T cell receptors targeting col6a3 peptides and uses thereof

Abstract

The present application relates to the technical field of biological medicine, in particular to a T cell receptor of a peptide targeting COL6A3 and use thereof. The present application further provides a T cell receptor, a corresponding nucleic acid molecule, a vector and a host cell, and a method for preparing the T cell receptor. The TCR-T cell obtained by the present application can effectively treat COL6A3-positive tumors, and provides more diversified treatment options for patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a T cell receptor targeting COL6A3 peptide and use thereof. BACKGROUND

[0002] TCR-T is a T cell expressing a tumor antigen-specific receptor, the alpha chain and beta chain of which are generated from a high-quality, high-affinity antigen-specific T cell clone. TCR-T cells are generated by introducing a TCR that specifically recognizes a tumor antigen into T cells through genetic engineering technology, so that they can specifically recognize and attack tumor cells. For TCR-T cell therapy, the selection of antigens is of the utmost importance. The ideal antigen should have tumor specificity, immunogenicity and other characteristics. The tumor antigens currently studied are divided into two categories: tumor-associated antigens (TAA) and tumor-specific antigens (TSA). TAA is an antigen that is overexpressed in tumor tissue and has limited expression in normal tissue, including cancer-testis antigens, tissue differentiation antigens and overexpressed antigens. TSA is a protein uniquely expressed by tumor cells, including viral antigens and neoantigens. The toxicity risk of immunotherapy targeting these neoantigens is lower.

[0003] Most of the current TCR-T cell therapy research is directed at solid tumors, among which the most common solid tumor is melanoma, followed by intestinal cancer, gastric cancer, lung cancer, etc. According to the statistics of target points, NY-ESO-1 and MAGE-A4 are the most targeted target points in clinical trials. In these clinical trials, the most common restrictive alleles are HLA-A*02, HLA-A*11 and HLA-A*24, respectively. On August 1, 2024, Adaptimmune announced that its TCR-T therapy, Afami-cel (trade name: Tecelra), was approved for marketing by the FDA and was used for the treatment of advanced synovial sarcoma.

[0004] Compared with the currently popular chimeric antigen receptor (CAR) cell therapy, T cell receptor (TCR) has some obvious advantages in T cell-based therapy, mainly including: 1. There are more subunits in its receptor structure, more tyrosine-based activation motifs (ITAM) in the immune receptor, and less dependence on antigens; 2. There are more costimulatory receptors (CD3, CD4, CD28, etc.), which enable TCRs with low MHC affinity ranges to effectively activate T cells; 3. Since it can target intracellular tumor-specific antigens, TCR has more potential in the treatment of solid tumors.

[0005] Collagen is a protein superfamily that plays a role in maintaining the integrity of various tissues. Collagen is an extracellular matrix protein and has a triple helix domain as its common important structure. The alpha-3 chain encoded by the COL6A3 gene is one of the three alpha chains of collagen type VI. This collagen is widely present in connective tissue. The alpha-3 chain of collagen type VI is larger than the alpha-1 chain and the alpha-2 chain, and the main difference is the increase in the number of subdomains - these structures similar to von Willebrand Factor type A domains exist in the amino-terminal globular domain of all alpha chains. Studies have shown that these domains can bind to extracellular matrix proteins, and this interaction explains the important role of this collagen in regulating matrix components.

[0006] Currently, many studies have shown that COL6A3 protein is a component of collagen type VI, which exists in connective tissue throughout the body, but exon 6 is only expressed in stromal cells in the tumor microenvironment. COL6A3 is related to diseases such as myotonic dystrophy, colorectal cancer, and pancreatic cancer. It is a relatively promising research target.

[0007] Currently, the tumor-related research on COL6A3 is still in its early stages. Based on the published data, there is currently only one drug in development (IMA-204) developed by Immatics, which is still in the preclinical research stage. Therefore, it is extremely important to develop a cell therapy product targeting the same target, which can provide patients with more diverse treatment options and provide important reference and data support for subsequent cell therapy research and development targeting COL6A3. SUMMARY

[0008] The technical problem to be solved by the present application is that there are currently few cell therapy products targeting COL6A3.

[0009] Accordingly, the technical solution adopted by the present application to solve its technical problem is to screen for a T cell receptor that specifically targets a COL6A3 peptide and use it for the treatment of COL6A3-positive cancer, especially gastrointestinal cancer and gastric cancer.

[0010] Specifically, part of the COL6A3 peptide segment (such as COL6A3-FLNV, sequence: FLLDGSANV) can bind to specific HLA molecules such as HLA-A*02:01, form an antigen peptide-MHC complex and be recognized by TCR, and induce an anti-tumor immune response.

