85% homology with SEQ ID NO: 8, wherein the SEQ ID NO: 8 is:

[0024]

[0025] the amino acid sequence of the constant region of the beta chain thereof comprises SEQ ID NO: 9, or comprises an amino acid sequence having at least 85% homology with SEQ ID NO: 9, wherein the SEQ ID NO: 9 is:

[0026]

[0027] (3) the amino acid sequence of the constant region of the alpha chain thereof comprises SEQ ID NO: 10, or comprises an amino acid sequence having at least 85% homology with SEQ ID NO: 10, wherein the SEQ ID NO: 10 is:

[0028]

[0029] The amino acid sequence of its constant region of the β chain contains SEQ ID NO: 11, or contains an amino acid sequence having at least 85% homology with SEQ ID NO: 11, wherein SEQ ID NO: 11 is:

[0030]

[0031] (4) The amino acid sequence of the constant region of its α chain contains SEQ ID NO: 12, or contains an amino acid sequence having at least 85% homology with SEQ ID NO: 12, wherein SEQ ID NO: 12 is:

[0032]

[0033] The amino acid sequence of its constant region of the β chain contains SEQ ID NO: 13, or contains an amino acid sequence having at least 85% homology with SEQ ID NO: 13, wherein SEQ ID NO: 13 is:

[0034]

[0035] (5) The amino acid sequence of the constant region of its α chain contains SEQ ID NO: 14, or contains an amino acid sequence having at least 85% homology with SEQ ID NO: 14, wherein SEQ ID NO: 14 is:

[0036]

[0037] The constant region of its β chain contains an amino acid sequence as shown in SEQ ID NO: 15, or contains an amino acid sequence having at least 85% homology with SEQ ID NO: 15, wherein SEQ ID NO: 15 is:

[0038] (6) The amino acid sequence of the constant region of its α chain contains SEQ ID NO: 16, or contains an amino acid sequence having at least 85% homology with SEQ ID NO: 16, wherein SEQ ID NO: 16 is:

[0039]

[0040] The constant region of its β chain contains an amino acid sequence as shown in SEQ ID NO: 17, or contains an amino acid sequence having at least 85% homology with SEQ ID NO: 17, wherein SEQ ID NO: 17 is:

[0041]

[0042] In the preferred technical solution of the present application, the variable region of the alpha chain comprises an alpha-CDR3, and the variable region of the beta chain comprises a beta-CDR3, wherein the amino acid sequence of the alpha-CDR3 is AVRGGADGLT (SEQ ID NO: 18), and the amino acid sequence of the beta-CDR3 is ASSPPNEKLF (SEQ ID NO: 19).

[0043] In the preferred technical solution of the present application, the variable region of the alpha chain comprises any one or a combination of alpha-CDR1 and alpha-CDR2, wherein the amino acid sequence of the alpha-CDR1 is DSVNN (SEQ ID NO: 20), and the amino acid sequence of the alpha-CDR2 is IPSGT (SEQ ID NO: 21).

[0044] In the preferred technical solution of the present application, the variable region of the beta chain comprises any one or a combination of beta-CDR1 and beta-CDR2, wherein the amino acid sequence of the beta-CDR1 is MGHRA (SEQ ID NO: 22), and the amino acid sequence of the beta-CDR2 is YSYEKL (SEQ ID NO: 23).

[0045] In the preferred technical solution of the present application, the amino acid sequence of the variable region of the alpha chain is as shown in SEQ ID NO: 24, and the SEQ ID NO: 24 is as follows:

[0046]

[0047]

[0048] The amino acid sequence of the variable region of the beta chain is as shown in SEQ ID NO: 25, and the SEQ ID NO: 25 is as follows:

[0049] In the preferred technical solution of the present application, the amino acid sequence of the variable region of the alpha chain comprises SEQ ID NO: 26, or comprises an amino acid sequence having at least 85% homology with SEQ ID NO: 26, and the SEQ ID NO: 26 is as follows:

[0050]

[0051] In the preferred technical solution of the present application, the amino acid sequence of the variable region of the beta chain comprises SEQ ID NO: 27, or comprises an amino acid sequence having at least 85% homology with SEQ ID NO: 27, and the SEQ ID NO: 27 is as follows:

[0052]

[0053] Preferably, the homology is selected from any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0054] In a preferred technical scheme of the present application, the amino acids of the alpha chain and the beta chain are linked by a connecting sequence as shown in SEQ ID NO: 28, wherein the SEQ ID NO: 28 is GSRAKRSGSGATNFSLLKQAGDVEENPGP.

[0055] In a preferred technical scheme of the present application, the T cell antigen receptor comprises an amino acid sequence selected from any one of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 or a combination thereof, or comprises an amino acid sequence having at least 85% homology with any one of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35.

[0056] Another object of the present application is to provide a nucleic acid comprising a nucleotide sequence encoding the TCR of the present application or a complementary sequence thereof.

[0057] In a preferred technical scheme of the present application, the nucleotide sequence or the complementary sequence thereof is selected from any one of single-stranded, double-stranded, DNA, RNA or a combination thereof.

[0058] In a preferred technical scheme of the present application, the nucleotide sequence or the complementary sequence thereof is codon-optimized.

[0059] In a preferred technical scheme of the present application, the codon optimization includes any one of changing a large number of rare codons used by viruses into corresponding mammalian codons, removing mRNA instability motifs, hidden splice sites or a combination thereof.

[0060] In the preferred technical solution of the present application, the nucleic acid sequence is selected from any one of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 or a combination thereof, or a nucleotide sequence having at least 85% homology with any one of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 or a combination thereof.

[0061] Another object of the present application is to provide an expression vector comprising the nucleic acid according to the present application.

[0062] In the preferred technical solution of the present application, the expression vector can be expressed under any condition in vivo, in vitro or ex vivo.

[0063] In the preferred technical solution of the present application, the expression vector is expressed at a high level in the cells in vivo.

[0064] In the preferred technical solution of the present application, the expression vector is selected from any one of a prokaryotic expression vector, a retroviral vector.

