T cell receptor for recognizing KRAS G12V antigen oligopeptide, compound, pharmaceutical composition and application

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a T cell receptor capable of recognizing KRAS G12V antigen oligopeptide, a compound, a pharmaceutical composition and application. The T cell receptor can be used for recognizing a KRAS G12V-HLA-A * 11: 01 compound with high affinity; the T cell receptor is an alpha-beta heterodimer consisting of an alpha chain and a beta chain; the amino acid sequence of the alpha chain is as shown in SEQ ID NO.10, or an amino acid sequence with at least 90% of sequence identity with the alpha chain; the amino acid sequence of the beta chain is as shown in SEQ ID NO.14, or has an amino acid sequence with at least more than 90% of sequence identity with the beta chain. The T cell receptor provided by the invention can be used for preparing medicines for treating tumors or other immune diseases.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a T cell receptor, a complex, a pharmaceutical composition and uses thereof that recognizes a KRAS G12V antigen short peptide. Background Art

[0002] T cell immunotherapy is a crucial approach in tumor immunotherapy. By isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue, cloning and expanding them in vitro, and then infusing them back into the patient, it can achieve lasting remissions in patients with advanced cancer who have developed resistance to radiotherapy and chemotherapy, resulting in excellent clinical therapeutic outcomes. However, the isolation and culture of TILs requires demanding conditions, the time required to reach a therapeutic cell count, and, more importantly, the limited availability of tumor tissue from which TILs can be successfully isolated. This has limited the clinical application of TILs in oncology.

[0003] T cells recognize tumors primarily through the T cell receptor (TCR) on their surface. TCRs recognize human major histocompatibility complex (MHC)-antigen peptide complexes on tumor cells. TCRs form a heterodimeric structure consisting of two peptide chains: an α and a β. Each peptide chain comprises a variable region, a joining region, and a constant region. The β chain typically also contains a short multivariable region between the variable and joining regions, but this multivariable region is often considered part of the joining region. Each variable region consists of three complementarity-determining regions (CDRs) (CDR1, CDR2, and CDR3) embedded within a framework. The CDRs determine TCR binding to the MHC complex. CDR3, formed by the recombination of the variable and joining regions and known as the hypervariable region, directly determines the antigen specificity of the TCR. When the TCR recognizes the MHC-antigen peptide complex, CDR3 directly binds to the antigen peptide. CDR1 and CDR2 primarily contact two α helices in the binding groove of the MHC molecule. These interactions ensure accurate TCR recognition of the MHC-antigen peptide complex. TCR α and β chains are generally considered to each have two "domains": a variable region and a constant region, with the variable region containing the joining region. TCR constant region sequences can be found in the public database of the International Immunogenetics Information System (IMGT), such as "TRAC" for the TCR α chain and "TRBC1" or "TRBC2" for the TCR β chain. In addition, the TCR α and β chains contain a transmembrane region and a short cytoplasmic region.

[0004] By using gene transduction to transfer TCRs that can recognize tumor cells into the patient's immune T cells, the patient's T cells can be transformed into tumor-specific cytotoxic T cells (TCR-T). When these genetically engineered TCR-T cells are delivered into the patient's body, these tumor-specific TCR-T cells will be activated by recognizing the MHC-peptide complex on the tumor cells, thereby proliferating in the patient's body and achieving the effect of treating the tumor by killing the tumor cells.

[0005] The protein encoded by the KRAS gene is a small GTPase that belongs to the RAS gene family. It is the most commonly mutated gene in various tumor types, including pancreatic cancer (90%), colorectal cancer (46%), endometrial cancer (17%), non-small cell lung cancer (12%), bile duct cancer, cervical cancer, bladder cancer, liver cancer, and breast cancer. KRAS mutations are most common at codons 12 and 13, which can promote tumor cell proliferation and metabolic reprogramming. Across all tumor types, the three most common KRAS codon 12 (G12) mutations are G12D, G12V, and G12C. The KRAS G12V mutation is a missense mutation in which the codon 12 of the KRAS coding region changes from G to V. The resulting KRAS G12V protein is a tumor-specific antigen (TSA) expressed exclusively in tumor cells. Therefore, studies have suggested that KRAS G12V is an excellent target antigen for tumor immunotherapy. After intracellular production, KRAS G12V is degraded into small peptides that, after binding to MHC molecules, are presented on the cell surface. VVGAVGVGK, a short peptide derived from the KRAS G12V antigen, is a target for the treatment of KRAS G12V-associated tumors. Traditional treatments for these KRAS G12V-related malignancies include radiotherapy and chemotherapy, both of which damage the patient's normal cells. The Institute of Microbiology, Chinese Academy of Sciences, has identified two TCRs specific for the KRAS-G12V-9 peptide in HLA-A*11:01 transgenic mice. T cells engineered with these TCRs exhibit specific responses to different tumor cells harboring the KRAS G12V mutation, providing a promising candidate for the treatment of KRAS G12V-mutant tumors. The TCRs reported in this paper were isolated from T cells of transgenic mice. Their variable regions are species-specific in both sequence and structure. Furthermore, based on immunological principles, they are not expected to be high-affinity TCRs. This is because KRAS G12V is a self-antigen, so in order to avoid autoimmune diseases, the cytotoxic T lymphocytes (CTLs) with high affinity for KRAS G12V in HLA-A*11:01 transgenic mice are deleted during the negative selection process of thymocytes. As a result, among the mouse's own T cells, only KRAS G12V-CTLs that are tolerant to the self-antigen KRAS G12V and have low affinity can survive. Since low-affinity CTLs often have poor anti-tumor activity, a very important goal of T cell-based tumor immunotherapy is to be able to obtain CTLs with high affinity and specificity for tumor antigens.

[0006] Research has described a non-self-restricting, or allogeneic, CTL strategy for obtaining high-affinity CTLs. By employing this allogeneic CTL strategy, they generated CTLs specific for the tumor-associated antigen tyrosinase and possessing high-affinity TCRs. This technique relies on stimulating allogeneic CTLs with autologous MHC-peptide complexes. Because allogeneic CTLs have not undergone negative selection against autologous MHC, they contain CTLs highly sensitive to autologous MHC and high-affinity TCRs. However, this allogeneic approach also has a significant drawback: only a small fraction of T cells that respond to autologous MHC-peptide complexes are both MHC-restricted and specific for the antigenic peptide, while the majority of cells recognize only MHC and lack specificity for the antigenic peptide. This means that the majority of CTLs stimulated using this approach are nonspecific. If such CTLs are used for tumor treatment, they can cause severe nonspecific immune responses. In summary, in order to address the problems of low affinity, poor anti-tumor activity and non-specificity of previously obtained KRAS G12V TCRs, it is necessary to develop a TCR with high affinity and specificity for KRAS G12V. Summary of the Invention

[0007] To address the problems of low affinity, poor anti-tumor activity, and non-specificity of KRAS G12V TCRs obtained in the prior art, the present invention has found a CTL with both high affinity and specificity for the tumor antigen KRAS G12V, and further isolated and obtained TCRs with high affinity and specificity for tumor antigens, providing a T cell receptor, complex, pharmaceutical composition, and use thereof that recognizes a short peptide of the KRAS G12V antigen.