[0011] In a first aspect, the present application relates to an antigen recognizing construct directed against the COL6A3 antigen. The construct is selective and specific for the tumor expressed antigen COL6A3. The amino acid sequence of the antigen COL6A3 is shown in SEQ ID NO: 1.

[0012] In a second aspect, the present application provides a T cell receptor targeting a COL6A3 peptide, the sequence of the COL6A3 peptide being FLLDGSANV (SEQ ID NO: 1), and the T cell receptor comprising a TCR a chain variable domain and a TCR β chain variable domain,

[0013] wherein the TCR a chain variable domain comprises:

[0014] a CDR1a of sequence TTMRA (SEQ ID NO: 2);

[0015] a CDR2a of sequence LASGT (SEQ ID NO: 3); and

[0016] a CDR3a of sequence AAEAGYQNFY (SEQ ID NO: 4),

[0017] wherein the TCR β chain variable domain comprises:

[0018] a CDR1β of sequence MSHET (SEQ ID NO: 5);

[0019] a CDR2β of sequence SYDVDS (SEQ ID NO: 6); and

[0020] a CDR3β of sequence ASSSARTISGNTLY (SEQ ID NO: 7).

[0021] In some embodiments, the T cell receptor is soluble.

[0022] In some embodiments, the T cell receptor comprises an artificial disulfide bond between the a chain constant region and the β chain constant region.

[0023] In some embodiments, the sequence of the TCR a chain variable domain is:

[0024] GDQVEQSPSALSLHEGTGSALRCNFTTTMRAVQWFQQNSRGSLINLFYLASGTKENGRLKSTFNSKESYSTLHIRDAQLEDSGTYFCAAEAGYQNFYFGKGTSLTVIPN (SEQ ID NO: 22);

[0025] the sequence of the TCR β chain variable domain is:

[0026] DMKVTQMPRYLIKRMGENVLLECGQDMSHETMYWYRQDPGLGLQLIYISYDVDSNSEGDIPKGYRVSRKKREHFSLILDSAKTNQTSVYFCASSSARTISGNTLYFGEGSRLIVVE (SEQ ID NO: 23).

[0027] In some embodiments, the T cell receptor is a fusion protein.

[0028] In some embodiments, the TCR of the application is capable of recognizing a COL6A3 antigen in a major histocompatibility complex (MHC) class I dependent manner; "MHC class I dependent manner" refers to the TCR triggering an immune response upon binding to the COL6A3 antigen in the context of a MHC class I molecule. The MHC class I molecule can be any MHC class I molecule known in the art, for example an HLA-A molecule.

[0029] In some embodiments, the MHC class I molecule is an HLA-A2 molecule.

[0030] In some embodiments, the TCR of the application can further comprise a constant region derived from any suitable species, for example any mammal, for example a human, a rat, a monkey, a rabbit, a donkey or a mouse.

[0031] In one embodiment of the application, the TCR of the application further comprises a human constant region.

[0032] In some preferred embodiments, the constant region of the TCR of the application can be modified, for example by introducing a heterologous sequence, preferably a mouse sequence, which can increase TCR expression and stability.

[0033] In a third aspect, the application provides a synthetic nucleic acid molecule, the synthetic nucleic acid molecule encoding a T cell receptor of the second aspect of the application.

[0034] In a fourth aspect, the application provides a vector, the vector comprising a synthetic nucleic acid molecule of the application.

[0035] In some embodiments, the vector of the application can be any suitable recombinant expression vector and can be used to transform or transfect any suitable host.

[0036] In some embodiments, the vector is an animal expression vector, for example: pEUK-Cl, pMAM and pMAMneo.

[0037] In some embodiments, the vector is a viral vector, for example a retroviral vector.

[0038] In some embodiments, the vector can comprise one or more marker genes that allow selection of transformed or transfected hosts.

[0039] In a fifth aspect, the present application provides a host cell comprising the vector of the present application.

[0040] In some embodiments, the host cell of the present application is a lymphocyte, preferably a T lymphocyte, such as a CD4-positive or CD8-positive T cell; further, the host cell is preferably a tumor-reactive T cell specific for tumor cells expressing COL6A3.

[0041] In some embodiments, the present application provides a host cell having integrated into its chromosome the synthetic nucleic acid molecule of the present application.

[0042] In a sixth aspect, the present application provides a method of producing a T cell receptor, comprising:

[0043] (i) culturing the host cell of the present application to express the T cell receptor of the second aspect of the present application; and

[0044] (ii) isolating the T cell receptor.

[0045] In a seventh aspect, the present application relates to a composition comprising a fusion polypeptide comprising the aforementioned TCR a chain and / or TCR β chain.

[0046] In some embodiments, the cell is genetically modified by introduction of an isolated nucleic acid molecule encoding a polypeptide comprising at least one of the aforementioned TCR a chain and TCR β chain.

[0047] In some embodiments, the cell is an immune cell.

