MAGE-A10 specific TCR and application thereof

Abstract

The invention discloses a MAGE-A10 specific TCR (T cell receptor) and an application thereof. The invention provides a TCR (T cell receptor) which has high antigen sensitivity and can remarkably kill target cells, and also provides nucleic acid for coding the TCR, a carrier comprising the nucleic acid, a cell, a pharmaceutical composition and application of the TCR in preparation of the pharmaceutical composition for treating tumors and screening of 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 application. Background Art

[0002] Melanoma associated antigen A10 (MAGE-A10) is a testicular cancer antigen associated with a variety of cancers. It is expressed in different histological types of tumors, including lung cancer, head and neck squamous cell carcinoma, liver cancer, melanoma, etc. MAGE-A10 protein has limited expression in normal healthy human tissues, but is highly expressed in specific cancers with obvious immunogenicity. Therefore, it has become an ideal target for immunotherapy and a new direction for cancer biomarkers.

[0003] Adoptive cell therapy is a cancer immunotherapy method that uses the patient's own T lymphocytes, which may be genetically modified to recognize specific cancer antigens, to expand in vitro and infuse back into the patient 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 triggering signal cascade reactions, 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 transforms T cells and infuses them back into the patient's body by screening and identifying TCR sequences that specifically bind to target antigens, enabling them to specifically kill tumor cells. Therefore, identifying specific TCRs for 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 applications thereof.

[0005] To achieve the above purpose, the present invention adopts the following technical solution.

[0006] The first aspect of the present invention provides a TCR targeting a melanoma-associated antigen, the TCR comprising an α chain and a β chain, the amino acid sequences of CDR1, CDR2 and CDR3 of the α chain are respectively as shown in SEQ ID NOs: 1-3 or one or more amino acids are substituted, deleted or inserted into the amino acid sequences shown in SEQ ID NOs: 1-3; the amino acid sequences of CDR1, CDR2 and CDR3 of the β chain are respectively as shown in SEQ ID NOs: 4-6 or one or more amino acids are substituted, deleted or inserted into the amino acid sequences shown in SEQ ID NOs: 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 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.

[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 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.

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

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

[0013] Furthermore, the linkage is indirect via a self-cleaving peptide.

[0014] Furthermore, the connection order of the α chain and the β chain may 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 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 includes the TCR described in the first aspect of the present invention.

[0020] Further, 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] Further, the TCR is conjugated via a linker molecule.

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

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

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

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

[0027] Furthermore, the vector also includes elements for controlling expression.

[0028] Furthermore, the vector may also include materials that facilitate its entry into cells.

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

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

[0031] Furthermore, the cells are selected from T cells.

[0032] The seventh aspect of the present invention provides a pharmaceutical composition, which includes 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.

[0033] Furthermore, the pharmaceutical composition also includes 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] Further, 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's lymphoma, multiple myeloma, melanoma, hepatocellular carcinoma, head and neck cancer, gastric cancer, endometrial cancer, cervical cancer, colorectal cancer, gastric adenocarcinoma, bile duct cancer, 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 for in vitro antigen detection or in the preparation of antigen detection products.

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

[0039] Further, the MAGE-A10.

[0040] Advantages and beneficial effects of the present invention: 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a graph showing the results of inducing MAGE-A10-specific T cells after DC cells were loaded with MAGE-A10 antigen peptide (GLYDGMEHL, SEQ ID NO: 14) and co-incubated with T cells; Figure 2 It is a structural diagram of TCR; Figure 3 It is a structural diagram of the HLA expression vector; Figure 4 This is a flow cytometry result of pMHC tetramer staining of NFAT-CD8-luc Jurkat cells transduced with different TCRs by electroporation; Figure 5 This is a graph showing the results of the NFAT-CD8-luc Jurkat reporter system assay expressing different TCRs; Figure 6 This is a flow cytometry result of pMHC tetramer staining of peripheral blood activated T cells transduced with different TCRs by electroporation; Figure 7 The results of the expression of the T cell activation marker 4-1BB and EC were detected after the same donor peripheral blood activated T cells transduced with different TCRs by electroporation were co-incubated with T2 cells loaded with different concentrations of gradient antigen peptides overnight. 50 Value graph; Figure 8 This is a flow cytometry result of T cell pMHC tetramer staining using knock-in TCR format; Fig. 9 This is a graph showing the results of a knock-in TCR-based T cell killing test on target cells expressing MAGE-10; Fig.10 This is the recognition motif diagram of the G60-01 sequence. DETAILED DESCRIPTION

[0042] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0043] The present invention provides a TCR targeting melanoma-associated antigens.