[0008] To achieve the above objectives, the present invention adopts the following technical solutions.

[0009] To obtain the small number of beneficial CTLs that are both MHC-restricted and specific for the antigenic peptide, the present invention performs single-cell cloning of mixed T cells that generate an immune response during stimulation. Since culturing T cells is inherently challenging, placing them in an environment with only one T cell per well is even more challenging, as a single T cell is difficult to survive. Therefore, ensuring its survival and expansion greatly increases the difficulty of the entire experiment, or in other words, significantly reduces the likelihood of success. Through repeated experiments and extensive screening, the present invention obtained a cytotoxic CTL strain with high affinity and specificity for KRAS G12V, and further isolated and obtained its TCR with high affinity and specificity for KRAS G12V.

[0010] The first object of the present invention is to provide a T cell receptor that recognizes a KRAS G12V antigen peptide. The T cell receptor is capable of recognizing the KRAS G12V-HLA-A*11:01 complex with high affinity, and the amino acid sequence of the KRAS G12V antigen peptide is shown in SEQ ID NO. 1.

[0011] The T cell receptor is an αβ heterodimer composed of an α chain and a β chain, wherein the α chain and the β chain are linked by a covalent bond or a disulfide bond.

[0012] The amino acid sequence of the α chain is shown in SEQ ID NO. 10, or an amino acid sequence having at least 90% sequence identity thereto.

[0013] The amino acid sequence of the β chain is shown in SEQ ID NO. 14, or an amino acid sequence having at least 90% sequence identity thereto.

[0014] The T cell receptor provided by the present invention is capable of recognizing the KRAS G12V-HLA-A*11:01 complex with high affinity. The T cell receptor is composed of an α chain (amino acid sequence SEQ ID NO. 10) and a β chain (amino acid sequence SEQ ID NO. 14), and recognizes the KRAS G12V antigen short peptide VVGAVGVGK (SEQ ID NO. 1) presented by the HLA-A*11:01 allele with high affinity. The T cell receptor provided by the present invention has both high affinity and specific CTL binding to the tumor antigen KRAS G12V, exhibiting high anti-tumor activity. This addresses the low affinity, poor anti-tumor activity, and non-specificity of previously obtained KRAS G12V TCRs.

[0015] Preferably, the α chain includes a TCR α chain variable region, and the TCR α chain variable region includes three complementarity determining regions CDR1α to CDR3α, and the nucleotide sequences thereof are shown in SEQ ID NO.2 to SEQ ID NO.4, respectively.

[0016] The β chain includes a TCR β chain variable region, and the TCR β chain variable region includes three complementarity determining regions CDR1β to CDR3β, and the nucleotide sequences thereof are shown in SEQ ID NO.5 to SEQ ID NO.7, respectively.

[0017] Preferably, the α chain further comprises a TCR α chain constant region, and the β chain further comprises a TCR β chain constant region.

[0018] Preferably, the T cell receptor is a single chain, comprising the variable region of the α chain of TCR and the variable region of the β chain of TCR.

[0019] The amino acid sequence of the α chain variable region of the TCR is shown in SEQ ID NO.26.

[0020] The amino acid sequence of the TCR β chain variable region is shown in SEQ ID NO.28.

[0021] The T cell receptor is formed by connecting the α chain variable region of the TCR and the β chain variable region of the TCR via a connecting peptide chain; the amino acid sequence of the connecting peptide chain is shown in SEQ ID NO.32.

[0022] Preferably, the T cell receptor is TCR-mRNA, and the TCR-mRNA includes the α chain of TCR-mRNA and the β chain of TCR-mRNA.

[0023] The amino acid sequence of the α chain of the TCR-mRNA is shown in SEQ ID NO.34.

[0024] The amino acid sequence of the β chain of the TCR-mRNA is shown in SEQ ID NO.36.

[0025] The TCR-mRNA is formed by connecting the α chain of the TCR-mRNA and the β chain of the TCR-mRNA via a connecting peptide chain; the amino acid sequence of the connecting peptide chain is shown in SEQ ID NO.40.

[0026] A second object of the present invention is to provide a multivalent T cell receptor complex comprising at least three T cell receptors, at least one of which is the above-mentioned T cell receptor.

[0027] The third object of the present invention is to provide a nucleic acid molecule comprising a nucleotide sequence encoding the T cell receptor or its complementary sequence, or a codon-optimized nucleotide sequence corresponding to the amino acid sequence of the T cell receptor.

[0028] A fourth object of the present invention is to provide a vector comprising the nucleic acid molecule. Preferably, the vector is a viral vector and / or a lipid nanoparticle vector; preferably, the vector is a lentiviral vector and / or a retroviral vector; more preferably, the vector is a retroviral vector.

[0029] The fifth object of the present invention is to provide an isolated host cell, wherein the host cell contains the vector or the exogenous nucleic acid molecule integrated into the chromosome.

[0030] The sixth object of the present invention is to provide a cell, which is a cell transduced and transfected by the nucleic acid molecule or the vector, wherein the TCR is heterologous to the cell; preferably, the cell is a T cell or a stem cell; more preferably, the cell is a primary T cell or stem cell from a subject.

[0031] The seventh object of the present invention is to provide a pharmaceutical composition, which comprises the T cell receptor, the complex, the nucleic acid molecule, or the vector or host cell, and a pharmaceutically acceptable carrier.

[0032] The eighth object of the present invention is to provide use of the T cell receptor, the complex, the vector or host cell, or the pharmaceutical composition in the preparation of drugs for treating tumors or other immune diseases.

[0033] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention provides a T cell receptor that recognizes a short peptide of the KRAS G12V antigen. The T cell receptor provided by the present invention can recognize the KRAS G12V-HLA-A*11:01 complex with high affinity. The T cell receptor is composed of an α chain (amino acid sequence SEQ ID NO. 10) and a β chain (amino acid sequence SEQ ID NO. 14), and recognizes the KRAS G12V antigen short peptide VVGAVGVGK (SEQ ID NO. 1) presented by the HLA-A*11:01 allele with high affinity. The T cell receptor provided by the present invention has both high affinity and specific CTL binding to the tumor antigen KRAS G12V, exhibiting high anti-tumor activity. This solves the problems of low affinity, poor anti-tumor activity, and nonspecificity of previously obtained KRAS G12V TCRs.

