CN118620059A - A T cell antigen receptor and its preparation method and application - Google Patents

Abstract

The present 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 of (a)-(b) or a combination thereof, (a) the constant region comprises 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 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

A T cell antigen receptor and its preparation method and application

Technical Field

The present invention belongs to the field of biomedicine, and specifically relates to a T cell antigen receptor, a preparation method thereof and an application thereof.

Background Art

T cell receptor (TCR) is a molecule that T lymphocytes specifically recognize antigens and initiate immune responses. It is a heterodimeric cell surface protein of the immunoglobulin superfamily, which is associated with the non-variant 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 crucial to the cellular immune function of the immune system. The binding of antigen-specific TCR to the MHC complex triggers direct physical contact and interaction between T cells and antigen-presenting cells, leading to subsequent cell signaling and other physiological reactions, so that T cells with different antigen specificities exert immune effects on their target cells.

The correct assembly of TCR is the key to the generation of TCR-T cells. TCR mismatching can lead to problems such as cross-reactive toxicity and become a technical difficulty in TCR-T cell therapy. In order to reduce the mismatch rate, mouse amino acids are introduced, which may lead to excessive immunogenicity and affect drugability. For this reason, it is necessary to develop a TCR modification program with good biological activity functions to effectively reduce the TCR mismatch rate and immunogenicity.

Summary of the invention

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),

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

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

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

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

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

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).

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.

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).

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

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

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;

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

DLNKVFPPEVAVFEPS X5 AEI X6 HTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCG 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.

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

(1) The amino acid sequence of the constant region of the α chain 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:

IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS;

The amino acid sequence of the constant region of the β 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:

DLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVCTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYQQGVLSATILYEILLGKATLYAVLVSTLVVMAMVKRKNS;

(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 with SEQ ID NO: 8, wherein the SEQ ID NO: 8 is:

IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSSDVPCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS;

The amino acid sequence of the constant region of the β 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:

DLNKVFPPEVAVFEPSKAEIAHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF;

(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 with SEQ ID NO: 10, wherein the SEQ ID NO: 10 is:

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 with SEQ ID NO: 11, wherein the SEQ ID NO: 11 is:

DLNKVFPPEVAVFEPSKAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF;

(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 with SEQ ID NO: 12, wherein the SEQ ID NO: 12 is:

IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKCVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS;

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

(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 with SEQ ID NO: 14, wherein the SEQ ID NO: 14 is:

IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSSPGGRIARLEEKVKTLKAQNSELASTANMLREQVAQLKQKVMNY;

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 with SEQ ID NO: 15, wherein SEQ ID NO: 15 is:

DLNKVFPPEVAVFEPSKAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFPGGLTDTLQAETDQLEDKKSALQTEIANLLKE KEKLEFILAAY;

(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 with SEQ ID NO: 16, wherein the SEQ ID NO: 16 is:

IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSSAQCKKKLQALKKKNAQLKWKLQALKKKLAQ;

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 with SEQ ID NO: 17, wherein SEQ ID NO: 17 is:

DLNKVFPPEVAVFEPSKAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAAWGRADCGFTSVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDFAQCEKELQALEKENAQLEWELQALEKELA Q.

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),

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).

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).

In a preferred technical solution of the present invention, the amino acid sequence of the variable region of the α chain is as shown in SEQ ID NO: 24, and the SEQ ID NO: 24 is:

IQVEQSPPDLI LQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVATERYSLLY ISSSQTTDSGVYFCAVRGGADGLTFGKGTHLI IQPY;

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

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 with SEQ ID NO: 26, and the SEQ ID NO: 26 is:

IQVEQSPPDLI LQEGANSTLRCNFSDSVNNLQWFHQNPWGQLINLFYIPSGTKQNGRLSATTVATERYSLLY ISSSQ L TDSGVYFCAVRGGADGLTFGKGTHLI IQPY;

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 the SEQ ID NO: 27 is:

EVTQTPRYLVMGMTNKKSLKCEQHMGHRAMYWYKQKAKKPPELMFVYSYEKLSINESVPSRFSPECPNSSLL NLHLHALQPEDSALYLCASSPPNEKLFFGSGTQLSVLE;

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%.

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.

In a preferred technical scheme of the present invention, the amino acid sequence contained in 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 contains 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.

