A T cell antigen receptor and its preparation method and application

Abstract

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

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a T cell antigen receptor, a preparation method thereof, and an application thereof. Background Art

[0002] The T cell receptor (TCR) is a molecule that specifically recognizes antigens and initiates immune responses in T lymphocytes. It is a heterodimeric cell surface protein of the immunoglobulin superfamily that associates with the unmodified protein of the CD3 complex, which is involved in regulating signal transduction. The TCR is the only receptor for specific antigenic peptides presented on the major histocompatibility complex (MHC) and is crucial for the cellular immunity function of the immune system. The binding of the antigen-specific TCR to the MHC complex triggers direct physical contact and interaction between the T cell and the antigen-presenting cell, leading to subsequent cell signaling and other physiological reactions, allowing T cells with different antigen specificities to exert immune effects on their target cells.

[0003] Correct TCR assembly is crucial for TCR-T cell generation. TCR mispairing can lead to cross-reactive toxicity and other issues, creating a technical challenge for TCR-T cell therapy. Introducing mouse-derived amino acids to reduce mispairing rates can lead to excessive immunogenicity, compromising drugability. Therefore, developing TCR modification strategies with robust bioactive functions is crucial to effectively reduce TCR mispairing rates and immunogenicity. Summary of the Invention

[0004] The object of the present invention is to provide a T cell antigen receptor, wherein the T cell antigen receptor comprises an α chain and / or a β chain, wherein the α chain comprises any one or a combination of (a)-(b),

[0005] (a) the constant region comprises the amino acid segment shown in SEQ ID NO: 1 (LVIVL);

[0006] (b) the constant region contains one or more cysteine residues capable of forming non-native disulfide bonds with β chains;

[0007] The constant region of the β chain comprises one or more cysteine residues capable of forming non-native disulfide bonds with the α chain;

[0008] Preferably, the amino acid sequence of the constant region of the T cell antigen receptor is completely replaced by mouse amino acids;

[0009] Preferably, the amino acid sequence of the constant region of the T cell antigen receptor is substituted by a mouse amino acid at least at one site, more preferably the amino acid sequence of the constant region of the T cell antigen receptor is substituted by 1 or 9 mouse amino acids.

[0010] In a preferred technical solution of the present invention, the transmembrane hydrophobic region of the constant region contains an amino acid segment as shown in SEQ ID NO: 1 (LVIVL).

[0011] In a preferred technical solution of the present invention, the constant regions of the α chain and the β chain further include at least one leucine zipper.

[0012] In a preferred technical solution of the present invention, the leucine zipper is selected from any one of SEQ ID NO: 2 (PGGRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNY), SEQ ID NO: 3 (PGGLTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAY), SEQ ID NO: 4 (AQCEKELQALEKENAQLEWELQALEKELAQ), and SEQ ID NO: 5 (AQCEKELQALEKENAQLEWELQALEKELAQ).

[0013] In a preferred technical solution of the present invention, a leucine zipper containing a sequence of SEQ ID NO: 2 is used in pair with a leucine zipper containing a sequence of SEQ ID NO: 3, and a leucine zipper containing a sequence of SEQ ID NO: 4 is used in pair with a leucine zipper containing a sequence of SEQ ID NO: 5.

[0014] In a preferred technical solution of the present invention, the amino acid sequence of the constant region of the α chain comprises the following sequence:

[0015] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPS X1X2X3X4 CDVKLVEKSFETDTNLNFQNL LVIVL RILLLKVAGFNLLMTLRLWSS, wherein X1 is selected from any one of S and P, X2 is selected from any one of D and E, X3 is selected from any one of V and S, and X4 is selected from any one of P and S, or an amino acid sequence having at least 85% homology with the above sequence;

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

[0017] DLNKVFPPEVAVFEPS X5 AEI X6HTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCG X7 TS X8 SY X9 QGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF, wherein X5 is selected from any one of E and K, X6 is selected from any one of A and S, X7 is selected from any one of I and F, X8 is selected from any one of A and V, and X9 is selected from any one of H and Q; or an amino acid sequence having at least 85% homology with the above sequence.

[0018] In a preferred embodiment of the present invention, the T cell antigen receptor comprises an amino acid sequence selected from any one of (1) to (7):

[0019] (1) The amino acid sequence of the constant region of its α chain comprises SEQ ID NO: 6, or comprises an amino acid sequence having at least 85% homology to SEQ ID NO: 6, wherein said SEQ ID NO: 6 is:

[0020] IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS;

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

[0022] DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS;

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

[0024] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVPCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS;

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

[0026] DLNKVFPPEVAVFEPSKAEIAHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF;

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

[0028] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS; the amino acid sequence of the constant region of its β chain comprises SEQ ID NO: 11, or comprises an amino acid sequence having at least 85% homology to SEQ ID NO: 11, wherein SEQ ID NO: 11 is:

[0029] DLNKVFPPEVAVFEPSKAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF;

[0030] (4) The amino acid sequence of the constant region of the α chain comprises SEQ ID NO: 12, or comprises an amino acid sequence having at least 85% homology to SEQ ID NO: 12, wherein SEQ ID NO: 12 is:

[0031] IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS;

[0032] The amino acid sequence of the constant region of the beta chain comprises SEQ ID NO: 13, or an amino acid sequence having at least 85% homology to SEQ ID NO: 13, wherein SEQ ID NO: 13 is: DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS;

[0033] (5) The amino acid sequence of the constant region of the α chain comprises SEQ ID NO: 14, or comprises an amino acid sequence having at least 85% homology to SEQ ID NO: 14, wherein SEQ ID NO: 14 is:

[0034] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSSPGGRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNY;

[0035] The sequence of the constant region of the β chain comprises the amino acid sequence shown in SEQ ID NO: 15, or comprises an amino acid sequence having at least 85% homology to SEQ ID NO: 15, wherein SEQ ID NO: 15 is:

[0036] DLNKVFPPEVAVFEPSKAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQ DRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFPGGLTDTLQAETDQLEDKKSALQTEIANLLKEKEKLEFILAAY;

[0037] (6) The amino acid sequence of the constant region of the α chain comprises SEQ ID NO: 16, or comprises an amino acid sequence having at least 85% homology to SEQ ID NO: 16, wherein SEQ ID NO: 16 is:

[0038] IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSSAQCKKKLQALKKKNAQLKWKLQALKKKLAQ;

[0039] The sequence of the constant region of the β chain comprises the amino acid sequence shown in SEQ ID NO: 17, or comprises an amino acid sequence having at least 85% homology to SEQ ID NO: 17, wherein SEQ ID NO: 17 is:

[0040] DLNKVFPPEVAVFEPSKAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLS ENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFAQCEKELQALEKENAQLEWELQALEKELAQ.