[0034] 2. The host cell provided by the present invention, which is composed of a TCR that recognizes a short peptide of the KRAS G12V antigen, has a strong killing effect on tumor cells that simultaneously express HLA-A*11:01 and KRAS G12V. The TCR (molecule) of the present invention is transferred into the T cells of a patient with a malignant tumor expressing KRAS G12V (or T cells from a donor), and then these TCR genetically engineered cells are injected into the patient to achieve treatment of the patient. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 CD8 KRAS G12V-specific CTL in the present invention + and HLA-A*11:01-KRAS G12V-tetramer-PE double-positive staining results.

[0036] Figure 2 The flow cytometry method was used to detect the CD8 + and HLA-A*11:01-KRAS G12V-tetramer-PE double positive staining results; among them, Figure 2 (A) shows the staining results of mock-transduced (Mock) T cells; Figure 2 (B) shows the staining results of T cells transduced with KRAS G12V TCR.

[0037] Figure 3 The flow cytometry method of the present invention is to detect the secretion of INF-γ by KRAS G12V-TCR-T cells (F194) under the stimulation of T2-A11 cells loaded with control antigen peptide and KRAS G12V antigen peptide: wherein, Figure 3 (A) shows the secretion of INF-γ by F194 in T2-A11 cells loaded with 10 μM KRAS WT (wild-type) control antigen peptide; Figure 3 (B) shows the secretion of INF-γ by F194 in T2-A11 cells stimulated by 10 μM KRAS G12D control antigen peptide; Figure 3 (C) shows the secretion of INF-γ by F194 in T2-A11 cells loaded with 10 μM KRAS G12V antigen peptide; Figure 3 (D) shows the secretion of INF-γ by F194 in T2-A11 cells loaded with 1 μM KRAS G12V antigen peptide; Figure 3 (E) shows the secretion of INF-γ by F194 in T2-A11 cells stimulated by 100 nM KRAS G12V antigen peptide; Figure 3 (F) in the middle shows the secretion of INF-γ by F194 when T2-A11 cells were stimulated by 10 nM KRAS G12V antigen peptide; Figure 3 (G) in the figure shows the secretion of INF-γ by F194 in T2-A11 cells when they were stimulated by 1 nM KRAS G12V antigen peptide. Figure 3 (H) shows the secretion of INF-γ by F194 in T2-A11 cells stimulated by 100 pM of KRASG12V antigen peptide.

[0038] Figure 4 The transfection efficiency of HLA-A*11:01-CD34-puro in K562, THP-1, PANC-1 and H460 cells was detected by flow cytometry in the present invention; wherein, Figure 4 (A) shows the percentage of positive cells in K562 cells after transfection with HLA-A*11:01-CD34-puro; Figure 4(B) shows the percentage of positive cells after THP-1 cells were transfected with HLA-A*11:01-CD34-puro; Figure 4 (C) in the middle shows the percentage of positive cells after PANC-1 cells were transfected with HLA-A*11:01-CD34-puro; Figure 4 (D) in the middle shows the percentage of positive cells after H460 cells were transfected with HLA-A*11:01-CD34-puro.

[0039] Figure 5 The transfection efficiency of KRAS WT-GFP and KRAS G12V-GFP in K562-A11, THP-1-A11, PANC-1-A11 and H460-A11 cells was detected by flow cytometry in the present invention: wherein, Figure 5 (A) shows the percentage of positive cells in K562-A11 cells after transfection with KRAS WT-GFP and KRAS G12V-GFP; Figure 5 (B) shows the percentage of positive cells in THP-1-A11 cells after transfection with KRAS WT-GFP and KRAS G12V-GFP; Figure 5 (C) shows the percentage of positive cells in PANC-1-A11 cells after transfection with KRAS WT-GFP and KRAS G12V-GFP; Figure 5 (D) shows the percentage of positive cells in H460-A11 cells after transfection with KRASWT-GFP and KRAS G12V-GFP.

[0040] Figure 6 The present invention uses flow cytometry and sulforhodamine B (SRB) colorimetric analysis to detect the killing effect of KRAS G12V-TCR-T cells and nonspecific T cells (mock) on target cells at different effector-target ratios (E:T); wherein, Figure 6 A in the figure is the target cell survival rate detected by flow cytometry after co-culture of KRAS G12V-TCR-T cells and mock cells with positive target cells K562-A11-G12V-GFP and negative target cells K562-A11-WT-GFP at different effector-target ratios; Figure 6 B in the figure is the target cell survival rate detected by flow cytometry after co-culture of KRAS G12V-TCR-T cells and mock cells with positive target cells THP-1-A11-G12V-GFP and negative target cells THP-1-A11-WT-GFP at different effector-target ratios; Figure 6Figure C shows the target cell survival rate detected by sulforhodamine B (SRB) colorimetric analysis after co-culture of KRAS G12V-TCR-T cells and mock cells with positive target cells PANC-1-A11-G12V-GFP and negative target cells PANC-1-A11-WT-GFP at different effector-target ratios.

[0041] Figure 7 The ELISA method of the present invention detects the secretion of INF-γ after co-culture of KRAS G12V-TCR-T cells and nonspecific T cells (mock) with target cells at different effector-target ratios; wherein, Figure 7 A in the figure shows the secretion of INF-γ after co-culture of KRAS G12V-TCR-T cells and mock cells with positive target cells K562-A11-G12V-GFP and negative target cells K562-A11-WT-GFP at different effector-target ratios; Figure 7 Figure B shows the secretion of INF-γ after co-culture of KRAS G12V-TCR-T cells and mock cells with positive target cells PANC-1-A11-G12V-GFP and negative target cells PANC-1-A11-WT-GFP at different effector-target ratios. DETAILED DESCRIPTION

[0042] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0043] All features disclosed in this specification, or all steps in the disclosed methods or processes, except mutually exclusive features and / or steps, can be combined in any manner.

[0044] Unless otherwise stated, any feature disclosed in the specification (including any appended claims, abstract, and drawings) of the present invention may be replaced by other equivalent or similar features. In other words, unless otherwise stated, each feature is merely an example of a series of equivalent or similar features.

[0045] After repeated, in-depth and meticulous research, the inventors of the present invention found a T cell receptor (TCR) that can specifically bind to the KRAS G12V antigen short peptide VVGAVGVGK (SEQ ID NO.1). The antigen short peptide VVGAVGVGK can form a complex with HLA-A*11:01 and be presented on the cell surface together.

[0046] The present invention also provides nucleic acid molecules encoding the TCR, vectors containing the nucleic acid molecules, and cells transduced with the TCR. The present invention also provides the use of the TCR, nucleic acid molecules, vectors, and cells in the preparation of drugs for treating tumors or other immune diseases.

[0047] In a preferred embodiment of the present invention, (1) the TCR α chain variable region comprises a CDR having the following amino acid sequence.

[0048] A CDR1 comprising the amino acid sequence listed in SEQ ID NO.2; a CDR2 comprising the amino acid sequence listed in SEQ ID NO.3; and a CDR3 comprising the amino acid sequence listed in SEQ ID NO.4.