[0065] In the preferred technical solution of the present application, the expression vector is selected from any one of Rous sarcoma virus (RSV), lentivirus, human immunodeficiency virus (HIV), murine leukemia virus (MLV), equine infectious anemia virus (EIAV), mouse mammary tumor virus (MMTV), Fujinami sarcoma virus (FuSV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine leukemia virus (Mo-MLV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), avian myelocytomatosis virus 29 (MC29), avian erythroblastosis virus (AEV) or a combination thereof.

[0066] Another object of the present application is to provide a host cell comprising any of the nucleic acid or the expression vector according to the present application.

[0067] In the preferred technical solution of the present application, the host cell is selected from any one of a eukaryotic cell or a prokaryotic cell.

[0068] In the preferred technical solution of the present application, the eukaryotic cell is selected from any one of a yeast cell, a 293 cell, a CHO cell or a combination thereof.

[0069] Another object of the present invention is to provide an immune cell that expresses the T-cell antigen receptor described in the present invention.

[0070] In a preferred embodiment of the present invention, the immune cells comprise one or more nucleic acid sequences as described in any of the present invention.

[0071] In a preferred embodiment of the present invention, the immune cells are selected from any one of stem cells, lymphocytes, T cells, B cells, and NK cells.

[0072] In a preferred embodiment of the present invention, the T cell antigen receptor structure of the T cell is as defined above.

[0073] In a preferred embodiment of the present invention, the T cells are selected from CD4. + T, CD8 + Any one or a combination of T.

[0074] In a preferred embodiment of the present invention, the immune cells are isolated from autologous T cells or allogeneic T cells.

[0075] Another object of the present invention is to provide a method for preparing immune cells, comprising transducing the nucleic acid sequence encoding the above-mentioned T cell antigen receptor into immune cells for expression.

[0076] In a preferred embodiment of the present invention, the immune cells are selected from any one of stem cells, lymphocytes, T cells, B cells, and NK cells.

[0077] In a preferred embodiment of the present invention, the T cell antigen receptor structure of the T cell is as defined above.

[0078] In a preferred embodiment of the present invention, the T cells are selected from CD4. + T, CD8 + Any one or a combination of T.

[0079] In a preferred embodiment of the present invention, the immune cells are isolated from autologous T cells or allogeneic T cells.

[0080] In a preferred embodiment of the present invention, the method further includes the step of knocking out endogenous TCRs in cells.

[0081] In a preferred embodiment of the present invention, the step of knocking out endogenous TCR in cells involves constructing a guide RNA (gRNA) targeting endogenous TCR into a lentiviral vector, and then co-transfecting it into T cells with a packaging plasmid and transfection reagent.

[0082] Another object of the present invention is to provide a method for preparing recombinant T cells, comprising the following steps:

[0083] 1) obtaining the nucleic acid of any one of the present application from positive T cells;

[0084] 2) isolating and culturing T cells;

[0085] 3) delivering the nucleic acid obtained in step 1) into the primary T cells of step 2) to obtain recombinant T cells expressing the T cell antigen receptor of any one of the present application,

[0086] In preferred embodiments of the present application, the T cells are selected from any one of hematopoietic stem cells or peripheral blood lymphocyte (PBL) derived T cells.

[0087] Another object of the present application is to provide a method for preparing a T cell antigen receptor, comprising the following steps:

[0088] (1) obtaining the nucleic acid of any one of the present application from positive T cells;

[0089] (2) ligating the nucleic acid obtained in step (1) to a vector backbone to obtain an expression vector;

[0090] (3) transforming the expression vector obtained in step (2) into a host cell and inducing its expression;

[0091] (4) obtaining an antibody or an antigen-binding fragment thereof or a T cell antigen receptor.

[0092] In preferred embodiments of the present application, the positive T cells specifically bind to the antigenic peptide.

[0093] Another object of the present application is to provide a pharmaceutical composition comprising any one of 1) to 5) or a combination thereof,

[0094] 1) the T cell antigen receptor of the present application;

[0095] 2) the nucleic acid of the present application;

[0096] 3) the expression vector of the present application;

[0097] 4) the host cell of the present application; or

[0098] 5) the immune cell of the present application.

[0099] In preferred embodiments of the present application, the composition further comprises a pharmaceutically acceptable carrier.

[0100] In preferred embodiments of the present application, the composition is optionally used in combination with other therapeutic agents.

[0101] In preferred embodiments of the present application, the other therapeutic agents are immunomodulators.

[0102] Another object of the present application is to provide the use of any one of the T cell antigen receptors or nucleic acids, expression vectors, host cells, immune cells, pharmaceutical compositions of the present application for the manufacture of a medicament for the diagnosis or prevention of a tumor.

[0103] In preferred embodiments of the present application, the tumor is selected from the group consisting of pancreatic cancer, liver cancer, oral squamous cell carcinoma, colon cancer, ovarian cancer, gastric cancer, rectal cancer, lymphoma, basal cell carcinoma, non-small cell lung cancer, leukemia, nasopharyngeal carcinoma, breast cancer, endometrial cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, cholangiocarcinoma, esophageal cancer, renal cancer, sarcoma, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, or complications thereof.

[0104] In preferred embodiments of the present application, the leukemia is selected from the group consisting of acute lymphoblastic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, chronic myeloid leukemia, or complications thereof.

[0105] In preferred embodiments of the present application, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, Waldenstrom macroglobulinemia, or complications thereof.

[0106] In preferred embodiments of the present application, the sarcoma is selected from the group consisting of osteosarcoma, Ewing's sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, chondrosarcoma, or complications thereof.

[0107] In preferred embodiments of the present application, the renal cancer is selected from the group consisting of clear cell renal cell carcinoma, papillary renal cell carcinoma, renal collecting duct carcinoma, renal medullary carcinoma, MiT family translocation renal cell carcinoma, succinate dehydrogenase-deficient renal cell carcinoma, mucinous tubular and spindle cell carcinoma, unclassified renal cell carcinoma, oncocytoma, and multilocular cystic renal tumors of low malignant potential, or combinations thereof.