[0041] In a preferred technical solution of the present invention, the variable region of the α chain comprises α-CDR3, and the variable region of the β chain comprises β-CDR3, wherein the amino acid sequence of the α-CDR3 is AVRGGADGLT (SEQ ID NO: 18), and the amino acid sequence of the β-CDR3 is ASSPPNEKLF (SEQ ID NO: 19).

[0042] In a preferred technical solution of the present invention, the variable region of the α chain comprises any one or a combination of α-CDR1 and α-CDR2, wherein the amino acid sequence of the α-CDR1 is DSVNN (SEQ ID NO: 20) and the amino acid sequence of the α-CDR2 is IPSGT (SEQ ID NO: 21).

[0043] In a preferred technical solution of the present invention, the variable region of the β chain comprises any one or a combination of β-CDR1 and β-CDR2, wherein the amino acid sequence of the β-CDR1 is MGHRA (SEQ ID NO: 22) and the amino acid sequence of the β-CDR2 is YSYEKL (SEQ ID NO: 23).

[0044] In a preferred embodiment of the present invention, the amino acid sequence of the variable region of the α chain is shown in SEQ ID NO: 24, and the SEQ ID NO: 24 is:

[0045] IQVEQSPPDLI LQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVATERYSLLY ISSSQTTDSGVYFCAVRGGADGLTFGKGTHLI IQPY;

[0046] The amino acid sequence of the variable region of the β chain is shown in SEQ ID NO: 25, which is: EVTQTPKHLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLLNLHLHALQPEDSALYLCASSPPNEKLFFGSGTQLSVLE.

[0047] In a preferred embodiment of the present invention, the amino acid sequence of the variable region of the α chain comprises SEQ ID NO: 26, or comprises an amino acid sequence having at least 85% homology to SEQ ID NO: 26, wherein SEQ ID NO: 26 is:

[0048] IQVEQSPPDLI LQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVATERYSLLY ISSSQ L TDSGVYFCAVRGGADGLTFGKGTHLI IQPY;

[0049] In a preferred embodiment of the present invention, the amino acid sequence of the variable region 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, wherein SEQ ID NO: 27 is:

[0050] EVTQTPRYLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLL NLHLHALQPEDSALYLCASSPPNEKLFFGSGTQLSVLE;

[0051] 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%.

[0052] In a preferred technical solution of the present invention, the amino acids of the α chain and the β chain are linked by a connecting sequence as shown in SEQ ID NO: 28, wherein the SEQ ID NO: 28 is GSRAKRSGSGATNFSLLKQAGDVEENPGP.

[0053] In a preferred technical solution of the present invention, the amino acid sequence comprised by the T cell antigen receptor is 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.

[0054] Another object of the present invention is to provide a nucleic acid comprising a nucleotide sequence encoding the TCR of the present invention or its complementary sequence.

[0055] In a preferred technical solution of the present invention, the nucleotide sequence or its complementary sequence is selected from any one of single-stranded, double-stranded, DNA, and RNA, or a combination thereof.

[0056] In a preferred technical solution of the present invention, the nucleotide sequence or its complementary sequence is codon optimized.

[0057] In a preferred technical solution of the present invention, the codon optimization includes converting a large number of rare codons used by viruses into corresponding mammalian codons, removing mRNA unstable motifs, and any one or a combination of hidden splicing sites.

[0058] In a preferred technical solution of the present invention, 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 comprises 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.

[0059] Another object of the present invention is an expression vector comprising the nucleic acid of the present invention.

[0060] In a preferred technical solution of the present invention, the expression vector can be expressed under any conditions, in vivo, in vitro or in vitro.

[0061] In a preferred technical solution of the present invention, the expression vector is continuously expressed at a high level in cells in vivo.

[0062] In a preferred technical solution of the present invention, the expression vector is selected from any one of a prokaryotic expression vector and a retroviral vector.

[0063] In a preferred technical solution of the present invention, 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 myelocytic virus 29 (MC29), and avian erythroblastosis virus (AEV), or a combination thereof.

[0064] Another object of the present invention is to provide a host cell, wherein the host cell comprises any one of the nucleic acids or expression vectors of the present invention.

[0065] In a preferred technical solution of the present invention, the host cell is selected from any one of a eukaryotic cell and a prokaryotic cell.

[0066] In a preferred technical solution of the present invention, the eukaryotic cells are selected from any one of yeast cells, 293 cells, and CHO cells, or a combination thereof.

[0067] Another object of the present invention is to provide an immune cell, wherein the immune cell expresses the T cell antigen receptor of the present invention.

[0068] In a preferred technical solution of the present invention, the immune cells contain one or more nucleic acid sequences described in any one of the present invention.

[0069] In a preferred technical solution of the present invention, the immune cells are selected from any one of stem cells, lymphocytes, T cells, B cells, and NK cells.

[0070] In a preferred technical solution of the present invention, the T cell antigen receptor structure of the T cell is as defined above.

[0071] In the preferred technical solution of the present invention, the T cells are selected from CD4 + T, CD8 + Any one or combination of T.

[0072] In a preferred technical solution of the present invention, the immune cells are separated from autologous T cells or allogeneic T cells.

[0073] 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.

[0074] In a preferred technical solution of the present invention, the immune cells are selected from any one of stem cells, lymphocytes, T cells, B cells, and NK cells.

[0075] In a preferred technical solution of the present invention, the T cell antigen receptor structure of the T cell is as defined above.

[0076] In the preferred technical solution of the present invention, the T cells are selected from CD4 + T, CD8 + Any one or combination of T.

[0077] In a preferred technical solution of the present invention, the immune cells are separated from autologous T cells or allogeneic T cells.

[0078] In a preferred technical solution of the present invention, the method further comprises the step of knocking out endogenous TCR of the cell.

[0079] In a preferred technical solution of the present invention, the step of knocking out the endogenous TCR of the cell is to construct a guide RNA (gRNA) targeting the endogenous TCR into a lentiviral vector, and then co-transfect it into T cells together with the packaging plasmid and transfection reagent.