[0049] Among them, the amino acid sequence of the TCR α chain variable region CDR1α (CDR1) is shown in SEQ ID NO. 2: TSDPSYG.

[0050] The amino acid sequence of the TCR α chain variable region CDR2α (CDR2) is shown in SEQ ID NO. 3: QGSYDQQN.

[0051] The amino acid sequence of the TCR α chain variable region CDR3α (CDR3) is shown in SEQ ID NO. 4: CAMREGDIYNQGGKLIF.

[0052] (2) The TCR β chain variable region comprises a CDR having the following amino acid sequence.

[0053] A CDR1 comprising the amino acid sequence listed in SEQ ID NO.5; a CDR2 comprising the amino acid sequence listed in SEQ ID NO.6; and a CDR3 comprising the amino acid sequence listed in SEQ ID NO.7.

[0054] Among them, the amino acid sequence of the TCR β chain variable region CDR1βCDR1 is shown in SEQ ID NO.5: SGDLS.

[0055] The amino acid sequence of the TCR β chain variable region CDR2β (CDR2) is shown in SEQ ID NO. 6: YYNGEE.

[0056] The amino acid sequence of the TCR β chain variable region CDR3β (CDR3) is shown in SEQ ID NO. 7: CASSVGTALAYEQYF.

[0057] Up to three (preferably one or two) amino acid residues in one or more CDRs may be replaced with another amino acid residue. Typically, in these variants, some amino acids are replaced with conservative amino acids. These conservative amino acids include the following groups: G, A; S, A, T; F, Y, W; D, E; N, Q; and I, L, V.

[0058] The amino acid sequences of the CDR regions of the present invention can be embedded into any suitable framework structure to create a chimeric TCR. As long as the framework structure is compatible with the CDR regions of the TCR of the present invention, those skilled in the art can design or synthesize a TCR (molecule) with corresponding functions based on the CDR regions disclosed herein. Therefore, the TCR (molecule) provided herein refers to a TCR (molecule) comprising the sequences of the CDRs in the α chain variable region and / or the β chain variable region and any suitable framework structure.

[0059] The TCR α chain variable region provided by the present invention is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO.8; the TCR β chain variable region provided by the present invention is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO.12.

[0060] In a preferred embodiment of the present invention, the TCR provided by the present invention is a heterodimer composed of an α chain and a β chain.

[0061] Specifically, in one aspect, the TCR α chain of the heterodimeric structure comprises a variable region and a constant region, and the amino acid sequence of the variable region of the TCR α chain comprises CDR1α (SEQ ID NO. 2), CDR2α (SEQ ID NO. 3), and CDR3α (SEQ ID NO. 4) of the aforementioned α chain. More preferably, the amino acid sequence of the variable region of the TCR α chain is as shown in SEQ ID NO. 8.

[0062] In another aspect, the TCR β chain in the heterodimeric structure comprises a variable region and a constant region, wherein the amino acid sequence of the variable region of the TCR β chain comprises CDR1β (SEQ ID NO. 5), CDR2β (SEQ ID NO. 6), and CDR3β (SEQ ID NO. 7) of the aforementioned β chain. More preferably, the amino acid sequence of the variable region of the TCR β chain is as shown in SEQ ID NO. 12.

[0063] In a preferred embodiment of the present invention, the TCR provided by the present invention is a single-chain TCR (molecule) composed of part or all of the α chain and / or part or all of the β chain.

[0064] The α and β chains of the single-chain TCR (molecule) are present in the same polypeptide chain, which comprises Vα, Vβ, and Cβ, preferably linked in order from N-terminus to C-terminus. In order to express a single-chain TCR, it is very useful to provide a construct encoding the constant region of the TCR α chain.

[0065] The amino acid sequence of the α chain variable region of the single-chain TCR (molecule) comprises the aforementioned α chain CDR1α (SEQ ID NO. 2), CDR2α (SEQ ID NO. 3), and CDR3α (SEQ ID NO. 4). Preferably, the single-chain TCR (molecule) comprises the α chain variable region amino acid sequence of SEQ ID NO. 8. More preferably, the amino acid sequence of the α chain variable region of the single-chain TCR (molecule) is SEQ ID NO. 8. The amino acid sequence of the β chain variable region of the single-chain TCR (molecule) comprises the aforementioned β chain CDR1β (SEQ ID NO. 5), CDR2β (SEQ ID NO. 6), and CDR3β (SEQ ID NO. 7). Preferably, the single-chain TCR (molecule) comprises the β chain variable region amino acid sequence of SEQ ID NO. 12. More preferably, the amino acid sequence of the β chain variable region of the single-chain TCR (molecule) is SEQ ID NO. 12.

[0066] In a preferred embodiment of the present invention, the TCR (molecule) provided herein is a TCR-mRNA molecule composed of part or all of the α chain and / or part or all of the β chain. Specifically, the α chain and β chain of the TCR-mRNA molecule are connected together by a linker sequence, which includes Vα and Vβ.

[0067] The amino acid sequence of the α chain of the TCR-mRNA molecule comprises the CDR1 (SEQ ID NO. 2), CDR2 (SEQ ID NO. 3), and CDR3 (SEQ ID NO. 4) of the aforementioned α chain. Preferably, the TCR-mRNA molecule comprises the α variable region amino acid sequence of SEQ ID NO. 8. The amino acid sequence of the β chain of the TCR-mRNA molecule comprises the CDR1 (SEQ ID NO. 5), CDR2 (SEQ ID NO. 6), and CDR3 (SEQ ID NO. 7) of the aforementioned β chain. Preferably, the TCR-mRNA molecule comprises the β chain variable region amino acid sequence as shown in SEQ ID NO. 12.

[0068] In a preferred embodiment of the present invention, the constant region of the TCR provided herein is a human constant region. Those skilled in the art are aware of or can obtain human constant region amino acid sequences by consulting relevant literature or the public database of IMGT (International Immunogenetics Information System). For example, the constant region sequence of the α chain of the TCR (molecule) of the present invention may be "TRAC," and the constant region sequence of the β chain of the TCR (molecule) may be "TRBC1" or "TRBC2." IMGT specifies that the amino acid sequence at position 50 in TRAC is Leu, which is represented herein as "Leu50 of TRAC," and so on. Preferably, the amino acid sequence of the α chain of the TCR (molecule) of the present invention is SEQ ID NO. 10, and / or the amino acid sequence of the β chain is SEQ ID NO. 14. More preferably, the amino acid sequence of the α chain of the TCR (molecule) of the present invention with a leader sequence is SEQ ID NO. 22, and / or the amino acid sequence of the β chain with a leader sequence is SEQ ID NO. 24.

