MAGE-A10 specific TCR and its applications

Abstract

The present invention discloses a MAGE-A10-specific TCR and its applications. The present invention provides a TCR with high antigen sensitivity and capable of significantly killing target cells. At the same time, it also provides nucleic acids encoding the TCR, vectors comprising the nucleic acids, cells, pharmaceutical compositions, and their applications in the preparation of pharmaceutical compositions for treating tumors and screening antigen detection products.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to MAGE-A10 specific TCR and its applications. Background Art

[0002] Melanoma associated antigen A10 (MAGE-A10) is a testicular cancer antigen associated with various cancers, and is expressed in tumors of different histological types such as lung cancer, head and neck squamous cell carcinoma, liver cancer, melanoma, etc. Due to the limited expression of MAGE-A10 protein in normal healthy human tissues and its high expression in specific cancers with obvious immunogenicity, it has become a new direction for people to study immunotherapy and cancer biomarkers.

[0003] Adoptive cell therapy is a cancer immunotherapy method that uses a patient's own T lymphocytes, which may be genetically modified to recognize specific cancer antigens, expanded in vitro and infused back into the patient's body to stimulate and expand antigen-specific T cell immunity. T cell adoptive immunotherapy based on T cell receptor (TCR) engineering has great prospects in the treatment of solid tumors. The role of TCR is to recognize antigens by recognizing MHC-epitope peptide complexes and trigger a signal cascade reaction, thereby activating T cells and playing an anti-tumor immune surveillance role. The anti-tumor immune process of T cells in tumor patients is often inhibited by tumor cells. TCR therapy screens and identifies TCR sequences that specifically bind to target antigens, modifies T cells and infuses them back into the patient's body, enabling them to specifically kill tumor cells. Therefore, identifying specific TCRs against MAGE-A10 is of great significance for the treatment of MAGE-A10 positive solid tumors. Summary of the Invention

[0004] To make up for the deficiencies of the prior art, the present invention provides TCR and its applications.

[0005] To achieve the above object, the present invention adopts the following technical solutions.

[0006] The first aspect of the present invention provides a TCR targeting melanoma-associated antigen, the TCR comprising an α-chain and a β-chain, wherein the amino acid sequences of CDR1, CDR2, and CDR3 of the α-chain are respectively as shown in SEQ ID NO: 1-3 or undergo one or more amino acid substitutions, deletions, or insertions on the amino acid sequences shown in SEQ ID NO: 1-3; the amino acid sequences of CDR1, CDR2, and CDR3 of the β-chain are respectively as shown in SEQ ID NO: 4-6 or undergo one or more amino acid substitutions, deletions, or insertions on the amino acid sequences shown in SEQ ID NO: 4-6.

[0007] Furthermore, the variable region of the α-chain of the TCR has at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO: 7, and the variable region of the β-chain of the TCR has at least 75% sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0008] Furthermore, the amino acid sequence of the variable region of the α-chain of the TCR is as shown in SEQ ID NO: 7, and the amino acid sequence of the variable region of the β-chain of the TCR is as shown in SEQ ID NO: 8.

[0009] Furthermore, the constant region of the α-chain of the TCR has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 9, and the constant region of the β-chain of the TCR has at least 70% sequence identity with the amino acid sequence shown in SEQ ID NO: 10.

[0010] Furthermore, the amino acid sequence of the constant region of the α-chain of the TCR is as shown in SEQ ID NO: 9, and the amino acid sequence of the constant region of the β-chain of the TCR is as shown in SEQ ID NO: 10.

[0011] Furthermore, the α-chain and the β-chain of the TCR are connected directly or indirectly.

[0012] Furthermore, the α-chain and the β-chain of the TCR are connected indirectly.

[0013] Furthermore, they are indirectly connected through a self-cleaving peptide.

[0014] Furthermore, the connection order of the α-chain and the β-chain can be α-chain - self-cleaving peptide - β-chain, or β-chain - self-cleaving peptide - α-chain.

[0015] Furthermore, the connection order of the α-chain and the β-chain is β-chain - self-cleaving peptide - α-chain.

[0016] Furthermore, the complete sequence of the TCR has at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO: 11.

[0017] Furthermore, the complete sequence of the TCR is as shown in SEQ ID NO: 11.

[0018] Furthermore, the melanoma-associated antigen is melanoma-associated antigen A10.

[0019] The second aspect of the present invention provides a multivalent TCR complex, which complex comprises the TCR of the first aspect of the present invention.

[0020] Furthermore, the complex comprises 2, 3, 4 or more TCRs.

[0021] Furthermore, the complex is present in a lipid bilayer or attached to a particle.

[0022] Furthermore, the TCRs are conjugated via a linker molecule.

[0023] The third aspect of the present invention provides a chimeric molecule, which chimeric molecule comprises the TCR of the first aspect of the present invention or a part thereof conjugated to a non-cellular substrate, a toxin and / or an antibody.

[0024] Furthermore, the non-cellular substrate comprises nanoparticles, exosomes.

[0025] The fourth aspect of the present invention provides a nucleic acid encoding the TCR of the first aspect of the present invention, encoding the complex of the second aspect of the present invention or encoding the chimeric molecule of the third aspect of the present invention.

[0026] The fifth aspect of the present invention provides a vector comprising the nucleic acid of the fourth aspect of the present invention.

[0027] Furthermore, the vector further comprises elements for controlling expression.

[0028] Furthermore, the vector further comprises materials that facilitate its entry into cells.

[0029] The sixth aspect of the present invention provides a cell, which cell comprises the nucleic acid of the fourth aspect of the present invention or the vector of the fifth aspect of the present invention.

[0030] Furthermore, the cell comprises T cells, lymphocytes or stem cells.

[0031] Furthermore, the cell is selected from T cells.