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.

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, RNA or a combination thereof.

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

In the 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 any one of unstable mRNA motifs and hidden splicing sites or a combination thereof.

In a preferred technical scheme 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.

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

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

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

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.

In a preferred technical scheme of the present invention, the expression vector is selected from Rous sarcoma virus (RSV), lentivirus, human immunodeficiency virus (HIV), murine leukemia virus (MLV), equine infectious anemia virus (EIAV), mouse mammary cancer 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 myeloerythroblastosis virus (AEV), any one or a combination thereof.

Another object of the present invention is to provide a host cell, wherein the host cell comprises any nucleic acid or expression vector described in the present invention.

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.

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.

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.

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.

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.

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

In a preferred technical solution of the present invention, the T cells are selected from any one of CD4 ⁺ T cells and CD8 ⁺ T cells or a combination thereof.

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

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.

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.

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

In a preferred technical solution of the present invention, the T cells are selected from any one of CD4 ⁺ T cells and CD8 ⁺ T cells or a combination thereof.

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

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

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

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

1) Obtaining any nucleic acid of the present invention 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 any T cell antigen receptor described in the present invention,

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.

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

(1) obtaining any nucleic acid described in the present invention 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.

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

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

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

2) the nucleic acid of the present invention;

3) the expression vector of the present invention;

4) the host cell of the present invention; or

5) The immune cells of the present invention.

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

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

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

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

In the preferred technical scheme 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 carcinoma, 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.

In the preferred technical scheme 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.

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.

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 its complications.

In a preferred technical scheme 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.

Another object of the present invention is to provide a kit, which comprises any one or a combination of 1)-5):

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

2) the nucleic acid of the present invention;

3) the expression vector of the present invention;

4) The host cell of the present invention;

5) The immune cells of the present invention.

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 described in the present invention.

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.

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.

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.

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.

In the preferred technical scheme 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 carcinoma, 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.

In the preferred technical scheme 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.

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.

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 its complications.

In a preferred technical scheme 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.

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.

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

In the preferred technical scheme 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 carcinoma, 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.

In the preferred technical scheme 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.

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.

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 its complications.

In a preferred technical scheme 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.

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

Table 1

Unless otherwise specified, the terms of 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.

The "T cell antigen receptor" of the present invention is a molecule that can recognize a peptide when presented by an MHC molecule or its tetramer, and is usually present on the surface of a T cell in the form of a complex with a CD3 molecule. The TCR of most T cells is composed of α and β peptide chains, and the TCR of a few T cells is composed of γ and δ peptide chains.

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.

The term "prevention" as used in the present invention refers to any action to suppress symptoms or delay the occurrence of specific symptoms by administering the product of the present invention.

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

The term "treatment" as used herein means to slow down, interrupt, prevent, control, stop, alleviate, reverse the progression or severity of a sign, symptom, disorder, symptom, or disease, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, symptoms, 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.

The "effective amount" described in 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.

The "product" described in the present invention 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 pharmaceutical compositions such as reagents, kits, chips, antibody conjugates or multifunctional antibodies that assist or cooperate with the above products.

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

The "homology" mentioned in the present invention means that the technicians in this field 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%.

The "paired use" described in the present invention refers to the interaction of proteins through their leucine zipper domains to form a stable dimer.

The real-time label-free dynamic cell analysis technology of the present invention integrates the microelectronic cell sensor chip into the bottom of the cell detection plate to construct a cell impedance detection sensing system that can track changes in cell morphology, proliferation and differentiation in real time, dynamically and quantitatively. When cells attached to the surface of the microelectrode cause changes in the impedance of the attached electrode interface, the change is correlated with the real-time functional state of the cell. Through real-time dynamic electrode impedance detection, biological information related to the physiological function of the cell can be obtained, including cell growth, extension, morphological changes, death and attachment.

Compared with the prior art, the present invention has the following beneficial technical effects: the T cell antigen receptor obtained by modifying the TCR constant region of the present invention significantly reduces the mismatch 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 reducing its immunogenicity and improving its biological activity. The TCR-T cell of the present invention maintains effective anti-tumor activity while reducing the mismatch rate, can specifically recognize and kill target cells expressing the corresponding antigen, and has broad application prospects in immunotherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a result of detecting the mismatch rate of T cell antigen receptors according to the present invention;

FIG2 shows the killing effect of the T cell antigen receptor of the present invention on target cells;

FIG3 Effect of the T cell antigen receptor of the present invention on the change of tumor volume in experimental animals;

FIG. 4 shows the effect of the T cell antigen receptor of the present invention on the weight change of experimental animals.

