A NY-ESO-1 TCR and its application

Abstract

The present invention belongs to the technical field of tumor immunotherapy, discloses a NY-ESO-1TCR and its application, and specifically discloses a TCR variant, comprising an α chain variable region and a β chain variable region, wherein the α chain variable region is obtained by replacing at least one amino acid with the amino acid shown in SEQ ID NO:2; and / or the β chain variable region is obtained by replacing at least one amino acid with the amino acid shown in SEQ ID NO:4. The TCR variant provided by the present invention is mainly obtained by introducing various mutations into the natural 1G4TCR as a template. The TCR variant of the present invention can bind to a variety of SLLMWITQC-HLA complexes. After the TCR variant of the present invention is constructed to form TCR T in T cells, better anti-tumor effects are exhibited, and it has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of tumor immunotherapy, and specifically relates to a NY-ESO-1 TCR and an application thereof. Background Art

[0002] T cell receptor (TCR) T cell (TCR T) therapy is an emerging cancer immunotherapy that genetically modifies the patient's own T cells to express specific TCRs, thereby recognizing and attacking cancer cells carrying specific antigens. In recent years, TCRT therapy has shown encouraging results in clinical trials, especially in the treatment of hematological malignancies. However, there are still many challenges in the treatment of solid tumors, such as immunosuppression in the tumor microenvironment and insufficient T cell infiltration. TCR T therapy targeting NY-ESO-1 is expected to overcome some of the existing obstacles and bring new treatment hope to patients with solid tumors.

[0003] TCR belongs to the immunoglobulin superfamily and is a functional unit of T lymphocytes that recognize antigens. At present, αβT cells are the most studied in the field of TCR-T cells. The TCR of αβT cells is composed of α chain and β chain, and recognizes pMHC in the form of heterodimers. The extramembrane parts of the α chain and β chain each contain two Ig-like domains, one is the constant region (C region) proximal to the membrane, and the other is the variable region (V region) distal to the membrane. The variable region determines the binding of TCR to the major histocompatibility complex (MHC). After the different V, D, and J rearrangements of different germline genes on TCR, the TCR α chain and β chain sequences are highly diverse, which determines the specificity of TCR.

[0004] TCR is a specific receptor on the surface of T cells that can recognize and bind to protein antigens. It is a characteristic mark of all T cells. In the immune system, antigen-specific TCR binds to the major histocompatibility complex to trigger direct physical contact between T cells and APCs, initiating the first transduction signal. Subsequently, other cell membrane surface molecules of both T cells and APCs interact, thereby inducing the activation of T cells and exerting adaptive immune effector functions.

[0005] MHC is a general term for a group of genes that encode animal major histocompatibility antigens. The main function of MHC molecules is to participate in antigen presentation, that is, MHC molecules bind to antigen peptides through their peptide binding grooves and present them on the cell surface for T cells to recognize and trigger immune responses. Human MHC is called HLA (human leukocyte antigen, HLA), that is, human leukocyte antigen.

[0006] NY-ESO-1 is a cancer-testis antigen that is expressed in a variety of cancers, including melanoma, lung cancer, breast cancer, and ovarian cancer. Because its expression in normal tissues is strictly restricted, but it is highly expressed in a variety of tumors, NY-ESO-1 has become an attractive immunotherapy target. NY-ESO-1 TCR T therapy aims to develop TCRs against the NY-ESO-1 antigen to treat a variety of cancers that express this antigen.

[0007] The SLLMWITQC peptide is derived from the NY-ESO-1 protein. In normal tissues, NY-ESO-1 is only expressed in tissues with immune exemption characteristics such as the testis and placenta. In cancer patients, NY-ESO-1 is widely expressed in a variety of tumors, such as neuroblastoma, sarcoma, and malignant melanoma. Therefore, NY-ESO-1 is a good target for tumor treatment. The SLLMWITQC peptide of the NY-ESO-1 protein is displayed on the cell surface through the HLA molecule, binds to its specific TCR, activates T cells, promotes T cells to kill target cells, and thus realizes its anti-tumor function. There are TCR-T immunotherapy products targeting the SLLMWITQC-HLA-A*02:01 target that are undergoing clinical trials, and the safety of the NY-ESO-1 target and its effectiveness against solid tumors have been preliminarily verified.

[0008] At present, improving the affinity of TCR-HLA peptides, eliminating non-specific reactions, and increasing the diversity of HLA typing are important issues that need to be urgently addressed in TCR-T cell therapy. Summary of the invention

[0009] The present invention aims to provide a TCR having a high binding ability to the SLLMWITQC-HLA-(A*02:01, A*02:02, A*02:03, A*02:05, A*02:06, A*02:09, A*02:10) complex, as well as a preparation method and application of the above-mentioned type of TCR.

[0010] The purpose of the first aspect of the present invention is to provide a TCR variant.

[0011] The second aspect of the present invention aims to provide a multivalent TCR complex.

[0012] The third aspect of the present invention aims to provide a biological material related to the TCR variant of the first aspect of the present invention or the multivalent TCR complex of the second aspect of the present invention.

[0013] The fourth aspect of the present invention aims to provide an immune cell.

[0014] The fifth aspect of the present invention is to provide a pharmaceutical composition.

[0015] The purpose of the sixth aspect of the present invention is to provide the application of the TCR variant of the first aspect of the present invention, the multivalent TCR complex of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, the immune cell of the fourth aspect of the present invention or the drug combination of the fifth aspect of the present invention.

[0016] The seventh aspect of the present invention aims to provide a reagent, a test kit or a medicine kit.

[0017] The eighth aspect of the present invention provides a method for delivering a therapeutic drug to a target cell.

[0018] In order to achieve the above object, the technical solution adopted by the present invention is:

[0019] In a first aspect of the present invention, a TCR variant is provided, comprising an α chain variable region and a β chain variable region, wherein:

[0020] The α chain variable region is obtained by replacing at least one amino acid with the amino acids shown in SEQ ID NO: 2; and / or

[0021] The beta chain variable region is obtained by replacing at least one amino acid with the amino acids shown in SEQ ID NO: 4.

[0022] In some embodiments of the present invention, at least one alanine amino acid in the amino acids shown in SEQ ID NO: 2 is replaced.

[0023] In some embodiments of the present invention, the alanine at position 51 in the amino acid sequence shown in SEQ ID NO: 2 is replaced.

[0024] In some embodiments of the present invention, the alanine is replaced by at least one of threonine, histidine, lysine, glutamine and serine.

[0025] In some embodiments of the present invention, at least one threonine and / or serine in the amino acids shown in SEQ ID NO: 4 is replaced.

[0026] In some embodiments of the present invention, the threonine at position 95 in the amino acid sequence of SEQ ID NO:4 is replaced.

[0027] In some embodiments of the present invention, the serine at position 96 in the amino acid sequence of SEQ ID NO:4 is replaced.

[0028] In some embodiments of the present invention, the threonine is replaced by lysine, and the serine is replaced by tyrosine.

[0029] Such substitutions may be made by any suitable method, including but not limited to polymerase chain reaction, restriction cloning or ligation-independent cloning methods.