[0069] In a specific embodiment, the TCR is a single chain; preferably, the amino acid sequence of the TCR α chain variable region is shown as SEQ ID NO.26, and the amino acid sequence of the TCR β chain variable region is SEQ ID NO.28; more preferably, the TCR is composed of the α chain variable region and the β chain variable region connected by a connecting peptide chain SEQ ID NO.32, and the amino acid sequence of the TCR is SEQ ID NO.30.

[0070] The amino acid sequence of the single-chain TCR connecting peptide chain is shown in SEQ ID NO.32: GTSGSSGSGSGGSGSGCSG.

[0071] In a specific embodiment, the TCR is TCR-mRNA; preferably, the TCR α chain amino acid sequence is SEQ ID NO.34, and the TCR β chain amino acid sequence is SEQ ID NO.36; more preferably, the TCR is composed of the α chain and the β chain connected by a connecting peptide chain SEQ ID NO.40, and the amino acid sequence of the TCR is SEQ ID NO.38.

[0072] In another preferred embodiment of the present invention, amino acids in the Vα and Vβ domains outside the antigen-binding CDR loops of the TCR provided herein are replaceable. Simple variable region modifications are performed at a distance from the antigen-binding loops (Thomas, S. et al., Nat Commun, 2019, 10, 4451). Therefore, the TCRs of the present invention can contain amino acid substitutions in residues in their α and β chain variable regions exposed to the solvent, the hydrophobic core, the Vα-Vβ interface, the Vα-Cα, or the Vβ-Cβ interface to increase TCR expression and improve effector function. Preferably, the amino acid residues are substituted at one or more positions selected from the following: amino acid positions 5, 8, 19, 20, 24, 39, 50, 55, 66, 86, or 96 of TRAV; amino acid positions 9, 10, or 43 of TRBV.

[0073] In another preferred embodiment of the present invention, a new artificial disulfide bond can be introduced between Thr48 of the α chain constant region and Ser57 of the β chain constant region (this is achieved by replacing these residues with cysteine. The original natural disulfide bond in the TCR connecting peptide can be retained in place or removed). Therefore, the TCR provided by the present invention can include an artificial disulfide bond formed by cysteine ​​introduced between residues in its α and β chain constant regions. It should be noted that the constant region contains or does not contain the artificial disulfide bond introduced as described above, and the TCR provided by the present invention can contain a TRAC constant region sequence and a TRBC1 or TRBC2 constant region sequence. A set of natural disulfide bonds exists between the Cα and Cβ chains in the membrane-proximal region of the natural TCR, which is referred to as a "natural interchain disulfide bond" in the present invention. The interchain covalent disulfide bonds artificially introduced in the present invention, which are located at a different position from the natural interchain disulfide bonds, are referred to as "artificial interchain disulfide bonds" in the present invention. Preferably, the cysteine ​​residues of the artificial disulfide bond replace one or more groups of sites selected from the following: Thr48 of TRAC and Ser57 of TRBC1 or TRBC2; Tyr10 of TRAC and Ser17 of TRBC1 or TRBC2; Ser15 of TRAC and Val13 of TRBC1 or TRBC2; Thr45 of TRAC and Ser77 of TRBC1 or TRBC2; Thr45 of TRAC and Asp59 of TRBC1 or TRBC2; Leu50 of TRAC and Ser57 of TRBC1 or TRBC2; Arg53 of TRAC and Ser54 of TRBC1 or TRBC2; Ser61 of TRAC and Arg79 of TRBC1 or TRBC2; Pro89 of TRAC and Ala19 of TRBC1 or TRBC2.

[0074] Introducing an artificial interchain disulfide bond between the TCR α chain variable region and the β chain constant region can improve TCR stability. Therefore, the TCR α chain variable region and the β chain constant region provided herein may also contain an artificial interchain disulfide bond. In the present invention, the position numbering of the amino acid sequences of the variable regions TRAV and TRBV follows the position numbering listed in IMGT. Specifically, the cysteine ​​residues that form the artificial interchain disulfide bond between the TCR α chain variable region and the β chain constant region are substituted with: amino acid position 46 of TRAV and amino acid position 60 of TRBC1 or TRBC2; amino acid position 47 of TRAV and amino acid position 61 of TRBC1 or TRBC2; amino acid position 46 of TRAV and amino acid position 61 of TRBC1 or TRBC2; or amino acid position 47 of TRAV and amino acid position 60 of TRBC1 or TRBC2.

[0075] In another embodiment, the cysteine ​​residue of the artificial disulfide bond is also substituted with one or more sites selected from the following groups: the amino acid at position 48, or position 49, or position 50 of TRAV, the amino acid at position 17, or position 18, or position 19 of the connecting peptide chain between the α chain variable region and the β chain variable region.

[0076] In addition, the TCR provided by the present invention may also be a hybrid TCR comprising sequences derived from more than one species. The TCR of the present invention may comprise a hybrid TCR consisting of a human variable region and a mouse constant region.

[0077] Transduction of the dual-chain TCR (molecule) of the present invention (e.g., α and β chain molecules comprising the amino acid sequences given in SEQ ID NOs. 10 and 14) or chimeric TCR (molecule) comprising the specific CDRs described above into human T cells can yield antigen-specific CTLs. Similarly, transduction of single-chain TCRs can also be used to obtain antigen-specific CTLs, and single-chain TCRs have the advantage of not pairing with endogenous TCRs. Single-chain TCRs can also be prepared as soluble TCRs in a manner similar to antibodies. In soluble TCRs, single-chain TCRs do not contain a transmembrane region.

[0078] It should be understood that the amino acid names herein are represented by single letters or three letters in the internationally accepted English alphabet, and the correspondence between single letters and three letters of the amino acid names is as follows: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), Val (V).

[0079] The fourth aspect of the present invention provides a nucleic acid molecule encoding the TCR (molecule) or part thereof of the first and second aspects of the present invention, wherein the part may be one or more CDRs, the variable region of the α and / or β chain, and the α chain and / or β chain.

[0080] The nucleotide sequence encoding the CDR region of the α chain of the TCR (molecule) of the first aspect of the present invention is as follows: A CDR1 comprising the nucleotide sequence listed in SEQ ID NO.16; a CDR2 comprising the nucleotide sequence listed in SEQ ID NO.17; and a CDR3 comprising the nucleotide sequence listed in SEQ ID NO.18.

[0081] The nucleotide sequence encoding the CDR region of the β chain of the TCR (molecule) of the first aspect of the present invention is as follows: A CDR1 comprising the nucleotide sequence listed in SEQ ID NO.19; a CDR2 comprising the nucleotide sequence listed in SEQ ID NO.20; and a CDR3 comprising the nucleotide sequence listed in SEQ ID NO.21.

[0082] Therefore, the nucleotide sequence of the nucleic acid molecule encoding the TCR α chain of the present invention includes SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, and / or the nucleotide sequence of the nucleic acid molecule encoding the TCR β chain of the present invention includes SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21.