[0044] In some embodiments, T cell receptor (TCR) refers to a molecule found on the surface of T cells (or T lymphocytes) that, in conjunction with CD3, is generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In most T cells, the TCR has a heterodimer of highly variable α and β chains (also referred to as TCRα and TCRβ, respectively) linked by disulfide bonds. The variable regions of the TCR α and β chains have three hypervariable or complementary determining regions (CDRs).

[0045] The amino acid sequences of CDR1, CDR2 and CDR3 of the α chain include one or more amino acid substitutions, deletions or insertions in 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 in the amino acid sequences shown in SEQ ID NOs: 4-6.

[0046] In some embodiments, the substitution, deletion or insertion of one or more amino acids may be, for example, a substitution, deletion or insertion of one, two or three amino acids, which does not affect the function of the amino acids.

[0047] The present invention provides a chimeric molecule comprising the above-mentioned TCR or a portion thereof conjugated to a non-cellular substrate, a toxin and / or an antibody.

[0048] In some embodiments, the chimeric molecule comprises a TCR or a portion thereof described herein conjugated to a non-cellular substrate, which includes nanoparticles, exosomes, or any other non-cellular substrate known in the art.

[0049] In some embodiments, the chimeric molecule includes a TCR or a portion thereof described herein conjugated to a toxin and / or an antibody. The toxin or antibody may be cytotoxic. The toxin may be a cytotoxic molecule or compound, such as a radioactive molecule or compound. The TCR portion of the chimeric molecule may confer the ability to recognize cells expressing antigenic proteins or peptides. Therefore, the chimeric molecule may specifically recognize and / or bind to tumor cells expressing antigens. Therefore, the chimeric molecule of the present application may provide antigen-targeted delivery of cytotoxic toxins, antibodies and / or compounds.

[0050] The present invention provides a multivalent TCR complex, which includes the above-mentioned TCR.

[0051] In some embodiments, the multivalent TCR complex includes a polymer of two or three or four or more TCRs connected by a linker molecule that are associated with each other (e.g., covalently or otherwise connected). 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. Therefore, biotinylated TCR molecules can form a polymer of TCRs with multiple TCR binding sites. The number of TCR molecules in the polymer will depend on the amount of TCR associated with the amount of the linker molecule used to make the polymer, and also depends on whether there are any other biotinylated molecules. Preferred polymers are dimers, trimers, or tetramers TCR complexes.

[0052] In some embodiments, a label or another part (e.g., a toxic or therapeutic part) may be included in the multivalent TCR complex of the present application. For example, a label or another part may be included in a mixed molecular multimer. An example of such a multimeric molecule is a tetramer, which contains three TCR molecules and one peroxidase molecule. This can be achieved by mixing the TCR and the enzyme in a molar ratio of 3:1 to produce a tetramer complex and separating the desired complex from any complex that does not contain the correct ratio of molecules. These mixed molecules may contain any combination of molecules, provided that steric hindrance does not impair or significantly impair the desired function of the molecule. Since steric hindrance is unlikely to occur, the positioning of the binding site on the streptavidin molecule is suitable for mixed tetramers.

[0053] The present invention provides a nucleic acid encoding the above-mentioned TCR or encoding the above-mentioned complex.

[0054] In some embodiments, nucleic acid or nucleic acid molecule or polynucleotide is used interchangeably, and refers to any deoxyribonucleic acid (DNA), ribonucleic acid (RNA), oligonucleotide, for example, by polymerase chain reaction (PCR), or by in vitro translation, and by any connection, breakage, endonuclease action or exonuclease action. In some embodiments, the nucleic acid of the present application is produced by PCR. Nucleic acid can be composed of monomers as naturally occurring nucleotides (such as deoxyribonucleotides and ribonucleotides), analogs of naturally occurring nucleotides (such as α-enantiomeric forms of naturally occurring nucleotides), or a combination of the two. The modified nucleotides can have modifications or substitutions of sugar moieties or pyrimidine or purine base moieties. Nucleic acid monomers can be connected by phosphodiester bonds or analogs of such bonds. Analogs of phosphodiester bonds include thiophosphates, dithiophosphates, selenophosphates, diselenosephosphates, anilinothiophosphates, aniline phosphates, aminophosphorothioates, etc. Nucleic acid molecules can be single-stranded or double-stranded.