[0030] In some embodiments of the present invention, the amino acids in the variable region of the β chain are shown in SEQ ID NO:4, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.

[0031] In some embodiments of the present invention, the amino acids in the α chain variable region are shown in SEQ ID NO:2 and SEQ ID NO:21.

[0032] In some embodiments of the present invention, the TCR variant contains the α chain variable region of the amino acid sequence shown in SEQ ID NO: 2 and the β chain variable region of the amino acid sequence shown in SEQ ID NO: 16; or

[0033] The TCR variant contains an α chain variable region of the amino acid sequence shown in SEQ ID NO: 2 and a β chain variable region of the amino acid sequence shown in SEQ ID NO: 17; or

[0034] The TCR variant contains an α chain variable region of the amino acid sequence shown in SEQ ID NO: 2 and a β chain variable region of the amino acid sequence shown in SEQ ID NO: 18; or

[0035] The TCR variant contains the α chain variable region of the amino acid sequence shown in SEQ ID NO: 2 and the β chain variable region of the amino acid sequence shown in SEQ ID NO: 19; or

[0036] The TCR variant contains the α chain variable region of the amino acid sequence shown in SEQ ID NO: 2 and the β chain variable region of the amino acid sequence shown in SEQ ID NO: 20; or

[0037] The TCR variant contains an α chain variable region of the amino acid sequence shown in SEQ ID NO: 21 and a β chain variable region of the amino acid sequence shown in SEQ ID NO: 4; or

[0038] The TCR variant contains an α chain variable region of the amino acid sequence shown in SEQ ID NO: 21 and a β chain variable region of the amino acid sequence shown in SEQ ID NO: 19; or

[0039] The TCR variant contains an α chain variable region with an amino acid sequence as shown in SEQ ID NO:21 and a β chain variable region with an amino acid sequence as shown in SEQ ID NO:16.

[0040] In some embodiments of the present invention, the α chain of the TCR variant further comprises an α chain constant region. The α chain constant region is derived from germline TRAC1.

[0041] In some embodiments of the present invention, the β chain of the TCR variant further comprises a β chain constant region. The β chain constant region is derived from germline TRBC2.

[0042] In some embodiments of the present invention, the α chain and / or β chain of the TCR further includes an extramembrane region, a transmembrane region and an intracellular sequence.

[0043] In some embodiments of the present invention, the TCR variant contains the α chain of the amino acid sequence shown in SEQ ID NO:6 and the β chain of the amino acid sequence shown in SEQ ID NO:10; or the TCR variant contains the α chain of the amino acid sequence shown in SEQ ID NO:6 and the β chain of the amino acid sequence shown in SEQ ID NO:11; or the TCR variant contains the α chain of the amino acid sequence shown in SEQ ID NO:6 and the β chain of the amino acid sequence shown in SEQ ID NO:12; or the TCR variant contains the α chain of the amino acid sequence shown in SEQ ID NO:6 and the β chain of the amino acid sequence shown in SEQ ID NO:13; or the TCR variant contains the α chain of the amino acid sequence shown in SEQ ID NO:6 and the β chain of the amino acid sequence shown in SEQ ID NO:14; or the TCR variant contains the α chain of the amino acid sequence shown in SEQ ID NO:15 and the β chain of the amino acid sequence shown in SEQ ID NO:8; or the TCR variant contains the α chain of the amino acid sequence shown in SEQ ID NO:15 and the β chain of the amino acid sequence shown in SEQ ID NO:13; or the TCR variant contains the α chain of the amino acid sequence shown in SEQ ID NO:15 and the β chain of the amino acid sequence shown in SEQ ID NO: β chain of the amino acid sequence shown in NO:10.

[0044] In some embodiments of the present invention, the α chain of the TCR variant is connected to the N-terminus or C-terminus of the β chain through a linking element.

[0045] In some embodiments of the present invention, the linking element comprises a polypeptide having self-cleavage ability.

[0046] In some embodiments of the present invention, the self-cleavage-capable polypeptide comprises a 2A peptide.

[0047] In some embodiments of the present invention, the 2A peptide comprises at least one of T2A, E2A and F2A. The amino acid sequence of T2A is shown in SEQ ID NO:34, and the nucleotide sequence is shown in SEQ ID NO:35. The amino acid sequence of P2A is shown in SEQ ID NO:36, and the nucleotide sequence is shown in SEQ ID NO:37.

[0048] The TCR variants provided by the present invention are double-chain TCRs. The forms include:

[0049] (1) A first segment consisting of a TCRα chain amino acid sequence (TCRα chain variable region and TCRα chain constant region), a second segment consisting of a TCRβ chain amino acid sequence (TCRβ chain variable region and TCRβ chain constant region), and a linker sequence (linking element) connecting the C-terminus of the first segment and the N-terminus of the second segment.

[0050] (2) A first segment composed of a TCRβ chain amino acid sequence (TCRβ chain variable region and TCRβ chain constant region), a second segment composed of a TCRα chain amino acid sequence (TCRα chain variable region and TCRα chain constant region), and a linker sequence (linking element) connecting the C-terminus of the first segment and the N-terminus of the second segment.

[0051] In the above TCR variant forms, a linker sequence may connect the C-terminus of the first segment and the N-terminus of the second segment.

[0052] The present invention provides a TCR variant with high binding properties to SLLMWITQC-HLA-(A*02:01, A*02:02, A*02:03, A*02:05, A*02:06, A*02:09, A*02:10). The TCR variant has strong binding ability to SLLMWITQC-HLA-A*02:02, SLLMWITQC-HLA-A*02:03, SLLMWITQC-HLA-A*02:05, SLLMWITQC-HLA-A*02:06, SLLMWITQC-HLA-A*02:09, and SLLMWITQC-HLA-A*02:10 complexes loaded with multiple subtypes of HLA. The TCR of the present invention has extremely high tumor killing ability and good safety, and has wide applicability.

[0053] The second aspect of the present invention provides a multivalent TCR complex comprising at least two TCR molecules, wherein at least one TCR molecule is a TCR variant of the first aspect of the present invention.

[0054] In some embodiments of the invention, at least one of the TCRs is associated with a therapeutic agent.

[0055] The third aspect of the present invention provides a biomaterial related to the TCR variant of the first aspect of the present invention or the multivalent TCR complex of the second aspect of the present invention, wherein the biomaterial is any one of a1) to a12):

[0056] a1) a nucleic acid molecule encoding the TCR variant of the first aspect of the present invention or the multivalent TCR complex of the second aspect of the present invention; a2) an expression cassette containing the nucleic acid molecule described in a1); a3) a recombinant vector containing the nucleic acid molecule described in a1); a4) a recombinant vector containing the expression cassette described in a2); a5) a recombinant microorganism containing the nucleic acid molecule described in a1); a6) a recombinant microorganism containing the expression cassette described in a2); a7) a recombinant microorganism containing the recombinant vector described in a3); a8) a recombinant microorganism containing the recombinant vector described in a4); a9) a transgenic cell line containing the nucleic acid molecule described in a1); a10) a transgenic cell line containing the expression cassette described in a2); a11) a transgenic cell line containing the recombinant vector described in a3); a12) a transgenic cell line containing the recombinant vector described in a4).

