T-cell receptors, complexes, pharmaceutical compositions and uses recognizing krass g12v antigenic short peptides

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a T cell receptor for recognizing a KRAS G12V antigen short peptide, a complex, a pharmaceutical composition and purposes. The T cell receptor can recognize a KRAS G12V-HLA-A*11:01 complex with high affinity; the T cell receptor is an alpha-beta heterodimer composed of an alpha chain and a beta chain; the amino acid sequence of the alpha chain is shown as SEQ ID NO. 10, or an amino acid sequence with at least 90% sequence identity; the amino acid sequence of the beta chain is shown as SEQ ID NO. 14, or an amino acid sequence with at least 90% sequence identity. The T cell receptor provided in the application has purposes for preparing a drug for treating tumors or other immune diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to T-cell receptors, complexes, pharmaceutical compositions, and uses that recognize short peptides of the KRAS G12V antigen. Background Technology

[0002] T-cell immunotherapy is a crucial method in tumor immunotherapy. By isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue, cloning and expanding them in vitro, and then reinfusing them into patients, it can achieve sustained remission in patients with advanced cancer who have developed resistance to radiotherapy and chemotherapy, producing good clinical therapeutic effects. However, the isolation and culture of TILs are demanding, requiring a long time to reach the required cell count for clinical treatment. More importantly, the number of tumor tissues from which TILs can be successfully isolated is still very limited, thus restricting the clinical application of TILs in oncology.

[0003] T cells primarily recognize tumors through T cell receptors (TCRs) on their surface. TCRs have the ability to recognize the major human histocompatibility complex (MHC)-antigen peptide complex on tumor cells. The TCR consists of a heterodimeric structure formed by two peptide chains, α and β. Each peptide chain contains a variable region, a linker region, and a constant region. The β chain usually also contains a short, variable region between the variable and linker regions, but this variable region is often considered part of the linker region. Each variable region contains three CDRs (complementarity-determining regions), namely CDR1, CDR2, and CDR3, embedded in the framework regions. The CDR regions determine the binding of the TCR to the MHC complex. CDR3, formed by the recombination of the variable and linker regions, is called the hypervariable region and directly determines the antigen specificity of the TCR. When the TCR recognizes the MHC-antigen peptide complex, CDR3 can directly bind to the antigen peptide. CDR1 and CDR2 mainly contact the two α-helices of the MHC molecule's binding groove. These interactions ensure that the TCR accurately recognizes the MHC-antigen peptide complex. The 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 a linker region. Sequences of the TCR constant region can be found in the public database of the International Immunogenetic Information System (IMGT), such as "TRAC" for the TCR (molecular) α chain and "TRBC1" or "TRBC2" for the TCR (molecular) β chain. In addition, the TCR α and β chains also 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, these tumor-specific TCR-T cells are activated upon encountering MHC-peptide complexes on tumor cells, thereby proliferating in the patient's body and achieving a therapeutic effect by killing tumor cells.

[0005] The KRAS gene encodes a small GTPase belonging to the RAS gene family. It is one of the most commonly mutated genes in various cancers, including pancreatic cancer (90%), colorectal cancer (46%), endometrial cancer (17%), non-small cell lung cancer (12%), cholangiocarcinoma, cervical cancer, bladder cancer, liver cancer, and breast cancer. KRAS mutations are most commonly found at codons 12 and 13, and these mutations can promote tumor cell proliferation and metabolic reprogramming. Among 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 where the twelfth codon (G) in the KRAS coding region is changed to V. Its product, the KRAS G12V protein, is a tumor-specific antigen (TSA) and is expressed only in tumor cells. Therefore, some studies have indicated that KRAS G12V is an excellent target antigen for tumor immunotherapy. After being generated intracellularly, KRAS G12V is degraded into small polypeptides, which, after binding with MHC molecules to form a complex, are presented to the cell surface. VVGAVGVGK, a short peptide derived from the KRAS G12V antigen, is a target for the treatment of KRAS G12V-related tumors. Traditional therapies for these KRAS G12V-related malignancies involve radiotherapy and chemotherapy, but these methods damage the patient's normal cells. The Institute of Microbiology, Chinese Academy of Sciences, identified two specific TCRs targeting the KRAS-G12V-9 peptide in HLA-A*11:01 transgenic mice. T cells constructed from these two specific TCRs showed specific responses to different tumor cells with KRAS-G12V mutations. This study provides a potential drug candidate for treating tumors carrying KRAS G12V mutations. The TCRs reported in this literature were isolated from T cells of transgenic mice, and their variable regions exhibit species specificity in gene sequence and structure. Furthermore, based on immunological principles, it should not be a high-affinity TCR. This is because KRAS G12V is an autoantigen. Therefore, to avoid autoimmune diseases, cytotoxic T lymphocytes (CTLs) with high affinity for KRAS G12V in HLA-A*11:01 transgenic mice were deleted during the negative selection process of thymocytes. As a result, in the mouse's own T cells, only KRAS G12V-CTLs, which are resistant to the KRAS G12V autoantigen and have low affinity, can survive. Since low-affinity CTLs often have poor anti-tumor activity, a crucial goal of T-cell-based tumor immunotherapy is to obtain CTLs with high affinity and specificity for tumor antigens.