[0080] Another object of the present invention is to provide a method for preparing recombinant T cells, comprising the following steps:

[0081] 1) obtaining any nucleic acid of the present invention from a positive T cell clone;

[0082] 2) Isolation and culture of T cells;

[0083] 3) delivering the nucleic acid obtained in step 1) into the primary T cells described in step 2) to obtain recombinant T cells expressing any of the T cell antigen receptors of the present invention,

[0084] In a preferred technical solution of the present invention, the T cells are selected from any one of hematopoietic stem cells or peripheral blood lymphocyte (PBL)-derived T cells.

[0085] Another object of the present invention is to provide a method for preparing a T cell antigen receptor, comprising the following steps:

[0086] (1) obtaining any nucleic acid of the present invention from a positive T cell clone;

[0087] (2) connecting the nucleic acid obtained in step (1) to a vector backbone to obtain an expression vector;

[0088] (3) transforming the expression vector obtained in step (2) into a host cell and inducing its expression;

[0089] (4) Obtaining antibodies or antigen-binding fragments thereof or T cell antigen receptors.

[0090] In a preferred technical solution of the present invention, the positive T cells specifically bind to the antigen peptide.

[0091] Another object of the present invention is to provide a pharmaceutical composition comprising any one of 1) to 5) or a combination thereof,

[0092] 1) The T cell antigen receptor of the present invention;

[0093] 2) the nucleic acid of the present invention;

[0094] 3) the expression vector of the present invention;

[0095] 4) the host cell of the present invention; or

[0096] 5) The immune cells of the present invention.

[0097] In a preferred technical solution of the present invention, the composition further contains a pharmaceutically acceptable carrier.

[0098] In a preferred technical solution of the present invention, the composition is optionally used in combination with other therapeutic agents.

[0099] In a preferred technical solution of the present invention, the other therapeutic agent is an immunomodulator.

[0100] Another object of the present invention is to provide a use of any one of the T cell antigen receptor or nucleic acid, expression vector, host cell, immune cell, and pharmaceutical composition of the present invention in preparing a product for diagnosing or preventing tumors.

[0101] In a preferred technical solution of the present invention, the tumor is selected from 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 cancer, breast cancer, endometrial cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, bile duct cancer, esophageal cancer, kidney cancer, sarcoma, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome or any one thereof or its complications.

[0102] In a preferred technical solution of the present invention, the leukemia is selected from any one of acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, chronic myeloid leukemia or its complications.

[0103] In a preferred technical solution of the present invention, 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's macroglobulinemia, or its complications.

[0104] In a preferred technical solution of the present invention, the sarcoma is selected from any one of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, chondrosarcoma, or their complications.

[0105] In a preferred technical solution of the present invention, 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 multilocular cystic renal tumor of low malignant potential, or a combination thereof.

[0106] Another object of the present invention is to provide a kit comprising any one or a combination of 1) to 5):

[0107] 1) The T cell antigen receptor of the present invention;

[0108] 2) the nucleic acid of the present invention;

[0109] 3) the expression vector of the present invention;

[0110] 4) The host cell of the present invention;

[0111] 5) The immune cells of the present invention.

[0112] Another object of the present invention is to provide a detection method, which comprises contacting a sample to be detected with the T cell antigen receptor of the present invention.

[0113] Another object of the present invention is to provide a method for preventing and treating tumors, which comprises administering to an individual an effective amount of the T cell antigen receptor or any one of its nucleic acid, expression vector, host cell, immune cell, and pharmaceutical composition of the present invention.

[0114] In a preferred technical solution of the present invention, the method comprises the step of adoptively transferring T cells expressing the T cell antigen receptor of the present invention to a subject.

[0115] In a preferred technical solution of the present invention, the T cells expressing the T cell antigen receptor of the present invention are derived from a subject.

[0116] In a preferred technical solution of the present invention, the T cells expressing the T cell antigen receptor of the present invention and the hematopoietic stem cells, organs, tissues, cells or stem cells are derived from the same donor.

[0117] In a preferred technical solution of the present invention, the tumor is selected from 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 cancer, breast cancer, endometrial cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, bile duct cancer, esophageal cancer, kidney cancer, sarcoma, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome or any one thereof or its complications.

[0118] In a preferred technical solution of the present invention, the leukemia is selected from any one of acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, chronic myeloid leukemia or its complications.

[0119] In a preferred technical solution of the present invention, 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's macroglobulinemia, or its complications.

[0120] In a preferred technical solution of the present invention, the sarcoma is selected from any one of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, chondrosarcoma, or their complications.

[0121] In a preferred technical solution of the present invention, 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 multilocular cystic renal tumor of low malignant potential, or a combination thereof.

[0122] Another object of the present invention is to provide a method for diagnosing tumors, comprising the following steps: contacting a patient's tumor tissue sample with the T cell antigen receptor of the present invention.

[0123] In a preferred technical solution of the present invention, the T cell antigen receptor includes a detectable marker.

[0124] In a preferred technical solution of the present invention, the tumor is selected from 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 cancer, breast cancer, endometrial cancer, bladder cancer, lung cancer, bronchial cancer, bone cancer, prostate cancer, bile duct cancer, esophageal cancer, kidney cancer, sarcoma, thyroid cancer, head and neck cancer, testicular cancer, glioblastoma, astrocytoma, melanoma, myelodysplastic syndrome or any one thereof or its complications.

[0125] In a preferred technical solution of the present invention, the leukemia is selected from any one of acute lymphocytic (lymphoblastic) leukemia, acute myeloid leukemia, myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, plasma cell leukemia, chronic myeloid leukemia or its complications.

[0126] In a preferred technical solution of the present invention, 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's macroglobulinemia, or its complications.

[0127] In a preferred technical solution of the present invention, the sarcoma is selected from any one of osteosarcoma, Ewing sarcoma, leiomyosarcoma, synovial sarcoma, soft tissue sarcoma, angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma, chondrosarcoma, or their complications.

[0128] In a preferred technical solution of the present invention, 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 multilocular cystic renal tumor of low malignant potential, or a combination thereof.

[0129] Unless otherwise indicated, the sequence numbers and names of the present invention are shown in Table 1.

[0130] Table 1

[0131]

[0132]

[0133]

[0134] Unless otherwise specified, the terms used in the present invention have the common meanings in the art, such as Sambrook et al., "Molecular Cloning: A Laboratory Manual", Lewin, "Genes VIII", Roitt et al., "Immunology" (8th edition), etc.

[0135] The "T cell antigen receptor" as used herein is a molecule capable of recognizing peptides when presented by MHC molecules or their tetramers, and is typically present on the surface of T cells in the form of a complex with a CD3 molecule. The TCR of most T cells is composed of α and β peptide chains, while the TCR of a few T cells is composed of γ and δ peptide chains.