[0057] In some embodiments of the invention, the transgenic animal cell line does not contain reproductive material.

[0058] In some embodiments of the present invention, the expression cassette comprises a 5' transcription control region, an open reading frame encoding the fusion protein of the first aspect of the present invention, a translation control signal, a 3' untranslated region (3'UTR) and a transcription termination signal.

[0059] In some embodiments of the present invention, the 5' transcription control region comprises a promoter (a universal promoter, such as a viral promoter (SV40 promoter) or a mammalian "housekeeping" promoter, may be used), a transcription start site, an enhancer and / or a silencer element.

[0060] In some embodiments of the invention, the 3'UTR may encode an AU-rich element, which is a common regulator of mRNA stability through the 3'-5' exosome pathway and is usually located in the 3'UTR. The AU-rich element may contain one or more repeats of the sequence AUUUA. It may also contain one or more so-called US2B elements having the sequence AUAUAU.

[0061] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0062] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a viral vector.

[0063] In some embodiments of the present invention, the viral vector comprises at least one of a lentiviral vector, an adenoviral vector, a baculoviral vector, a retroviral vector, a poxvirus vector, a Sendai virus vector, and a herpes simplex virus vector.

[0064] In some embodiments of the present invention, the non-viral vector includes at least one of a plasmid vector, a cationic polymer vector, chitosan, polyethyleneimine, a nanoparticle vector, and a liposome.

[0065] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a bacteriophage, a cosmid, a F cosmid, or an artificial chromosome.

[0066] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0067] In some embodiments of the present invention, the recombinant expression vector uses pET-28a(+) as the original expression vector.

[0068] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells; the cells are not new species of plants or animals.

[0069] In some embodiments of the present invention, the prokaryotic cells include Escherichia coli, Streptomyces, Bacillus subtilis and other bacteria well known in the art that can be used to express target proteins.

[0070] In some embodiments of the present invention, the eukaryotic cell comprises at least one of a yeast cell, a mammalian cell, a plant cell and an insect cell.

[0071] The fourth aspect of the present invention provides an immune cell, wherein the immune cell expresses the TCR variant of the first aspect of the present invention.

[0072] In some embodiments of the present invention, the immune cells include T cells, lymphocytes or stem cells.

[0073] In some embodiments of the present invention, the T cells, lymphocytes or stem cells are selected from one or more of CD4 cells, CD8 cells, naive T cells, memory stem T cells, central memory T cells, double negative T cells, effector memory T cells, effector T cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, γ / δT cells, natural killer cells, natural killer T cells, dendritic cells, hematopoietic stem cells and pluripotent stem cells.

[0074] In some embodiments of the present invention, the immune cells can be obtained by introducing a nucleic acid molecule encoding the TCR variant into an immune cell. Specifically, 293T cells are infected with a lentiviral vector containing a nucleic acid molecule encoding the TCR variant, and specific T cells are obtained by culture. The SLLMWITQC-HLA complex can activate the T cells at a SLLMWITQC peptide concentration of 0.01 nM. That is, after the TCR variant is transduced into the T cell by lentivirus, it exhibits specific killing activity against tumors.

[0075] The fifth aspect of the present invention provides a pharmaceutical composition comprising the TCR variant of the first aspect of the present invention, the multivalent TCR complex of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, or the immune cell of the fourth aspect of the present invention.

[0076] In some embodiments of the present invention, the pharmaceutical composition further comprises a combined drug.

[0077] In some embodiments of the present invention, the combination drug includes at least one of an immune effector molecule, a cytotoxic drug, and an anticancer drug.

[0078] In some embodiments of the present invention, the immune effector molecule includes at least one of IL-2, IL-7, IL-15 and IL-21.

[0079] In some embodiments of the present invention, the anticancer drug includes at least one of lenvatinib, sorafenib, cabozantinib, apatinib, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and azacytidine.

[0080] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or diluent. A pharmaceutically acceptable carrier and / or diluent refers to a method of preparing the above-mentioned TCR or the above-mentioned antigen binding fragment or the above-mentioned polynucleotide or the above-mentioned expression vector or the above-mentioned engineered cell into various desired dosage forms. For example, tablets, powders, pills, powders, granules, fine granules, soft / hard capsules, film coatings, pellets, sublingual tablets, ointments, etc. as oral agents can be listed, and injections, suppositories, transdermal agents, ointments, plasters, external liquids, etc. can be listed as non-oral agents. Those skilled in the art can select appropriate dosage forms according to the route of administration and the subject of administration.

[0081] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, and the pharmaceutically acceptable excipient comprises at least one of a disintegrant, a dispersant, a stabilizer, a lubricant, a binder, a wetting agent, a flavoring agent, a solubilizing agent, a suspending agent, a solvent, a sustained-release agent, an emulsifier, an absorption enhancer, a surfactant, a preservative, a pigment, a flavor and a solvent.

[0082] The sixth aspect of the present invention provides a use of the TCR variant of the first aspect of the present invention, the multivalent TCR complex of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, the immune cell of the fourth aspect of the present invention, or the drug combination of the fifth aspect of the present invention in any one of (1) to (3):

[0083] (1) Preparation of drugs for preventing and / or treating tumors; (2) Preparation of tumor diagnostic reagents or kits; (3) Preparation of reagents for increasing the level of IFN-γ and / or TNF-α cytokine secretion by T cells.

[0084] In some embodiments of the present invention, the tumors described in (1) and (2) are NYESO-1 positive tumors, including at least one of sarcoma (synovial sarcoma, liposarcoma), prostate cancer, uterine cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, multiple myeloma, melanoma (malignant melanoma), neuroblastoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, hepatobiliary carcinoma, breast cancer, bladder cancer, oral squamous cell carcinoma, myeloid leukemia and acute lymphoblastic leukemia.

[0085] The seventh aspect of the present invention provides a reagent, a test kit or a medicine kit, comprising the TCR variant of the first aspect of the present invention, the multivalent TCR complex of the second aspect of the present invention, the biomaterial of the third aspect of the present invention, the immune cell of the fourth aspect of the present invention or the drug combination of the fifth aspect of the present invention.

[0086] The eighth aspect of the present invention provides a method for delivering a therapeutic drug to a target cell, the method comprising contacting a potential target cell with a TCR variant of the first aspect of the present invention under conditions where the two are bound, wherein the TCR has specificity for the SLLMWITQC-HLA-(A*02:01, A*02:02, A*02:03, A*02:05, A*02:06, A*02:09, A*02:10) complex and is bound to a therapeutic drug. Specifically, the TCR variant of the present invention can be used to deliver a therapeutic drug to an antigen presenting cell. The therapy can be applied to the treatment of a variety of diseases, particularly in anti-tumor aspects.

[0087] The beneficial effects of the present invention are:

[0088] The TCR variants provided by the present invention are mainly obtained by introducing various mutations into the natural 1G4 TCR as a template. The TCR variants of the present invention can bind to a variety of SLLMWITQC-HLA complexes, including SLLMWITQC-HLA-(A*02:01, A*02:02, A*02:03, A*02:05, A*02:06, A*02:09, A*02:10). After the TCR variants of the present invention are constructed into T cells to form TCR T, better anti-tumor effects are exhibited, and good application prospects are provided. Compared with the natural 1G4 TCR dimer, the TCR of the present invention has better SLLMWITQC-HLA complex binding ability and anti-tumor effect, and has good application prospects.