[0032] The seventh aspect of the present invention provides a pharmaceutical composition, which pharmaceutical composition comprises the TCR of the first aspect of the present invention, the complex of the second aspect of the present invention, the chimeric molecule of the third aspect of the present invention, the nucleic acid of the fourth aspect of the present invention, the vector of the fifth aspect of the present invention or the cell of the sixth aspect of the present invention.

[0033] Furthermore, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0034] The eighth aspect of the present invention provides the use of the TCR described in the first aspect of the present invention, the complex described in the second aspect of the present invention, the chimeric molecule described in the third aspect of the present invention, the nucleic acid described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, or the cell described in the sixth aspect of the present invention in the preparation of a pharmaceutical composition for treating tumors.

[0035] Furthermore, the tumor is selected from MAGE-related tumors.

[0036] Furthermore, the MAGE-related tumors include prostate cancer, uterine cancer, thyroid cancer, testicular cancer, kidney cancer, pancreatic cancer, ovarian cancer, esophageal cancer, non-small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, non-Hodgkin lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, cervical cancer, colorectal cancer, gastric adenocarcinoma, cholangiocarcinoma, breast cancer, bladder cancer, myeloid leukemia and acute lymphoblastic leukemia, sarcoma or osteosarcoma.

[0037] The ninth aspect of the present invention provides the use of the TCR described in the first aspect of the present invention, the complex described in the second aspect of the present invention, the chimeric molecule described in the third aspect of the present invention, the nucleic acid described in the fourth aspect of the present invention, the vector described in the fifth aspect of the present invention, or the cell described in the sixth aspect of the present invention in in vitro antigen detection or in the preparation of an antigen detection product.

[0038] Furthermore, the antigen is selected from MAGE.

[0039] Furthermore, the MAGE-A10.

[0040] Advantages and beneficial effects of the present invention:

[0041] The TCR-transduced T cells provided by the present invention have high antigen sensitivity and can significantly kill target cells, and have broad application prospects. Description of the Drawings

[0042] Figure 1 It is a result diagram of inducing MAGE-A10-specific T cells after DC cells are loaded with MAGE-A10 antigen peptide (GLYDGMEHL, SEQ ID NO:14) and co-incubated with T cells;

[0043] Figure 2 It is a structural diagram of TCR;

[0044] Figure 3 It is a structural diagram of the HLA expression vector;

[0045] Figure 4It is a flow cytometry detection result diagram of pMHC tetramer staining of NFAT-CD8-luc Jurkat cells transduced with different TCRs in an electroporation form;

[0046] Figure 5 It is a detection result diagram of the NFAT-CD8-luc Jurkat reporter system expressing different TCRs;

[0047] Figure 6 It is a flow cytometry detection result diagram of pMHC tetramer staining of activated peripheral blood T cells transduced with different TCRs in an electroporation form;

[0048] Figure 7 It is the detection result of the expression of the T cell activation marker 4-1BB and the EC 50 value diagram after overnight co-incubation of activated peripheral blood T cells from the same donor transduced with different TCRs in an electroporation form and T2 cells loaded with different concentration gradients of antigenic peptides;

[0049] Figure 8 It is a flow cytometry detection result diagram of pMHC tetramer staining of T cells in the knock-in TCR form;

[0050] Figure 9 It is a detection result diagram of the killing of target cells expressing MAGE-10 by T cells in the knock-in TCR form;

[0051] Figure 10 It is the recognition motif diagram of the G60-01 sequence. Detailed implementation manners

[0052] The following provides definitions of some terms used in this specification. Unless otherwise specified, all technical and scientific terms used herein generally have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs.

[0053] The present invention provides a TCR targeting a melanoma-associated antigen.

[0054] In some embodiments, the T cell receptor (TCR) refers to a molecule found on the surface of T cells (or T lymphocytes), which associates with CD3 and is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In most T cells, the TCR is a heterodimer of highly variable α and β chains (also called TCRα and TCRβ, respectively) linked by disulfide bonds. The variable regions of the TCRα and β chains have three hypervariable or complementarity-determining regions (CDRs).

[0055] The amino acid sequences of CDR1, CDR2, and CDR3 of the α chain include one or more amino acid substitutions, deletions, or insertions on the amino acid sequences shown in SEQ ID NOs: 1-3; the amino acid sequences of CDR1, CDR2, and CDR3 of the β chain include one or more amino acid substitutions, deletions, or insertions on the amino acid sequences shown in SEQ ID NOs: 4-6.

[0056] In some embodiments, one or more amino acid substitutions, deletions, or insertions can be, for example, one, two, or three amino acid substitutions, deletions, or insertions that do not affect the function of the amino acids.

[0057] The present invention provides a chimeric molecule that includes the above TCR or a portion thereof conjugated to a non-cellular substrate, toxin, and / or antibody.

[0058] In some embodiments, the chimeric molecule includes the TCR or a portion thereof described in the present application conjugated to a non-cellular substrate, and the non-cellular substrate includes nanoparticles, exosomes, or any other non-cellular substrate known in the art.

[0059] In some embodiments, the chimeric molecule includes the TCR or a portion thereof described in the present application conjugated to a toxin and / or antibody. The toxin or antibody can be cytotoxic. The toxin can be a cytotoxic molecule or compound, such as a radioactive molecule or compound. The TCR portion of the chimeric molecule can confer the ability to recognize cells expressing an antigenic protein or peptide. Thus, the chimeric molecule can specifically recognize and / or bind tumor cells expressing an antigen. Therefore, the chimeric molecule of the present application can provide antigen-targeted delivery of cytotoxic toxins, antibodies, and / or compounds.

[0060] The present invention provides a multivalent TCR complex that includes the above TCR.