[0083] The nucleotide sequence of the nucleic acid molecule of the present invention can be single-stranded or double-stranded, can be DNA or RNA, and may or may not contain introns. Preferably, the nucleotide sequence of the nucleic acid molecule of the present invention does not contain introns but is capable of encoding a polypeptide of the TCR of the present invention, for example, the nucleotide sequence of the nucleic acid molecule encoding the variable region of the TCR α chain of the present invention comprises either SEQ ID NO. 9 or SEQ ID NO. 27 and / or the nucleotide sequence of the nucleic acid molecule encoding the variable region of the TCR β chain of the present invention comprises either SEQ ID NO. 13 or SEQ ID NO. 29. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable region of the TCR α chain of the present invention comprises SEQ ID NO. 27 and / or the nucleotide sequence of the nucleic acid molecule of the present invention encoding the variable region of the TCR β chain of the present invention comprises SEQ ID NO. 29. Alternatively, the messenger ribonucleotide sequence of the nucleic acid molecule of the present invention encoding the TCR α chain of the present invention comprises SEQ ID NO. 35 and / or the messenger ribonucleotide sequence of the nucleic acid molecule of the present invention encoding the TCR β chain of the present invention comprises SEQ ID NO. 37. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present invention comprises the nucleotide sequence encoding the TCR α chain of SEQ ID NO. 11 and / or comprises the nucleotide sequence encoding the TCR β chain of SEQ ID NO. 15. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present invention is SEQ ID NO. 31. Alternatively, the messenger ribonucleotide sequence of the nucleic acid molecule of the present invention is SEQ ID NO. 39.

[0084] Example 1: Obtaining KRAS G12V antigen short peptide-specific T cell clones Peripheral blood mononuclear cells (PBMCs) from HLA-A11-negative healthy volunteers were stimulated with T2-A11 cells (T2 cells were transferred with HLA-A*11:01 and named T2-A11 cells. Since these cells lack the antigen processing-related factor TAP, they can be effectively loaded with exogenous peptides) loaded with the KRAS G12V synthetic short peptide VVGAVGVGK (as shown in SEQ ID NO.1, synthesized by Jiangsu GenScript Biotechnology Co., Ltd.). T cells in PBMCs that can recognize the HLA-A*11:01 / VVGAVGVGK complex will be activated and expanded.

[0085] T cells specific for the KRAS G12V antigen peptide VVGAVGVGK were detected using PE-labeled HLA-A*11:01-VVGAVGVGK tetramer (MBL International). Expanded T cells were stained with tetramer-PE and anti-CD8-APC and analyzed by flow cytometry to confirm the presence of double-positive cells.

[0086] In order to obtain true tumor-specific CTLs that are restricted by HLA-A*11:01 and can recognize the antigen peptide VVGAVGVGK, the present invention used the limiting dilution method to culture the double-positive cells obtained by tetramer-PE and anti-CD8-APC staining sorting (for the limited dilution method monoclonal isolation technology, please refer to Gross A, et al. Int J Mol Sci. 2015 Jul 24; 16(8): 16897-919.). After screening dozens of monoclonals, the present invention obtained a tumor-specific T cell clone (named F194) that is restricted by HLA-A*11:01 and can recognize the antigen peptide VVGAVGVGK. After staining with CD8 and tetramer, the flow cytometry FACS data of this monoclonal T cell are as follows: Figure 1 shown.

[0087] Example 2: Obtaining the TCR gene of KRAS G12V antigen short peptide-specific T cell clones and constructing vectors Methods for single-cell VDJ sequencing have been developed in recent years and are well known in the art, and are described in detail in manuals such as 10×Genomics based on microfluidics and oil droplet encapsulation technology.

[0088] Specifically, gel beads with barcodes and primers were encapsulated in oil droplets with single cells of the T cell clone specific for the antigenic short peptide VVGAVGVGK screened in Example 1 and restricted to HLA-A*11:01. The gel beads dissolved in each oil droplet, the cells were lysed to release mRNA, and barcoded cDNA for sequencing was produced by reverse transcription, followed by TCR library construction; wherein the V(D)J sequence of the TCR was enriched by nested PCR primers designed in the C region of the TCR. The library was then sequenced and tested using the Illumina sequencing platform to obtain the TCR data of the T cell clone in Example 1. After α and β pairing and verification, the T cell clone expressed a TCR α chain comprising a CDR with the following amino acid sequence:

[0089] CDR1α-SEQ ID NO.2; CDR2α-SEQ ID NO.3; CDR3α-SEQ ID NO.4.

[0090] The beta chain contains a CDR with the following amino acid sequence: CDR1β-SEQ ID NO.5; CDR2β-SEQ ID NO.6; CDR3β-SEQ ID NO.7.

[0091] The TCR α chain and TCR β chain partial or full-length genes were connected to the P2A sequence through the Furin GSG linker by artificial gene synthesis to obtain the TCRβ-2A-TCRα fragment. This fragment was synthesized by the standard method described in J Sambrook, David Russell. et al., Molecular Cloning: A Laboratory Manual, 2016, Third Edition. Not I+ EcoR I double-digested and cloned into the retroviral expression vector pMP71 to obtain the recombinant plasmid pMP71-TCRβ-2A-TCRα. This recombinant plasmid can express the amino acid sequences of the TCR α and TCR β chain variable regions shown in SEQ ID NO. 8 and SEQ ID NO. 12, as well as the amino acid sequence of the single-chain TCR shown in SEQ ID NO. 30. Alternatively, the TCR-mRNA molecule can be cloned into the in vitro transcription vector pcDNA3.1(+). This recombinant plasmid can express the amino acid sequences of the TCR α and β chains shown in SEQ ID NO. 34 and SEQ ID NO. 36. TCR mRNA can be obtained through in vitro transcription, 5' capping with Cap1, pseudo-UTP (Ψ) substitution, 5-Methyl-CTP substitution, and 3' poly(A) tailing.

[0092] In order to enable the α and β chains of the TCR (molecule) of the present invention to form correct pairing more effectively during the transduction process, a cysteine ​​residue is introduced into the constant region of the α and β chains of the TCR (molecule) of the present invention to form an artificial interchain disulfide bond, and the positions of the introduced cysteine ​​residues are Thr48 of TRAC and Ser57 of TRBC, respectively.

[0093] Example 3: Preparation of TCR-retrovirus (1) Preparation of recombinant plasmid: The recombinant plasmid pMP71-TCR obtained in Example 2 was transformed into Stbl3 competent cells and evenly spread on LB solid culture medium plates containing 0.1% by volume of ampicillin. After incubation at 37°C for 14 h, a single colony was picked and transferred to LB liquid culture medium containing 0.1% by volume of ampicillin. The culture was shaken at 37°C and 220 rpm / min for 14 h, and the plasmid was extracted to obtain the pMP71-TCR recombinant plasmid.