[0136] When the term "comprising" as used in the present invention is used to describe a protein or nucleic acid sequence, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention.

[0137] The term "prevention" as used in the present invention refers to any action that suppresses symptoms or delays the onset of specific symptoms by administering the product of the present invention.

[0138] The term "diagnosis" as used herein refers to determining whether a patient has a disease or its progression, or evaluating a patient's response to treatment.

[0139] The term "treat" as used herein means to slow down, interrupt, prevent, control, stop, alleviate, or reverse the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, conditions, or disorders, and refers to therapeutic intervention that improves the signs, symptoms, and the like of a disease or pathological state after the disease has begun to develop.

[0140] The "effective amount" of the present invention refers to a dose that provides the desired therapeutic or preventive effect after being administered to a patient or an organ in a single dose or multiple doses.

[0141] The "products" described in the present invention include but are not limited to the T cell antigen receptors, nucleic acids, expression vectors, host cells, immune cells described in the present invention, and other pharmaceutical compositions such as reagents, kits, chips, antibody conjugates or multifunctional antibodies that assist or cooperate with the above products.

[0142] The "subject" described in the present invention is selected from any one of humans or other mammals.

[0143] The "homology" mentioned in the present invention means that those skilled in the art can adjust the sequence as needed without changing the main structure or function of the amino acid sequence or nucleotide sequence so that its homology is selected from any one of 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100%.

[0144] The "paired use" mentioned in the present invention refers to the interaction of proteins through their leucine zipper domains to form stable dimers.

[0145] The present invention's real-time, label-free, dynamic cell analysis technology integrates a microelectronic cell sensor chip into the bottom of a cell detection plate, creating a cell impedance detection sensing system that dynamically and quantitatively tracks changes in cell morphology, proliferation, and differentiation in real time. When cells attached to the microelectrode surface cause changes in the impedance of the adherent electrode interface, these changes correlate with the cells' real-time functional status. Through real-time, dynamic electrode impedance detection, biological information related to the cell's physiological functions can be obtained, including cell growth, extension, morphological changes, death, and adherence.

[0146] Compared with the existing technology, the present invention has the following beneficial technical effects: the T cell antigen receptor obtained by modifying the TCR constant region significantly reduces the mismatch rate of the TCR molecule, ensuring the correct expression of the TCR, thereby improving the T cell's specific recognition ability for tumor cells or pathogens and enhancing the therapeutic effect; significantly reducing its immunogenicity while increasing its biological activity. The TCR-T cells of the present invention maintain effective anti-tumor activity while reducing the mismatch rate, can specifically recognize and kill target cells expressing the corresponding antigen, and have broad application prospects in immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0147] Figure 1 T cell antigen receptor mismatch rate detection results of the present invention;

[0148] Figure 2 The T cell antigen receptor of the present invention has a killing effect on target cells;

[0149] Figure 3 The effect of the T cell antigen receptor of the present invention on the change of tumor volume in experimental animals;

[0150] Figure 4 The T cell antigen receptor of the present invention affects the weight changes of experimental animals. DETAILED DESCRIPTION

[0151] The methods, steps, techniques and operations not described in detail in the present invention are all conventional operations in the art. The present invention is described below with reference to examples, but the present invention is not limited to the examples.

[0152] The plasmids and lentivirus of the present invention were purchased from Pharosvaccine Inc., Gyeonggi, Republic of Korea. DMEM medium was purchased from Gibco Life Sciences; PEI MAX polyetherimide was purchased from Polysciences, Inc. F108 was purchased from Sigma-Aldrich Fine Chemicals Biosciences; IL-2 was purchased from Beijing Sihuan Biopharmaceutical Co., Ltd.; X-VIVO15 was purchased from Lonza Group AG; Dextramer reagent was purchased from Immudex ApS; CD8 antibody was purchased from BD; Vβ7.1 was purchased from Miltenyi Biopharmaceuticals; Dextramer was purchased from immudex; HERV-E positive target cells with HLA subtype A*1101 were purchased from T-CURE BIOSCIENCE, INC.

[0153] Example 1 Construction of the recombinant lentiviral plasmid of the present invention

[0154] According to the relevant content of the "Molecular Cloning Experiment Guide", the nucleic acid sequences of TCRs of groups 1 to 7 and TCRs of control groups 1-3 were constructed into the pPVLV4 plasmid through the restriction sites XbaI and XhoI to obtain the pPVLV4 shuttle plasmid for lentiviral packaging.

[0155] The TCR nucleic acid sequences for groups 1-7 and controls 1-2 were excised from the synthetic cloning vector pUC57 (purchased from Jiangsu GenScript Biotechnology Co., Ltd.) using XbaI and XhoI. The target fragments were then excised from gels and ligated with the lentiviral plasmids digested with XbaI and XhoI using T4 DNA ligase, and then transformed into competent cells. The next day, single clones were selected and cultured in LB liquid medium containing antibiotics for 12-16 hours. The plasmids were extracted, their concentration and purity were tested, and the correct clones were identified by enzyme digestion. DNA sequencing confirmed the correct plasmids, yielding the shuttle plasmid pPVLV4_HERV-E containing the TCR nucleic acid sequences for groups 1-7 and controls 1-2, respectively.

[0156] Example 2 Production of the lentiviral vector of the present invention

[0157] The preparation of lentiviral vectors includes the following steps:

[0158] (1) The revived 293T cells were cultured in DMEM medium containing 10% FBS. When the cell confluence reached 85%, the 293T cells were digested with recombinant trypsin, the digested cells were plated, and then cultured at 37°C and 5% CO2;

[0159] (2) The shuttle plasmid pPVLV4_HERV-E:pMDLg / pRRE(K):pRSV-Rev(K):pMD.G(K) prepared in Example 1 was mixed in a mass ratio of 4:2:2:1 and added to 3 ml of DMEM medium. The mixture was allowed to stand at room temperature for 5 min and the mixture was plated at 225 cm 2 Add 175.5μl 1mg / ml PEI MAX solution and 3ml DMEM medium to the cell culture flask and let it stand at room temperature for 5 minutes. Thoroughly mix the PEI MAX mixture and the plasmid mixture at a ratio of 1:1 to form a PEI-DNA mixture, and let it stand at room temperature for 20 minutes. Add 6ml of the PEI-DNA mixture to 60ml of DMEM medium containing 10% FBS, mix well to form a plasmid transfection solution. Remove the culture supernatant of 293T cells, replace it with 66ml of plasmid transfection solution, and incubate it at 37℃ and 5% CO2 for 18±2h; discard the culture supernatant, add 60ml of DMEM medium, and culture it at 37℃ and 5% CO2 for 24h;

[0160] (3) The collected culture supernatant was centrifuged at 500g*4min (room temperature), and the precipitate was discarded. The collected supernatant was centrifuged at high speed at 35000g*4h (4°C), and the supernatant was discarded. The harvested lentiviral vector was resuspended with an appropriate amount of DMEM culture medium and centrifuged. The collected virus precipitate was mixed evenly by pipetting, and then divided into EP tubes and stored at -80°C to obtain lentiviral vectors containing the TCR nucleic acid sequences of groups 1-7 and control groups 1-2, respectively.