[0055] In some embodiments, nucleic acid can be a variant of a polynucleotide encoding a modified TCR. Polynucleotide variants can have substantial consistency with the nucleic acid sequence encoding the modified TCR described in the present application. For example, polynucleotides can be polynucleotides comprising at least 70% sequence identity, preferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or higher sequence identity compared to a reference polynucleotide sequence (e.g., a sequence encoding the TCR described in the present application). Those skilled in the art will recognize that these values ​​can be appropriately adjusted so as to determine the corresponding consistency of the protein encoded by the two nucleotide sequences by considering code degeneracy, amino acid similarity, reading frame positioning, etc.

[0056] The present invention provides a vector comprising the above nucleic acid.

[0057] In some embodiments, a vector is a tool that allows or facilitates the transfer of an entity from one environment to another. A vector can be used for the purpose of maintaining a heterologous nucleic acid (DNA or RNA) within a cell, facilitating the replication of a vector containing a nucleic acid segment, or facilitating the expression of a protein encoded by a nucleic acid segment.

[0058] In some embodiments, the carrier can be, for example, a plasmid, a cosmid, a virus, an RNA vector, or a linear or circular DNA or RNA molecule, which can include a chromosomal, non-chromosomal, semisynthetic or synthetic nucleic acid molecule. Exemplary carriers are carriers (expression vectors) that can replicate autonomously (episomal vectors) or express nucleic acid molecules connected to them.

[0059] In a preferred embodiment, the vector is selected from a plasmid vector.

[0060] The present invention provides a cell, comprising the above nucleic acid or the above vector.

[0061] In some embodiments, the cell can be a T cell, a lymphocyte, or a stem cell.

[0062] In a preferred embodiment, the cell is selected from T cells. The T cell may 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 may 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 may also be enriched or purified. The T cell may be any type of T cell and may be of any developmental stage, including but not limited to CD4 + / CD8 + Double positive T cells, CD4 + Helper T cells (such as Th1 and Th2 cells), CD4 + T cells, CD8 + T cells (such as cytotoxic T cells), tumor infiltrating lymphocytes (TIL), memory T cells (such as central memory T cells and effector memory T cells), naive T cells, etc.

[0063] In some embodiments, a vector comprising a nucleic acid used in the present application can be introduced into a cell using various techniques known in the art, such as transformation, transfection, and transduction.

[0064] 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 preparing a pharmaceutical composition for treating melanoma-related antigen diseases.

[0065] The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0066] In some embodiments, a pharmaceutically acceptable carrier refers to a carrier for administering a therapeutic agent. The term refers to such pharmaceutical carriers: they do not induce the production of antibodies harmful to the individual receiving the composition, and there is no excessive toxicity 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., NJ 1991). Such carriers include, but are not limited to, saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and combinations thereof.

[0067] Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. In addition, auxiliary substances such as wetting agents or emulsifiers, pH buffer substances, etc. may also be present in these carriers.

[0068] Typically, therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in liquid vehicles prior to injection can also be prepared.

[0069] Once the pharmaceutical composition of the present application is formulated, it can be administered by conventional routes, including but not limited to: intraocular, intramuscular, intravenous, subcutaneous, intradermal, or topical administration, preferably parenteral including subcutaneous, intramuscular or intravenous. The subject to be prevented or treated can be an animal; especially a human.

[0070] When the pharmaceutical composition of the present application is used for actual treatment, the pharmaceutical composition can be used in various dosage forms according to the usage, such as injection, oral preparation, etc.

[0071] These pharmaceutical compositions can be formulated by mixing, diluting or dissolving according to conventional methods, and appropriate pharmaceutical additives such as excipients, disintegrants, binders, lubricants, diluents, buffers, isotonicities, preservatives, wetting agents, emulsifiers, dispersants, stabilizers and solubilizers are occasionally added, and the formulation process can be carried out in a conventional manner according to the dosage form.