[0089] The TCR variants provided by the present invention can be combined with a detectable portion of a therapeutic drug or a diagnostic agent. For example, a detectable portion, a marker for diagnostic purposes, can be used to label the TCR chain of the present invention. Such labeled TCRs can be used to detect SLLMWITQC-HLA-(A*02:01, A*02:02, A*02:03, A*02:05, A*02:06, A*02:09, A*02:10) complexes. For example, a tetrameric TCR complex formed by a biotinylated heterodimer can be provided with a detectable label using fluorescent streptavidin. This fluorescently labeled TCR tetramer is suitable for FACS analysis, such as detecting antigen presenting cells carrying these high-affinity TCR-specific SLLMWITQC-HLA-(A*02:01, A*02:02, A*02:03, A*02:05, A*02:06, A*02:09, A*02:10) complexes. BRIEF DESCRIPTION OF THE DRAWINGS

[0090] Figure 1 Schematic diagram of the 1G4 TCR gene structure.

[0091] Figure 2 It is the positive rate of T cells infected by the TCR (Blank) virus of the present invention.

[0092] Figure 3 It is the positive rate of T cells infected by the TCR (1G4α chain + 1G4β chain) virus of the present invention.

[0093] Figure 4 It is the positive rate of T cells infected by the TCR (1G4α chain (T95L, S96Y) + 1G4β chain) virus of the present invention.

[0094] Figure 5 It is the positive rate of T cells infected by the TCR (1G4α chain + 1G4β chain) virus of the present invention.

[0095] Figure 6It is the positive rate of T cells infected by the TCR (1G4α chain (T95L, S96Y) + 1G4β chain) virus of the present invention.

[0096] Figure 7 It is the positive rate of T cells infected by the TCR (1G4α chain (T95L, S96Y) + 1G4β chain) virus of the present invention.

[0097] Figure 8 Elispot was used to detect the effect of peptide-HLA complex on T cell activation.

[0098] Fig. 9 This is the in vitro killing function of the TCR T cells of the present invention.

[0099] Fig.10 This is the in vivo anti-tumor efficacy of the TCR T cells of the present invention.

[0100] Fig.11 The TCR T cells of the present invention respond to multiple HLA types. DETAILED DESCRIPTION

[0101] The present invention is further described in detail below through specific examples.

[0102] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0103] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0104] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0105] Example 1 Modification of 1G4 TCR

[0106] The natural 1G4 TCR gene was synthesized by entrusting Sangon Biotech Co., Ltd., which mainly includes the 1G4 TCRβ chain variable region (nucleotide sequence as shown in SEQ ID NO:3, amino acid sequence as shown in SEQ ID NO:4) connected to the mouse β chain constant region, 2A, 1G4TCRα chain variable region (nucleotide sequence as shown in SEQ ID NO:1, amino acid sequence as shown in SEQ ID NO:2) connected to the α chain mouse constant region, and the TCRα chain and TCRβ chain are connected with a P2A linker base sequence. The specific structure is as follows Figure 1The nucleotide sequence of the α chain of the natural 1G4 TCR is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6. The nucleotide sequence of the β chain of the natural 1G4 TCR is shown in SEQ ID NO:7, and the amino acid sequence is shown in SEQ ID NO:8. The amino acid sequence of the natural 1G4 TCR is shown in SEQ ID NO:9.

[0107] Since the natural 1G4 TCR can only recognize the SLLMWITQC-HLA-A*02:01 complex, tumor-targeted cell therapy can be performed for patients with HLA A*02:01, and its affinity is not optimal. After affinity optimization, the 1G4 TCR is more suitable for delivering cytotoxic agents or immunostimulants to target cells, and for immunotherapy for tumor patients expressing HLA A*02:02, A*02:03, A*02:05, A*02:06, A*02:09, and A*02:10, expanding the coverage of patients. In this example, the 1G4 TCR is further modified to obtain different mutant 1G4 TCRs, such as direct mutation of the β chain of the 1G4 TCR to obtain A51E, A51H, A51K, A51Q and A51S mutant 1G4 TCRs, wherein A51E, A51H, A51K, A51Q and A51S represent that the A at position 51 of the amino acid sequence of the natural 1G4 TCR β chain (i.e., the amino acid shown in SEQ ID NO: 8) is mutated to E, H, K, Q and S respectively; the α chain of the 1G4 TCR is directed to obtain a (T95L, S96Y) mutant 1G4 TCR, i.e., the α chain of the natural 1G4 TCR (i.e., the amino acid shown in SEQ ID NO: 8) is directed to obtain a (T95L, S96Y) mutant 1G4 TCR, i.e., the α chain of the natural 1G4 TCR (i.e., the amino acid shown in SEQ ID NO: 8) is directed to obtain a (T95L, S96Y) mutant 1G4 TCR, NO:6), the T at position 95 of its amino acid sequence was mutated to L, and the S at position 96 was mutated to Y; and the α chain and β chain were mutated simultaneously to obtain (T95L, S96Y)+A51Q and (T95L, S96Y)+A51E mutant 1G4 TCRs.

[0108] The mutant IG4 TCR gene is as follows:

[0109] The amino acid sequence of the α chain of the A51E mutant IG4 TCR (i.e., 1G4α+A51Eβ) is shown in SEQ ID NO:6, and the amino acid sequence of the β chain is shown in SEQ ID NO:10 (the amino acid sequence of the variable region of the β chain is shown in SEQ ID NO:16);

[0110] The amino acid sequence of the α chain of the A51H mutant IG4 TCR (i.e., 1G4α+A51Hβ) is shown in SEQ ID NO:6, and the amino acid sequence of the β chain is shown in SEQ ID NO:11 (the amino acid sequence of the variable region of the β chain is shown in SEQ ID NO:17);

[0111] The amino acid sequence of the α chain of the A51K mutant IG4 TCR (i.e., 1G4α+A51Kβ) is shown in SEQ ID NO:6, and the amino acid sequence of the β chain is shown in SEQ ID NO:12 (the amino acid sequence of the variable region of the β chain is shown in SEQ ID NO:18);

[0112] The amino acid sequence of the α chain of the A51Q mutant IG4 TCR (i.e., 1G4α+A51Qβ) is shown in SEQ ID NO:6, and the amino acid sequence of the β chain is shown in SEQ ID NO:13 (the amino acid sequence of the variable region of the β chain is shown in SEQ ID NO:19);

[0113] The amino acid sequence of the α chain of the A51S mutant IG4 TCR (i.e., 1G4α+A51Sβ) is shown in SEQ ID NO:6, and the amino acid sequence of the β chain is shown in SEQ ID NO:14 (the amino acid sequence of the variable region of the β chain is shown in SEQ ID NO:20);

[0114] The amino acid sequence of the α chain of the (T95L, S96Y) mutant IG4 TCR (i.e., 1G4α(T95L, S96Y)+1G4β, or 95LY) is shown in SEQ ID NO: 15 (the amino acid sequence of the variable region of the α chain is shown in SEQ ID NO: 21), and the amino acid sequence of the β chain is shown in SEQ ID NO: 8

[0115] The amino acid sequence of the α chain of the (T95L, S96Y)+A51Q mutant IG4 TCR (i.e., 1G4α(T95L, S96Y)+A51Qβ) is shown in SEQ ID NO: 15, and the amino acid sequence of the β chain is shown in SEQ ID NO: 13;

[0116] The amino acid sequence of the α chain of the (T95L, S96Y)+A51E mutant IG4 TCR (i.e., 1G4α(T95L, S96Y)+A51Eβ) is shown in SEQ ID NO:15, and the amino acid sequence of the β chain is shown in SEQ ID NO:10.