DETAILED DESCRIPTION

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.

The plasmid 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.

Example 1 Construction of the recombinant lentiviral plasmid of the present invention

According to the relevant contents 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.

XbaI and XhoI were used to cut the TCR nucleic acid sequences of groups 1 to 7 and control groups 1-2 from the synthetic cloning vector pUC57 (purchased from Jiangsu GenScript Biotechnology Co., Ltd.), the target fragments were cut and recovered, and then connected to the lentiviral plasmids cut by XbaI and XhoI with T4 DNA ligase to transform competent cells. The next day, a single clone was picked and cultured in LB liquid medium containing antibiotics for 12-16h, the plasmid was extracted, the concentration and purity of the plasmid were detected, the correct clones were identified by enzyme cutting, and the correct plasmids were identified by DNA sequencing to obtain the shuttle plasmid pPVLV4_HERV-E containing the TCR nucleic acid sequences of groups 1-7 and control groups 1-2, respectively.

Example 2 Production of the Lentivirus Vector of the Present Invention

The preparation of lentiviral vector comprises the following steps:

(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 ;

(2) The shuttle plasmid pPVLV4_HERV-E: pMDLg/pRRE(K): pRSV-Rev(K): pMD.G(K) prepared in Example 1 was mixed at a mass ratio of 4:2:2:1, and then added to 3 ml of DMEM medium, and allowed to stand at room temperature for 5 min. 175.5 μl of 1 mg/ml PEI MAX solution and 3 ml of DMEM medium were added to each 225 ^cm2 cell culture flask, and allowed to stand at room temperature for 5 min. The PEI MAX mixed solution and the plasmid mixed solution were fully mixed at a ratio of 1:1 to form a PEI-DNA mixed solution, and allowed to stand at room temperature for 20 min. 6 ml of the PEI-DNA mixed solution was added to 60 ml of DMEM medium containing 10% FBS, and mixed evenly to form a plasmid transfection solution. Remove the culture supernatant of 293T cells, replace with 66 ml of plasmid transfection solution, and incubate at 37°C, 5% CO ₂ for 18±2 h; discard the culture supernatant, add 60 ml of DMEM medium, and incubate at 37°C, 5% CO ₂ for 24 h;

(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 dispensed 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.

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

1. PBMC recovery and T cell sorting and activation

(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 (1500 rpm for 5 min). The supernatant was removed and the cells were resuspended with cell freezing solution. The cells were transferred to 4°C for precooling and then frozen at -80°C.

(2) PBMC recovery: After the cryopreserved PBMCs were recovered, they were placed in 30 mL of X-VIVO15 medium and centrifuged at 526 g for 5 min. The supernatant was discarded and the cells were resuspended in 12 mL of X-VIVO15 medium. The cell density was adjusted to 5 × 10 ⁶ cells/ml. TransAct (T cell activation reagent, from Miltenyi Biotec), the main components of which were CD3 and CD28 antibodies, was added according to the instructions and the cells were incubated at 37°C, 5% CO2 for 24 h.

2. Lentiviral transduction of 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 (adding the lentiviral vector containing the TCR nucleic acid sequence of Group 1 prepared in Example 2), and cultured at 37°C, 5% _CO2 for 24 hours to obtain lentiviral transduced T cells, centrifuged at 526g*5min, resuspended with 1ml of X-VIVO15 medium containing 200IU/mL IL-2, and the cell density was adjusted to 5× ¹⁰⁵ /mL. It was placed at 37°C, 5% _CO2 and incubated for 48h. After elution, T cells containing the TCR nucleic acid sequence of Group 1 were obtained.