[0072] 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 a pill or microcapsule with a sustained-release polymer as a carrier, and then the pill or microcapsule is surgically implanted into the tissue to be treated. As examples of sustained-release polymers, there can be cited ethylene-vinyl acetate copolymers, polyhydroxymethylacrylate (polyhydrometaacrylate), polyacrylamide, polyvinyl pyrrolidone, methylcellulose, lactic acid polymers, lactic acid-glycolic acid copolymers, etc., such as biodegradable polymers such as lactic acid polymers and lactic acid-glycolic acid copolymers.

[0073] When the pharmaceutical composition of the present application is used for actual treatment, the TCR or TCR complex of the present application or the cells presenting the TCR of the present application as the active ingredient can be reasonably determined based on the weight, age, gender, and symptom severity of each patient to be treated, and the reasonable dosage is ultimately determined by the physician.

[0074] In some embodiments, the melanoma-associated antigenic disease includes but is not limited to melanoma, liver cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, head and neck squamous cell carcinoma.

[0075] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0076] Example

[0077] Example 1 Expansion of MAGEA10-specific T cells Step 1: Isolation of CD14 from PBMC + Monocytes and differentiation induction HLA-A*02:01 type cryopreserved PBMCs were purchased from Miaoshun Biotechnology. The resuscitated PBMCs were resuspended in RPMI 1640 medium and the cell count was completed and the cell viability was recorded. Then, they were centrifuged at 350g for 5 minutes, the supernatant was fully discarded, and the magnetic bead sorting buffer was used according to the counting results at 1×10 8 / mL resuspend the cells, and then 8 Add 100 μL of EasySep TM Human CD14 Positive Selection Cocktail Ⅱ, pipette to mix well and let stand at room temperature for 15 minutes; TM Dextran RapidSpheres™ 50100) were vortexed to ensure thorough mixing. 8Add 100 μL of magnetic beads to the cell suspension, mix well by pipetting, and let 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. Let stand for 15 minutes to complete the sorting. The sorted CD14 + Monocytes were resuspended in 1640 medium containing 10% FBS and supplemented with 20 ng / mL IL-4 and 100 ng / mL GM-CSF. 6 / mL density was inoculated into six-well plates and placed in a 37°C CO2 incubator for culture. After 3 days of cell culture, the culture medium was adjusted and supplemented with 50ng / mL TNF-α for 16 hours. Mature DC cells were loaded with antigen peptides.

[0078] Step 2 Isolation of T cells from CD14- PBMCs T cells were enriched from frozen CD14- PBMCs by negative selection. The sorted T cells were resuspended in X-VIVO 15 medium containing 5% human serum and supplemented with 5 ng / mL IL-7. They were cultured in a 37°C CO2 incubator for 1 day before being prepared for co-culture with DC cells.

[0079] Step 3 Co-culture of DCs and T cells DC cells and T cells were co-cultured for two rounds at a ratio of 1:2. The co-culture medium was X-VIVO15 medium containing 5% human serum and supplemented with 10ng / mL IL-7, 10ng / mL IL-15, and 20ng / mL IL-2. After 14 days of culture, the specific T cells induced by each group of samples were detected by flow cytometry.

[0080] Figure 1 The results show that MAGE-A10-specific T cells were induced. The ratios of specific T cells induced by co-culturing DC cells loaded with antigen peptides and T cells were 1.72%, 0.27%, 0.03% and 0.20%, respectively.

[0081] MAGEA10-specific T cells in each group were sorted into 96-well plates as monoclonal clones by flow cytometry, RNA was extracted, and cDNA was obtained by reverse transcription. TCRα and TCRβ genes were amplified by multiplex PCR, and high-frequency clones were selected for plasmid synthesis.

[0082] Example 2 Construction of in vitro transcription plasmid vector expressing MAGE-A10 specific TCR and preparation of mRNA The α and β variable region sequences of the MAGE-A10 specific TCR obtained by sequencing were fused with the α constant region and β constant region of the mouse, respectively. The TCRα and TCRβ chains were connected by the P2A sequence, as shown in the structural diagram Figure 2The TCR sequences are shown in Table 1.