[0006] Previous studies have described a non-self-restricting, or allogeneic-restricted, CTL protocol for obtaining high-affinity CTLs. By employing this protocol, researchers obtained CTLs specific to tumor-associated antigen tyrosinase (TAI) and high-affinity TCRs. The principle behind this technique is based on stimulating allogeneic CTLs with the patient's own MHC-peptide complex. Because the allogeneic CTLs have not undergone the negative selection process of their own MHC, they contain CTLs highly sensitive to their own MHC and possess high-affinity TCRs. However, the allogeneic restriction protocol also has a significant drawback: among the T cells that generate an immune response to their own MHC-peptide complex, only a small fraction are both MHC-restricted and specific to the antigenic peptide; the majority of cells only recognize MHC and lack specificity for the antigenic peptide. In other words, most of the CTLs stimulated using this method are non-specific T cells. If such CTLs are used to treat tumors, they can induce severe non-specific immune responses. In summary, given the problems of low affinity, poor antitumor activity, and nonspecificity 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 antitumor activity, and nonspecificity of previously obtained KRAS G12V TCRs in the prior art, this invention identifies a CTL with both high affinity and specificity for the tumor antigen KRAS G12V, thereby isolating and obtaining a TCR with high affinity and specificity for the tumor antigen, and providing a T cell receptor, complex, pharmaceutical composition, and uses for recognizing short peptides of the KRAS G12V antigen.

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

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

[0010] The first objective of this invention is to provide a T-cell receptor that recognizes a short peptide of the KRAS G12V antigen. 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, which are connected by covalent bonds or disulfide bonds.

[0012] The amino acid sequence of the α chain is as shown in SEQ ID NO.10, or is an amino acid sequence that has at least 90% sequence identity with it.

[0013] The amino acid sequence of the β chain is as shown in SEQ ID NO.14, or is an amino acid sequence that has at least 90% sequence identity with it.

[0014] The T-cell receptor provided by this invention can recognize the KRAS G12V-HLA-A*11:01 complex with high affinity, and 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), recognizing the short peptide VVGAVGVGK (SEQ ID NO. 1) of the KRAS G12V antigen presented by the HLA-A*11:01 allele with high affinity. The T-cell receptor provided by this invention has both high affinity and specificity for the tumor antigen KRAS G12V CTLs, exhibiting high anti-tumor activity, and can solve the problems of low affinity, poor anti-tumor activity, and non-specificity of previously obtained KRAS G12V TCRs in the prior art.

[0015] Preferably, the α chain includes a TCR α chain variable region, which includes three complementarity-determining regions CDR1α~CDR3α, the nucleotide sequences of which are shown in SEQ ID NO.2~SEQ ID NO.4.

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

[0017] Preferably, the α chain further includes a TCRα chain constant region, and the β chain further includes a TCRβ chain constant region.

[0018] Preferably, the T cell receptor is a single chain, comprising the α-chain variable region and the β-chain variable region of the 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 β-chain variable region of the TCR is shown in SEQ ID NO.28.

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

[0022] Preferably, the T cell receptor is TCR-mRNA, which includes the α chain 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 linking the α chain and the β chain of the TCR-mRNA through a linker peptide chain; the amino acid sequence of the linker peptide chain is shown in SEQ ID NO.40.

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

[0027] A third objective 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 carrier containing the nucleic acid molecule. Preferably, the carrier is a viral carrier and / or a lipid nanoparticle carrier; more preferably, the carrier is a lentiviral carrier and / or a retroviral carrier; more preferably, the carrier is a retroviral carrier.

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

[0030] A sixth object of the present invention is to provide a cell that is 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 a stem cell derived from a subject.

[0031] A seventh object of the present invention is to provide a pharmaceutical composition comprising the T cell receptor, the complex, the nucleic acid molecule, or the carrier or host cell, and a pharmaceutically usable carrier.