[0117] Example 2 Plasmid construction (including target fragment mutation)

[0118] The primers containing mutant amino acids were artificially designed to amplify the different mutant 1G4 TCRs in Example 1, and the double-fragment homologous recombination technology was used for connection. The large fragment amplification product was 1608 bp, the small fragment PCR product was 252 bp, and the annealing temperature was 64° C. The fragments and the backbone vector were spliced ​​using the GIBSON connection system. The connected vectors were identified by colony PCR and sequenced after enzyme digestion identification.

[0119] The primers for the amino acid mutations are as follows:

[0120] 1G4-A51E:

[0121] F1: 5'-ATTTCAGGTGTCGTGAAGCGGC-3' (SEQ ID NO: 22);

[0122] R1: 5'-GGTGATGCCCTCGCCCAC-3' (SEQ ID NO: 23);

[0123] F2: 5'-GTGGGCGAGGGCATCAC-3' (SEQ ID NO: 24);

[0124] R2: 5'-CAGAGGTTGATTGTCGACTCAGC-3' (SEQ ID NO: 25);

[0125] 1G4-A51H:

[0126] F1: 5'-ATTTCAGGTGTCGTGAAGCGGC-3' (SEQ ID NO: 22);

[0127] R1: 5'-GGGTGATGCCGTGGCCCAC-3' (SEQ ID NO: 26);

[0128] F2: 5'-GTGGCCACGGCATCAC-3' (SEQ ID NO: 27);

[0129] R2: 5'-CAGAGGTTGATTGTCGACTCAGC-3' (SEQ ID NO: 25);

[0130] 1G4-A51K:

[0131] F1: 5'-ATTTCAGGTGTCGTGAAGCGGC-3' (SEQ ID NO: 22);

[0132] F2: 5'-GTGGGCAAGGGCATCAC-3' (SEQ ID NO: 28);

[0133] R1: 5'-GGTGATGCCCTTGCCCAC-3' (SEQ ID NO: 29);

[0134] R2: 5'-CAGAGGTTGATTGTCGACTCAGC-3' (SEQ ID NO: 25);

[0135] 1G4-A51Q

[0136] F1: 5'-ATTTCAGGTGTCGTGAAGCGGC-3' (SEQ ID NO: 22);

[0137] R1: 5'-GGTGATGCCCTGGCCCAC-3' (SEQ ID NO: 30);

[0138] F2: 5'-GTGGCCAGGGCATCAC-3' (SEQ ID NO: 31);

[0139] R2: 5'-CAGAGGTTGATTGTCGACTCAGC-3' (SEQ ID NO: 25);

[0140] 1G4-A51S

[0141] F1: 5'-ATTTCAGGTGTCGTGAAGCGGC-3' (SEQ IDNO: 22);

[0142] R1: 5'-GGGTGATGCCGCTGCCCAC-3' (SEQ IDNO: 32);

[0143] F2: 5'-GTGGGCCAGCGGCATCAC-3' (SEQ IDNO: 33);

[0144] R2: 5'-CAGAGGTTGATTGTCGACTCAGC-3' (SEQ IDNO: 25);

[0145] (1) Single fragment amplification reaction

[0146] The single fragment amplification reaction system was prepared according to Table 1, and the PCR reaction was performed according to the reaction procedure shown in Table 2.

[0147] Table 1 Single fragment amplification reaction system

[0148] 5×Q5 Reaction Buffer 10μL 10mM dNTPs 1μL Q5 High-Fidelity DNA Polymerase 0.5μL Template plasmid 0.1~2ng Primer F 1~2μL Primer R 1~2μL <![CDATA[UltraPure TM Distilled Water]]> to 50μL

[0149] Table 2 Single fragment amplification reaction procedure

[0150] temperature time Number of cycles Pre-denaturation 98℃ 30s 1 transsexual 98℃ 10s 30 annealing 60～70℃ 20s extend 72℃ 1min extend 72℃ 2min 1

[0151] (2) Multi-fragment homologous recombination

[0152] The multi-fragment homologous recombination reaction system was prepared as shown in Table 3, and the multi-fragment homologous recombination was performed. The reaction procedure was as follows: single-fragment recombination reaction, 50°C, 5 min; cool to 4°C or immediately cool on ice; multi-fragment recombination reaction, 50°C, 15 min; cool to 4°C or immediately cool on ice.

[0153] Table 3 Multi-fragment homologous recombination reaction system

[0154] Backbone plasmid digestion and purification product 50~100ng Target gene amplification recovery product 1:1 molar ratio of the same backbone plasmid digestion and purification product 2×ClonExpress Mix 5μL UltraPureTM Distilled Water To 10μL

[0155] Example 3 Plasmid Extraction

[0156] (1) Plasmid transformation was performed on the vector that was correctly identified by sequence alignment at 37°C for 14 h; (2) Single clone colonies were picked and cultured in a small amount of bacterial solution with a culture volume of 4 mL Amp+LB (100 mg / L Amp+) at 37°C and 220 rpm for 10 h; (3) Large-scale bacterial culture: The next day, the seed solution was inoculated into 250 mL Amp+LB (100 mg / L Amp+) at 37°C and 220 rpm for 14 h; (4) Plasmid extraction was performed using the Tiangen Plasmid Extraction Kit:

[0157] a. Add the bacterial solution to the collection bottle (500mL), centrifuge at 4000rpm for 30min, and remove the supernatant as much as possible. b. Column balancing step: Add 2.5mL of balancing solution BL to the adsorption column CP6 (the adsorption column is placed in a 50mL collection tube). c. Add 10mL of solution P1 to the bacterial precipitate to resuspend the bacteria. d. Add 10mL of solution P2 to the bacterial suspension, gently flip 6 to 8 times to fully lyse the bacteria. e. Add 10mL of solution P4 to the centrifuge tube, gently flip 6 to 8 times, and mix thoroughly until the solution has white dispersed flocculent precipitation. f. Centrifuge the lysate at 3580×g for 30min. g. Carefully pour the supernatant lysate into the filter CS1, slowly push the push handle to filter, and collect the filtrate in a clean 50mL tube. h. Add 3mL of red endotoxin-removing solution ER respectively, and after inversion and mixing, the solution presents a uniform and transparent yellow color. i. Add 0.3 times the volume of isopropanol in the above filtrate, mix by inverting, and transfer to the adsorption column CP6. j. Centrifuge at 8228×g for 2 minutes at room temperature, discard the waste liquid in the collection tube, and put the adsorption column CP6 back into the collection tube. k. Add 10mL of buffer ED to the adsorption column CP6, centrifuge at 8228×g for 2 minutes, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. l. For low-copy plasmids, repeat k. m. Add 10mL of rinse solution PW to the adsorption column CP6, centrifuge at 8228×g for 2 minutes, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube. n. Repeat operation m. o. Put the adsorption column CP6 back into the collection tube, centrifuge at 8228×g for 5 minutes, then open the adsorption column CP6 and leave it at room temperature for a few minutes to completely dry the residual rinse solution in the adsorption material. p. Place the adsorption column CP6 in a clean 50mL collection tube, add 1-2mL of elution buffer TB to the middle part of the adsorption membrane, place it at room temperature for 5 minutes, and then centrifuge it at 8228×g for 5 minutes. q. Take 1μL of plasmid DNA to determine the concentration (adjust Buffer TE to zero), divide the plasmid DNA into 1.5mL centrifuge tubes, mark the name, concentration, batch number and volume with a marker, and store at -20℃.

[0158] Example 4 Virus packaging and concentration

[0159] (1) Observe the 293T cells for packaging under a microscope and select cells with a cell density of 80% to 95%. (2) Prepare 10% packaging culture medium. Open a new bottle of 500mL DMEM culture medium, add 55mL FBS, 550μL 1mol / L sodium pyruvate solution and 550μL 25mmol / L chloroquine phosphate solution, and mix well. (3) Take out the selected cells from the incubator, pour the old culture medium in the culture bottle into the waste liquid tank, add 17mL 10% packaging culture medium, and then put it back into the carbon dioxide incubator to adapt. (4) According to the number of culture bottles that need to be packaged, calculate the expression plasmid, packaging auxiliary plasmid, and envelope plasmid that need to be added. The plasmid-to-plasmid ratio is 10:8.9:3.5, and the total amount of plasmid added is 30μg / bottle T175 and 0.125mol / L calcium chloride solution to prepare DNA-CaCl 2 Mix the solution. Take an equal volume of 2×HBS solution and add it dropwise to the DNA-CaCl 2 Add DNA-CaCl to the mixture while vortexing 2 Mixed solution. After the addition, let stand at room temperature for 20 minutes until a white precipitate is formed. (5) Take out the cells from step (3) from the incubator, add 8 mL of calcium phosphate-DNA precipitation complex to each bottle, mark the virus name and operation date on the bottle, return it to the incubator, and replace the medium with 5% packaging medium after 4 to 6 hours. (6) Prepare 5% packaging medium, open a new bottle of 500 mL DMEM medium, add 27.5 mL FBS and 27.5 mL serum substitute, then add 550 μL 1 mol / L sodium pyruvate solution and 550 μL 25 mmol / L chloroquine phosphate solution, and mix well. (7) Take out the 293T cells from step (5) from the incubator, discard the old medium, add 25 mL of 5% packaging medium to each bottle, and then return it to the incubator to continue culturing. Collect the virus after 42 to 48 hours of culture. (8) Divide the lentiviral solution into 50mL centrifuge tubes and place them in a centrifuge for centrifugation at 4000g, 25min, and 4°C. (9) Transfer the supernatant of the lentiviral solution after centrifugation to a 0.45μm vacuum filtration system for filtration; (10) Divide the lentiviral supernatant into high-speed centrifuge tubes and place them in a centrifuge for centrifugation at 19500rpm, 4h, and 4°C. (11) After centrifugation, discard the supernatant in a biosafety cabinet, add 200μL of HBSS to each high-speed centrifuge tube, gently resuspend the virus precipitate, transfer it to a 50mL centrifuge tube, label the bottle with the virus name and batch number, and store it in a medical refrigerator.

[0160] Example 5 Virus infection of T cells

[0161] After the modified 1G4 TCR construct plasmid of the present invention is packaged with a virus, the virus infects T cells, and the virus is integrated into the T cell genome to construct the 1G4 TCR stably transfected T cells of the present invention. The details are as follows:

[0162] (1) Take PBMC separated by Ficoll separation solution and adjust the cell density to 1x10 6 T cells / mL. (2) Add Anti-Human CD3e and Anti-Human CD28 antibodies at a final concentration of 500 ng / mL and 1000 IU / mL hIL-2. (3) Perform virus infection 24 hours after stimulation. Add the virus to the culture system at an MOI of 5 and gently pipette to mix. (4) Place in a 37°C CO 2 Culture in an incubator. After the cells have expanded to 200 to 500 times, conduct subsequent functional experiments.

[0163] The results are as follows Figure 2 to Figure 7 The figure shows the transduction efficiency of the modified 1G4 TCR T cell D7. The infection efficiency is more than 80%, which proves that the modified 1G4 TCR of the present invention has a good infection effect when transfected into T cells.

[0164] Example 6 Elispot detection of T cell activation

[0165] Verify that the T cells infected by the modified 1G4 TCR of the present invention can be specifically activated by target cells. Elispot technology is used to detect the production of IFN-γ as a marker molecule for T cell activation. The details are as follows:

[0166] Experimental culture medium: RPMI1640, 10% FBS, 1000 IU / mL hIL-2.

[0167] Peptide: synthetic SLLMWITQC, working concentrations (0.01 nM, 0.05 nM, 0.1 nM, 0.3 nM, 0.6 nM, 1 nM).

[0168] Elispot kit (BD, 6t8155): Follow the instructions provided with the reagent.

[0169] operate:

[0170] (1) Activation of pre-coated plates: Add 200 μL RPMI-1640 medium to each well, let stand at room temperature for 5-10 min, then remove the medium. (2) Add the 1G4 TCR T cell suspension obtained in Example 5: Add the cell suspension with adjusted concentration to each experimental well, 50 μL / well. Positive control well: Cell concentration 1×10 5cells / well; background control: medium for resuspended cells; experimental well: cell concentration 1×10 5 (3) Adding stimuli: K562 expressing HLA-0201 loaded with peptides at different concentrations, 1×10 5 cells / 50μL / well. Positive control well: add positive stimulant (SLLMWITQC-HLA-A*02:01) working solution. Negative control well: resuspend cells in culture medium. Experimental well: stimulants (SLLMWITQC-HLA*02:02, A*02:03, A*02:05, A*02:06, A*02:09, A*02:10). (4) Incubation: After all samples and stimulants are added, cover the plate. Place in 37°C, 5% CO 2 Incubate in the incubator for 16 to 20 hours. (5) Lyse cells: Pour out the cells and culture medium in the wells. Add ice-cold deionized water, 200 μL / well, and place in a 4°C refrigerator for 10 minutes to hypotonic lyse the cells. (6) Wash the plate: Pour out the liquid in the wells, add 1×Washing buffer, 200 μL / well, and wash 5 to 7 times. Stay for 30 to 60 seconds each time. After the last wash, tap dry on absorbent paper. (7) Incubate with detection antibodies: Add the diluted biotin-labeled anti-INFγ antibody (Elispot kit (BD, 6t8155)) with a working concentration of 1 μg / mL to each experimental well, 100 μL / well. Incubate at 37°C for 1 hour. (8) Wash the plate: Pour out the liquid in the wells, add 1×Washing buffer, 200 μL / well, and wash 5 times. Stay for 30 to 60 seconds each time. After the last wash, tap dry on absorbent paper. (9) Enzyme-linked avidin incubation: Add the diluted enzyme-linked avidin to each experimental well at a dilution of 1:1000, 100 μL / well. Incubate at 37°C for 1 hour. (10) Plate washing: Pour the liquid in the well, 1×Washing buffer, 200 μL / well, wash 5 times. Stay for 30-60 seconds each time. After the last wash, tap dry on absorbent paper. (11) Color development: Add the freshly prepared AEC color development solution to each experimental well, 100 μL / well. Incubate at room temperature away from light for 15-45 minutes (at 20-25°C, 25 minutes of color development is more appropriate). If the room temperature is lower than 20°C, it is recommended to perform color development in a 37°C incubator and check every 5-10 minutes. (12) Stop color development: Pour the liquid in the well, uncover the base of the plate, wash the front and back sides and the base with deionized water / tap water 3-5 times, and stop color development. Place the plate in a cool place at room temperature and allow it to dry naturally before closing the base. (13) Count the spots on the ELISPOT plate and record various parameters of the spots for statistical analysis.