[0083] The codon-optimized exogenous TCR gene of TCRα / β was synthesized and digested with BamHI and SacI restriction endonucleases, and then cloned into 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 in vitro transcription plasmid vector was constructed, mRNA synthesis was performed, and the synthesized mRNA was temporarily stored at -80°C in preparation for in vitro functional verification.

[0084] Table 1 TCR sequences 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 series 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 Activation verification of MAGE-A10 specific TCR Step 1 Preparation of K562 cells overexpressing HLA-A*02:01 The plasmid constructed by HLA expression gene was constructed using 293FT tool cells (see the map for details) Figure 3 ) was packaged into lentivirus, and HLA-A*02:01 was integrated into the K562 cell genome by lentivirus infection to obtain stable inheritance and expression. K562-HLA-A*02:01 OE Serves as target cells for validation of specific TCR activation.

[0085] Step 2 Preparation of NFAT-CD8-Luc Jurkat cells expressing TCR The activation state of the TCR pathway can be verified by detecting the fluorescence value of Luciferase in genetically engineered NFAT-CD8-Luc Jurkat cells. TCR mRNA was transduced into NFAT-CD8-Luc Jurkat cells by electroporation to express specific TCR. The detailed steps are as follows: NFAT-CD8-Luc Jurkat cells were collected and centrifuged at 350g for 5 minutes. After washing once with DPBS (Cytiva), the cells were resuspended in R solution (Thermo Fisher Scientific) at 2×10 7 / mL, take 100μL of cell suspension and add it to a 1.5mL EP tube, add 5μg TCR mRNA, and mix thoroughly; add 3~5mL E2 solution (Thermo Fisher Scientific) to the electroporation cup, place it in the cup slot of the electroporator (Thermo Fisher Scientific), carefully absorb the cell suspension mixed with mRNA with a 100μL electroporation gun tip to avoid bubbles, insert the electroporation gun into the E2 solution of the electroporator, set the electroporation conditions to 1400V, 20ms, 2pulse, start electroporation, transduce TCR mRNA into NFAT-CD8-LucJurkat cells, and obtain cells expressing TCR. 24 hours after electroporation, NFAT-CD8-Luc Jurkat cells were stained with MAGE-A10 tetramers, and the results of transduction were analyzed on a flow cytometer ( Figure 4 ). The experimental results showed that Jurkat cells transduced with TCR of G60-01, G60-02, G60-03 and G60-05 all had Tetramer-positive cells, and the positive rates were 45.96%, 43.67%, 36.87% and 29.81% respectively.

[0086] Step 3: TCR pathway activation status detection results NFAT-CD8-Luc Jurkat cells electroporated for 24 hours have been shown to express TCR and were incubated with K562-HLA-A*02:01 loaded with antigen peptide (10 μM) overnight. OE The samples were co-incubated in a 96-well plate at a 20:1 effector-target ratio and incubated in a 37°C carbon dioxide incubator for 4 hours. At the same time, the Bio-Lite detection reagent (Nanjing Novozyme Biotech Co., Ltd.) stored at -20°C was thawed in a dark environment and allowed to return to room temperature before use. After incubation, the samples were centrifuged and washed once with DPBS, the supernatant was removed, 100 μL of Bio-Lite detection reagent was added to each well, and the mixture was kept away from light for 3 to 5 minutes before detection on an ELISA instrument.

[0087] Figure 5 The fluorescence values ​​of NFAT-CD8-Luc Jurkat cells transduced with different TCRs are shown, and the control group is untransduced cells. The activation results of different TCRs are different. The fluorescence value of NFAT-Luc Jurkat cells transduced with G60-01 is the highest.