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

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

2. Lentivirus transduction of 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 (adding the lentiviral vector containing the TCR nucleic acid sequence of the second group prepared in Example 2), and cultured at 37°C, 5% _CO2 for 24 hours to obtain lentivirus-transduced T cells, centrifuged at 526g*5min, resuspended with 1ml of X-VIVO15 culture medium containing 200IU/mL IL-2, and the cell density was adjusted to 5× ¹⁰⁵ /mL. It was placed at 37°C, 5% _CO2 and incubated for 48h. After elution, T cells containing the TCR nucleic acid sequence of the second group were obtained.

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

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

2. Lentivirus transduction of 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 (adding the lentiviral vector containing the TCR nucleic acid sequence of the third group prepared in Example 2), and cultured at 37°C, 5% _CO2 for 24 hours to obtain lentivirus-transduced T cells, centrifuged at 526g*5min, resuspended with 1ml of X-VIVO15 culture medium containing 200IU/mL IL-2, and the cell density was adjusted to 5× ¹⁰⁵ /mL. It was placed at 37°C, 5% _CO2 and incubated for 48h. After elution, T cells containing the TCR nucleic acid sequence of the third group were obtained.

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

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

2. Lentiviral transduction of 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 (adding the lentiviral vector containing the TCR nucleic acid sequence of the fourth group prepared in Example 2), and cultured at 37°C, 5% _CO2 for 24 hours to obtain lentiviral transduced T cells, centrifuged at 526g*5min, resuspended with 1ml of X-VIVO15 culture medium containing 200IU/mL IL-2, and the cell density was adjusted to 5× ¹⁰⁵ /mL. It was placed at 37°C, 5% _CO2 and incubated for 48h. After elution, T cells containing the TCR nucleic acid sequence of the fourth group were obtained.

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

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

2. Lentivirus transduction of 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 (adding the lentiviral vector containing the TCR nucleic acid sequence of Group 5 prepared in Example 2), and cultured at 37°C, 5% _CO2 for 24 hours to obtain lentivirus-transduced T cells, centrifuged at 526g*5min, resuspended with 1ml of X-VIVO15 culture medium containing 200IU/mL IL-2, and the cell density was adjusted to 5× ¹⁰⁵ /mL. It was placed at 37°C, 5% _CO2 and incubated for 48h. After elution, T cells containing the TCR nucleic acid sequence of Group 5 were obtained.

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

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

2. Lentivirus transduction of 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 (adding the lentiviral vector containing the TCR nucleic acid sequence of Group 6 prepared in Example 2), and cultured at 37°C, 5% _CO2 for 24 hours to obtain lentivirus-transduced T cells, centrifuged at 526g*5min, resuspended with 1ml of X-VIVO15 culture medium containing 200IU/mL IL-2, and the cell density was adjusted to 5× ¹⁰⁵ /mL. It was placed at 37°C, 5% _CO2 and incubated for 48h. After elution, T cells containing the TCR nucleic acid sequence of Group 6 were obtained.

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

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

2. Lentivirus transduction of 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 (adding the lentiviral vector containing the TCR nucleic acid sequence of Group 7 prepared in Example 2), and cultured at 37°C, 5% _CO2 for 24 hours to obtain lentivirus-transduced T cells, centrifuged at 526g*5min, resuspended with 1ml of X-VIVO15 culture medium containing 200IU/mL IL-2, and the cell density was adjusted to 5× ¹⁰⁵ /mL. It was placed at 37°C, 5% _CO2 and incubated for 48h. After elution, T cells containing the TCR nucleic acid sequence of Group 7 were obtained.

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

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

2. Lentivirus transduction of 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 (adding the lentiviral vector containing the TCR nucleic acid sequence of the control group 1 prepared in Example 2), and cultured at 37°C, 5% _CO2 for 24 hours to obtain lentivirus-transduced T cells, centrifuged at 526g*5min, and resuspended with 1ml of X-VIVO15 culture medium containing 200IU/mL IL-2, adjusting the cell density to 5× ¹⁰⁵ /mL, and incubated at 37°C, 5% _CO2 for 48 hours. After elution, T cells containing the TCR nucleic acid sequence of the control group 1 were obtained.

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

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

2. Lentivirus transduction of 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 (adding the lentiviral vector containing the TCR nucleic acid sequence of the control group 2 prepared in Example 2), and cultured at 37°C, 5% _CO2 for 24 hours to obtain lentivirus-transduced T cells, centrifuged at 526g*5min, and resuspended with 1ml of X-VIVO15 culture medium containing 200IU/mL IL-2, adjusting the cell density to 5× ¹⁰⁵ /mL, and incubated at 37°C, 5% _CO2 for 48 hours. After elution, T cells containing the TCR nucleic acid sequence of the control group 2 were obtained.