[0094] (2) Packaging of recombinant plasmid: 293Vec-RD114 cells in the logarithmic growth phase were used as packaging cells and seeded into a 10 cm dish containing culture medium (DMEM medium containing 10% by volume FBS). When the cell density reached 80% by area, the recombinant plasmid pMP71-TCR described in Example 2 was transfected using the standard polyethyleneimine (PEI) method. After 6 hours of culture, the culture medium containing the transfection reagent was removed and replaced with fresh complete culture medium. After 48 hours, the culture medium was collected and filtered with a 0.45 μm filter membrane to remove cell debris. The TCR-retroviral suspension was obtained and stored at -80°C.

[0095] Example 4: Preparation of KRAS G12V-specific TCR-T cells and analysis of their TCR expression Peripheral blood was collected from healthy donors, lysed with red blood cell lysis buffer (Solebo), and CD8 cells were sorted using CD8 magnetic beads (Miltenyi Biotec). + T cells. Adjust the cell density to 1×10 6 cells / mL, and anti-CD3 / CD28-coupled magnetic beads (Miltenyi Biotec) were added to the cell culture medium to activate the T cells. After 48 hours, the TCR-retroviral suspension was removed from the -80°C freezer and slowly thawed in a 4°C refrigerator. 0.5×10 cells / mL were placed in each well of a 24-well plate pre-coated with RetroNectin (Takara). 6 CD8 + To the T cells, add 1.5 mL of viral supernatant and IL-2 (600 U / mL), gently pipette to mix, and centrifuge at 2200 rpm at 32°C for 90 minutes. Then, continue to culture in a 37°C, 5% CO2 incubator. On the 4th day, FACS was used to detect the expression of KRAS G12V-TCR on T cells. Figure 2 As shown, mock-transduced T cells could not be stained with KRAS G12V-tetramers, indicating that they had no KRAS G12V specificity; whereas KRAS G12V-TCR-transduced T cells could be stained with KRAS G12V-tetramers, indicating that they acquired KRAS G12V specificity through TCR transduction. Figure 2 The freshly transduced KRAS G12V-TCR-T cells shown were stimulated with T2-A11 cells loaded with the KRAS G12V antigen peptide VVGAVGVGK, and the KRAS G12V-specific T cells in them were significantly expanded.

[0096] Among them, the source of healthy donor peripheral blood is the mobilized peripheral blood of hematopoietic stem cell transplant donors.

[0097] Example 5: Intracellular immune factor staining was used to detect the function of KRAS G12V-specific TCR-T cells.

[0098] The artificially synthesized KRAS G12V antigen peptide VVGAVGVGK and control peptides (unmutated wild-type WT antigen peptide VVVGAGGVGK and mutated KRAS G12D antigen peptide VVVGADGVGK) were incubated with T2-A11 cells at 37°C and 5% CO2 for 2 hours (the concentrations of KRAS G12V polypeptide in the experimental group were 10μM, 1μM, 100nM, 10nM, 1nM and 100pM, and the concentration of polypeptide in the control group was consistent with the highest concentration of 10μM in the experimental group). The unbound antigen peptide and control peptide were washed to remove the cells, and then the cells were collected to obtain T2-A11 cells loaded with antigen peptides and control peptides.

[0099] The KRAS G12V-specific TCR-T cells obtained in Example 4 were co-cultured with T2-A11 target cells loaded with the specific antigen peptide VVGAVGVGK and control antigen peptides (WT antigen peptide VVVGAGGVGK and KRAS G12D antigen peptide VVVGADGVGK) in a 96-well plate at 37°C and 5% CO2, where the concentrations of T cells and target cells were both 0.4×10 6 / well, and add BFA (the function of BFA is to retain the immune factors in T cells in the cells without being released, so as to facilitate staining monitoring by FACS) to make the final concentration of 1.5μg / mL.

[0100] After 24 h of coculture, the cells were collected and first stained for cell surface staining with anti-CD3 / CD8-APC. Then, the Fix and Perm kit (Invitrogen) was used to stain intracellular immune factors according to the manufacturer's instructions. The stained cells were then tested for the production of various immune factors by FACS.

[0101] Figure 3The results showed that after co-culturing KRAS G12V-TCR-T cells with T2-A11 target cells, T2-A11 cells loaded with KRAS G12V antigen peptides could stimulate TCR-T cells to secrete IFN-γ, while T2-A11 cells loaded with control antigen peptides could not induce TCR-T cells to express IFN-γ. Furthermore, the concentration of the specific antigen peptide recognized by KRAS G12V-TCR-T cells can be as low as picomolar (pM). These results indicate that the high-affinity TCR obtained by the present invention can specifically recognize the KRAS G12V antigen peptide VVGAVGVGK at the picomolar (pM) level. T cells transduced with the TCR of the present invention can secrete IFN-γ after recognizing target cells, thereby killing the target cells. However, when encountering control target cells, no IFN-γ is produced, thus avoiding unnecessary side effects.

[0102] Example 6: Preparation of target cells K562 / THP-1 / PANC-1 / H460-A11-WT-GFP and K562 / THP-1 / PANC-1 / H460-A11-G12V-GFP Artificial gene synthesis was used to obtain HLA-A*11:01-CD34-puro, KRAS WT-GFP, and KRASG12V-GFP, in which A11 was linked to CD34 via a P2A sequence, CD34 was linked to puro via a T2A sequence, and KRAS WT / G12V was linked to GFP via a T2A sequence. The recombinant plasmids pMP71-HLA-A*11:01-CD34-puro, pMP71-KRAS WT-GFP, and pMP71-KRAS G12V-GFP were obtained using the same method as in Example 2. The same method as in Examples 3 and 4 was used to transfer HLA-A*11:01-CD34-puro, KRAS WT-GFP, and KRAS G12V-GFP into the leukemia cell lines K562 and THP-1, the lung cancer cell line H460, and the pancreatic cancer cell line PANC-1 to obtain the positive target cells K562 / THP-1 / PANC-1 / H460-A11-G12V-GFP and the control target cells K562 / THP-1 / PANC-1 / H460-A11-WT-GFP required by the present invention. The HLA-A11-CD34 staining and GFP expression of these target cells are shown in FIG. Figure 4 and Figure 5 .

[0103] Depend on Figure 4 and Figure 5As can be seen, K562 / THP-1 / PANC-1 / H460-A11-G12V-GFP expresses both HLA-A*11:01 and the KRAS G12V antigen linked to GFP. The expression of HLA-A*11:01 and KRAS G12V enables recognition by the KRAS G12V-TCR-T of the present invention, while GFP expression can be used to indicate whether the target cells have been killed. K562 / THP-1 / PANC-1 / H460-A11-WT-GFP cells express HLA-A*11:01 and unmutated wild-type KRAS, but lack the G12V mutation, and serve as control target cells.