[0061] In some embodiments, the multivalent TCR complex includes a multimer in which two or three or four or more TCRs are associated (e.g., covalently or otherwise linked) to each other through a linker molecule. Suitable linker molecules include, but are not limited to, multivalent attachment molecules such as avidin, streptavidin, neutravidin, and extravidin, each of which has four binding sites for biotin. Thus, biotinylated TCR molecules can form a multimer of TCRs with multiple TCR binding sites. The number of TCR molecules in the multimer will depend on the amount of TCR relative to the amount of linker molecule used to make the multimer, and also on the presence of any other biotinylated molecules. Preferred multimers are dimeric, trimeric, or tetrameric TCR complexes.

[0062] In some embodiments, a label or additional moiety (e.g., a toxic or therapeutic moiety) can be included in the multivalent TCR complexes of the present application. For example, the label or additional moiety can be included in a mixed molecular multimer. An example of such a multimeric molecule is a tetramer that includes three TCR molecules and one peroxidase molecule. This can be achieved by mixing TCR and enzyme in a 3:1 molar ratio to produce a tetrameric complex and separating the desired complex from any complex that does not contain molecules in the correct ratio. These mixed molecules can include any combination of molecules provided that steric hindrance does not impair or does not significantly impair the desired function of the molecules. Since steric hindrance is unlikely to occur, the positioning of the binding sites on the streptavidin molecule is suitable for the mixed tetramer.

[0063] The present invention provides a nucleic acid encoding the above TCR or encoding the above complex.

[0064] In some embodiments, nucleic acid, nucleic acid molecule, or polynucleotide can be used interchangeably and refers to any deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotide, such as a fragment produced by polymerase chain reaction (PCR), or by in vitro translation, and fragments produced by any ligation, cleavage, endonuclease action, or exonuclease action. In certain embodiments, the nucleic acids of the present application are produced by PCR. The nucleic acid can be composed of monomers that are naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (such as the α-enantiomeric form of a naturally occurring nucleotide), or a combination of both. Modified nucleotides can have modifications or substitutions in the sugar moiety or the pyrimidine or purine base moiety. Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include phosphorothioates, dithiophosphates, selenophosphates, diselenophosphates, anilinothiophosphates, anilinophosphates, aminophosphates, and the like. The nucleic acid molecule can be single-stranded or double-stranded.

[0065] In some embodiments, the nucleic acid can be a variant of a polynucleotide encoding a modified TCR. The polynucleotide variant can have substantial identity with the nucleic acid sequence encoding the modified TCR described in the present application. For example, the polynucleotide can be a polynucleotide that, using the methods described in the present application (e.g., BLAST analysis using standard parameters, as described below), has at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity when compared to a reference polynucleotide sequence (such as the sequence encoding the TCR described in the present application). Those skilled in the art will recognize that these values can be adjusted appropriately to determine the corresponding identity of the proteins encoded by two nucleotide sequences by considering codon degeneracy, amino acid similarity, reading frame positioning, and the like.

[0066] The present invention provides a vector comprising the above nucleic acid.

[0067] In some embodiments, a vector is a tool that allows or facilitates the transfer of an entity from one environment to another. Vectors can be used for the purpose of maintaining a heterologous nucleic acid (DNA or RNA) within a cell, promoting the replication of a vector containing a nucleic acid segment, or promoting the expression of a protein encoded by a nucleic acid segment.

[0068] In some embodiments, the vector can be, for example, a plasmid, cosmid, virus, RNA vector, or linear or circular DNA or RNA molecule, which may comprise chromosomal, extrachromosomal, semisynthetic, or synthetic nucleic acid molecules. Exemplary vectors are vectors capable of autonomous replication (episomal vectors) or expressing nucleic acid molecules ligated thereto (expression vectors).

[0069] In a preferred embodiment, the vector is selected from plasmid vectors.

[0070] The present invention provides a cell comprising the above nucleic acid or the above vector.

[0071] In some embodiments, the cell can be a T cell, lymphocyte, or stem cell.

[0072] In a preferred embodiment, the cell is selected from T cells. The T cell can be any T cell, such as a cultured T cell (e.g., a primary T cell, or a T cell from a cultured T cell line (e.g., Jurkat, SupT1, etc.)), or a T cell obtained from a mammal. If obtained from a mammal, the T cell can be obtained from a variety of sources, including but not limited to blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cell can also be enriched or purified. The T cell can be any type of T cell and can be at any stage of development, including but not limited to CD4 + / CD8 + double positive T cells, CD4 + helper T cells (e.g., Th1 and Th2 cells), CD4 + T cells, CD8 + T cells (e.g., cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells (e.g., central memory T cells and effector memory T cells), naive T cells, etc.

[0073] In some embodiments, a variety of techniques known in the art, such as transformation, transfection, and transduction, can be used to introduce a vector comprising the nucleic acid for use in the present application into a cell.

[0074] The present invention provides the use of the above-mentioned TCR, the above-mentioned complex, the above-mentioned nucleic acid, the above-mentioned vector or the above-mentioned cell in the preparation of a pharmaceutical composition for treating diseases related to melanoma-associated antigens.

[0075] The pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0076] In some embodiments, a pharmaceutically acceptable carrier refers to a carrier for administering a therapeutic agent. The term refers to such pharmaceutical carriers that do not themselves induce the production of antibodies harmful to the individual receiving the composition and are not unduly toxic after administration. These carriers are well known to those of ordinary skill in the art. A full discussion of pharmaceutically acceptable excipients can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Such carriers include, but are not limited to: saline, buffers, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.

[0077] The pharmaceutically acceptable carrier in a therapeutic composition may contain liquids such as water, saline, glycerol, and ethanol. Additionally, auxiliary substances such as wetting agents or emulsifying agents, pH buffering substances, etc. may also be present in these carriers.

[0078] Generally, the therapeutic composition can be made into an injectable, such as a liquid solution or suspension; it can also be made into a solid form suitable for formulation into a solution or suspension in a liquid carrier before injection.