[0048] In some embodiments, the immune cell is selected from the group consisting of an antigen presenting cell, a B cell, a dendritic cell, a macrophage, a Langerhans cell, a T cell, an NK cell, an NK T cell.

[0049] In an eighth aspect, the present application relates to a method of producing a specific CTL targeting a COL6A3-positive tumor, comprising the steps of:

[0050] 1) preparing a mixture of peptides from a COL6A3 short peptide (FLLDGSANV);

[0051] 2) adding dendritic cells (DCs) to the mixture of polypeptides of step 1) and culturing to obtain antigen presenting cells;

[0052] 3) co-culturing the antigen presenting cells and peripheral blood mononuclear cells obtained in step 2) to obtain specific CTLs targeting COL6A3 positive tumors.

[0053] In some embodiments, the method for preparing the specific CTLs targeting COL6A3 positive tumors comprises the steps of:

[0054] 1) preparing a short peptide (FLLDGSANV) of COL6A3 to obtain a peptide mixture;

[0055] 2) mixing and co-culturing the DCs, the short peptide obtained in step 1) and peripheral blood mononuclear cells to obtain specific CTLs targeting COL6A3 positive tumors.

[0056] In some embodiments, the method for preparing the specific CTLs targeting COL6A3 positive tumors comprises the steps of:

[0057] 1) transfecting an mRNA encoding COL6A3 amino acid (SEQ ID NO: 1) into DC cells;

[0058] 2) mixing and co-culturing the DCs obtained in step 1) and peripheral blood mononuclear cells to obtain specific CTLs targeting COL6A3 positive tumors.

[0059] In a ninth aspect, the present application relates to a pharmaceutical composition comprising the T cell receptor according to the preceding aspect, or the fusion protein according to the preceding aspect, or the nucleic acid molecule according to the preceding aspect, or the vector according to the preceding aspect, or the host cell according to the preceding aspect, and a pharmaceutically acceptable carrier, stabilizer and / or excipient.

[0060] In a tenth aspect, the present application relates to the use of the antigen recognizing construct, the T cell receptor, the nucleic acid molecule, the vector, the host cell, the pharmaceutical composition according to the first to ninth aspects in medicine.

[0061] In a preferred embodiment, the use in medicine comprises the use in the diagnosis, prevention and / or treatment of a tumor disease, such as a malignant or benign tumor disease; the tumor disease refers to a tumor disease characterized by overexpression of COL6A3.

[0062] In some embodiments, the tumor comprises: acute lymphocytic cancer, acute myeloid leukemia, bone cancer, brain cancer, breast cancer, eye cancer, intrahepatic bile duct cancer, joint cancer, neck cancer, gallbladder cancer or pleural cancer, nasal cancer, nasal cavity cancer or middle ear cancer, oral cavity cancer, vaginal cancer, vulvar cancer, chronic lymphocytic leukemia, chronic myelocytic cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid, glioma, Hodgkin's lymphoma, hypopharyngeal cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, oropharyngeal cancer, ovarian cancer, penile cancer, pancreatic cancer, peritoneal cancer, omental cancer, mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, uterine cancer, ureteral cancer, and bladder cancer. Preferred cancers are cervical cancer, oropharyngeal cancer, anal cancer, anal canal cancer, anorectal cancer, vaginal cancer, vulvar cancer, or penile cancer.

[0063] In some embodiments, preferred tumors are COL6A3-positive cancers, including gastrointestinal cancer and stomach cancer.

[0064] The beneficial effects of the present application are that by screening with COL6A3 short peptides, a T cell receptor with high affinity to COL6A3 peptide and stable expression on cell membrane is obtained, which can specifically bind to FLLDGSANV-HLA A0201 complex, and the T cell receptor exhibits excellent specific killing activity on antigen-positive tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 A graph showing the experimental results of the binding affinity of TCR to COL6A3 peptide.

[0066] Figure 2 A graph showing the experimental results of the stability of TCR expression on cell membrane.

[0067] Figure 3 A graph showing the experimental results of the effect of T cells overexpressing TCR on specific IFN-γ secretion of antigen-positive target cells.

[0068] Figure 4 A graph showing the experimental results of the effect of T cells overexpressing TCR on specific IL-2 secretion of antigen-positive target cells.

[0069] Figure 5 A graph showing the experimental results of the effect of T cells overexpressing TCR on CD137 expression.

[0070] Figure 6 A graph showing the experimental results of the specific killing activity of T cells overexpressing TCR on antigen-positive tumor cells. DETAILED DESCRIPTION

[0071] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, exemplary methods and materials are described.

[0072] As used herein, "CDR" is defined as the complementarity determining region amino acid sequence of a TCR or TCR chain.