[0161] Example 3 Preparation of T cells containing TCR nucleic acid sequences of the present invention

[0162] 1. PBMC recovery and T cell sorting and activation

[0163] (1) Isolation of PBMC: 50 ml of blood from an HLA*A1101 volunteer was collected and placed in a 250 ml centrifuge tube. Sodium chloride injection was added to dilute the blood at a volume ratio of 1:1, and the blood was centrifuged at 400 g for 30 min (18-20°C). The middle layer cells were collected and placed in a 250 ml centrifuge tube. The volume was adjusted to 200 ml with sodium chloride injection, and the blood was centrifuged at 400 g for 10 min. The supernatant was removed and the cells were resuspended with 200 ml of sodium chloride injection. The blood was centrifuged at 400 g for 10 min, and the supernatant was removed. After the cells were dispersed, the volume was adjusted to 50 ml with X-VIVO15 serum-free medium (Lonza) and mixed. After counting the cells, the PBMC cell suspension was centrifuged at 1500 rpm for 5 min. The supernatant was removed and the cells were resuspended with cell freezing medium. The cells were transferred to 4°C for pre-cooling and then frozen at -80°C.

[0164] (2) PBMC recovery: After PBMC recovery, place them in 30 mL of X-VIVO15 medium, centrifuge at 526 g for 5 min, discard the supernatant, and resuspend the cells in 12 mL of X-VIVO15 medium to adjust the cell density to 5 × 10 6 After adding TransAct (T cell activation reagent, from Miltenyi Biotec), the main components of which are CD3 and CD28 antibodies, according to the instructions, the cells were incubated at 37°C and 5% CO2 for 24 hours.

[0165] 2. Lentiviral Transduction of T Cells

[0166] The activated cells were removed and placed in a cell culture plate. They were divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (added with the lentiviral vector containing the TCR nucleic acid sequence of Group 1 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 and resuspended in 1 ml of X-VIVO15 medium containing 200 IU / mL IL-2. The cell density was adjusted to 5 × 10 5 / mL, incubate it at 37°C, 5% CO2 for 48 hours, and after elution, obtain T cells containing the TCR nucleic acid sequence of group 1.

[0167] Example 4 Preparation of T cells containing TCR nucleic acid sequences of the present invention

[0168] 1. Recovery of PBMC and sorting and activation of T cells (same as Example 3)

[0169] 2. Lentiviral transduction of T cells: The activated cells were removed and placed in a cell culture plate, and divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (adding the lentiviral vector containing the TCR nucleic acid sequence of Group 2 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 526g for 5 min and resuspended in 1 ml of X-VIVO15 medium containing 200 IU / mL IL-2. The cell density was adjusted to 5 × 10 5 / mL, incubate it at 37°C, 5% CO2 for 48 hours, and after elution, obtain T cells containing the TCR nucleic acid sequence of the second group.

[0170] Example 5 Preparation of T cells containing TCR nucleic acid sequences of the present invention

[0171] 1. Recovery of PBMC and sorting and activation of T cells (same as Example 3)

[0172] 2. Lentiviral transduction of T cells: The activated cells were removed and placed in a cell culture plate, and divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (added with the lentiviral vector containing the TCR nucleic acid sequence of Group 3 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 and resuspended in 1 ml of X-VIVO15 medium containing 200 IU / mL IL-2. The cell density was adjusted to 5 × 10 5 / mL, incubate it at 37°C, 5% CO2 for 48 hours, and after elution, obtain T cells containing the TCR nucleic acid sequence of the third group.

[0173] Example 6 Preparation of T cells containing TCR nucleic acid sequences of the present invention

[0174] 1. Recovery of PBMC and sorting and activation of T cells (same as Example 3)

[0175] 2. Lentiviral transduction of T cells: The activated cells were removed and placed in a cell culture plate. They were divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (adding the lentiviral vector containing 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 526g for 5 min and resuspended in 1 ml of X-VIVO15 medium containing 200 IU / mL IL-2. The cell density was adjusted to 5 × 10 5 / mL, incubate it at 37°C, 5% CO2 for 48 hours, and after elution, obtain T cells containing the TCR nucleic acid sequence of the fourth group.

[0176] Example 7 Preparation of T cells containing TCR nucleic acid sequences of the present invention

[0177] 1. Recovery of PBMC and sorting and activation of T cells (same as Example 3)

[0178] 2. Lentiviral transduction of T cells: The activated cells were removed and placed in a cell culture plate, and divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (adding the lentiviral vector containing 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 526g for 5 min and resuspended in 1 ml of X-VIVO15 medium containing 200 IU / mL IL-2. The cell density was adjusted to 5 × 10 5 / mL, incubate it at 37°C, 5% CO2 for 48 hours, and after elution, obtain T cells containing the TCR nucleic acid sequence of group 5.

[0179] Example 8 Preparation of T cells containing TCR nucleic acid sequences of the present invention

[0180] 1. Recovery of PBMC and sorting and activation of T cells (same as Example 3)

[0181] 2. Lentiviral transduction of T cells: The activated cells were removed and placed in a cell culture plate, and divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (adding the lentiviral vector containing the TCR nucleic acid sequence of Group 6 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 526g for 5 min and resuspended in 1 ml of X-VIVO15 medium containing 200 IU / mL IL-2. The cell density was adjusted to 5 × 10 5 / mL, incubate it at 37°C, 5% CO2 for 48 hours, and after elution, obtain T cells containing the TCR nucleic acid sequence of group 6.