[0079] Once formulated into the pharmaceutical composition of the present application, it can be administered by conventional routes, including but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably parenterally including subcutaneous, intramuscular, or intravenous. The subject to be prevented or treated can be an animal; especially a human.

[0080] When the pharmaceutical composition of the present application is used for actual treatment, pharmaceutical compositions of various different dosage forms can be adopted according to the usage. For example, injections, oral preparations, etc.

[0081] These pharmaceutical compositions can be formulated by conventional methods by mixing, diluting, or dissolving, and occasionally adding suitable pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifying agents, dispersants, stabilizers, and solubilizers, and the formulation process can be carried out in a conventional manner according to the dosage form.

[0082] The pharmaceutical composition of the present application can also be administered in the form of a sustained-release agent. For example, the TCR of the present application can be incorporated into pills or microcapsules with a sustained-release polymer as a carrier, and then the pills or microcapsules are surgically implanted into the tissue to be treated. Examples of the sustained-release polymer include ethylene-vinyl acetate copolymer, polyhydrometaacrylate, polyacrylamide, polyvinylpyrrolidone, methylcellulose, lactic acid polymer, lactic acid-glycolic acid copolymer, etc., such as biodegradable polymers like lactic acid polymer and lactic acid-glycolic acid copolymer.

[0083] When the pharmaceutical composition of the present application is used for actual treatment, the TCR or TCR complex of the present application as the active ingredient or the cells presenting the TCR of the present application can be reasonably determined according to the weight, age, gender, and degree of symptoms of each patient to be treated, and finally the reasonable dosage is determined by the physician.

[0084] In some embodiments, melanoma-associated antigen diseases include but are not limited to melanoma, liver cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, and head and neck squamous cell carcinoma.

[0085] The present invention will be further described below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the present invention. Without departing from the scope of the present invention, the main features of the present invention can be used in various embodiments.

[0086] Examples

[0087] Example 1 Amplification of MAGEA10-Specific T Cells

[0088] Step 1 Sort CD14 from PBMC + Monocytes and Induce Differentiation

[0089] Cryopreserved PBMC of HLA-A*02:01 type was purchased from Miaoshun Biology. The resuscitated PBMC was resuspended in RPMI 1640 medium, the cell count was completed and the cell viability was recorded. Then, it was centrifuged at a centrifugal force of 350g for 5 minutes, and the supernatant was completely discarded. According to the counting result, the cells were resuspended with magnetic bead sorting buffer at 1×10 8 / mL, and then 100 μL of EasySep 8 Human CD14 Positive Selection Cocktail Ⅱ was added at a ratio of per 1×10 TM cells. After pipetting and mixing evenly, it was left standing at room temperature for 15 minutes; the magnetic beads (EasySep TMDextran RapidSpheres™ 50100) vortex oscillation to ensure thorough mixing. Add magnetic beads to the cell suspension at a ratio of 100 μL per 1×10 8 cells. After pipetting and mixing, let it stand at room temperature for 6 minutes. Add an appropriate volume of sorting buffer, mix well, transfer to a sorting tube, and place it on a sorting rack. After standing for 15 minutes, the sorting is completed. The sorted CD14 + monocytes are resuspended in RPMI 1640 medium containing 10% FBS and supplemented with 20 ng / mL of IL-4 and 100 ng / mL of GM-CSF. The cells are seeded in a six-well plate at a density of 1×10 6 / mL and placed in a 37°C carbon dioxide incubator for culture. After 3 days of cell culture, adjust the medium and supplement 50 ng / mL of TNF-α for 16 hours. Mature DC cells are loaded with antigen peptides.

[0090] Step 2 Sorting of T cells from CD14− PBMCs

[0091] Enrich T cells from cryopreserved CD14− PBMCs by negative selection. The sorted T cells are resuspended in X-VIVO 15 medium containing 5% human serum and supplemented with 5 ng / mL of IL-7, and placed in a 37°C carbon dioxide incubator for 1 day to prepare for co-culture with DC cells.

[0092] Step 3 Co-culture of DC and T cells

[0093] DC cells and T cells are co-cultured in two rounds at a ratio of 1:2. The medium during the co-culture stage is X-VIVO15 medium containing 5% human serum and supplemented with 10 ng / mL of IL-7, 10 ng / mL of IL-15, and 20 ng / mL of IL-2. After 14 days of culture, the specific T cells induced in each group of samples are detected by flow cytometry.

[0094] Figure 1 The induced MAGE-A10-specific T cells are shown. The proportions of specific T cells induced by co-culture of DC cells loaded with antigen peptides and T cells are 1.72%, 0.27%, 0.03%, and 0.20% respectively.

[0095] Sort each group of MAGEA10-specific T cells into 96-well plates by flow sorting for monoclonal sorting, extract RNA, and reverse transcribe to obtain cDNA. Apply multiplex PCR to amplify the TCRα and TCRβ genes, and select high-frequency clones for plasmid synthesis.

[0096] Example 2 Construction of an in vitro transcription plasmid vector expressing MAGE-A10-specific TCR and preparation of mRNA

[0097] The α and β variable region sequences of the MAGE-A10-specific TCR obtained by sequencing were respectively fused with the α constant region and β constant region of mice. The TCRα and TCRβ chains were linked with the P2A sequence, and the structural diagram is as shown in Figure 2 Figure [not provided in the original, assumed to be missing], and the TCR sequences are shown in Table 1.

[0098] The TCRα / β in the codon-optimized exogenous TCR gene was synthesized by gene synthesis and digested with BamHI and SacI restriction endonucleases, and then cloned between the BamHI and SacI restriction sites of the in vitro transcription (IVT) plasmid vector. A Kozak (GCCACC) sequence was added downstream of the BamHI restriction site, and a double stop codon (TGATAA) sequence was added upstream of the SacI restriction site. After the construction of the in vitro transcription plasmid vector, mRNA was synthesized, and the synthesized mRNA was temporarily stored at -80 °C for in vitro functional verification.