[0073] In the context of the present application, the following common nucleic acid base abbreviations are used. "A" means adenosine, "C" means cytidine, "G" means guanosine, "T" means thymidine, and "U" means uridine.

[0074] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to a compound composed of amino acid residues connected to one another by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids to the sequence that can make up a protein or peptide. A polypeptide includes any peptide or protein comprising two or more amino acids connected to one another by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as, e.g., peptides, oligopeptides, and oligomers) and long chains (which are commonly referred to in the art as proteins, which have many types).

[0075] As used herein, "vector" can mean a nucleic acid sequence that contains an origin of replication. The vector can be a plasmid, a bacteriophage, a bacterial artificial chromosome, or a yeast artificial chromosome. The vector can be a DNA or RNA vector. The vector can be a self-replicating extrachromosomal vector or a vector that integrates into the host genome.

[0076] In some embodiments (Y24074-B9), the TCR comprises a TCR a chain variable domain and a TCR β chain variable domain, the 3 complementarity determining regions (CDRs) of the TCR a chain variable domain are:

[0077] CDR1a - TTMRA (SEQ ID NO: 2);

[0078] CDR2a - LASGT (SEQ ID NO: 3); and

[0079] CDR3a - AAEAGYQNFY (SEQ ID NO: 4), and

[0080] the 3 complementarity determining regions (CDRs) of the TCR β chain variable domain are:

[0081] CDR1β - MSHET (SEQ ID NO: 5);

[0082] CDR2β-SYDVDS (SEQ ID NO: 6); and

[0083] CDR3β-ASSSARTISGNTLY (SEQ ID NO: 7).

[0084] In some embodiments (Y23160F-16), the TCR comprises a TCR a chain variable domain and a TCR β chain variable domain, the three complementarity determining regions (CDRs) of the TCR a chain variable domain are:

[0085] CDR1 a of the sequence DRGSQS (SEQ ID NO: 8);

[0086] CDR2 a of the sequence IYSNGD (SEQ ID NO: 9); and

[0087] CDR3 a of the sequence AVNTGYGNKLV (SEQ ID NO: 10),

[0088] wherein the TCR β chain variable domain comprises:

[0089] CDR1 β of the sequence SGDLS (SEQ ID NO: 11);

[0090] CDR2 β of the sequence YYNGEE (SEQ ID NO: 12); and

[0091] CDR3 β of the sequence ASSGQYNEQF (SEQ ID NO: 13).

[0092] In some embodiments (Y23121-10), the TCR comprises a TCR a chain variable domain and a TCR β chain variable domain, the three complementarity determining regions (CDRs) of the TCR a chain variable domain are:

[0093] CDR1 a-DRVSQS (SEQ ID NO: 14);

[0094] CDR2 a-IYSNGD (SEQ ID NO: 15); and

[0095] CDR3 a-AVPGTNSGGYQKVT (SEQ ID NO: 16), and

[0096] the three complementarity determining regions (CDRs) of the TCR β chain variable domain are:

[0097] CDR1 β-SGHTA (SEQ ID NO: 17);

[0098] CDR2β-FQGNSA (SEQ ID NO: 18); and

[0099] CDR3β-ASSLGGPGVGYT (SEQ ID NO: 19).

[0100] In some embodiments (Y23160F-16), the TCR comprises an alpha chain variable domain amino acid sequence as set forth in SEQ ID NO: 20:

[0101] QKEVEQNSGPLSVPEGAIASLNCTYSDRGSQSFFWYRQYSGKSPELIMFIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVNTGYGNKLVFGAGTILRVKSY;

[0102] and the TCR comprises a beta chain variable domain amino acid sequence as set forth in SEQ ID NO: 21:

[0103] DSGVTQTPKHLITATGQRVTLRCSPRSGDLSVYWYQQSLDQGLQFLIQYYNGEERAKGNILERFSAQQFPDLHSELNLSSLELGDSALYFCASSGQYNEQFFGPGTRLTVLE.

[0104] In some embodiments (Y24074-B9), the TCR comprises an alpha chain variable domain amino acid sequence as set forth in SEQ ID NO: 22:

[0105] GDQVEQSPSALSLHEGTGSALRCNFTTTMRAVQWFQQNSRGSLINLFYLASGTKENGRLKSTFNSKESYSTLHIRDAQLEDSGTYFCAAEAGYQNFYFGKGTSLTVIPN;

[0106] and the TCR comprises a beta chain variable domain amino acid sequence as set forth in SEQ ID NO: 23:

[0107] DMKVTQMPRYLIKRMGENVLLECGQDMSHETMYWYRQDPGLGLQLIYISYDVDSNSEGDIPKGYRVSRKKREHFSLILDSAKTNQTSVYFCASSSARTISGNTLYFGEGSRLIVVE.