[0108] Another object of the present application is to provide a kit comprising any one of 1) to 5), or a combination thereof:

[0109] 1) the T cell antigen receptor of the present application;

[0110] 2) the nucleic acid of the present application;

[0111] 3) the expression vector of the present application;

[0112] 4) the host cell of the present application;

[0113] 5) the immune cell of the present application.

[0114] Another object of the present application is to provide a method for detecting, said method comprising contacting a sample to be detected with the T cell antigen receptor of the present application.

[0115] Another object of the present application is to provide a method for preventing and treating a tumor, said method comprising administering to an individual an effective amount of any one of the T cell antigen receptor, nucleic acid, expression vector, host cell, immune cell, or pharmaceutical composition of the present application.

[0116] In a preferred embodiment of the present application, the method comprises the step of adoptively transferring T cells expressing the T cell antigen receptor of the present application to a subject.

[0117] In a preferred embodiment of the present application, the T cells expressing the T cell antigen receptor of the present application are derived from the subject.

[0118] In a preferred embodiment of the present application, the T cells expressing the T cell antigen receptor of the present application are derived from the same donor as the hematopoietic stem cell, organ, tissue, cell, or stem cell.

[0119] In a preferred embodiment of the present application, the tumor is selected from the group consisting of pancreatic cancer, liver cancer, oral squamous cell carcinoma, colon cancer, ovarian cancer, gastric cancer, rectal cancer, lymphoma, basal cell carcinoma, non-small cell lung cancer, leukemia, nasopharyngeal carcinoma, breast cancer, endometrial cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, cholangiocarcinoma, esophageal cancer, renal cancer, sarcoma, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, and complications thereof.

[0120] In a preferred embodiment of the present application, the leukemia is selected from the group consisting of acute lymphoblastic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, chronic myeloid leukemia, and complications thereof.

[0121] In a preferred embodiment of the present application, the lymphoma is selected from the group consisting of Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, Waldenstrom's macroglobulinemia, and complications thereof.

[0122] In the preferred technical solution of the present application, the sarcoma is selected from any one of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, chondrosarcoma, or a complication thereof.

[0123] In the preferred technical solution of the present application, the renal cancer is selected from any one of clear cell renal cell carcinoma, papillary renal cell carcinoma, renal collecting duct carcinoma, renal medullary carcinoma, MiT family translocation renal cell carcinoma, succinate dehydrogenase-deficient renal cell carcinoma, mucinous tubular and spindle cell carcinoma, unclassified renal cell carcinoma, oncocytoma, and low potential for malignancy multilocular cystic renal tumor, or a combination thereof.

[0124] Another object of the present application is to provide a method for diagnosing tumors, comprising the step of contacting a tumor tissue sample of a patient with the T cell antigen receptor of the present application.

[0125] In the preferred technical solution of the present application, the T cell antigen receptor comprises a detectable label.

[0126] In the preferred technical solution of the present application, the tumor is selected from any one of pancreatic cancer, liver cancer, oral squamous cell carcinoma, colon cancer, ovarian cancer, gastric cancer, rectal cancer, lymphoma, basal cell carcinoma, non-small cell lung cancer, leukemia, nasopharyngeal carcinoma, breast cancer, endometrial cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, cholangiocarcinoma, esophageal cancer, renal cancer, sarcoma, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome, or a complication thereof.

[0127] In the preferred technical solution of the present application, the leukemia is selected from any one of acute lymphoblastic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, chronic myeloid leukemia, or a complication thereof.

[0128] In the preferred technical solution of the present application, the lymphoma is selected from any one of Hodgkin's lymphoma, non-Hodgkin's lymphoma, B-cell lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, marginal zone B-cell lymphoma, T-cell lymphoma, Waldenstrom macroglobulinemia, or a complication thereof.

[0129] In the preferred technical solution of the present application, the sarcoma is selected from any one of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, chondrosarcoma, or a complication thereof.

[0130] In the preferred technical solution of the present application, the renal cancer is selected from any one of clear cell renal cell carcinoma, papillary renal cell carcinoma, renal collecting duct carcinoma, renal medullary carcinoma, MiT family translocation renal cell carcinoma, succinate dehydrogenase-deficient renal cell carcinoma, mucinous tubular and spindle cell carcinoma, unclassified renal cell carcinoma, oncocytoma, and low potential malignancy multilocular cystic renal tumor, or a combination thereof.

[0131] Unless otherwise indicated, the sequence numbers and names of the present application are shown in Table 1.

[0132] Table 1

[0133]

[0134]

[0135]

[0136] Unless otherwise indicated, the terms of the present application are of the usual meaning in the art. As Sambrook et al., "Molecular Cloning: A Laboratory Manual", Lewin, "Genes VIII", Roitt et al., "Immunology" (8th edition) and the like.

[0137] The "T cell antigen receptor" according to the present application is a molecule capable of recognizing a peptide when presented by a MHC molecule or tetramer thereof, usually present in complex with CD3 molecules on the surface of T cells. The TCR of most T cells consists of alpha and beta peptide chains, while the TCR of a minority of T cells consists of gamma and delta peptide chains.

[0138] The "comprising" according to the present application is used to describe the sequence of a protein or nucleic acid, which can consist of the sequence, or can have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still has the activity described in the present application.

[0139] The "prevention" according to the present application means all actions to inhibit symptoms or delay the tension of specific symptoms by administering the product according to the present application.

[0140] The "diagnosis" according to the present application means to find out whether a patient has a disease or its progression, or to evaluate the patient's response to treatment.

[0141] The "treatment" according to the present application means to slow down, interrupt, stop, control, stop, alleviate, reverse the progression or severity of a sign, symptom, disorder, condition, disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, disorders, and refers to therapeutic intervention to improve signs, symptoms, etc. of a disease or pathological state after the disease has begun to develop.

[0142] The "effective amount" of the present application refers to the dosage that provides the desired therapeutic or prophylactic effect after administration to the patient or organ in a single dose or multiple doses.