[0099] Table 1 TCR sequences

[0100] Name Amino acid sequence G60-01-α-CDR1 TTLSN (SEQ ID NO:1) G60-01-α-CDR2 LVKSGEV (SEQ ID NO:2) G60-01-α-CDR3 CAGWTISNFGNEKLTF (SEQ ID NO:3) G60-01-β-CDR1 MDHEN (SEQ ID NO:4) G60-01-β-CDR2 SYDVKM (SEQ ID NO:5) G60-01-β-CDR3 CASRSRQGGTGELFF (SEQ ID NO:6) G60-01-α-Variable region MLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGWTISNFGNEKLTFGTGTRLTIIP (SEQ ID NO:7) G60-01-β-Variable region MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASRSRQGGTGELFFGEGSRLTVL (SEQ ID NO:8) G60-01-α-Constant region NIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:9) G60-01-β-Constant region EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNS (SEQ ID NO:10) G60-01 Complete sequence MGIRLLCRVAFCFLAVGLVDVKVTQSSRYLVKRTGEKVFLECVQDMDHENMFWYRQDPGLGLRLIYFSYDVKMKEKGDIPEGYSVSREKKERFSLILESASTNQTSMYLCASRSRQGGTGELFFGEGSRLTVLEDLRNVTPPKVSLFEPSKAEIANKQKATLVCLARGFFPDHVELSWWVNGKEVHSGVSTDPQAYKESNYSYCLSSRLRVSATFWHNPRNHFRCQVQFHGLSEEDKWPEGSPKPVTQNISAEAWGRADCGITSASYHQGVLSATILYEILLGKATLYAVLVSGLVLMAMVKKKNSRAKRSGSGATNFSLLKQAGDVEENPGPMLLITSMLVLWMQLSQVNGQQVMQIPQYQHVQEGEDFTTYCNSSTTLSNIQWYKQRPGGHPVFLIQLVKSGEVKKQKRLTFQFGEAKKNSSLHITATQTTDVGTYFCAGWTISNFGNEKLTFGTGTRLTIIPNIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINVPKTMESGTFITDKTVLDMKAMDSKSNGAIAWSNQTSFTCQDIFKETNATYPSSDVPCDATLTEKSFETDMNLNFQNLSVMGLRILLLKVAGFNLLMTLRLWSS (SEQ ID NO:11) G60-01-α variable region DNA sequence ATGCTGCTGATCACCTCTATGCTGGTGCTTTGGATGCAACTGTCTCAGGTCAACGGCCAGCAGGTTATGCAAATTCCTCAGTATCAGCACGTCCAGGAGGGCGAGGACTTTACAACGTACTGTAACTCCTCGACCACACTGAGCAACATCCAGTGGTACAAGCAGCGCCCCGGCGGCCACCCCGTGTTCCTGATTCAGCTGGTGAAGTCTGGTGAGGTGAAGAAGCAGAAGCGCCTCACGTTCCAGTTCGGGGAGGCCAAGAAGAACAGCTCTCTGCACATTACTGCTACCCAGACCACCGATGTGGGCACCTATTTCTGCGCCGGCTGGACCATTAGCAACTTCGGCAACGAGAAGCTGACCTTCGGTACCGGTACTCGCCTCACCATCATCCCC (SEQ ID NO:12) G60-01-β-Variable region DNA sequence ATGGGCATCCGCTTGCTGTGCCGTGTGGCGTTCTGTTTCCTGGCCGTGGGATTGGTTGATGTAAAGGTGACCCAGAGCAGCCGCTACCTCGTCAAGCGCACCGGCGAGAAAGTATTCTTGGAGTGCGTGCAGGACATGGATCATGAGAACATGTTCTGGTACAGGCAGGACCCGGGGCTGGGCCTCCGCCTCATCTACTTCAGCTACGACGTGAAGATGAAGGAAAAGGGCGACATCCCTGAAGGCTATTCCGTATCCCGCGAGAAAAAGGAGAGGTTCTCTCTTATCCTGGAGAGCGCCTCCACGAACCAGACTTCCATGTATCTGTGCGCCTCCCGTAGTCGCCAGGGTGGCACCGGAGAGCTGTTCTTTGGAGAGGGCTCCCGTCTGACAGTGCTG (SEQ ID NO:13) Example 3 Verification of the activation of MAGE-A10-specific TCR

[0101] Step 1 Preparation of K562 cells overexpressing HLA-A*02:01

[0102] Using 293FT tool cells, the plasmid constructed with the HLA expression gene (the map is shown in Figure 3 Figure [not provided in the original, assumed to be missing]) was packaged into lentivirus, and HLA-A*02:01 was integrated into the genome of K562 cells by lentivirus infection, enabling it to obtain stable inheritance and expression. K562-HLA-A*02:01 OE was used as the target cell for the verification of specific TCR activation.