[0108] In some embodiments (Y23121-10), the TCR comprises an alpha chain variable domain amino acid sequence as set forth in SEQ ID NO: 24:

[0109] QKEVEQNSGPLSVPEGAIASLNCTYSDRVSQSFFWYRQYSGKSPELIMSIYSNGDKEDGRFTAQLNKASQYVSLLIRDSQPSDSATYLCAVPGTNSGGYQKVTFGTGTKLQVIPN;

[0110] and the TCR comprises a beta chain variable domain amino acid sequence as set forth in SEQ ID NO: 25:

[0111] GAGVSQSPSNKVTEKGKDVELRCDPISGHTALYWYRQSLGQGLEFLIYFQGNSAPDKSGLPSDRFSAERTGGSVSTLTIQRTQQEDSAVYLCASSLGGPGVGYTFGSGTRLTVVE.

[0112] In some embodiments, the TCR is single chain.

[0113] In some embodiments, the TCR is linked by a peptide linker between the alpha chain variable region and the beta chain variable region.

[0114] In some embodiments, a cysteine residue forms an artificial disulfide bond between the alpha and beta chain constant domains of the TCR.

[0115] In some embodiments, the C- or N-terminus of the alpha chain and / or beta chain of the TCR is conjugated to a conjugate. Preferably, the conjugate is a detectable label, a therapeutic agent, a PK modifying moiety, or a combination of any of these.

[0116] In some embodiments, the TCR is a murine TCR, a human-murine chimeric TCR, or a humanized TCR.

[0117] In some embodiments, the vector comprises an expression vector, i.e., a construct that is capable of expression in vivo or in vitro. Commonly used vectors include bacterial plasmids, bacteriophage, and viral vectors.

[0118] In some embodiments, the viral vector includes, but is not limited to, an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes viral vector, a retroviral vector, a lentiviral vector, a baculoviral vector. Preferably, the vector can transfer the nucleotide of the present application into a cell, e.g., a T cell, such that the cell expresses a TCR specific for the COL6A3 antigen. The vector should be capable of expressing at a high level and persistently in the T cell.

[0119] In some embodiments, the lentiviral vector can include: lentiviral expression vector pLenti (addgene).

[0120] In some embodiments, the host cell is a mammalian cell. For example, the host cell is a human cell. While the host cell can be a cell of any cell type, can be derived from any type of tissue, and can be a cell of any developmental stage, the host cell is preferably a peripheral blood lymphocyte (PBL) or a peripheral blood mononuclear cell (PBMC). More preferably, the host cell is a T cell.

[0121] In some embodiments, the host cell can be a cell that is allogeneic to the mammal or autologous to the mammal when the host cell or related cell population is administered. Preferably, the cell is autologous to the mammal.

[0122] In some embodiments, the mammal refers to any mammal, including but not limited to: rodents such as mice and hamsters, and lagomorphs such as rabbits. Preferably, the mammal is from the order Carnivora, including felines (e.g., cats) and canines (e.g., dogs). More preferably, the mammal is from the order Artiodactyla, including bovids (e.g., cattle) and suids (e.g., pigs), or from the order Perissodactyla, including equids (e.g., horses). Most preferably, the mammal is from the order Primates, the order Simiiformes, or the suborder Anthropoidea (humans and anthropoids). It is particularly preferred that the mammal is a human.

[0123] The principles and implementations of the present application are described in detail with specific examples in this document, and the description of the examples is only used to help understand the method of the present application and its central idea. It should be noted that for those skilled in the art, without departing from the principles of the present application, some improvements and modifications can be made to the present application, and these improvements and modifications also fall within the protection scope of the claims of the present application.

[0124] Example 1: Cloning of antigenic short peptide-specific T cells

[0125] Peripheral blood lymphocytes (PBLs) from healthy volunteers with genotype HLA-A*02:01 were stimulated with synthetic short peptide COL6A3 (FLLDGSANV). The short peptide was complexed with biotin-labeled HLA-A*02:01 to prepare pHLA haplotype. These haplotypes were combined with streptavidin labeled with PE (BD company) into PE-labeled tetramer, and the tetramer and anti-CD8-APC double positive cells were sorted using BD Melody flow sorting instrument, and the positive cells were sorted into 96-well plates, one cell per well.