[0143] The "product" of the present application includes, but is not limited to, the T cell antigen receptor, the nucleic acid, the expression vector, the host cell, the immune cell, and other auxiliary or synergistic agents, kits, chips, antibody conjugates or multifunctional antibodies and other pharmaceutical compositions.

[0144] The "subject" of the present application is selected from any one of humans or other mammals.

[0145] The "homology" of the present application refers to the adjustment of the sequence by the skilled person in the art without changing the main structure or function of the amino acid sequence or nucleotide sequence, so that the homology is selected from any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%.

[0146] The "pairing use" of the present application refers to the interaction of proteins through their leucine zipper domains to form stable dimers.

[0147] The real-time label-free dynamic cell analysis technology of the present application integrates a microelectronic cell sensor chip into the bottom of a cell detection plate to construct a real-time, dynamic and quantitative tracking cell impedance detection sensor system for changes in cell morphology, proliferation and differentiation. When cells adherently growing on the surface of the microelectrode cause changes in the interfacial impedance of the adherent electrode, the real-time functional state of the cells is changed, and by real-time dynamic electrode impedance detection, biological information related to the physiological function of the cells, including cell growth, stretching, morphological changes, death and adhesion, can be obtained.

[0148] Compared with the prior art, the present application has the following beneficial technical effects: the T cell antigen receptor obtained by modifying the TCR constant region significantly reduces the mispairing rate of the TCR molecule, ensures the correct expression of the TCR, thereby improving the specific recognition ability of the T cell to tumor cells or pathogens, and improving the therapeutic effect; significantly reduces its immunogenicity, while improving its biological activity. The TCR-T cell of the present application reduces the mispairing rate while maintaining effective anti-tumor activity, can specifically recognize and kill target cells expressing the corresponding antigen, and has a broad application prospect in immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0149] Figure 1 The T cell antigen receptor mispairing rate detection result of the present application;

[0150] Figure 2 The T cell antigen receptor of the present application has an effect on target cell killing;

[0151] Figure 3 The T cell antigen receptor of the present application has an effect on tumor volume change in experimental animals;

[0152] Figure 4 The T cell antigen receptor of the present application has an effect on body weight change in experimental animals. DETAILED DESCRIPTION

[0153] Methods, steps, techniques and operations not described in detail in the present application are conventional operations in the art. The present application is explained below with reference to examples, but the present application is not limited to the examples.

[0154] The plasmid and lentivirus of the present application are purchased from Pharosvaccine Inc., Gyeonggi, Republic of Korea. DMEM medium is purchased from Gibco Life Sciences; PEI MAX polyetherimide is purchased from Polysciences, Inc; F108 is purchased from Sigma-Aldrich Fine Chemicals Biosciences; IL-2: is purchased from Beijing Sihuan Biopharmaceutical Co., Ltd.; X-VIVO 15 is purchased from Lonza Group AG; Dextramer reagent is purchased from Immudex ApS; CD8 antibody is purchased from BD; Vβ7.1 is purchased from Meitianyi; Dextramer is purchased from immudex; HERV-E positive target cells with HLA subtype A*1101 are purchased from T-CURE BIOSCIENCE, INC.

[0155] Example 1 Construction of recombinant lentivirus plasmid of the present application

[0156] According to the relevant contents of "Molecular Cloning Laboratory Guide", the nucleic acid sequences of TCR of the first group to the seventh group and TCR of control group 1-3 are constructed into pPVLV4 plasmid through enzyme cutting site XbaI and enzyme cutting site XhoI, to obtain pPVLV4 shuttle plasmid for lentivirus packaging.

[0157] The TCR nucleic acid sequences of the 1st group to the 7th group and the control group 1-2 are cut from the synthetic cloning vector pUC57 (purchased from Jiangsu Jinershui Biological Technology Co., Ltd.) using XbaI and XhoI, the target fragments are recovered by cutting the gel, and then the fragments are connected with the XbaI and XhoI enzyme cut lentiviral plasmid using T4 DNA ligase, and the competent cells are transformed. The next day, single colonies are picked and cultured in LB liquid medium containing antibiotics for 12-16h, plasmid extraction, detection of plasmid concentration and purity, enzyme digestion identification of correct clones, and DNA sequencing identification of correct plasmids, to obtain TCR nucleic acid sequence shuttle plasmid pPVLV4_HERV-E containing the 1st-7th group and the control group 1-2, respectively.

[0158] Example 2 Production of the lentiviral vector of the application

[0159] Preparation of the lentiviral vector, comprising the following steps:

[0160] (1) The recovered 293T cells are cultured in DMEM medium containing 10% FBS, and when the cell confluence is 85%, the 293T cells are digested with recombinant trypsin, and then plated and cultured at 37°C, 5% CO2.

[0161] (2) The shuttle plasmid pPVLV4_HERV-E: pMDLg / pRRE(K): pRSV-Rev(K): pMD.G(K) prepared in Example 1 is mixed according to the mass ratio of 4:2:2:1, and then added to 3ml DMEM medium, and then placed at room temperature for 5min. 2 The cell culture bottle is added with 175.5ul of 1mg / ml PEI MAX solution and 3ml of DMEM medium, and then placed at room temperature for 5min. The PEI MAX mixture and the plasmid mixture are mixed at a ratio of 1:1 to form a PEI-DNA mixture, and then placed at room temperature for 20min. 6ml of PEI-DNA mixture is added to 60ml of DMEM medium containing 10% FBS, and then mixed uniformly to form a plasmid transfection solution. The culture supernatant of the 293T cells is removed and replaced with 66ml of plasmid transfection solution, and then incubated at 37°C, 5% CO2 for 18±2h; the culture supernatant is discarded, and then 60ml of DMEM medium is added, and then cultured at 37°C, 5% CO2 for 24h.

[0162] (3) Centrifuge the collected culture supernatant at 500g for 4 min (room temperature), discard the precipitate, centrifuge the collected supernatant at 35000g for 4 h (4℃), discard the supernatant, resuspend the harvested lentiviral vector in an appropriate amount of DMEM medium, centrifuge, collect the virus precipitate, mix it evenly by pipetting, dispense it into EP tubes, and store it at -80℃ to obtain lentiviral vectors containing the TCR nucleic acid sequences of groups 1-7 and control groups 1-2, respectively.