[0103] Step 2 Preparation of NFAT-CD8-Luc Jurkat cells expressing TCR

[0104] Genetically engineered NFAT-CD8-Luc Jurkat cells can verify the activation status of the TCR pathway by detecting the Luciferase fluorescence value. The TCR mRNA was transfected into NFAT-CD8-Luc Jurkat cells by electroporation to make it express specific TCR. The detailed steps are as follows: Collect NFAT-CD8-Luc Jurkat cells, centrifuge at a centrifugal force of 350 g for 5 minutes, wash once with DPBS (Cytiva), and then resuspend the cells with R solution (Thermo Fisher Scientific) to a concentration of 2×10 7 / mL. Take 100 μL of the cell suspension and add it to a 1.5 mL EP tube. Add 5 μg of TCR mRNA and mix well. After adding 3 - 5 mL of E2 solution (Thermo Fisher Scientific) to the electroporation cuvette, place it in the cuvette slot of the electroporator (Thermo Fisher Scientific). Carefully aspirate the cell suspension mixed with mRNA using a 100 μL electroporation pipette tip, avoiding the generation of air bubbles. Insert the electroporation pipette into the E2 solution in the electroporator. Set the electroporation conditions to 1400 V, 20 ms, 2 pulses, turn on the electroporation, and transduce TCR mRNA into NFAT-CD8-Luc Jurkat cells to obtain cells expressing TCR. 24 hours after the electroporation is completed, perform MAGE-A10 tetramer staining on NFAT-CD8-Luc Jurkat cells and analyze the transduction results by flow cytometry ( Figure 4 ) The experimental results demonstrate that Jurkat cells transduced with G60-01, G60-02, G60-03, and G60-05 TCRs all have Tetramer-positive cells, and the positive rates are 45.96%, 43.67%, 36.87%, and 29.81% in sequence.

[0105] Step 3 Detection results of the activation status of the TCR pathway

[0106] NFAT-CD8-Luc Jurkat cells that have been electroporated for 24 hours have been proven to be able to express TCR. Incubate them with K562-HLA-A*02:01 OE loaded with antigenic peptides (10 μM) overnight at an effector-to-target ratio of 20:1 in a 96-well plate and incubate for 4 hours in a 37°C carbon dioxide incubator. At the same time, place the Bio-Lite detection reagent (Nanjing Novoprotein Science & Technology Co., Ltd.) stored at -20°C in a dark environment to thaw and use it after it returns to room temperature. After the incubation is completed, centrifuge the samples, wash them once with DPBS, remove the supernatant, add 100 μL of the Bio-Lite detection reagent to each well, let it stand in the dark for 3 - 5 minutes, and detect it using an enzyme-linked immunosorbent assay reader.

[0107] Figure 5 Shows the fluorescence values of NFAT-CD8-Luc Jurkat cells transduced with different TCRs. The control group is untransduced cells. There are differences in the activation results of different TCRs. The fluorescence value of NFAT-Luc Jurkat cells transduced with G60-01 is the highest.

[0108] Example 4 Peptide sensitivity detection of MAGE-A10-specific TCR

[0109] Step 1 Preparation of T cells expressing TCR

[0110] Cryopreserved PBMCs from the peripheral blood of healthy donors (Miaoshun Biology) were thawed and resuspended in X-VIVO15 medium. T cells were purified and separated using magnetic beads, and the T cells were activated with CD3 / CD28 magnetic beads. The cells were resuspended in X-VIVO15 medium containing 2.5% human serum and 30 IU / mL IL-2 and cultured for three days. On the third day, the magnetic beads were removed and the culture was continued. On the fourth day, the cells were collected and centrifuged at 350 g for 5 minutes. After washing once with DPBS, the cells were resuspended in R solution at a density of 2×10 7 / mL. 100 μL of the cell suspension was added to a 1.5 mL EP tube, and 5 μg of TCR mRNA was added simultaneously. The mixture was thoroughly mixed. After adding 3 - 5 mL of E2 solution to the electroporation cuvette, the cuvette was placed in the cuvette slot of the electroporator. 100 μL of the cell suspension mixed with mRNA was carefully aspirated using an electroporation pipette tip to avoid generating air bubbles. The electroporation pipette was inserted into the E2 solution in the electroporator, and the electroporation conditions were set to 1400 V, 10 ms, 3 pulses. Electroporation was initiated to transduce TCR mRNA into the activated T cells, and T cells expressing TCR were obtained. 24 hours after the completion of electroporation, the T cells were stained with MAGE-A10 tetramer, and the transduction results were analyzed by flow cytometry ( Figure 6 ). The experimental results demonstrated that G60-01, G60-02, G60-03, and G60-05 TCRs were successfully transduced into the activated T cells, and CD4 + and CD8 + T cells were both able to detect tetramer-positive cells. The proportions of tetramer-positive cells in CD3 + T cells were 86.96%, 54.30%, 54.01%, and 28.32% respectively.

[0111] Step 2: Prepare T2 cells

[0112] T2 cells lack the peptide transporter associated with antigen processing (TAP) and are unable to transfer endogenous peptides to the MHC loading site. Their HLA type is HLA-A*02:01, making them suitable as target cells for TCR peptide sensitivity verification. The cultured T2 cells were collected, washed once, and then resuspended in RPMI 1640 medium containing 10% FBS. The cells were seeded in a 24-well plate at a density of 1×10 6 / mL, and different concentrations of antigen peptides were added respectively. The concentration gradient was 10 -15 M - 10 -6 M, with a total of 4 groups. The antigen peptide loading time was 4 hours.

[0113] Step 3: Results of polypeptide sensitivity detection

[0114] The activated T cells expressing TCR were co-cultured with T2 cells loaded with different concentrations of antigen peptides at a density of 1×105 Mix and inoculate the cells with different numbers of holes in a 96-well plate and place them in a 37°C carbon dioxide incubator for overnight culture. Collect the cells of each group the next day, stain the cells with flow antibodies CD3-Violet786, CD8-BV510, CD4-APC-Cy7, and 4-1BB-BV421, and detect them on the machine. The expression level of the CD8 + T cell activation marker 4-1BB can reflect the activation effect of target cells loaded with different concentrations of antigen peptides on T cells, and calculate to obtain the EC 50 value ( Figure 7 ), and the EC 50 value can reflect the polypeptide sensitivity of TCR. The experimental results show that the EC 50 value of G60-01 is 1.199×10 -10 M, the EC 50 value of G60-02 is 7.530×10 -10 M, the EC 50 value of G60-03 is 5.361×10 -9 M, and the EC 50 value of G60-05 is 6.496×10 -10 M.