[0126] Example 2: Obtaining TCR genes of COL6A3 antigenic short peptide-specific T cell clones and construction of vectors

[0127] After the cells obtained in Example 1 were lysed using 0.1% Triton-X (Shanghai Yingqiu Biotech Co., Ltd.), the SMARTRACE cDNA amplification kit from Clontech was used, and the primers were designed in the C-terminal conserved region of the human TCR gene. The downstream primer for the C-terminal conserved region of the alpha chain of TCR: TCAGCTGGACCACAGC (SEQ ID NO: 26); the downstream primer for the C-terminal conserved region of the beta chain of TCR: AATCCTTTCTCTTGACCATGGCCATC (SEQ ID NO: 27). The full-length genes of TCR alpha chain and beta chain were cloned into the lentiviral expression vector pCDH (SBI) by overlap PCR, respectively. The specific steps are as follows: the full-length genes of TCR alpha chain and TCR beta chain were connected by overlap PCR to obtain the TCRa-2A-TCRb fragment. The lentiviral expression vector and TCRa-2A-TCRb were digested and ligated to obtain the pCDH-TRA-2A-TRB plasmid, which was sequenced and confirmed (IMGT) to obtain three TCR clone plasmids: Y23160F-16, Y24074-B9 and Y23121-10.

[0128] The sequencing results of CDR1a, CDR2a and CDR3a of the variable domain of the TCR alpha chain of Y24074-B9 were SEQ ID NOs: 2-4, respectively; the sequencing results of CDR1b, CDR2b and CDR3b of the variable domain of the TCR beta chain were SEQ ID NOs: 5-7, respectively. The sequencing result of the variable domain of the TCR alpha chain was SEQ ID NO: 22, and the sequencing result of the variable domain of the TCR beta chain was SEQ ID NO: 23.

[0129] The sequencing results of CDR1a, CDR2a and CDR3a of the variable domain of the TCR alpha chain of Y23160F-16 were SEQ ID NOs: 8-10, respectively; the sequencing results of CDR1b, CDR2b and CDR3b of the variable domain of the TCR beta chain were SEQ ID NOs: 11-13, respectively. The sequencing result of the variable domain of the TCR alpha chain was SEQ ID NO: 20, and the sequencing result of the variable domain of the TCR beta chain was SEQ ID NO: 21.

[0130] The sequencing results of CDR1a, CDR2a and CDR3a of the variable domain of the TCR alpha chain of Y23121-10 were SEQ ID NOs: 14-16, respectively; the sequencing results of CDR1b, CDR2b and CDR3b of the variable domain of the TCR beta chain were SEQ ID NOs: 17-19, respectively. The sequencing result of the variable domain of the TCR alpha chain was SEQ ID NO: 24, and the sequencing result of the variable domain of the TCR beta chain was SEQ ID NO: 25.

[0131] The 293T was then used to package the pseudovirus. Specifically, the above-mentioned plasmids were mixed with the VSVG plasmid, the RRE plasmid, and the Rev plasmid (purchased from Addgene) in a ratio of 20:7:13:5, and 20 μg was diluted in DMEM medium (0.5 mL) as a DNA solution. 60 μL of polyetherimide (PEI 1 μg / μL) was added to DMEM (0.5 mL), and the PEI / DMEM mixed solution was added to the DNA solution, which was incubated at room temperature for 20 minutes before being added to 293T cells cultured in a 10 cm dish and mixed. After 6 hours, fresh DMEM medium was replaced. After 72 hours, the supernatant containing the lentivirus was collected, which was the lentivirus supernatant of each TCR.

[0132] Example 3: Construction of overexpression cell lines of antigen short peptide specific TCR

[0133] The NFAT-GFP element (Addgene) was inserted into the expression vector pCDH (SBI) by the standard method described in the Molecular Cloning Laboratory Manual, and the fragment was sequenced to confirm that it was correct. The 293T (Pronex CL-0130) was then used to package the pseudovirus. The Jurkat cell line was infected with the pseudovirus containing the NFAT-GFP element and the pseudovirus containing the TCR element, and through limiting dilution and monoclonal amplification, each Jurkat-NFAT-GFP-TCR overexpression cell line was obtained, namely: Jurkat-NFAT-GFP-Y23160F-16-TCR, Jurkat-NFAT-GFP-Y24074-B9-TCR, and Jurkat-NFAT-GFP-Y23121-10-TCR.

[0134] Example 4: Experiment on the binding affinity of TCR to mutant peptide and wild-type peptide or tumor-associated antigen peptide

[0135] (1) Construction of K562-CD80-HLA-A*02:01 and 293T-CD80-HLA-A*02:01

[0136] The HLA-A*02:01 element (IMGT / HLA Acc No: HLA00043) and CD80 (NP_005182.1) were synthesized and inserted into the expression vector pCDH (SBI). After sequencing, the fragments were confirmed to be correct. Then the 293T cell line was used to package the lentivirus. Specifically, the plasmid containing the CD80-HLA-A*02:01 element was mixed with the VSVG plasmid, the RRE plasmid, and the Rev plasmid (purchased from Addgene) at a ratio of 20:7:13:5. Then 20 μg of the mixture was diluted in DMEM medium (0.5 mL) as a DNA solution. 20 μL of polyetherimide (PEI 1 μg / μl) was added to DMEM (0.5 mL), and the PEI / DMEM mixture was added to the DNA solution. After incubation at room temperature for 20 minutes, the mixture was added to 293T cells in a 10 cm dish and mixed well. After 6 hours, the medium was replaced with fresh DMEM medium. After 72 hours, the supernatant containing the lentivirus was collected, which was the CD80-HLA-A*02:01 lentivirus supernatant. The K562 or 293T cell line was infected with the CD80-HLA-A*02:01 element-containing pseudovirus, and through limiting dilution and monoclonal expansion, the K562-CD80-HLA-A*02:01 and 293T-CD80-HLA-A*02:01 overexpression cell lines were obtained.