[0171] The results are as follows Figure 8As shown, compared with the natural 1G4 TCR group, the A51E group, A51Q group, A51H group, (T95L, S96Y) + A51E group, and (T95L, S96Y) + A51Q group can all be activated by the SLLMWITQC-HLA-A*02:01 complex target cells. The minimum action concentration of the 1G4 group can reach 0.01nM, and the minimum action concentration of the 1G4-95LY group can reach 0.05nM. Among them, the two mutations of the β chain A52E and A52Q of the TCR of the present invention have better binding ability with the SLLMWITQC-HLA-A*02:01 complex.

[0172] Example 7 In vitro T cell killing

[0173] The T cells stably transfected with 1G4 TCR in Example 5 were verified to have the ability to kill target cells expressing NY-ESO-1. The details are as follows:

[0174] (1) T cell preparation: After successful culture of the T cells in Example 5, use them as T cells for functional detection in this experiment. (2) Target cell preparation: A375 tumor cell line. (3) Experimental wells: T cells and target cells are plated in 48-well plates in different proportions. Control wells: Only target cells are plated. Observe the proliferation of target cells in the experimental wells compared with the control wells. (4) Observe for 24h and 72h. Count and statistically analyze the target cells respectively.

[0175] The results are as follows Fig. 9 As shown, compared with the natural 1G4 TCR group, 1G4α(T95L, S96Y)+1G4β, 1G4α+A51Qβ, 1G4α(T95L, S96Y)+A51Qβ, and 1G4α(T95L, S96Y)+A51Eβ all showed stronger killing function against target cells A375 (naturally having SLLMWITQC-HLA-A*02:01).

[0176] Example 8 Drug efficacy in tumor-bearing mice

[0177] It was verified that the TCR stably transferred T cells in Example 5 had anti-tumor function in animals.

[0178] Experimental animals: B-NDG immunodeficient mice, 5-6 weeks old, female.

[0179] Experimental groups: control group and different experimental groups, 6 to 8 mice in each group.

[0180] T cells: The T cells obtained in Example 5 were successfully cultured and used as T cells for functional detection in this experiment.

[0181] Tumor-bearing cells: A375 cells.

[0182] Experimental operation:

[0183] (1) Collect tumor cells in the exponential phase, remove the culture medium by centrifugation, wash the cells once with sterile PBS, resuspend them in serum-free culture medium, count the live cells, and adjust the cell density to 2.5×10 7 Select B-NDG mice that have passed the adaptive observation and inoculate subcutaneously. Each animal is inoculated with 0.1 mL, that is, the inoculated cell volume is 2.5 x 10 6 (2) Observe every day after inoculation. When the average tumor volume is 80-120mm 3 (3) After grouping, the TCR T cells obtained in Example 5 were injected into the groups at a dose of 5 x 10 6 (4) On the day of T cell injection, IL-2 was injected intraperitoneally at a dose of 50,000 IU / animal, once a day for 7 consecutive days. (5) After injection of TCR T cells, the animal's physical signs were observed every day. The tumor diameter was measured on the day of administration (D0), and the long diameter (a) and short diameter (b) of the tumor were measured with a vernier caliper every 3 days after administration. (6) According to the formula 1 / 2ab 2 Calculate the tumor volume. When the tumor in the control group grows to the humanitarian endpoint, the mice are killed. The tumor size data are counted and analyzed.

[0184] The results are as follows Fig.10 As shown, compared with the natural 1G4 TCR group, 1G4α+A51Qβ and 1G4α+A51Eβ have better tumor (A375) inhibition effects. Compared with the 1G4α(T95L, S96Y)+1G4β group, 1G4α(T95L, S96Y)+A51Qβ and 1G4α(T95L, S96Y)+A51Eβ have better tumor (A375) inhibition effects.

[0185] Example 9 HLA screening experiment

[0186] The 1G4 TCR stably transfected T cells in Example 5 were verified to be activated by various SLLMWITQC-HLA-(A*02:01, A*02:02, A*02:03, A*02:05, A*02:06, A*02:09, A*02:10) complexes. The production of IFN-γ was detected using Elisa technology as a marker molecule for T cell activation.

[0187] Target cells: K562 cells were used to construct cell lines expressing different HLAs, including HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, HLA-A*02:09, and HLA-A*02:10.

[0188] T cells: After the T cells in Example 5 were successfully cultured, they were used as T cells for functional detection in this experiment.

[0189] Small peptide: synthetic SLLMWITQC, working concentration (1 μg / mL, 0.1 μg / mL).

[0190] Experimental operation:

[0191] (1) 3x10 4 K562 cells, 50 μL volume, were plated in 96-well round-bottom plates. (2) The concentrations of the small peptides were adjusted to 2 μg / mL and 0.2 μg / mL, respectively, and plated in 100 μL volume in the well plate. (3) The T cells in Example 5, 3×10 4 Cells, 50 μL volume, were plated on a well plate. (4) The above system was incubated for 24 hours and the supernatant was collected. (5) The IFN-γ content in the supernatant was detected using an IFN-γ ELISA kit (MabTech, 3420-1H-20). The IFN-γ secretion was used as a marker of T cell activation. (6) The IFN-γ ELISA experiment was performed according to the instructions. The main operations are as follows: add 100 μL of capture antibody solution to each well, cover with sealing film, incubate overnight at 4°C, and discard the solution in the plate after overnight; add 300 μL of washing solution to each well and wash five times; dilute the sample with sample diluent at 1:5 and 1:20; prepare IFN-γ protein standard and draw a standard curve at this concentration; add samples, standards, and sample diluent to the corresponding wells at 100 μL / well as standard test group, standard group, and blank group, respectively, and incubate at room temperature for 2 hours; add 300 μL of washing solution to each well and wash five times; add biotin-conjugated antibodies to all wells and incubate at room temperature in the dark for 2 hours; add 300 μL of washing solution to each well and wash five times; add 100 μL of diluted streptavidin-HRP to all wells and incubate at room temperature in the dark for 1 hour; add 300 μL of washing solution to each well and wash five times; add 100 μL TMB colorimetric solution was added and incubated at room temperature in the dark for 30 min. 50 μL of stop solution was added to all wells and the A450 value was measured after mixing. The IFN-γ secretion of the experimental group samples was calculated based on the standard curve.