Experimental Example 1 Study on the effect of the T cell antigen receptor of the present invention on reducing TCR mismatch rate

The T cells prepared in Examples 3-5 and 7-9 and the T cells prepared in Comparative Examples 1-2 were taken, and their Dextramer positivity and Vβ positivity were detected. Two parallels were set up in each group (one with CD8 antibody and Dextramer reagent; the other with CD8 antibody and Vβ7.1 antibody), and the cell density was adjusted to 5×10 ⁵ cells/mL with X-VIVO15 medium containing 200 IU/mL IL-2, and then placed at 37°C, 5% CO ₂ for 6-10 d.

Mix the sample, take the cell suspension and add it to the marked flow tube, add 2mL PBS (1X) to each tube, mix, centrifuge at 526g*3min, discard the supernatant, shake and disperse the cells, incubate at room temperature for 15min, add 2mL PBS (1X) to each tube, mix, centrifuge at 526g*3min, discard the supernatant, shake and disperse the cells, add 50μL Resuspend cells in PBS, create a new experiment, test 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 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). The results are shown in Figure 1.

Experimental Example 2 Investigation of the cell killing rate of the T cell antigen receptor of the present invention

Target cells: HERV-E positive cells of 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, Examples 6-7, and Example 9 were selected.

The NC group was PBMC, and the extraction method was as follows: the blood of the volunteer (the HLA restriction type of the volunteer 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 aspirated, and sodium chloride injection was added. Resuspend the cells in 200ml and mix well; centrifuge at 400g for 10min, remove the supernatant, disperse the cells and make up to 50ml with X-VIVO15 serum-free medium (Lonza), mix well; take 0.5ml for cell number calculation, and centrifuge the remaining PBMC cell suspension at 1500rpm for 5min; remove the supernatant and resuspend the cells with cell freezing solution; quickly transfer the cryopreservation tube to a programmed cooling box precooled at 4℃; place the programmed cooling box in a -80℃ refrigerator overnight; take out the cells from the programmed cooling box the next day and transfer them to liquid nitrogen for storage.

In vitro killing experiment: 6×10 ³ target cells were plated per well, and the effector cells and target cells were mixed at a ratio of 1:1. They were incubated at 37°C for 4 h. Real Time Cell Analyzer (RTCA, Agilent) was used to obtain the killing efficiency.

HERV-E positive target cells with HLA subtype A*1101 (from T-CURE BIOSCIENCE, INC) were used for plating, with 6000 cells per well. On the second day, effector cells were added according to the ratio of CD8+DEX+ positive cells to target cells at 1:1, and the killing efficiency was evaluated by collecting data 47 hours after plating the effector cells. The specific killing results of each group are shown in Figure 2.

Experimental Example 3 Evaluation of the efficacy of the specific T cells of the present invention in mice

1. Experimental Materials

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 containing 5% CO2. When the cell density reached more than 80%, the cells were passaged for 3 times. When the total number of cells reached 6× ¹⁰⁷ , the cells were resuspended in a cell suspension with a concentration of 5× ¹⁰⁶ cells/ml, and the viability was 97%.

OS-RC-2-A11 tumor cells are tumor cells that overexpress the human histocompatibility complex HLA*A1101 protein. They are constructed by introducing genes encoding HLA*A1101 proteins into tumor cells OS-RC-2 (human renal cancer cells, purchased from the Concord Cell Bank): tumor cells OS-RC-2 are transduced with lentivirus containing genes encoding HLA*A1101 proteins, and the medium is replaced with complete medium for 1-2 days after 1 day, and then replaced with complete medium containing puromycin for continued culture, and the expression of HLA*A1101 proteins in the transduced group tumor cells is detected. Specifically, the following steps are included:

1) Lentiviral transduction of genes encoding HLA*A1101 protein: Use complete culture medium (RPMI1640 culture medium (Gibco) containing 10% FBS) to adjust the density of tumor cells OS-RC-2 to 4-6×10 ⁵ cells/ml, add 1 ml/well to a 6-well plate, add 8 μl Polybrene (1 mg/ml) to each well to a final concentration of 8 μg/ml, add 10-30 μl of lentiviral vector containing genes encoding HLA*A1101 protein to each well, set up cell control wells (non-transduced group), and add the control wells with the above-mentioned 1 ml tumor cells OS-RC-2 and 8 μl Polybrene, mix well, and incubate in a CO2 incubator (37°C, 5% CO2) for 1 day; discard the supernatant, add 2 ml of complete culture medium to each well, and incubate in a CO2 incubator for 2 days;

2) Puromycin selection of transduced tumor cells: discard the supernatant, add 2 ml of complete medium containing 1 μg/ml puromycin (Solarbio) to each well, and 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 fully grown; use the complete medium containing 1 μg/ml puromycin for selection and culture for 7 days. If there are still living cells in the control group, increase the puromycin concentration and continue culturing until all cells in the control group die;

3) Monoclonal culture of cells containing the gene encoding HLA*A1101 protein: Tumor cells transduced with the gene encoding HLA*A1101 protein and cultured for more than 7 days were plated according to the limiting dilution method, 1 cell/well, 3 cells/well, cultured in complete medium containing puromycin and the cells in the cell wells were observed, and monoclonal wells were marked and cultured continuously until the cell number reached at least 6×10 ⁷ cells and then frozen to obtain overexpression target cells OS-RC-2-A11.

Take 25 female mice aged 6-8 weeks (18-20g), 5 mice per cage, 20-26℃, 30-70%, 12h lighting and 12h darkness, corn cob bedding, change once a week, free access to food and water. Inoculation: After removing the hair at the inoculation site, disinfect the inoculation site of the mouse with iodine cotton balls, and use a 1mL syringe to subcutaneously inoculate 0.2mL of OS-RC-2-A11 tumor cell suspension (containing 1×10 ⁶ OS-RC-2-A11 tumor cells/mouse) on the right shoulder of the mouse.

After inoculation, the 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 the tumor volume and body weight, with 5 animals in each group. The drugs were reinfused through the tail vein according to Table 2. I L2 was injected intraperitoneally on the same day, 24h and 48h after reinfusion. The injection volume of I L2 was 200,000 IU/200μl/animal/time.

The start date of drug administration was considered as PG-Day 0. After the start of drug administration, the body weight and tumor volume of mice were measured twice a week. After the end of drug administration, the tumor growth trend and mouse body weight were continuously observed, and the experimental animals were directly euthanized without tissue collection.

Table 2 Grouping and dosing information

After inoculation, the animals were observed for morbidity and mortality daily, including tumor growth and the effects of drugs on experimental animals, such as changes in activity, changes in food and water intake, emaciation, changes in hair and eye appearance, death and other clinical symptoms. The weight of experimental animals was measured once a week from inoculation to grouping. The weight of experimental animals was measured twice a week after grouping, or the frequency of measuring mouse weight was changed according to customer requirements. From inoculation to grouping, when the tumor was visible, the tumor volume of experimental animals was measured once a week, and the tumor volume of experimental animals was measured twice a week after inoculation and grouping. The tumor volume was measured by two-way measurement method, and then the tumor volume (TV) = 0.5*a*b ² was calculated, where a is the long diameter of the tumor and b is the short diameter of the tumor. All data statistics were analyzed using SPSS24.0 software and one-way ANOVA method. The LSD method was used for analysis, and a p value less than 0.05 was considered to be significantly different, and a p value less than 0.01 was considered to be extremely significantly different.

The mice were observed for adverse conditions, body weight (weight loss greater than 10%), and death. On PG-Day 13, G1 and G3 were first observed to have hunched backs, and on PG-Day 17, G2 also had hunched backs. On PG-Day 20, almost all G1, G2, and G3 showed hunched backs with symptoms of erect hair, and deaths occurred one after another. The sample size of each group gradually decreased, so PG-Day 20 was used as the drug efficacy statistical node.

The changes in the tumor volume of mice were observed, and the results are shown in Figure 3. At PG-Day 20 of the experiment, 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). At PG-Day 59, all 5 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); at PG-Day 59, the tumor volume was 0 mm ^3. There was no significant decrease in body weight, and the safety was good (Figure 4).