[0137] (2) Jurkat-NFAT-GFP-TCR overexpression cell line co-cultured with K562-CD80-HLA-A*02:01

[0138] The cell lines (Jurkat-NFAT-GFP-Y23160F-16-TCR, Jurkat-NFAT-GFP-Y24074-B9-TCR, and Jurkat-NFAT-GFP-Y23121-10-TCR) were co-incubated with K562-CD80-HLA-A*02:01 loaded with different concentrations of the antigen peptide (COL6A3-FLLDGSANV) to be tested. Specifically, the K562 cells were incubated with different concentrations of the target antigen peptide at 37°C for 1 h, and then resuspended in medium after centrifugation. The Jurkat and K562 cells were counted, and 2 x 105 Jurkat cells and 2 x 105 K562 cells loaded with polypeptides were taken and mixed. Then the mixture was co-cultured in a 96-well plate for 24 h. After co-culturing, the Jurkat cell reporter gene activation level and CD69 cell activation level were detected by flow cytometry. 4 Cells, mixed and co-cultured in a 96-well plate for 24 h. The Jurkat cell reporter gene activation level and CD69 cell activation level were detected by flow cytometry.

[0139] The results are shown in FIG. 1. Figure 1Display: TCR T cells expressing Y23160F-16, Y24074-B9 and Y23121-10 have strong reactivity and specificity to the target antigen peptide COL6A3, and the relevant T cell receptor proteins are suitable for therapeutic use.

[0140] Example 5: Stability experiment of overexpressed TCR on cell membrane expression

[0141] TCR-T cells expressing TCR (Y24176, Y24329 and Y23195) were constructed using the TCR elements (TCR a-2A-TCR b fragment) of Y23160F-16, Y24074-B9 and Y23121-10, respectively, and the specific steps were as follows:

[0142] (1) Preparation of TCR lentivirus

[0143] Each TCR element was inserted into the expression vector pCDH (SBI) after synthesis by standard methods described in Molecular Cloning a Laboratory Manual (ISBN 978-1-936113-42-2; Chapter 3 Cloning and Transformation with Plasmid Vectors) together with GFP (addgene), and the fragments were sequenced to confirm that there were no errors. Then the pseudovirus was packaged using 293T (Pronova CL-0130) according to the specific operation steps of Example 2.

[0144] (2) Construction of TCR-T cells expressing TCR

[0145] After thawing the PBMC, an appropriate amount of X-VIVO 15 medium containing 100 IU / mL rhIL-2 was used to make the density 1 x 10 6 / mL. 2 x 10 6Cells were added with 10 μL MACS CD3 / CD28 T cell TransAct beads, and the culture medium was X-VIVO15 containing 100 IU / mL rhIL-2, and the cells were mixed gently and cultured in a cell incubator. After 24 hours, the supernatant was centrifuged and poured out to remove the magnetic beads, and resuspended in 1 mL of X-VIVO15 culture medium containing 100 IU / mL rhIL-2, and the target lentivirus was added according to the total number of cells and the determination of virus titer (infection at MOI=10), and part of the cells were not added with lentivirus as a control group without TCR transduction, and each was supplemented with culture medium to a final volume of 2 mL of X-VIVO15 containing 100 IU / mL IL-2, and 10 μg / mL of polybrene was added, and the plate hanger was centrifuged at 2000g for 60 minutes at 37°C, and the infected cells were placed in a carbon dioxide incubator for culture for 24h. Fresh culture medium was changed regularly and the cell density was adjusted, and cultured to the 14th day.

[0146] GFP was used to detect the expression rate of TCR-T. The results showed (see Figure 2 ) that each TCR could be stably expressed in the cell membrane.

[0147] Example 6: Specific IFN-γ and IL-2 secretion of TCR-T cells overexpressing antigen-positive target cells

[0148] 1. Using T cells expressing TCR (same as Example 5) as effector cells, and PBMC without TCR transduction as a control for parallel expansion of effector cells after culture.

[0149] 2. K562-CD80-HLA-A*02:01 cells loaded with 10 -7 M COL6A3-FLLDGSANV short peptide or irrelevant peptide were used as positive target cells (same as Example 4); the E:T effector target ratio (effector cell: target cell ratio) was 1:1, and the expression of IL-2 or / and IFN-γ in the cells was detected after incubation for 24h.