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

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] 1. This invention provides a T-cell receptor that recognizes a short peptide of the KRAS G12V antigen. The T-cell receptor provided by this 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 short peptide VVGAVGVGK (SEQ ID NO. 1) of the KRAS G12V antigen presented by the HLA-A*11:01 allele with high affinity. The T-cell receptor provided by this invention has both high affinity and specificity for the tumor antigen KRAS G12V CTLs, exhibiting high anti-tumor activity. This solves the problems of low affinity, poor anti-tumor activity, and non-specificity of previously obtained KRAS G12V TCRs in the prior art.

[0035] 2. The host cell provided in this invention, 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 this invention can be transferred into the patient's own T cells (or T cells from a donor) of a malignant tumor patient expressing KRAS G12V, and then these genetically engineered cells can be introduced into the patient to achieve treatment. Attached Figure Description

[0036] Figure 1 CD8 of the KRAS G12V specific CTL in this invention + And HLA-A*11:01-KRAS G12V-tetramer-PE double positive staining results.

[0037] Figure 2 The present invention uses flow cytometry to detect CD8+ in primary human T cells after KRAS G12V-TCR is transfected. + and HLA-A*11:01-KRAS G12V-tetramer-PE double positive staining results; among which, Figure 2 (A) in the image represents the staining results of mock T cells. Figure 2 (B) in the image shows the staining results of T cells transduced by KRAS G12V TCR.

[0038] Figure 3 To detect the secretion of INF-γ by KRAS G12V-TCR-T cells (F194) in T2-A11 cells stimulated with control antigen peptide and KRAS G12V antigen peptide in this invention using flow cytometry: wherein, Figure 3 In the figure, (A) represents the secretion of INF-γ by F194 cells in T2-A11 cells stimulated with 10 μM of KRAS WT (wild-type) control antigen peptide. Figure 3 (B) represents the secretion of INF-γ by F194 cells in T2-A11 cells stimulated with 10 μM of KRAS G12D control antigen peptide. Figure 3 In the figure, (C) represents the secretion of INF-γ by F194 in T2-A11 cells stimulated with 10 μM of KRAS G12V antigen peptide. Figure 3 In the figure (D), F194 secretes INF-γ in T2-A11 cells stimulated with 1 μM of KRAS G12V antigen peptide. Figure 3 In the figure (E), F194 secretes INF-γ in T2-A11 cells stimulated with 100 nM of KRAS G12V antigen peptide. Figure 3 In this context, (F) represents the secretion of INF-γ by F194 in T2-A11 cells stimulated with 10 nM of KRAS G12V antigen peptide. Figure 3 In this context, (G) represents the secretion of INF-γ by F194 in T2-A11 cells stimulated with 1 nM of KRAS G12V antigen peptide. Figure 3 (H) in the figure represents the secretion of INF-γ by F194 in T2-A11 cells stimulated with 100 pM of KRASG12V antigen peptide.

[0039] Figure 4 This invention utilizes flow cytometry to detect the transfection efficiency of HLA-A*11:01-CD34-puro in K562, THP-1, PANC-1, and H460 cells; wherein, Figure 4 In this context, (A) represents the percentage of positive K562 cells after transfection with HLA-A*11:01-CD34-puro; Figure 4 (B) represents the percentage of positive THP-1 cells after transfection with HLA-A*11:01-CD34-puro; Figure 4 In the figure, (C) represents the percentage of positive cells in PANC-1 cells after transfection with HLA-A*11:01-CD34-puro; Figure 4 (D) represents the percentage of positive cells in H460 cells after transfection with HLA-A*11:01-CD34-puro.

[0040] Figure 5 To detect the transfection efficiency of KRAS WT-GFP and KRAS G12V-GFP in K562-A11, THP-1-A11, PANC-1-A11 and H460-A11 cells using flow cytometry in this invention: Figure 5 In the figure, (A) represents the percentage of positive cells in K562-A11 cells after transfection with KRAS WT-GFP and KRAS G12V-GFP; Figure 5 (B) represents the percentage of positive THP-1-A11 cells after transfection with KRAS WT-GFP and KRAS G12V-GFP; Figure 5 In the figure, (C) represents the percentage of positive cells in PANC-1-A11 cells after transfection with KRAS WT-GFP and KRAS G12V-GFP; Figure 5 In the figure (D), the percentage of positive cells in H460-A11 cells after transfection with KRASWT-GFP and KRAS G12V-GFP is represented.