[0182] Example 9 Preparation of T cells containing TCR nucleic acid sequences of the present invention

[0183] 1. Recovery of PBMC and sorting and activation of T cells (same as Example 3)

[0184] 2. Lentiviral transduction of T cells: The activated cells were removed and placed in a cell culture plate, and divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (adding the lentiviral vector containing the TCR nucleic acid sequence of Group 7 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 526g for 5 min and resuspended in 1 ml of X-VIVO15 medium containing 200 IU / mL IL-2. The cell density was adjusted to 5 × 10 5 / mL, incubate it at 37°C, 5% CO2 for 48 hours, and after elution, obtain T cells containing the TCR nucleic acid sequence of group 7.

[0185] Comparative Example 1 Preparation of T cells containing TCR nucleic acid sequences

[0186] 1. Recovery of PBMC and sorting and activation of T cells (same as Example 3)

[0187] 2. Lentiviral transduction of T cells: The activated cells were removed and placed in a cell culture plate, and divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (added with the lentiviral vector containing the TCR nucleic acid sequence of control group 1 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 and resuspended in 1 ml of X-VIVO15 medium containing 200 IU / mL IL-2. The cell density was adjusted to 5 × 10 5 / mL, incubate at 37°C, 5% CO2 for 48 hours, and after elution, obtain T cells containing the TCR nucleic acid sequence of control group 1.

[0188] Comparative Example 2 Preparation of T cells containing TCR nucleic acid sequences

[0189] 1. Recovery of PBMC and sorting and activation of T cells (same as Example 3)

[0190] 2. Lentiviral transduction of T cells: The activated cells were removed and placed in a cell culture plate, and divided into a non-transduced T cell group (NC, without lentivirus) and a transduced T cell group (added with the lentiviral vector containing the TCR nucleic acid sequence of control group 2 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 and resuspended in 1 ml of X-VIVO15 medium containing 200 IU / mL IL-2. The cell density was adjusted to 5 × 10 5 / mL, incubated at 37°C, 5% CO2 for 48 hours, and eluted to obtain T cells containing the TCR nucleic acid sequence of control group 2.

[0191] Test Example 1 Study on the effect of the T cell antigen receptor of the present invention on reducing TCR mismatch rate

[0192] The T cells prepared in Examples 3-5, 7-9 and the T cells prepared in Comparative Examples 1-2 were taken, and their Dextramer positive rates and Vβ positive rates were detected. Two parallel groups were set up for each group (one group was added with CD8 antibody and Dextramer reagent; the other group was added with CD8 antibody and Vβ7.1 antibody). The cell density was adjusted to 5×10 5 After the cells were grown to 500 μg / mL, they were placed at 37°C and 5% CO2 for 6-10 days.

[0193] Mix the sample, take the cell suspension and add it to the marked flow tube, add 2mL PBS (1X) to each tube, mix, centrifuge 526g*3min, discard the supernatant, shake to disperse the cells, incubate at room temperature for 15min, add 2mL PBS (1X) to each tube, mix, centrifuge 526g*3min, discard the supernatant, shake to disperse the cells, and add 50μL to each tube Resuspend the cells in PBS, create a new experiment, test on the machine, and collect 20,000 cells in the cell gate; count the Dextramer positivity rate (D%) and Vβ7.1 positivity rate (V%) in the CD8 gate respectively, and calculate the mismatch rate = 1-D% / V%, where 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). See the results. Figure 1 .

[0194] Test Example 2 Investigation of the cell killing rate of the T cell antigen receptor of the present invention

[0195] Target cells: HLA*1101 HERV-E positive cells were used as target cells to evaluate the function of TCR-T cells. Effector cells: TCR-T cells prepared in Examples 3-4, Examples 6-7, and Example 9 were selected.

[0196] The NC group was PBMC, and the extraction method was as follows: the blood of the volunteer (the volunteer's HLA restriction type was HLA*A1101) was transferred to a 250ml centrifuge tube, sodium chloride injection and blood were mixed at a ratio of at least 1:1, 15ml / tube was added to a 50ml centrifuge tube, and 30ml of diluted blood was added to each tube; centrifuged at 400g for 30min (18-20℃); the middle layer cells were collected in a 250ml centrifuge tube, sodium chloride injection was made up to 200ml, and mixed; centrifuged at 400g for 10min, the supernatant was removed, and sodium chloride injection was added. Resuspend the cells in 200 ml and mix well; centrifuge at 400 g for 10 min, remove the supernatant, disperse the cells and make up to 50 ml with X-VIVO15 serum-free medium (Lonza) and mix well; take 0.5 ml for cell number calculation, and centrifuge the remaining PBMC cell suspension at 1500 rpm for 5 min; remove the supernatant and resuspend the cells in cell freezing solution; quickly transfer the cryovial to a programmed cooling box pre-cooled at 4°C; store the programmed cooling box in a -80°C refrigerator overnight; remove the cells from the programmed cooling box the next day and transfer them to liquid nitrogen for storage.

[0197] In vitro killing assay: Target cells were plated at 6×10 3 The cells were plated and mixed at a ratio of 1:1 between effector cells and target cells, and incubated at 37°C for 4 h. The killing efficiency was obtained by Real Time Cell Analyzer (RTCA, Agilent).

[0198] HERV-E positive target cells with HLA subtype A*1101 (from T-CURE BIOSCIENCE, INC) were plated, with 6000 cells per well. The next day, effector cells were added according to a 1:1 ratio of CD8+DEX+ positive cell to target cell number for detection. Data was collected 47 hours after effector cell plating to evaluate the killing efficiency. The specific killing results of each group are shown in the figure below. Figure 2 shown.

[0199] Test Example 3 Evaluation of the efficacy of the specific T cells of the present invention in mice

[0200] 1. Experimental Materials

[0201] OS-RC-2-A11 tumor cells were revived and cultured in RPMI-1640 medium containing 10% fetal bovine serum in an incubator at 37°C with 5% CO2. Cells were passaged three times when the cell density reached 80% or higher. The total number of cells reached 6 × 10 7 Resuspend at 5×10 6 Cell suspension with a concentration of cells / ml was used, and the viability was 97%.