[0163] Example 3 Preparation of T cells containing TCR nucleic acid sequence in this invention

[0164] 1. PBMC resuscitation and T cell sorting and activation

[0165] (1) Isolation of PBMCs: 50 ml of blood was collected from HLA*A1101 volunteers and placed in a 250 ml centrifuge tube. Sodium chloride injection solution was added at a volume ratio of 1:1 to dilute the blood, and the tube was centrifuged at 400 g for 30 min (18-20℃). Intermediate layer cells were collected and placed in a 250 ml centrifuge tube. Sodium chloride injection solution was added to bring the volume up to the target.

[0166] Centrifuge at 200ml for 400g for 10 minutes, remove the supernatant, resuspend the cells in 200ml of sodium chloride injection solution, and centrifuge again.

[0167] 400g*10min, aspirate the supernatant, disperse the cells, and bring the volume to 50ml with X-VIVO15 serum-free medium (Lonza) and mix well; after cell counting, centrifuge the PBMC cell suspension (1500rpm*5min); aspirate the supernatant, resuspend the cells with cell cryopreservation solution, transfer to 4℃ for pre-cooling, and then freeze at -80℃.

[0168] (2) PBMC thawing: After thawing, the frozen PBMCs were placed in 30 mL of X-VIVO15 medium, centrifuged at 526 g for 5 min, the supernatant was discarded, and the cells were resuspended in 12 mL of X-VIVO15 medium, adjusting the cell density to 5 × 10⁻⁶ cells / mL. 6 The cells were added at a concentration of 100 cells / ml according to the instructions. TransAct (T-cell activation reagent, derived from Miltenyi Biotec) with CD3 and CD28 antibodies as the main components was added, and the mixture was incubated at 37°C with 5% CO2 for 24 hours.

[0169] 2. Lentiviral transduction of T cells

[0170] The activated cells were taken out, placed in a cell culture plate, and divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (with the lentivirus vector containing the TCR nucleic acid sequence of Group 1 prepared in Example 2) and incubated at 37°C, 5% CO2 for 24 h to obtain lentivirus-transduced T cells, centrifuged at 526g*5 min, resuspended with 1 ml of X-VIVO 15 medium containing 200 IU / mL IL-2, adjusted to a cell density of 5x10

[0171] 526g*5min, resuspended with 1 ml of X-VIVO 15 medium containing 200 IU / mL IL-2, adjusted to a cell density of 5x10 5 After elution, T cells containing the TCR nucleic acid sequence of Group 1 were obtained.

[0172] Example 4 Preparation of T cells containing TCR nucleic acid sequences of the present application

[0173] 1. Resuscitation of PBMC and sorting and activation of T cells (same as Example 3)

[0174] 2. Lentivirus-transduced T cells: The activated cells were taken out, placed in a cell culture plate, and divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (with the lentivirus vector containing the TCR nucleic acid sequence of Group 2 prepared in Example 2) and incubated at 37°C, 5% CO2 for 24 h to obtain lentivirus-transduced T cells, centrifuged at 526g*5 min, resuspended with 1 ml of X-VIVO 15 medium containing 200 IU / mL IL-2, adjusted to a cell density of 5x10 5 After elution, T cells containing the TCR nucleic acid sequence of Group 2 were obtained.

[0175] Example 5 Preparation of T cells containing TCR nucleic acid sequences of the present application

[0176] 1. Resuscitation of PBMC and sorting and activation of T cells (same as Example 3)

[0177] 2. Lentivirus-transduced T cells: The activated cells were taken out, placed in a cell culture plate, and divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (with the lentivirus vector containing the TCR nucleic acid sequence of Group 3 prepared in Example 2) and incubated at 37°C, 5% CO2 for 24 h to obtain lentivirus-transduced T cells, centrifuged at 526g*5 min, resuspended with 1 ml of X-VIVO 15 medium containing 200 IU / mL IL-2, adjusted to a cell density of 5x10 5The cells were incubated at 37°C with 5% CO2 for 48 hours. After elution, T cells containing the TCR nucleic acid sequence of group 3 were obtained.

[0178] Example 6 Preparation of T cells containing TCR nucleic acid sequence in this invention

[0179] 1. PBMC resuscitation and T cell sorting and activation (same as Example 3)

[0180] 2. Lentiviral transduction of T cells: Activated cells were removed and placed in cell culture plates, and divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (containing the lentiviral vector with the TCR nucleic acid sequence of group 4 prepared in Example 2). The cells were cultured at 37°C and 5% CO2 for 24 h to obtain lentiviral transduced T cells. The cells were centrifuged at 526 g for 5 min, resuspended in 1 ml of X-VIVO15 medium containing 200 IU / mL IL-2, and the cell density was adjusted to 5 × 10⁻⁶ cells / mL. 5 The cells were incubated at 37°C and 5% CO2 for 48 hours. After elution, T cells containing the TCR nucleic acid sequence of group 4 were obtained.

[0181] Example 7 Preparation of T cells containing TCR nucleic acid sequence in this invention

[0182] 1. PBMC resuscitation and T cell sorting and activation (same as Example 3)

[0183] 2. Lentiviral transduction of T cells: Activated cells were removed and placed in cell culture plates, and divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (containing the lentiviral vector with the TCR nucleic acid sequence of group 5 prepared in Example 2). The cells were cultured at 37°C and 5% CO2 for 24 h to obtain lentiviral transduced T cells. The cells were centrifuged at 526 g for 5 min, resuspended in 1 ml of X-VIVO15 medium containing 200 IU / mL IL-2, and the cell density was adjusted to 5 × 10⁻⁶ cells / mL. 5 The cells were incubated at 37°C and 5% CO2 for 48 hours. After elution, T cells containing the TCR nucleic acid sequence of group 5 were obtained.