[0041] Figure 6 In this invention, flow cytometry and sulfonylrhodamine B (SRB) colorimetric analysis were used to detect the cytotoxic effects of KRAS G12V-TCR-T cells and non-specific T cell (mock) cells on target cells at different effector-to-target ratios (E:T). Figure 6 In the figure, A represents the target cell survival rate after KRAS G12V-TCR-T cells and mock cells were co-cultured with positive target cells K562-A11-G12V-GFP and negative target cells K562-A11-WT-GFP at different effector-to-target ratios, as detected by flow cytometry. Figure 6 B in the figure represents the target cell survival rate after KRAS G12V-TCR-T cells and mock cells were co-cultured with positive target cells THP-1-A11-G12V-GFP and negative target cells THP-1-A11-WT-GFP at different effector-to-target ratios, as detected by flow cytometry. Figure 6Figure C shows the target cell survival rate after co-culturing 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 under different effective-target ratios, as detected by sulfonylrhodamine B (SRB) colorimetric analysis.

[0042] Figure 7 In this invention, ELISA was used to detect the secretion of INF-γ by KRAS G12V-TCR-T cells and non-specific T cells (mock) co-cultured with target cells at different effector-to-target ratios; wherein, Figure 7 In the figure, A represents the secretion of INF-γ by KRAS G12V-TCR-T cells and mock cells co-cultured with positive target cells K562-A11-G12V-GFP and negative target cells K562-A11-WT-GFP under different effective-to-target ratios. Figure 7 In the figure, B represents the secretion of INF-γ by KRAS G12V-TCR-T cells and mock cells co-cultured with positive target cells PANC-1-A11-G12V-GFP and negative target cells PANC-1-A11-WT-GFP at different effective-to-target ratios. Detailed Implementation

[0043] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0044] All features disclosed in this specification, or steps in all methods or processes disclosed herein, may be combined in any way, except for mutually exclusive features and / or steps.

[0045] Any feature disclosed in the specification of this invention (including any appended claims, the abstract, and the drawings) may be replaced by other equivalent or similar features, unless specifically stated otherwise. That is, unless specifically stated otherwise, each feature is merely one example of a series of equivalent or similar features.

[0046] Through repeated, in-depth, and meticulous research, the inventors of this invention have discovered 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 together to the cell surface.

[0047] The present invention also provides a nucleic acid molecule encoding the TCR and a vector comprising the nucleic acid molecule, and cells transduced with the TCR. The present invention further provides the use of the TCR, nucleic acid molecule, vector, and cells for the preparation of medicaments for treating tumors or other immune diseases.

[0048] In a preferred embodiment of the present invention, (1) the variable region of the TCR α chain contains a CDR having the following amino acid sequence.

[0049] CDR1, which contains the amino acid sequence listed in SEQ ID NO.2; CDR2, which contains the amino acid sequence listed in SEQ ID NO.3; and CDR3, which contains the amino acid sequence listed in SEQ ID NO.4.

[0050] The amino acid sequence of the variable region CDR1α (CDR1) of the TCR α chain is shown in SEQ ID NO.2: TSDPSYG.

[0051] The amino acid sequence of the variable region CDR2α (CDR2) of the TCR α chain is shown in SEQ ID NO.3: QGSYDQQN.

[0052] The amino acid sequence of the variable region CDR3α (CDR3) of the TCR α chain is shown in SEQ ID NO.4: CAMREGDIYNQGGKLIF.

[0053] (2) The variable region of the TCR β chain contains a CDR having the following amino acid sequence.

[0054] CDR1, which contains the amino acid sequence listed in SEQ ID NO.5; CDR2, which contains the amino acid sequence listed in SEQ ID NO.6; and CDR3, which contains the amino acid sequence listed in SEQ ID NO.7.

[0055] The amino acid sequence of the variable region CDR1βCDR1 of the TCR β chain is shown in SEQ ID NO.5: SGDLS.

[0056] The amino acid sequence of the variable region CDR2β (CDR2) of the TCR β chain is shown in SEQ ID NO.6: YYNGEE.

[0057] The amino acid sequence of the variable region CDR3β (CDR3) of the TCR β chain is shown in SEQ ID NO.7: CASSVGTALAYEQYF.

[0058] In one or more of the CDRs, up to three (preferably one or two) amino acid residues can be replaced by another amino acid residue. Typically, in these variants, some amino acids are replaced by conserved amino acids. These conserved amino acids include the following groups: G, A; S, A, T; F, Y, W; D, E; N, Q and I, L, V.