[0202] OS-RC-2-A11 tumor cells are tumor cells that overexpress the human histocompatibility complex HLA*A1101 protein. They were constructed by introducing a gene encoding the HLA*A1101 protein into OS-RC-2 tumor cells (human renal carcinoma cells, purchased from the Kyowa Cell Bank): Lentivirus containing the gene encoding the HLA*A1101 protein was used to transduce OS-RC-2 tumor cells. After one day, the medium was replaced with complete culture medium for 1-2 days, and then replaced with complete culture medium containing puromycin for continued culture. The expression of the HLA*A1101 protein in the transduced tumor cells was detected. The specific steps include:

[0203] 1) Lentiviral transduction of the gene encoding HLA*A1101 protein: The density of OS-RC-2 tumor cells was adjusted to 4-6×10 using complete culture medium (RPMI1640 medium (Gibco) containing 10% FBS). 5 cells / ml, 1 ml / well was added to a 6-well plate, 8 μl of Polybrene (1 mg / ml) was added to each well to a final concentration of 8 μg / ml, 10-30 μl of a lentiviral vector containing a gene encoding the HLA*A1101 protein was added to each well, and a cell control well (untransduced group) was set up. The control wells were added with 1 ml of the above-mentioned tumor cells OS-RC-2 and 8 μl of Polybrene, mixed, and incubated in a CO2 incubator (37°C, 5% CO2) for 1 day; the supernatant was discarded, and 2 ml of complete culture medium was added to each well, and the cells were incubated in a CO2 incubator for 2 days;

[0204] 2) Puromycin selection of transduced tumor cells: Discard the supernatant and add 2 ml of complete medium containing 1 μg / ml puromycin (Solarbio) to each well. Incubate in a CO2 incubator. Observe the cells every 2 days and replace the complete medium containing 1 μg / ml puromycin. Passage the cells when they are confluent. Select and culture them in complete medium containing 1 μg / ml puromycin for 7 days. If viable cells are still present in the control group, increase the puromycin concentration and continue culturing until all cells in the control group die.

[0205] 3) Monoclonal culture of cells encoding the gene for HLA*A1101 protein: Tumor cells transduced with the gene encoding HLA*A1101 protein and cultured for more than 7 days were plated using the limiting dilution method at 1 cell / well or 3 cells / well. The cells were cultured in complete medium containing puromycin and the cells in the wells were observed. The monoclonal wells were marked and cultured continuously until the cell count reached at least 6×10 7 Cells were frozen when the cells were above 400, and the overexpressed target cells OS-RC-2-A11 were obtained.

[0206] Twenty-five female mice aged 6-8 weeks (18-20 g) were collected, 5 per cage, and housed at 20-26°C, 30-70% humidity, 12 hours of light and 12 hours of darkness. Corn cob bedding was changed weekly and food and water were freely available. Inoculation: After removing the hair at the inoculation site, the inoculation site was disinfected with iodine cotton balls. 0.2 mL of OS-RC-2-A11 tumor cell suspension (containing 1×10 6 OS-RC-2-A11 tumor cells / mouse).

[0207] After inoculation, tumor volume and body weight were measured once a week. When the tumor volume reached 70-100 mm 3 25 animals were randomly divided into 5 groups according to tumor volume and body weight, with 5 animals in each group. The drugs were reinfused into the tail vein according to Table 2. I L2 was injected intraperitoneally on the same day, 24 hours, and 48 hours after reinfusion. The injection volume of I L2 was 200,000 IU / 200 μl / animal / time.

[0208] The start date of dosing was considered PG-Day 0. After dosing began, body weight and tumor volume were measured twice weekly. After dosing ended, tumor growth trends and body weight were continuously observed. Animals were euthanized without tissue collection.

[0209] Table 2 Grouping and dosing information

[0210]

[0211] After inoculation, the animals are observed daily for morbidity and mortality, including tumor growth and the effects of drugs on experimental animals, such as changes in activity, changes in food and water intake, weight loss, changes in hair and eye appearance, death and other clinical symptoms. From inoculation to grouping, the body weight of experimental animals is measured once a week. After grouping, the body weight of experimental animals is measured twice a week, or the frequency of mouse weight measurement can be changed according to customer requirements. From inoculation to grouping, when the tumor is visible, the tumor volume of experimental animals is measured once a week, and after inoculation and grouping, the tumor volume of experimental animals is measured twice a week. Tumor volume is measured using a two-way measurement method, and then the tumor volume (TV) is calculated as 0.5*a*b 2 , where a is the long diameter of the tumor and b is the short diameter of the tumor. All statistical data were analyzed using SPSS 24.0 software using one-way analysis of variance (ANOVA). The LSD method was used for analysis; a p-value less than 0.05 was considered significant, and a p-value less than 0.01 was considered extremely significant.

[0212] Mice were observed for adverse conditions, weight loss (weight loss greater than 10%), and mortality. Arched backs were first observed in G1 and G3 mice on PG-Day 13, followed by G2 on PG-Day 17. By PG-Day 20, nearly all mice in G1, G2, and G3 exhibited arched backs with piloerection. Mortality subsequently occurred, and the sample size of each group gradually decreased. Therefore, PG-Day 20 was used as the endpoint for drug efficacy analysis.

[0213] Observe the changes in tumor volume in mice. Figure 3 On PG-Day 20, there was no significant difference between the G2 group and the control group G1 (p>0.05), and there was a very significant difference between the G3 group and the control group G1 (p<0.01). On PG-Day 59, all five mice in the G3 group had tumor recurrence. There were very significant differences between the G4 and G5 groups and the control group G1 (p<0.01). On PG-Day 59, the tumor volume was 0 mm 3 . And there is no significant weight loss, and the safety is good ( Figure 4 ).

[0214] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of protection of the claims of the present invention.

Claims

1. A T cell antigen receptor, comprising an α chain and a β chain, wherein the amino acid sequence of the constant region of the α chain of the T cell antigen receptor comprises SEQ ID NO: 8; and the amino acid sequence of the constant region of the β chain of the T cell antigen receptor comprises SEQ ID NO: 9; or, wherein the amino acid sequence of the constant region of the α chain of the T cell antigen receptor comprises SEQ ID NO: 16; and the amino acid sequence of the constant region of the β chain of the T cell antigen receptor comprises the amino acid sequence shown in SEQ ID NO:

17.

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

19.

3. The T cell antigen receptor according to claim 1, wherein the variable region of the α chain comprises any one of α-CDR1 and α-CDR2 or a combination thereof, wherein: The amino acid sequence of the α-CDR1 is SEQ ID NO: 20, and the amino acid sequence of the α-CDR2 is SEQ ID NO:

21.