[0115] Example 5 Detection of the killing function of MAGE-A10-specific TCR against natural antigen target cells

[0116] Step 1 Isolate and activate T cells

[0117] Resuscitate and count PBMC (Miaoshun Biology). Centrifuge at 360g for 5 min. After centrifugation, resuspend the cells by adding 100 μL of X-VIVO15, 20 μL of antibody and 20 μL of FBS per 1E7 cells, and let it stand at 4°C for 20 min. Pre-cool the centrifuge to 4°C in advance during the waiting time. Take 100 μL of negative selection magnetic beads per 1E7 cells (the magnetic beads need to be vortexed for 30 s before use), add 2 mL of X-VIVO15 to wash the magnetic beads, then let it stand on the magnetic stand for 1 min, discard the supernatant, and resuspend the magnetic beads with 2 mL of X-VIVO15. Wash the cells with 2 mL of X-VIVO15, centrifuge at 4°C and 360g for 8 min, then add the resuspended magnetic beads to the cells and resuspend, let it stand at room temperature for 15 min, place it on the magnetic stand, and aspirate the supernatant into a new 15 mL centrifuge tube. Take 25 μL of CD3 / CD28 activation magnetic beads per 1E6 cells (the magnetic beads need to be vortexed for 30 s before use), add 500 μL of X-VIVO15 to wash the magnetic beads, then let it stand on the stand for 1 min, discard the supernatant, and resuspend the magnetic beads with the same volume of X-VIVO15. Centrifuge at 360g for 5 min. After centrifuging the above cells, resuspend them with 1 mL of activation medium (X-VIVO15 + 5% FBS + 300 IU / mL IL-2 + 5 ng / mL IL-7 + 5 ng / mL IL-15), count, add CD3 / CD28 activation beads, supplement the activation medium (culture with 1 mL of activation medium per 1E6 cells), and place it in the incubator for culture.

[0118] Step 2: Prepare homologous recombination template

[0119] Dilute the G60-01 TCR homologous recombination template to 20 ng / μL, prepare the PCR system, mix well and load it onto the machine. Set the program as pre-denaturation at 94°C for 2 min; denaturation at 98°C for 10 sec, annealing at 70°C for 30 sec, extension at 68°C for 2 min, for 34 cycles; finally maintain at 10°C. After completion, take 1 μL of the PCR product, add 4 μL of water and 1 μL of 6X loading die, run on a 1% agarose gel at 130V for 20 min to identify whether the band is single. Then perform magnetic bead recovery. Add AMPure XP magnetic beads to the PCR product according to a volume ratio of 1:1, mix well and incubate for 5 min, place it on the magnetic stand and let it stand for 1 min to remove the supernatant, then wash it twice with 1 mL of 70% ethanol. After the ethanol volatilizes, elute with enzyme-free water for 10 min (elute according to 0.07 times the PCR volume), then measure the DNA concentration, dilute the concentration to 2000 ng / μL, and store the prepared DNA template in a -20°C refrigerator.

[0120] Step 3: Prepare T cells stably expressing TCR

[0121] Two days after T cell activation, collect the T cells using a 15 mL centrifuge tube, place it on a magnetic stand, and aspirate the supernatant into a new 15 mL centrifuge tube. Add 6 / 12 pmol TrueCut™ Cas9 Protein v2, 6 / 12 pmol TRAC-sgRNA, and 6 / 12 pmol TRBC-sgRNA to 5 μL of Solution R, gently mix, and let stand at room temperature for 15 minutes to form RNP. When adding PGA, note that PGA should be mixed with sgRNA first and then added to cas9 to form RNP, and 24 μg of PGA is added for every 6 pmol of Cas9. Aliquot 2E5 cells per 10 μL electroporation system, wash the cells once with DPBS, and then resuspend the cells with 6 μL of Solution R. Add 0.5 μg of dsDNA / tCTS dsDNA to the RNP solution, incubate for 30 s / 5 min respectively, and then add the cells resuspended with 5 μL of Solution R, gently mix. Aspirate the above mixed system using a Neon™ 10-μL tip, try to avoid generating air bubbles, perform electroporation using program #24 (1600 V / 10 ms / 3 pulses), and then quickly transfer it to a pre-warmed 24-well cell culture plate. Add fresh complete medium (X-VIVO15 + 5% FBS + 500 IU / mL IL-2) during the culture period. Five days later, perform MAGE-A10 tetramer staining on the T cells and analyze the transduction results by flow cytometry ( Figure 8 ), and the proportion of Tetramer-positive cells in CD8 + T cells is 19.56%.

[0122] Step 4 Detection of the killing function of T cells stably expressing TCR against natural antigen target cells

[0123] Select T cells stably expressing G60-01 TCR to detect the killing ability against target cells expressing MAGEA10 antigen. Inoculate the melanoma cell line A375 expressing MAGEA10 and the human non-small cell lung cancer cell line NCI H1703-MAGEA10 OE into a 96-well detection plate of Live-cell analysis system (Axion) for culture. After 24 hours, co-incubate the T cells transfected with TCR with the two types of target cells at an effector-to-target ratio of 4:1. After 36 hours, detect the killing effect ( Figure 9 ). The experimental results show that G60-01 has strong specific killing against both types of target cells, indicating that G60-01 has good ability to kill target cells.

[0124] Example 6 Recognition motif of MAGE-A10-specific TCR

[0125] Alanine Scanning is a conventional method used to identify specific amino acid sites that are closely related to TCR function, stability, and conformation. By replacing the amino acids at each position of the polypeptide with alanine, the active groups on the side chain are removed and replaced with a small, non-functional methyl group, which has less impact on the protein structure and can distinguish the effect of a specific amino acid on TCR recognition.