[0184] Example 8 Preparation of T cells containing TCR nucleic acid sequence in this invention

[0185] 1. PBMC resuscitation and T cell sorting and activation (same as Example 3)

[0186] 2. Lentivirus transduction of T cells: Take out the activated cells, put them in a cell culture plate, and divide them into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (add the lentivirus vector containing the TCR nucleic acid sequence of Group 6 prepared in Example 2), incubate at 37°C, 5% CO2 for 24h, obtain lentivirus transduced T cells, centrifuge at 526g*5min, resuspend with 1ml X-VIVO15 medium containing 200IU / mL IL-2, adjust the cell density to 5x10 5 6 / mL, incubate at 37°C, 5% CO2 for 48h, after elution, obtain T cells containing the TCR nucleic acid sequence of Group 6.

[0187] Example 9 Preparation of T cells containing TCR nucleic acid sequences of the present application

[0188] 1. Resuscitation of PBMC and sorting and activation of T cells (same as Example 3)

[0189] 2. Lentivirus transduction of T cells: Take out the activated cells, put them in a cell culture plate, and divide them into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (add the lentivirus vector containing the TCR nucleic acid sequence of Group 7 prepared in Example 2), incubate at 37°C, 5% CO2 for 24h, obtain lentivirus transduced T cells, centrifuge at 526g*5min, resuspend with 1ml X-VIVO15 medium containing 200IU / mL IL-2, adjust the cell density to 5x10 5 6 / mL, incubate at 37°C, 5% CO2 for 48h, after elution, obtain T cells containing the TCR nucleic acid sequence of Group 7.

[0190] Comparative Example 1 Preparation of T cells containing TCR nucleic acid sequences of the present application

[0191] 1. Resuscitation of PBMC and sorting and activation of T cells (same as Example 3)

[0192] 2. Lentivirus transduction of T cells: Take out the activated cells, put them in a cell culture plate, and divide them into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (add the lentivirus vector containing the TCR nucleic acid sequence of Group 7 prepared in Example 2), incubate at 37°C, 5% CO2 for 24h, obtain lentivirus transduced T cells, centrifuge at 526g*5min, resuspend with 1ml X-VIVO15 medium containing 200IU / mL IL-2, adjust the cell density to 5x10 5 6 / mL, incubate at 37°C, 5% CO2 for 48h, after elution, obtain T cells containing the TCR nucleic acid sequence of Group 7.

[0193] Comparative Example 2 Preparation of T cells containing TCR nucleic acid sequences

[0194] 1. Resuscitation of PBMC and sorting and activation of T cells (as in Example 3)

[0195] 2. Lentivirus transduction of T cells: Take the activated cells and place them in a cell culture plate and divide them into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (add the lentivirus vector containing the TCR nucleic acid sequences of Control Group 2 prepared in Example 2), and incubate at 37°C, 5% CO2 for 24h to obtain lentivirus transduced T cells, centrifuge at 526g*5min, resuspend with 1ml X-VIVO15 medium containing 200IU / mL IL-2, adjust the cell density to 5x10 5 6x106 / mL, incubate at 37°C, 5% CO2 for 48h, and after elution, obtain T cells containing the TCR nucleic acid sequences of Control Group 2.

[0196] Test Example 1 Study on the effect of the T cell antigen receptor of the application in reducing TCR mismatch rate

[0197] Take the T cells prepared in Examples 3-5, 7-9 and the T cells prepared in Comparative Examples 1-2, after detecting the Dextramer positive rate and the Vβ positive rate, set two parallels for each group (one added with CD8 antibody and Dextramer reagent; the other added with CD8 antibody and Vβ7.1 antibody), adjust the cell density to 5x10 5 6x106 / mL with X-VIVO15 medium containing 200IU / mL IL-2, and incubate at 37°C, 5% CO2 for 6-10d.

[0198] Mix the sample, add the cell suspension into the labeled flow tube, add 2 mL PBS (1X) per tube, mix, centrifuge at 526 g*3 min, discard the supernatant, shake the cells, incubate at room temperature for 15 min, then add 2 mL PBS (1X) per tube, mix, centrifuge at 526 g*3 min, discard the supernatant, shake the cells, add 50 μL PBS per tube to resuspend the cells, start a new experiment, detect on the machine, collect 20,000 cells in the cell gate; count the Dextramer positive rate (D%) and Vβ7.1 positive rate (V%) in the CD8 gate, and calculate the mismatch rate = 1-D% / V%, wherein V% is the proportion of cells expressing Vβ in the CD8 positive cell population (Vβ antibody detects the transduction efficiency of TCR gene, and the higher the value, the higher the transduction efficiency of TCR gene); D% is the proportion of cells expressing correctly paired TCR in the CD8 positive cell population (Dextramer detects correctly paired TCR, and the higher the value, the higher the proportion of correctly paired TCR). The results are shown in Figure 1 .

[0199] Test Example 2 Cell killing rate of T cell antigen receptor of the application

[0200] Target cells: HERV-E positive cells with HLA*1101 were used as target cells to evaluate the function of TCR-T cells. Effector cells: TCR-T cells prepared in Examples 3-4, 6-7, and 9 were selected.

[0201] The NC group is PBMC, and the extraction method is as follows: transfer the blood of a volunteer (the HLA restriction type of the volunteer is HLA*A1101) into a 250 ml centrifuge tube, mix sodium chloride injection with the blood at a ratio of at least 1:1, mix 15 ml / branch in a 50 ml centrifuge tube, add 30 ml of diluted blood to each tube; centrifuge at 400 g for 30 min (18-20°C); collect the middle layer cells into a 250 ml centrifuge tube, add sodium chloride injection to 200 ml, mix well; centrifuge at 400 g for 10 min, remove the supernatant, resuspend the cells with 200 ml of sodium chloride injection, mix well; centrifuge at 400 g for 10 min, remove the supernatant, disperse the cells, and then add X-VIVO15 serum-free medium (Lonza) to 50 ml, mix well; take 0.5 ml for cell number calculation, and the remaining PBMC cell suspension is centrifuged at 1500 rpm for 5 min; remove the supernatant, resuspend the cells with cell freezing solution; quickly transfer the freezing tube to a pre-cooled program cooling box at 4°C; the program cooling box is stored in a -80°C refrigerator overnight; the next day, the cells in the program cooling box are taken out and transferred to liquid nitrogen for storage.