4. The T cell antigen receptor according to claim 1, wherein the variable region of the β chain comprises any one of β-CDR1 and β-CDR2 or a combination thereof, wherein: The amino acid sequence of the β-CDR1 is SEQ ID NO: 22, and the amino acid sequence of the β-CDR2 is SEQ ID NO:

23. 5 . The T cell antigen receptor according to claim 1 , wherein the amino acid sequence of the variable region of the α chain is shown in SEQ ID NO: 24; and the amino acid sequence of the variable region of the β chain is shown in SEQ ID NO:

25. 6 . The T cell antigen receptor according to claim 1 , wherein the amino acids of the α chain and the β chain are linked by a linker sequence as shown in SEQ ID NO:

28.

7. The T cell antigen receptor according to claim 1, wherein the amino acid sequence comprised by the T cell antigen receptor is selected from any one of SEQ ID NO: 30, SEQ ID NO: 35, or a combination thereof.

8. A nucleic acid comprising a nucleotide sequence encoding the TCR according to any one of claims 1 to 7 or a complementary sequence thereof.

9. The nucleic acid according to claim 8, wherein the nucleotide sequence or its complementary sequence is selected from any one of single-stranded, double-stranded, DNA, and RNA, or a combination thereof.

10. The nucleic acid according to claim 9, wherein the nucleotide sequence or its complementary sequence is codon optimized.

11. The nucleic acid according to claim 10, wherein the codon optimization comprises changing a large number of rare codons used by viruses to corresponding mammalian codons, removing mRNA unstable motifs, and any one or a combination thereof.

12. The nucleic acid according to any one of claims 8 to 11, wherein the nucleic acid sequence is selected from any one of SEQ ID NO: 37, SEQ ID NO: 42 or a combination thereof.

13. An expression vector comprising the nucleic acid according to any one of claims 8 to 12. The expression vector according to claim 13 , wherein the expression vector can be expressed under any conditions in vivo, in vitro or in vitro. The expression vector according to claim 13 , wherein the expression vector is continuously expressed at a high level in cells in vivo. The expression vector according to claim 13 , wherein the expression vector is selected from any one of a prokaryotic expression vector and a retroviral vector.

17. The expression vector according to any one of claims 13 to 16, wherein 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 myeloproliferative virus 29 (MC29), and avian erythroblastosis virus (AEV), or a combination thereof.

18. A host cell, comprising the nucleic acid according to any one of claims 8 to 12 or the expression vector according to any one of claims 13 to 17. The host cell according to claim 18 , wherein the host cell is selected from any one of a eukaryotic cell and a prokaryotic cell.

20. The host cell according to claim 19, wherein the eukaryotic cell is selected from any one of yeast cells, 293 cells, and CHO cells, or a combination thereof.

21. An immune cell, wherein the immune cell expresses the T cell antigen receptor according to any one of claims 1 to 7.

22. The immune cell according to claim 21, comprising one or more nucleic acid sequences according to any one of claims 9 to 12.

23. The immune cell according to claim 21, wherein the immune cell is selected from any one of stem cells, lymphocytes, T cells, B cells, and NK cells. The immune cell according to claim 23 , wherein the T cell antigen receptor structure of the T cell is as defined above.

25. The immune cell according to claim 24, wherein the T cell is selected from CD4 + T, CD8 + Any one or combination of T.

26. The immune cell according to any one of claims 21 to 25, wherein the immune cell is isolated from autologous T cells or allogeneic T cells.

27. A method for preparing immune cells, comprising transducing the nucleic acid sequence according to any one of claims 8 to 12 into immune cells for expression.

28. The preparation method according to claim 27, wherein the immune cells are selected from any one of stem cells, lymphocytes, T cells, B cells, and NK cells. The preparation method according to claim 28 , wherein the T cell antigen receptor structure of the T cell is as defined above.

30. The preparation method according to claim 28, wherein the T cells are selected from CD4 + T, CD8 + Any one or combination of T.

31. The preparation method according to claim 28, wherein the immune cells are separated from autologous T cells or allogeneic T cells.

32. The preparation method according to any one of claims 27 to 31, further comprising the step of knocking out endogenous TCR of the cell.

33. The preparation method according to claim 32, wherein the step of knocking out the endogenous TCR of the cell is to construct a guide RNA targeting the endogenous TCR into a lentiviral vector, which is then co-transfected into T cells together with a packaging plasmid and a transfection reagent.

34. A method for preparing recombinant T cells, comprising the following steps: 1) obtaining the nucleic acid according to any one of claims 8 to 12 from a positive T cell clone; 2) Isolation and culture of T cells; 3) delivering the nucleic acid obtained in step 1) into the primary T cells described in step 2) to obtain recombinant T cells expressing the T cell antigen receptor according to any one of claims 13 to 17. The preparation method according to claim 34 , wherein the T cells are selected from any one of hematopoietic stem cells and peripheral blood lymphocyte-derived T cells.

36. A method for preparing a T cell antigen receptor, comprising the following steps: (1) obtaining the nucleic acid according to any one of claims 8 to 12 from a positive T cell clone; (2) connecting the nucleic acid obtained in step (1) to a vector backbone to obtain an expression vector; (3) transforming the expression vector obtained in step (2) into a host cell and inducing its expression; (4) Obtaining antibodies or antigen-binding fragments thereof or T cell antigen receptors. The preparation method according to claim 36 , wherein the positive T cells specifically bind to the antigen peptide.

38. A pharmaceutical composition comprising any one of 1) to 5) or a combination thereof, 1) The T cell antigen receptor according to any one of claims 1 to 7; 2) The nucleic acid according to any one of claims 8 to 12; 3) The expression vector according to any one of claims 13 to 17; 4) The host cell according to any one of claims 18 to 20; or 5) The immune cell according to any one of claims 21 to 26.

39. The pharmaceutical composition according to claim 38, further comprising a pharmaceutically acceptable carrier.

40. The pharmaceutical composition of claim 38, optionally in combination with other therapeutic agents.

41. The pharmaceutical composition according to claim 40, wherein the other therapeutic agent is an immunomodulator.

42. Use of the T cell antigen receptor according to any one of claims 1-7, the nucleic acid according to any one of claims 8-12, the expression vector according to any one of claims 13-17, the host cell according to any one of claims 18-20, the immune cell according to any one of claims 21-26, and the pharmaceutical composition according to any one of claims 38-41 for preparing a product for diagnosing, preventing and / or treating a tumor, wherein the tumor is renal cancer.

43. A kit comprising any one or a combination of 1) to 5): 1) The T cell antigen receptor according to any one of claims 1 to 7; 2) The nucleic acid according to any one of claims 8 to 12; 3) The expression vector according to any one of claims 13 to 17; 4) The host cell according to any one of claims 18 to 20; 5) The immune cell according to any one of claims 21 to 26.

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