[0126] In this experiment, an alanine scanning peptide library was constructed. First, the TCR mRNA to be verified was transfected into T cells by electroporation. Four hours later, it was co-incubated with T2 cells loaded with each mutant peptide (the mutant peptide was added one day in advance and loaded overnight). The secretion of IFN-γ in each group was detected by enzyme-linked immunospot (ELISPOT) assay. The ELISPOT operation steps are as follows: After washing both effector cells and target cells once, they were resuspended in serum-free ELISPOT medium, and the cell density was adjusted to 1×10 5 / mL. The effector cells and target cells were inoculated into the pre-washed ELISPOT-IFN-γ detection plate at a ratio of 1:1. After overnight incubation, color development was carried out, and then it was placed in a cool, dark place at room temperature. After it dried naturally, an ELISPOT analyzer (C.T.L S6) was used to image and read the immunospots. The ELISPOT detection results and statistical results of G60-01-transduced T cells are shown in Figure 10 . In the statistical results, the statistical values were corrected for the background values of the corresponding control groups. Therefore, the recognition motif of G60-01 was determined to be G-YD--E-- (SEQ ID NO:15).

[0127] In vivo experiments

[0128] On Day 0, 4×10 6 human malignant melanoma cells A375 (MAGEA10 + ; HLA-A*02:01 + ) were subcutaneously transplanted into severely immunodeficient mice. On Day 2, T cells stably expressing G60-01 TCR were infused via tail vein injection in three different dose groups (1×10 7 , 1×10 6 , 1×10 5 TCR-T cells). T cells not transduced with TCR were used as the negative control group. After that, the tumor volume was measured twice a week and recorded to observe the inhibitory effect of different doses of TCR-T cells on the tumor.

[0129] The description of the above embodiments is only for understanding the method and its core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A TCR targeting melanoma-associated antigen A10, characterized in that: The TCR comprises an α chain and a β chain, and the amino acid sequences of CDR1, CDR2 and CDR3 of the α chain are shown in SEQ ID NOs: 1-3, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 of the β chain are shown in SEQ ID NOs: 4-6, respectively.

2. The TCR according to claim 1, characterized in that The amino acid sequence of the variable region of the α chain of the TCR is shown in SEQ ID NO:7, and the amino acid sequence of the variable region of the β chain of the TCR is shown in SEQ ID NO:

8.

3. The TCR according to claim 1, characterized in that The amino acid sequence of the constant region of the α chain of the TCR is shown in SEQ ID NO:9, and the amino acid sequence of the constant region of the β chain of the TCR is shown in SEQ ID NO:

10.

4. The TCR according to claim 1, characterized in that The α chain and β chain of the TCR are directly or indirectly connected.

5. The TCR according to claim 4, characterized in that The α chain and β chain of the TCR are indirectly connected.

6. The TCR according to claim 5, characterized in that Indirect attachment via a self-cleaving peptide.

7. The TCR according to claim 6, characterized in that The connection order of the α chain and the β chain is α chain-self-cleaving peptide-β chain, β chain-self-cleaving peptide-α chain.

8. The TCR according to claim 7, characterized in that The connection order of the α chain and the β chain is β chain-self-cleaving peptide-α chain.

9. The TCR according to claim 1, characterized in that The complete sequence of the TCR is shown in SEQ ID NO:

11.

10. A multivalent TCR complex, characterized in that The complex comprises the TCR described in any one of claims 1-9.

11. The multivalent TCR complex according to claim 10, characterized in that The complex comprises 2, 3, 4 or more TCRs.

12. The multivalent TCR complex according to claim 11, characterized in that The complex is present in the lipid bilayer or attached to a particle.

13. The multivalent TCR complex according to claim 12, characterized in that The TCR is conjugated via a linker molecule.

14. A chimeric molecule, characterized in that The chimeric molecule comprises the TCR of any one of claims 1-9 conjugated to a non-cellular substrate, a toxin and / or an antibody.

15. The chimeric molecule according to claim 14, characterized in that The non-cellular substrates include nanoparticles and exosomes.

16. A nucleic acid encoding the TCR of any one of claims 1-9, encoding the complex of any one of claims 10-13, or encoding the chimeric molecule of any one of claims 14-15.

17. A vector comprising the nucleic acid of claim 16.

18. The carrier according to claim 17, characterized in that The vector also includes elements for controlling expression.

19. The carrier according to claim 17, characterized in that The vector may also include materials that facilitate its entry into cells.

20. A cell, characterized in that The cell comprises the nucleic acid of claim 16 or the vector of any one of claims 17-19.

21. The cell according to claim 20, characterized in that The cells include lymphocytes or stem cells.

22. The cell according to claim 20, characterized in that The cells are selected from T cells.

23. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the TCR of any one of claims 1-9, the complex of any one of claims 10-13, the chimeric molecule of any one of claims 14-15, the nucleic acid of claim 16, the vector of any one of claims 17-19 or the cell of any one of claims 20-22.

24. The pharmaceutical composition according to claim 23, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

25. Use of the TCR according to any one of claims 1 to 9, the complex according to any one of claims 10 to 13, the chimeric molecule according to any one of claims 14 to 15, the nucleic acid according to claim 16, the vector according to any one of claims 17 to 19 or the cell according to any one of claims 20 to 22 in the preparation of a pharmaceutical composition for treating tumors; The tumor is selected from MAGE-related tumors; The MAGE-related tumors are melanoma, liver cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, and head and neck squamous cell carcinoma.

26. Use of the TCR of any one of claims 1-9, the complex of any one of claims 10-13, the chimeric molecule of any one of claims 14-15, the nucleic acid of claim 16, the vector of any one of claims 17-19 or the cell of any one of claims 20-22 in detecting melanoma-associated antigen A10 for non-diagnostic purposes or in preparing a melanoma-associated antigen A10 detection product.

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