[0088] Example 4 Peptide-sensitive detection of MAGE-A10-specific TCR Step 1 Preparation of T cells expressing TCR Cryopreserved PBMC (Miaoshun Biotechnology) from healthy donors were revived and resuspended in X-VIVO15 medium. T cells were purified and isolated using magnetic beads, activated with CD3 / CD28 magnetic beads, and resuspended in X-VIVO15 medium containing 2.5% human serum and 30 IU / mL IL-2 for three days. On the third day, the magnetic beads were removed and cultured again. On the fourth day, the cells were collected and centrifuged at 350g for 5 minutes. After washing with DPBS, the cells were resuspended in R solution to 2×10 7 / mL, take 100μL of cell suspension and add it to a 1.5mL EP tube, add 5μg TCR mRNA at the same time, and mix thoroughly; add 3~5mL E2 solution to the electroporation cup, place it in the electroporation cup slot, carefully absorb the cell suspension mixed with mRNA with a 100μL electroporation gun tip to avoid bubbles, insert the electroporation gun into the E2 solution of the electroporation instrument, set the electroporation conditions to 1400V, 10ms, 3pulse, start electroporation, transduce TCR mRNA into activated T cells, and obtain TCR-expressing T cells. 24 hours after electroporation, the T cells were stained with MAGE-A10 tetramers, and the transduction results were analyzed on a flow cytometer ( Figure 6 ). The experimental results show that G60-01, G60-02, G60-03 and G60-05TCR were successfully transduced into activated T cells, and CD4 + and CD8 + Tetramer-positive cells can be detected by T cells. Tetramer-positive cells are in the CD3 + The proportions in T cells were 86.96%, 54.30%, 54.01% and 28.32%, respectively.

[0089] Step 2: Prepare T2 cells T2 cells lack peptide transporters involved in antigen processing (TAP) and cannot transfer endogenous peptides to the MHC loading site. Their HLA type is HLA-A*02:01, so they can be used as target cells for TCR peptide sensitivity verification. Collect the cultured T2 cells, wash them once, resuspend the cells in RPMI 1640 medium containing 10% FBS, and incubate the cells at 1×10 6 / mL were inoculated in a 24-well plate, and different concentrations of antigen peptides were added, with a concentration gradient of 10 -15 M~10 -6 M, 4 groups in total, antigen peptide loading time was 4 hours.

[0090] Step 3 Peptide sensitivity test results Activated T cells expressing TCR were incubated with T2 cells loaded with different concentrations of antigen peptides at a rate of 1×10 5The number of cells per well was mixed and inoculated in a 96-well plate and placed in a 37°C carbon dioxide incubator for overnight culture. The cells of each group were collected on the second day, stained with flow cytometry antibodies CD3-Violet786, CD8-BV510, CD4-APC-Cy7, and 4-1BB-BV421, and tested on the machine. + The expression level of T cell activation marker 4-1BB can reflect the activation effect of target cells loaded with different concentrations of antigen peptides on T cells. EC 50 value( Figure 7 ), E.C. 50 The value can reflect the peptide sensitivity of TCR. The experimental results show that the EC of G60-01 50 The value is 1.199×10 -10 M, EC of G60-02 50 The value is 7.530×10 -10 M, G60-03 EC 50 The value is 5.361×10 -9 M, EC of G60-05 50 The value is 6.496×10 -10 M.

[0091] Example 5 Detection of the killing function of MAGE-A10 specific TCR on natural antigen target cells Step 1: Isolate and activate T cells PBMC (Miaoshun Biotechnology) was revived and counted. Centrifuge at 360g for 5min. After centrifugation, 100μL X-VIVO15, 20μL antibody and 20μl FBS were added to each 1E7 cells to resuspend the cells. The cells were allowed to stand at 4℃ for 20min. The waiting time was precooled to 4℃ in advance. 100μL negative selection magnetic beads were taken for each 1E7 cells (the magnetic beads need to be vortexed for 30s before use), 2mL X-VIVO15 was added to wash the magnetic beads, and then placed on the magnetic rack for 1min, the supernatant was discarded, and the magnetic beads were resuspended with 2mL X-VIVO15. The cells were washed with 2mL X-VIVO15, centrifuged at 4℃, 360g for 8min, and then the resuspended magnetic beads were added to the cells to resuspend, and the cells were allowed to stand at room temperature for 15min, placed on the magnetic rack, and the supernatant was aspirated into a new 15ml centrifuge tube. Take 25μL CD3 / CD28 activation beads for every 1E6 cells (vortex mix for 30s before use), add 500μL X-VIVO15 to wash the beads, then place on a shelf for 1min, discard the supernatant, and resuspend the beads with an equal volume of X-VIVO15. Centrifuge at 360g for 5min, centrifuge the above cells, resuspend with 1mL 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 activation medium (use 1mL activation medium for every 1E6 cells), and place in an incubator for culture.