The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may 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 all fall within the scope of protection of the claims of the present invention.

Claims (20)

1. A T cell antigen receptor comprising an alpha chain and/or a beta chain, said alpha chain comprising any one of (a) - (b) or a combination thereof,

(A) The constant region comprises the sequence shown in SEQ ID NO:1 (LVIVL);

(b) The constant region comprises one or more cysteine residues capable of forming a non-native disulfide bond with the β chain;

the constant region of the β chain comprises one or more cysteine residues capable of forming a non-native disulfide bond with the α chain;

preferably, the amino acid sequence of the constant region of the T cell antigen receptor is completely replaced by amino acids of murine origin;

preferably, the amino acid sequence of the constant region of the T cell antigen receptor is substituted with a murine amino acid at least at one position, more preferably the amino acid sequence of the constant region of the T cell antigen receptor is substituted with 1 or 9 murine amino acids.

2. The T cell antigen receptor of claim 1, the constant regions of the alpha and beta chains further comprising at least one leucine zipper, preferably the leucine zipper is selected from the group consisting of SEQ ID NOs: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO:5, and any one of the following; more preferably, the polypeptide comprises SEQ ID NO:2 and a leucine zipper comprising the sequence of SEQ ID NO:3, comprising SEQ ID NO:4 and a leucine zipper comprising the sequence of SEQ ID NO: the leucine zipper of sequence 5 was used in pairs.

3. The T cell antigen receptor of any one of claims 1 or 2, wherein the constant region amino acid sequence of the alpha chain comprises the sequence:

IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFAC ANAFNNSI IPEDTFFPSX1X2X3X4CDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLMTLRLWSS, 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, X4 is selected from any one of P and S, or an amino acid sequence having at least 85% homology with the above sequence;

The constant region amino acid sequence of the beta chain comprises the following sequence:

DLNKVFPPEVAVFEPSX5AEIX6HTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVCTDPQPLKEQP ALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGX7TSX8SYX9QGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF, 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;

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

4. A T cell antigen receptor according to any one of claims 1 to 3, comprising an amino acid sequence selected from any one of (1) to (7):

(1) The amino acid sequence of the constant region of the alpha chain comprises SEQ ID NO:6, or comprises a sequence identical to SEQ ID NO:6 having at least 85% or more homology, the amino acid sequence of the constant region of the β chain comprising the amino acid sequence of SEQ ID NO:7, or comprises a sequence identical to SEQ ID NO:7 an amino acid sequence having at least 85% homology;

(2) The amino acid sequence of the constant region of the alpha chain comprises SEQ ID NO:8, or comprises a sequence identical to SEQ ID NO:8 an amino acid sequence having at least 85% homology; the amino acid sequence of the constant region of the β chain comprises SEQ ID NO:9, or comprises a sequence identical to SEQ ID NO:9 has an amino acid sequence having at least 85% homology;

(3) The amino acid sequence of the constant region of the alpha chain comprises SEQ ID NO:10, or comprises a sequence identical to SEQ ID NO:10 having an amino acid sequence with at least 85% homology; the amino acid sequence of the constant region of the β chain comprises SEQ ID NO:11, or comprises a sequence identical to SEQ ID NO:11 has an amino acid sequence having at least 85% homology;

(4) The amino acid sequence of the constant region of the alpha chain comprises SEQ ID NO:12, or comprises a sequence identical to SEQ ID NO:12, the amino acid sequence of the constant region of the β chain of which comprises the amino acid sequence of SEQ ID NO:13, or comprises a sequence identical to SEQ ID NO:13 having at least 85% or more homology;

(5) The amino acid sequence of the constant region of the alpha chain comprises SEQ ID NO:14, or comprises a sequence identical to SEQ ID NO:14 having at least 85% homology; the sequence of the constant region of the beta chain comprises the sequence as shown in SEQ ID NO:15, or comprises an amino acid sequence as set forth in SEQ ID NO:15 having at least 85% homology;

(6) The amino acid sequence of the constant region of the alpha chain comprises SEQ ID NO:16, or comprises a sequence identical to SEQ ID NO:16 having at least 85% homology; the sequence of the constant region of the beta chain comprises the sequence as shown in SEQ ID NO:17, or comprises an amino acid sequence as set forth in SEQ ID NO:17 having at least 85% or more homology;