[0104] Example 7: Killing effect of KRAS G12V-TCR-T cells on target cells The K562 / THP-1 / PANC-1-A11-G12V-GFP constructed in Example 6 was used as the positive target cell, and the K562 / THP-1 / PANC-1-A11-WT-GFP was used as the negative target cell control. 1×10 4 Positive target cells and control target cells (K562 line / THP-1 line) were added to each well of a flat-bottom 96-well plate in 100 μL of DMEM medium. 4 Positive target cells and control target cells (PANC-1 line) were then added with 100 μL of nonspecific T cells (mock) and KRAS G12V-TCR-T cells at different effector-target ratios (E:T) for mixed culture.

[0105] After 24 hours, all cells from each well of the K562 and THP-1 lines were aspirated and transferred to flow cytometry tubes for flow cytometry analysis of changes in the GFP-positive rate of positive and control target cells. For the PANC-1 line, the culture supernatant was discarded and the OD values ​​of positive and control target cells were measured using sulforhodamine B (SRB) colorimetric analysis. Changes in the GFP-positive rate of target cells and changes in the SRB OD values ​​indicated whether the target cells had been killed.

[0106] Figure 6 The results showed that when mock cells were added, both the control target cells and the positive target cells survived well. However, when KRAS G12V-TCR-T cells were added, the positive target cells were significantly killed, while the control target cells still survived well. This indicates that the KRAS G12V-TCR-T cells of the present invention can selectively kill cells that express both HLA-A*11:01 and KRASG12V.

[0107] Example 8: Elisa detection of INF-γ secretion after co-culture of KRAS G12V-TCR-T cells with target cells The K562 / PANC-1-A11-G12V-GFP constructed in Example 6 was used as the positive target cell, and the K562 / PANC-1-A11-WT-GFP was used as the negative target cell control. 1×10 4 Positive target cells and control target cells (K562 line) were added to each well of a flat-bottom 96-well plate in 100 μL of DMEM medium. 4 Positive target cells and control target cells (PANC-1 line) were then added with 100 μL of nonspecific T cells (mock) and KRAS G12V-TCR-T cells at different effector-target ratios (E:T) for mixed culture. After 24 hours, the supernatant from each well was aspirated for ELISA detection of INF-γ secretion.

[0108] like Figure 7 The results showed that under different effector-target ratios, neither the control target cells nor the positive target cell groups could stimulate mock cells to secrete IFN-γ. However, after co-culture of KRAS G12V-TCR-T cells with positive target cells, they could stimulate TCR-T to secrete large amounts of IFN-γ, while the control target cells could not stimulate KRAS G12V-TCR-T to produce immune factors, and there was a significant difference between the two. This result further indicates that the KRAS G12V-TCR can specifically recognize the antigen peptide VVGAVGVGK. After T cells transduced with the KRAS G12V-TCR recognize target cells, they can secrete IFN-γ, thereby killing target cells. However, when encountering control target cells, no immune factors are produced, thus avoiding unnecessary side effects.

[0109] As can be seen from the above, the T cell receptor for the KRAS G12V antigen peptide provided by the present invention recognizes the KRAS G12V-HLA-A*11:01 complex with high affinity, can recognize specific KRAS G12V antigen peptides at concentrations as low as picomolar levels, and has a strong killing effect on tumor cells that co-express HLA-A*11:01 and KRAS G12V. The KRAS G12V-specific T cells of the present invention can be used to treat KRAS G12V-related diseases that present the KRAS G12V antigen peptide VVGAVGVGK-HLA-A*11:01 complex.

[0110] It should be noted that when the present invention involves a numerical range, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes a preferred embodiment.

[0111] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once the basic inventive concepts become known, and all such changes and modifications fall within the scope of the present invention.

Claims

1. A T cell receptor that recognizes a short peptide of the KRAS G12V antigen, characterized in that: The T cell receptor can recognize the KRAS G12V-HLA-A*11:01 complex with high affinity, and the amino acid sequence of the KRAS G12V antigen short peptide is shown in SEQ ID NO.1; The T cell receptor is an αβ heterodimer composed of an α chain and a β chain; The amino acid sequence of the α chain is as shown in SEQ ID NO. 10, or an amino acid sequence having at least 90% sequence identity thereto; The amino acid sequence of the β chain is shown in SEQ ID NO. 14, or an amino acid sequence having at least 90% sequence identity thereto.

2. The T cell receptor according to claim 1, characterized in that The α chain includes a TCR α chain variable region, and the TCR α chain variable region includes three complementarity determining regions CDR1α to CDR3α, and the nucleotide sequences thereof are shown in SEQ ID NO. 2 to SEQ ID NO. 4, respectively; The β chain includes a TCR β chain variable region, and the TCR β chain variable region includes three complementarity determining regions CDR1β to CDR3β, and the nucleotide sequences thereof are shown in SEQ ID NO.5 to SEQ ID NO.7, respectively.

3. The T cell receptor according to claim 1, characterized in that The α chain further comprises a TCR α chain constant region, and the β chain further comprises a TCR β chain constant region.

4. The T cell receptor according to claim 1, characterized in that The T cell receptor is a single chain, comprising the variable region of the α chain of TCR and the variable region of the β chain of TCR; The amino acid sequence of the TCR α chain variable region is shown in SEQ ID NO. 26; The amino acid sequence of the TCR β chain variable region is shown in SEQ ID NO.28; The T cell receptor is formed by connecting the α chain variable region of the TCR and the β chain variable region of the TCR via a connecting peptide chain with an amino acid sequence as shown in SEQ ID NO.

32.

5. The T cell receptor according to claim 1, characterized in that The T cell receptor is TCR-mRNA, and the TCR-mRNA includes the α chain of TCR-mRNA and the β chain of TCR-mRNA; The amino acid sequence of the α chain of the TCR-mRNA is shown in SEQ ID NO. 34; The amino acid sequence of the β chain of the TCR-mRNA is shown in SEQ ID NO.36; The TCR-mRNA is formed by connecting the α chain of the TCR-mRNA and the β chain of the TCR-mRNA via a connecting peptide chain with an amino acid sequence as shown in SEQ ID NO.

40.

6. A multivalent T cell receptor complex, characterized in that The method comprises at least three T cell receptors, at least one of which is the T cell receptor according to any one of claims 1 to 5.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises a nucleotide sequence encoding the T cell receptor according to any one of claims 1 to 5 or a complementary sequence thereof, or a codon-optimized nucleotide sequence corresponding to the amino acid sequence of the T cell receptor.

8. A vector or host cell, characterized in that The vector or the host cell contains the nucleic acid molecule according to claim 7.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the T cell receptor according to any one of claims 1 to 5, the complex according to claim 6, the nucleic acid molecule according to claim 7, or the vector or host cell according to claim 8, and a pharmaceutically acceptable carrier.

10. Use of the T cell receptor according to any one of claims 1 to 5, the complex according to claim 6, the vector or host cell according to claim 8, or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating tumors or other immune diseases.

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