[0092] Step 2: Preparation of homologous recombination template Dilute the G60-01 TCR homologous recombination template to 20ng / μL, prepare the PCR system, mix well and load on the machine, set the program to 94℃, 2 min pre-denaturation; 98℃, 10 sec denaturation, 70℃, 30 sec annealing, 68℃, 2min extension, 34 cycles; finally maintain at 10℃. After completion, take 1μL of PCR product, add 4μL water and 1μL 6X loading die, 1% agarose gel, 130V, 20min run to identify whether the band is single. Then, magnetic beads were recovered. AMPure XP magnetic beads were added to the PCR product at a volume ratio of 1:1, mixed and incubated for 5 minutes, placed on a magnetic stand for 1 minute to remove the supernatant, and then washed twice with 1 mL of 70% ethanol. After the ethanol evaporated, it was eluted with enzyme-free water for 10 minutes (eluted at 0.07 times the PCR volume), and then the DNA concentration was measured and diluted to 2000 ng / μL. The prepared DNA template was stored in a -20°C refrigerator.

[0093] Step 3 Preparation of T cells stably expressing TCR After 2 days of T cell activation, collect T cells in a 15mL centrifuge tube, place on a magnetic rack, and aspirate the supernatant into a new 15mL centrifuge tube. Add 6 / 12pmol TrueCut™ Cas9 Protein v2, 6 / 12pmol TRAC-sgRNA, and 6 / 12pmol TRBC-sgRNA to 5μL R solution, mix gently, and let stand at room temperature for 15 minutes to form RNP. When adding PGA, pay attention to mixing PGA with sgRNA before adding cas9 to form RNP, and add 24μg PGA for every 6pmol Cas9. Aliquot 2E5 cells per 10μL electroporation system, wash the cells once with DPBS, and then resuspend the cells with 6μL R solution. Add 0.5μg dsDNA / tCTS dsDNA to the RNP solution, incubate for 30s / 5min respectively, then add 5μL R solution resuspended cells and mix gently. Use Neon™ 10-μL tip to absorb the above mixed system, try to avoid the generation of bubbles, use program #24 (1600 V / 10 ms / 3 pulses) program for electroporation, and then quickly transfer to a preheated 24-well cell culture plate. During the culture period, add fresh complete culture medium (X-VIVO15+5%FBS+500 IU / mL IL-2). After 5 days, T cells were stained with MAGE-A10 tetramers, and the results of transduction were analyzed by flow cytometry ( Figure 8 ), Tetramer-positive cells in CD8 + The proportion of T cells was 19.56%.

[0094] Step 4: Detection of the killing function of T cells stably expressing TCR against natural antigen target cells The killing ability of T cells stably expressing G60-01 TCR against target cells expressing MAGEA10 antigen was tested. The melanoma cell line A375 expressing MAGEA10 and the human non-small cell lung cancer cell line NCI H1703-MAGEA10 were OE The cells were inoculated and cultured in a 96-well assay plate of the Live-cell analysis system (Axion). After 24 hours, the TCR knocked-in T cells were co-incubated with two target cells at an effector-target ratio of 4:1. The killing effect was detected after 36 hours ( Figure 9 ). The experimental results showed that G60-01 had strong specific killing effect on both target cells, indicating that G60-01 had good ability to kill target cells.

[0095] Example 6 Recognition motif of MAGE-A10-specific TCR Alanine Scanning is a conventional method used to identify specific amino acid sites that are closely related to TCR function, stability and conformation. The amino acids at various positions of the peptide are replaced with alanine, removing the active groups on the side chain and replacing them with methyl groups that are small in size and have no other functional groups. This has little effect on the protein structure and can distinguish the effect of a specific amino acid on TCR recognition.

[0096] In this experiment, an alanine scanning peptide library was constructed. First, the TCR mRNA to be verified was transduced into T cells by electroporation. After 4 hours, it was co-incubated with T2 cells loaded with various mutant peptides (mutant peptides must be added one day in advance and loaded overnight). The secretion of IFN-γ in each group was detected by enzyme-linked immunosorbent spot (ELISPOT). The ELISPOT operation steps are as follows: After both the effector cells and the target cells were washed 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 in a pre-washed ELISPOT-IFN-γ test plate at a ratio of 1:1. After overnight incubation, the color was developed and then placed in a cool dark place at room temperature. After drying naturally, the immune spots were imaged and read using an ELISPOT analyzer (CTL S6). The ELISPOT test results and statistical results of G60-01-transduced T cells are shown in Figure 10 In the statistical results, the statistical values ​​removed the background values ​​of the corresponding control group. Therefore, it can be determined that the recognition motif of G60-01 is G-YD--E-- (SEQ ID NO: 15).