[0192] The results are as follows Fig.11 As shown, 1G4α(T95L, S96Y)+1G4β, 1G4α(T95L, S96Y)+A51Qβ, 1G4α(T95L, S96Y)+A51Eβ can all be activated by different SLLMWITQC-HLA stimulation. The main HLAs include but are not limited to HLA-A*02:01, HLA-A*02:02, HLA-A*02:03, HLA-A*02:05, HLA-A*02:06, HLA-A*02:09, and HLA-A*02:10.

[0193] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A TCR variant comprising an α chain variable region and a β chain variable region, wherein: The α chain variable region is obtained by replacing at least one amino acid with the amino acids shown in SEQ ID NO: 2; and / or The beta chain variable region is obtained by replacing at least one amino acid with the amino acids shown in SEQ ID NO:

4.

2. The TCR variant according to claim 1, characterized in that At least one alanine amino acid in the amino acids shown in SEQ ID NO: 2 is replaced; Preferably, the alanine at position 51 in the amino acid sequence of SEQ ID NO: 2 is replaced; Preferably, the alanine is replaced with at least one of threonine, histidine, lysine, glutamine and serine; preferably, at least one threonine and / or serine in the amino acids shown in SEQ ID NO: 4 is replaced; Preferably, the threonine at position 95 in the amino acid sequence of SEQ ID NO:4 is replaced; Preferably, the serine at position 96 in the amino acid sequence of SEQ ID NO:4 is replaced; Preferably, the threonine is replaced by lysine, and the serine is replaced by tyrosine.

3. The TCR variant according to claim 1 or 2, characterized in that The amino acids in the beta chain variable region are shown in SEQ ID NO:4, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20; and / or The amino acids in the α chain variable region are shown in SEQ ID NO:2 and SEQ ID NO:

21.

4. The TCR variant according to claim 3, characterized in that The TCR variant contains an α chain variable region of the amino acid sequence shown in SEQ ID NO: 2 and a β chain variable region of the amino acid sequence shown in SEQ ID NO: 16; or The TCR variant contains an α chain variable region of the amino acid sequence shown in SEQ ID NO: 2 and a β chain variable region of the amino acid sequence shown in SEQ ID NO: 17; or The TCR variant contains an α chain variable region of the amino acid sequence shown in SEQ ID NO: 2 and a β chain variable region of the amino acid sequence shown in SEQ ID NO: 18; or The TCR variant contains the α chain variable region of the amino acid sequence shown in SEQ ID NO: 2 and the β chain variable region of the amino acid sequence shown in SEQ ID NO: 19; or The TCR variant contains the α chain variable region of the amino acid sequence shown in SEQ ID NO: 2 and the β chain variable region of the amino acid sequence shown in SEQ ID NO: 20; or The TCR variant contains an α chain variable region of the amino acid sequence shown in SEQ ID NO: 21 and a β chain variable region of the amino acid sequence shown in SEQ ID NO: 4; or The TCR variant contains an α chain variable region of the amino acid sequence shown in SEQ ID NO: 21 and a β chain variable region of the amino acid sequence shown in SEQ ID NO: 19; or The TCR variant contains an α chain variable region with an amino acid sequence as shown in SEQ ID NO:21 and a β chain variable region with an amino acid sequence as shown in SEQ ID NO:

16.

5. A multivalent TCR complex comprising at least two TCR molecules, wherein at least one TCR molecule is a TCR variant according to any one of claims 1 to 4.

6. A biomaterial related to the TCR variant according to any one of claims 1 to 4 or the multivalent TCR complex according to claim 5, wherein the biomaterial is any one of a1) to a12): a1) a nucleic acid molecule encoding the TCR variant according to any one of claims 1 to 4 or the multivalent TCR complex according to claim 5; a2) an expression cassette containing the nucleic acid molecule described in a1); a3) a recombinant vector containing the nucleic acid molecule described in a1); a4) a recombinant vector containing the expression cassette described in a2); a5) a recombinant microorganism containing the nucleic acid molecule described in a1); a6) a recombinant microorganism containing the expression cassette described in a2); a7) a recombinant microorganism containing the recombinant vector described in a3); a8) a recombinant microorganism containing the recombinant vector described in a4); a9) a transgenic cell line containing the nucleic acid molecule described in a1); a10) a transgenic cell line containing the expression cassette described in a2); a11) a transgenic cell line containing the recombinant vector described in a3); a12) A transgenic cell line containing the recombinant vector described in a4).

7. An immune cell, the immune cell expressing the TCR variant according to any one of claims 1 to 4; Preferably, the immune cells include T cells, lymphocytes or stem cells; Preferably, the T cells, lymphocytes or stem cells are selected from one or more of CD4 cells, CD8 cells, naive T cells, memory stem T cells, central memory T cells, double negative T cells, effector memory T cells, effector T cells, Th0 cells, Tc0 cells, Th1 cells, Tc1 cells, Th2 cells, Tc2 cells, Th17 cells, Th22 cells, γ / δT cells, natural killer cells, natural killer T cells, dendritic cells, hematopoietic stem cells and pluripotent stem cells.

8. A pharmaceutical composition comprising the TCR variant according to any one of claims 1 to 4, the multivalent TCR complex according to claim 5, the biomaterial according to claim 6 or the immune cell according to claim 7; Preferably, the pharmaceutical composition further comprises a combined drug; Preferably, the combination drug includes at least one of an immune effector molecule, a cytotoxic drug, and an anticancer drug; Preferably, the immune effector molecule comprises at least one of IL-2, IL-7, IL-15 and IL-21.

9. Use of the TCR variant according to any one of claims 1 to 4, the multivalent TCR complex according to claim 5, the biomaterial according to claim 6, the immune cell according to claim 7 or the pharmaceutical composition according to claim 8 in any one of (1) to (3): (1) Preparation of drugs for preventing and / or treating tumors; (2) Preparation of tumor diagnostic reagents or kits; (3) preparing a reagent for increasing the level of IFN-γ and / or TNF-α cytokine secretion by T cells; Preferably, the tumors described in (1) and (2) include at least one of sarcoma, prostate cancer, uterine cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, lung cancer, non-Hodgkin's lymphoma, multiple myeloma, melanoma, neuroblastoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, colorectal cancer, hepatobiliary carcinoma, breast cancer, bladder cancer, myeloid leukemia and acute lymphoblastic leukemia.

10. A reagent, a test kit or a medicine kit, comprising the TCR variant according to any one of claims 1 to 4, the multivalent TCR complex according to claim 5, the biomaterial according to claim 6, the immune cell according to claim 7 or the pharmaceutical composition according to claim 8.

Images