[0202] In vitro killing experiment: the target cells were prepared at 6×10 3Plates, mixed at 1:1 effector cells:target cells, incubated at 37℃ for 4h, and the killing efficiency was obtained by real-time label-free cell function analyzer (Real Time Cell Analyzer, RTCA, Agilent). Real Time CellAnalyzer, RTCA, Agilent) to obtain the killing efficiency.

[0203] The HERV-E positive target cells with HLA subtype A*1101 (from T-CURE BIOSCIENCE, INC) were plated at 6000 cells per well. The next day, the effector cells were added at 1:1 ratio of CD8+ DEX+ positive cell number to target cell number, and the data collected 47h after plating the effector cells were used to evaluate the killing efficiency. The specific killing results of each group are shown in Table 1. Figure 2

[0204] Test Example 3 Pharmacodynamic evaluation of specific T cells of the present application in mice

[0205] 1. Experimental materials

[0206] OS-RC-2-A11 tumor cells were resuspended at a concentration of 5×10 7 cells / ml after resuscitation, and the total number of cells reached 6×10 6 cells / ml after resuscitation, and the total number of cells reached 6×10

[0207] OS-RC-2-A11 tumor cells are tumor cells that overexpress human histocompatibility complex HLA*A1101 protein, which are constructed by introducing a gene encoding HLA*A1101 protein into tumor cells OS-RC-2 (human renal cancer cells, purchased from the Union Cell Bank): tumor cells OS-RC-2 are transduced with lentivirus containing a gene encoding HLA*A1101 protein, and the medium is changed after 1 day to use complete culture medium for 1-2 days, and then the complete culture medium containing puromycin is used for continuous culture, and the expression of HLA*A1101 protein in the transduced tumor cells is detected. The specific steps include the following:

[0208] 1) Lentivirus transduction of gene encoding HLA*A1101 protein: the density of tumor cells OS-RC-2 is adjusted to 4-6×10 5 ​cells / ml, 1 ml / well, 6-well plate, 8 μl Polybrene (1 mg / ml) was added to each well to a final concentration of 8 μg / ml, 10-30 μl of lentiviral vector containing the gene encoding HLA*A1101 protein was added to each well, a cell control well (non-transduced group) was set up, 1 ml of tumor cell OS-RC-2 and 8 μl of Polybrene were added to the control well, the mixture was mixed and incubated in a CO2incubator (37°C, 5% CO2) for 1 day; the supernatant was discarded, 2 ml of complete medium was added to each well, and the mixture was incubated in a CO2incubator for 2 days;

[0209] 2) Puromycin screening of transduced tumor cells: the supernatant was discarded, 2 ml of complete medium containing 1 μg / ml of puromycin (Solarbio) was added to each well, and the mixture was incubated in a CO2incubator; the cells were observed and the complete medium containing 1 μg / ml of puromycin was replaced every 2 days, and the adherent cells were subcultured when they were fully grown; the complete medium containing 1 μg / ml of puromycin was used for screening and culture for 7 days, if there were still live cells in the control group, the concentration of puromycin was increased and the culture was continued until all cells in the control group died;

[0210] 3) Monoclonal culture of cells containing the gene encoding HLA*A1101 protein: the tumor cells transduced with the gene encoding HLA*A1101 protein that were cultured for more than 7 days were plated according to the limited dilution method, 1 cell / well, 3 cells / well, and the complete medium containing puromycin was used for culture and observation of the cells in the well, and the monoclonal well was marked, and the culture was continued until the number of cells reached at least 6×10 7 cells or more, and then the cells were frozen, thereby obtaining the target cell OS-RC-2-A11 overexpressing.

[0211] Twenty-five 6-8 week old female mice (18-20 g) were taken, 5 mice per cage, 20-26°C, 30-70%, 12h light and 12h dark, corn cob bedding, changed once a week, free access to food and water. Inoculation: after removing the body hair at the inoculation site, the inoculation site of the mouse was disinfected with an iodophor cotton ball, 0.2 mL of OS-RC-2-A11 tumor cell suspension (containing 1×10 6 cells per mouse) was inoculated subcutaneously on the right shoulder of the mouse using a 1 mL syringe.

[0212] After inoculation, the tumor volume and body weight were measured once a week, and when the tumor volume reached 70-100 mm 3 , the 25 animals were randomly divided into 5 groups according to the tumor volume and body weight, 5 animals per group, and the drugs were infused into the tail vein according to Table 2, and IL2 was injected intraperitoneally on the day of infusion, 24h and 48h, the injection amount of IL2 was 200,000 IU / 200 μl per mouse per time.

[0213] The date of the start of administration is taken as PG-Day 0. After the start of administration, the mice are measured for body weight and tumor volume twice a week. After the end of administration, the trend of tumor growth and the body weight of the mice is continued to be observed, and no tissue collection is performed, and the experimental animals are directly euthanized.

[0214] Table 2 Grouping and administration information

[0215]

[0216] After inoculation, the animals are observed daily for morbidity and mortality, including tumor growth and the effects of the drug on the experimental animals, such as changes in activity, changes in food and water intake, weight loss, hair, changes in the appearance of the eyes, death and other clinical symptoms. The body weight of the experimental animals is measured once a week from after inoculation to before grouping. The body weight of the experimental animals is measured twice a week after grouping or the frequency of measuring the body weight of the mice is changed according to the requirements of the customer. The tumor volume of the experimental animals is measured once a week when the tumor is visible from after inoculation to before grouping, and the tumor volume of the experimental animals is measured twice a week after inoculation and grouping. The tumor volume is measured by the two-way measurement method, and the tumor volume (TV) = 0.5 * a * b is calculated 2 , where a is the long diameter of the tumor and b is the short diameter of the tumor. All data statistics are analyzed using SPSS 24.0 software by the One-Way ANOVA method. The LSD method is used for analysis, and a p value less than 0.05 is considered to be a significant difference, and a p value less than 0.01 is considered to be a very significant difference.