[0097] In vivo experiments Day 0 will be 4×10 6 Human malignant melanoma cell line A375 (MAGEA10 + ; HLA-A*02:01 + ) were subcutaneously transplanted into severely immunodeficient mice. On Day 2, T cells stably expressing G60-01 TCR were divided into three different dose groups (1×10 7 , 1×10 6 , 1×10 5 TCR-T cells) were infused by tail vein injection, and T cells without TCR transduction were used as the negative control group. The tumor volume was measured and recorded twice a week to observe the inhibitory effect of different doses of TCR-T cells on tumors.

[0098] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A TCR targeting a melanoma-associated antigen, 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 respectively as shown in SEQ ID NOs: 1-3 or one or more amino acids are substituted, deleted or inserted into the amino acid sequences shown in SEQ ID NOs: 1-3; the amino acid sequences of CDR1, CDR2 and CDR3 of the β chain are respectively as shown in SEQ ID NOs: 4-6 or one or more amino acids are substituted, deleted or inserted into the amino acid sequences shown in SEQ ID NOs: 4-6.

2. The TCR according to claim 1, characterized in that 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 of SEQ ID NO:8; Preferably, 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; Preferably, 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; Preferably, 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; Preferably, the α chain and β chain of the TCR are directly or indirectly connected; Preferably, the α chain and β chain of the TCR are indirectly connected; Preferably, the linkage is indirect via a self-cleaving peptide; Preferably, the connection order of the α chain and the β chain can be α chain-self-cleaving peptide-β chain, β chain-self-cleaving peptide-α chain; Preferably, the connection order of the α chain and the β chain is β chain-self-cleaving peptide-α chain; Preferably, the complete sequence of the TCR has at least 60% sequence identity with the amino acid sequence shown in SEQ ID NO: 11; Preferably, the complete sequence of the TCR is shown in SEQ ID NO: 11; Preferably, the melanoma-associated antigen is melanoma-associated antigen A10.

3. A multivalent TCR complex, characterized in that The complex comprises the TCR of claim 1 or 2; Preferably, the complex comprises 2, 3, 4 or more TCRs; Preferably, the complex is present in a lipid bilayer or attached to a particle; Preferably, the TCR is conjugated via a linker molecule.

4. A chimeric molecule, characterized in that The chimeric molecule comprises the TCR or a portion thereof of claim 1 or 2 conjugated to a non-cellular substrate, a toxin and / or an antibody; Preferably, the non-cellular substrate comprises nanoparticles and exosomes.

5. A nucleic acid encoding the TCR of claim 1 or 2, encoding the complex of claim 3, or encoding the chimeric molecule of claim 4.

6. A vector comprising the nucleic acid of claim 5; Preferably, the vector further comprises an element for controlling expression; Preferably, the vector also includes materials that facilitate its entry into cells.

7. A cell, characterized in that The cell comprises the nucleic acid of claim 5 or the vector of claim 6; Preferably, the cells include T cells, lymphocytes or stem cells; Preferably, the cells are selected from T cells.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the TCR of claim 1 or 2, the complex of claim 3, the chimeric molecule of claim 4, the nucleic acid of claim 5, the vector of claim 6 or the cell of claim 7; Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

9. Use of the TCR according to claim 1 or 2, the complex according to claim 3, the chimeric molecule according to claim 4, the nucleic acid according to claim 5, the vector according to claim 6 or the cell according to claim 7 in the preparation of a pharmaceutical composition for treating tumors; Preferably, the tumor is selected from MAGE-related tumors; Preferably, the MAGE-related tumors include melanoma, liver cancer, lung cancer, breast cancer, ovarian cancer, colon cancer, and head and neck squamous cell carcinoma.

10. Use of the TCR according to claim 1 or 2, the complex according to claim 3, the chimeric molecule according to claim 4, the nucleic acid according to claim 5, the vector according to claim 6 or the cell according to claim 7 in antigen detection or in the preparation of antigen detection products; Preferably, the antigen is selected from MAGE; Preferably, the MAGE-A10.

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