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

5. The T cell antigen receptor of any one of claims 1-4, wherein 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 as set forth in SEQ ID NO:18, the amino acid sequence of the beta-CDR 3 is shown in SEQ ID NO: as indicated by the numeral 19,

The variable region of the alpha chain comprises any one or combination of alpha-CDR 1 and alpha-CDR 2, wherein the amino acid sequence of the alpha-CDR 1 is shown in SEQ ID NO:20, the amino acid sequence of the alpha-CDR 2 is shown in SEQ ID NO: as indicated by the reference numeral 21,

The variable region of the beta chain comprises any one or combination of beta-CDR 1 and beta-CDR 2, wherein the amino acid sequence of the beta-CDR 1 is shown in SEQ ID NO:22, the amino acid sequence of the beta-CDR 2 is shown in SEQ ID NO: 23.

6. The T cell antigen receptor of any one of claims 1-5, wherein the variable region of the alpha chain has an amino acid sequence as set forth in SEQ ID NO:24, the amino acid sequence of the variable region of the beta chain is shown in SEQ ID NO: shown at 25;

Preferably, the variable region amino acid sequence of the alpha chain comprises SEQ ID NO:26, or comprises a sequence identical to SEQ ID NO:26, and the variable region amino acid sequence of the β chain comprises the amino acid sequence of SEQ ID NO:27, or comprises a sequence identical to SEQ ID NO:27, preferably said homology is selected from any of 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, 100%.

7. The T cell antigen receptor of any one of claims 1-6, wherein the amino acids of the α and β chains pass through the amino acid sequence set forth in SEQ ID NO:28, and a linker sequence.

8. The T cell antigen receptor of any one of claims 1-7, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 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 comprising a sequence identical to 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, and an amino acid sequence having at least 85% or more homology.

9. A nucleic acid encoding the T cell antigen receptor of any one of claims 1-8.

10. The nucleic acid of claim 9, having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 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 comprises any one of the amino acid sequences set forth in 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, preferably said homology is selected from any of 86%,87%,88%,89%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%, 100%.

11. An expression vector comprising the nucleic acid of any one of claims 9 or 10.

12. A host cell comprising the nucleic acid of any one of claims 9 or 10 or the expression vector of claim 12.

13. An immune cell expressing the T cell antigen receptor of any one of claims 1-8.

14. A method for preparing an immune cell, comprising transduction of the nucleic acid sequence according to any one of claims 9 or 10 into an immune cell for expression.

15. A method of preparing a recombinant T cell comprising the steps of:

1) Preparing the nucleic acid of any one of claims 9 or 10;

2) Separating and culturing T cells;

3) Delivering the nucleic acid of step 1) into the T cell of step 2) to obtain a recombinant T cell expressing the T cell antigen receptor of any one of claims 1-8.

16. A method for preparing a T cell antigen receptor, comprising the steps of:

(1) Preparing the nucleic acid of any one of claims 9 or 10;

(2) Ligating the nucleic acid prepared in step 1) to a vector to obtain an expression vector;

(3) Transducing the expression vector produced in step 2) into a host cell and then inducing expression thereof;

(4) Obtaining the T cell antigen receptor.

17. Use of a T cell antigen receptor according to any one of claims 1 to 8, a nucleic acid according to any one of claims 9 or 10, an expression vector according to claim 11, a host cell according to claim 12, an immune cell according to any one of claims 13 for the preparation of a preparation for the diagnosis, prevention and/or treatment of a tumor.

18. A pharmaceutical composition comprising any one of or a combination of 1) -5),

1) The T cell antigen receptor of any one of claims 1-8;

2) The nucleic acid of any one of claims 9 or 10;

3) The expression vector of claim 11;

4) The host cell of claim 12; or (b)

5) The immune cell of claim 13.

19. A kit comprising any one of or a combination of 1) -5):

1) The T cell antigen receptor of any one of claims 1-8;

2) The nucleic acid of any one of claims 9 or 10;

3) The expression vector of claim 11;

4) The host cell of claim 12;

5) The immune cell of claim 13.

20. A method of detection comprising contacting a sample to be detected with the T cell antigen receptor of any one of claims 1-8.