[0217] The adverse states, body weight (weight loss greater than 10%) and death of the mice are observed. At PG-Day 13, mice in G1 and G3 are observed to be hunched, and at PG-Day 17, mice in G2 are also hunched. At PG-Day 20, almost all mice in G1, G2 and G3 are hunched and accompanied by piloerection symptoms, and deaths occur successively. The sample size of each group gradually decreases, so PG-Day 20 is taken as the node for efficacy statistics.

[0218] The change in the tumor volume of the mice is observed, and the results are shown in Table 3 Figure 3 . From the start of administration to PG-Day 20, there is no significant difference between G2 and the control group G1 (p>0.05), and there is a very significant difference between G3 and the control group G1 (p<0.01). By PG-Day 59, all 5 mice in G3 have tumor recurrence. There is a very significant difference between G4 and G5 and the control group G1 (p<0.01); by PG-Day 59, the tumor volume is 0 mm 3 . And there is no obvious decrease in body weight, and the safety is good Figure 4 .

[0219] The above description of the specific embodiments of the present application is not intended to limit the present application, and various changes or modifications can be made to the present application by those skilled in the art without departing from the spirit of the present application, and all such changes or modifications shall fall within the scope of protection of the claims of the present application.

Claims

1. A T-cell antigen receptor, said T-cell antigen receptor comprising an amino acid sequence selected from any one of (4) or (5): (4) The amino acid sequence of the constant region of its α chain contains SEQ ID NO: 12, or contains an amino acid sequence that has at least 85% homology with SEQ ID NO: 12, and the amino acid sequence of the constant region of its β chain contains SEQ ID NO: 13, or contains an amino acid sequence that has at least 85% homology with SEQ ID NO:

13. (5) The amino acid sequence of the constant region of its α chain contains SEQ ID NO: 14, or contains an amino acid sequence that has at least 85% homology with SEQ ID NO: 14; the sequence of the constant region of its β chain contains an amino acid sequence as shown in SEQ ID NO: 15, or contains an amino acid sequence that has at least 85% homology with SEQ ID NO:

15. Preferably, the homology is selected from any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

2. The T-cell antigen receptor according to claim 1, wherein the variable region of the α chain includes α-CDR3, and the variable region of the β chain includes β-CDR3, wherein, The amino acid sequence of α-CDR3 is shown in SEQ ID NO: 18, and the amino acid sequence of β-CDR3 is shown in SEQ ID NO:

19. The variable region of the α chain includes any one or a combination of α-CDR1 and α-CDR2, wherein the amino acid sequence of α-CDR1 is shown in SEQ ID NO: 20, and the amino acid sequence of α-CDR2 is shown in SEQ ID NO:

21. The variable region of the β chain includes any one or a combination of β-CDR1 and β-CDR2, wherein the amino acid sequence of β-CDR1 is shown in SEQ ID NO: 22 and the amino acid sequence of β-CDR2 is shown in SEQ ID NO:

23. Preferably, the variable region amino acid sequence of the α chain is as shown in SEQ ID NO: 24, and the variable region amino acid sequence of the β chain is as shown in SEQ ID NO: 25; Preferably, the variable region amino acid sequence of the α chain comprises SEQ ID NO: 26, or comprises an amino acid sequence having at least 85% homology with SEQ ID NO: 26, and the variable region amino acid sequence of the β chain comprises SEQ ID NO: 27, or comprises an amino acid sequence having at least 85% homology with SEQ ID NO:

27. Preferably, the homology is selected from any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. Preferably, the amino acids of its α chain and β chain are linked by a linking sequence as shown in SEQ ID NO: 28; preferably, the amino acid sequence of the T cell antigen receptor is selected from any one or a combination of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, or contains an amino acid sequence having at least 85% homology to any one of SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO:

35.

3. A nucleic acid encoding the T-cell antigen receptor according to any one of claims 1-2; Preferably, the nucleotide sequence is selected from any one of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42, or contains a nucleotide sequence having at least 85% homology with any one of SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO:

42. Preferably, the homology is selected from any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

4. An expression vector comprising the nucleic acid of claim 3.

5. A host cell comprising the nucleic acid of claim 3 or the expression vector of claim 4.

6. An immune cell expressing the T-cell antigen receptor as described in any one of claims 1-2.

7. A method for preparing immune cells, comprising transducing the nucleic acid sequence of claim 3 into immune cells for expression.

8. A method for preparing recombinant T cells, comprising the following steps: 1) Prepare the nucleic acid according to claim 3; 2) Isolate and culture T cells; 3) Deliver the nucleic acid from step 1) into the T cells described in step 2) to obtain recombinant T cells expressing the T cell antigen receptor described in any one of claims 1-2.

9. A method for preparing a T-cell antigen receptor, comprising the following steps: (1) Prepare the nucleic acid according to claim 3; (2) The nucleic acid obtained in step 1) is ligated into a vector to obtain an expression vector; (3) Transduc the expression vector obtained in step 2) into the host cell and then induce its expression; (4) Obtain T cell antigen receptors.

10. The use of the T-cell antigen receptor as described in any one of claims 1-2, the nucleic acid as described in claim 3, the expression vector as described in claim 4, the host cell as described in claim 5, and the immune cell as described in claim 6 in the preparation of articles for the diagnosis, prevention, and / or treatment of tumors.

11. A pharmaceutical composition comprising any one or a combination of 1)-5), 1) The T-cell antigen receptor according to any one of claims 1-2; 2) The nucleic acid as described in claim 3; 3) The expression vector as described in claim 4; 4) The host cell as described in claim 5; or 5) The immune cells as described in claim 6.

12. A kit comprising any one or a combination of 1)-5): 1) The T-cell antigen receptor according to any one of claims 1-2; 2) The nucleic acid according to any one of claims 3; 3) The expression vector as described in claim 4; 4) The host cell as described in claim 5; 5) The immune cells as described in claim 6.

13. A detection method, the method comprising contacting a sample to be tested with the T-cell antigen receptor as described in any one of claims 1-2.

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