[0150] 3. K562-CD80-HLA-A*02:01 cells loaded with 10 -10 M 10 -5 M COL6A3-FLLDGSANV short peptide at different concentrations were used as positive target cells (same as Example 4); the E:T effector target ratio (effector cell: target cell ratio) was 1:1, and the expression of CD137 in the cells was detected after incubation for 24h.

[0151] Results show that TCR-T overexpression group produces IL-2 and IFN-γ in the presence of positive target cells, and CD137 expression is up-regulated. The TCR-T overexpression group does not produce IFN-γ or IL-2 against negative target cells. Part of the results of INF-γ and IL-2 expression and secretion are shown in Figure 3 and Figure 4 respectively. Part of the results of CD137 up-regulation are shown in Figure 5 . The above data show that the T cells related to the overexpression of TCR have specific activation efficacy against antigen-positive target cells, and the TCR protein obtained by the present application is suitable for use for therapeutic purposes.

[0152] Example 7: Specific killing activity of TCR-T overexpression T cells against antigen-positive tumor cells

[0153] 1. Use T cells expressing TCR (same as Example 5) as effector cells, and use PBMC not transduced with TCR as blank control (Mock) for parallel expansion culture of effector cells.

[0154] 2. Use 293T-CD80-HLA-A*02:01 cells loaded with 10 -8 M~10 -6 COL6A3-FLLDGSANV short peptide (same as Example 5) as positive target cells (+); the E:T effector target (effector cell: target cell ratio) is 10:1, and the adhesion ability of target cells is detected in real time by RTCA (real-time label-free cell analysis system, which integrates microelectronic cell sensor chips into the bottom of the cell detection plate, and obtains biological information related to cell physiological function through real-time dynamic electrode impedance detection, including cell growth, stretching, morphological changes, death and adhesion, etc.). Specifically, the instrument collects the cell adhesion ability value (Cell Index) for each well every 15 min, and the data at the last time point before adding T cells is used as the normalized value for subsequent data processing, and the normalized cell adhesion ability value (Normalized Cell Index) at each time point in each group is calculated; the results are shown (see Figure 6 ): The TCR-T overexpression group only has significant killing activity against 293T-CD80-HLA-A*02:01 cells loaded with tumor-associated antigen polypeptides, and has no killing effect on 293T-CD80-HLA-A*02:01 cells without polypeptide loading, and Y24329 has better killing effect.

[0155] While the application has been disclosed with reference to particular embodiments, it is apparent that other embodiments and variations of the application can be devised by others skilled in the art without departing from the true spirit and scope of the application. The appended claims are intended to be construed to include all such embodiments and equivalents.

Claims

1. A T cell receptor targeting a COL6A3 peptide, characterized in that, The COL6A3 peptide has the sequence FLLDGSANV, and the T cell receptor comprises a TCR alpha chain variable domain and a TCR beta chain variable domain, wherein the TCR alpha chain variable domain comprises: a CDR1 alpha having the sequence TTMRA; a CDR2 alpha having the sequence LASGT; and a CDR3 alpha having the sequence AAEAGYQNFY, wherein the TCR beta chain variable domain comprises: a CDR1 beta having the sequence MSHET; a CDR2 beta having the sequence SYDVDS; and a CDR3 beta having the sequence ASSSARTISGNTLY.

2. The T cell receptor of claim 1, wherein, The T cell receptor is soluble.

3. The T cell receptor of claim 1, wherein, The T cell receptor comprises an artificial disulfide bond between the alpha chain constant region and the beta chain constant region.

4. The T cell receptor of claim 1, wherein the TCR alpha chain variable domain has the sequence: GDQVEQSPSALSLHEGTGSALRCNFTTTMRAVQWFQQNSRGSLINLFYLASGTKENGRLKSTFNSKESYSTLHIRDAQLEDSGTYFCAAEAGYQNFYFGKGTSLTVIPN, wherein the TCR beta chain variable domain has the sequence: DMKVTQMPRYLIKRMGENVLLECGQDMSHETMYWYRQDPGLGLQLIYISYDVDSNSEGDIPKGYRVSRKKREHFSLILDSAKTNQTSVYFCASSSARTISGNTLYFGEGSRLIVVE.

5. A synthetic nucleic acid molecule, characterized in that, The synthetic nucleic acid molecule encodes the T cell receptor of claim 1.

6. A vector, characterized in that, The vector comprises the synthetic nucleic acid molecule of claim 5.

7. A host cell, characterized in that, The host cell comprises the vector of claim 6.

8. A method of making a T cell receptor, comprising, comprises: (i) culturing the host cell of claim 7 to express the T cell receptor of claim 1; and (ii) isolating the T cell receptor.

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