[0059] The amino acid sequence of the CDR region of the present invention can be inserted into any suitable framework structure to prepare a chimeric TCR. As long as the framework structure is compatible with the CDR region of the TCR of the present invention, those skilled in the art can design or synthesize a TCR (molecule) with corresponding function based on the CDR region disclosed in the present invention. Therefore, the TCR (molecule) provided by the present invention refers to the TCR (molecule) containing the CDR sequence in the α-chain variable region and / or β-chain variable region and any suitable framework structure.

[0060] 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.

[0061] In a preferred embodiment of the present invention, the TCR provided by the present invention is a heterodimer composed of α chain and β chain.

[0062] Specifically, on the one hand, the TCR α chain of the heterodimer structure includes a variable region and a constant region, and the amino acid sequence of the variable region of the TCR α chain includes 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 shown in SEQ ID NO.8.

[0063] On the other hand, the TCR β chain of the heterodimer structure includes a variable region and a constant region, and the amino acid sequence of the variable region of the TCR β chain includes 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.

[0064] 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.

[0065] The α and β chains of the single-chain TCR (molecule) exist within the same polypeptide chain, comprising Vα, Vβ, and Cβ, preferably linked in order from the N-terminus to the C-terminus. To express the single-chain TCR, it is useful to provide a construct encoding the constant region of the TCR α chain.

[0066] The α-chain variable region amino acid sequence of the single-chain TCR (molecule) comprises CDR1α (SEQ ID NO. 2), CDR2α (SEQ ID NO. 3), and CDR3α (SEQ ID NO. 4) of the aforementioned α-chain. Preferably, the single-chain TCR (molecule) comprises the α-chain variable region amino acid sequence SEQ ID NO. 8. More preferably, the α-chain variable region amino acid sequence of the single-chain TCR (molecule) is SEQ ID NO. 8. The β-chain variable region amino acid sequence of the single-chain TCR (molecule) comprises CDR1β (SEQ ID NO. 5), CDR2β (SEQ ID NO. 6), and CDR3β (SEQ ID NO. 7) of the aforementioned β-chain. Preferably, the single-chain TCR (molecule) comprises the β-chain variable region amino acid sequence SEQ ID NO. 12. More preferably, the β-chain variable region amino acid sequence of the single-chain TCR (molecule) is SEQ ID NO. 12.

[0067] In a preferred embodiment of the present invention, the TCR (molecule) provided by the present invention 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 linked together by a linker sequence containing Vα and Vβ.

[0068] The amino acid sequence of the α chain of the TCR-mRNA molecule comprises CDR1 (SEQ ID NO. 2), CDR2 (SEQ ID NO. 3), and CDR3 (SEQ ID NO. 4) of the α chain. Preferably, the TCR-mRNA molecule comprises the α variable region amino acid sequence SEQ ID NO. 8. The amino acid sequence of the β chain of the TCR-mRNA molecule comprises CDR1 (SEQ ID NO. 5), CDR2 (SEQ ID NO. 6), and CDR3 (SEQ ID NO. 7) of the β chain. Preferably, the TCR-mRNA molecule comprises the β chain variable region amino acid sequence as shown in SEQ ID NO. 12.

[0069] In a preferred embodiment of the present invention, the constant region of the TCR provided by the present invention is the human constant region. Those skilled in the art know or can obtain the amino acid sequence of the human constant region by consulting relevant books or the publicly available database of IMGT (International Immunogenetic Information System). For example, the constant region sequence contained in the α chain of the TCR (molecule) of the present invention can be "TRAC", and the constant region sequence contained in the β chain of the TCR (molecule) can be "TRBC1" or "TRBC2". IMGT gives the 50th amino acid sequence of TRAC as Leu, which is here represented 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 having a leader sequence is SEQ ID NO.22, and / or the amino acid sequence of the β chain having a leader sequence is SEQ ID NO.24.

[0070] In one specific embodiment, the TCR is a single chain; preferably, the amino acid sequence of the variable region of the TCR α chain is as shown in SEQ ID NO.26, and the amino acid sequence of the variable region of the TCR β chain is as shown in SEQ ID NO.28; more preferably, the TCR is formed by linking the variable region of the α chain and the variable region of the β chain through a linking peptide chain SEQ ID NO.32, and the amino acid sequence of the TCR is as shown in SEQ ID NO.30.

[0071] The amino acid sequence of the single-chain TCR linking the peptide chain is shown in SEQ ID NO.32: GTSGSSGSGSGGSGSGCSG.

[0072] In one specific embodiment, the TCR is TCR-mRNA; preferably, the amino acid sequence of the TCR α chain is SEQ ID NO.34, and the amino acid sequence of the TCR β chain is SEQ ID NO.36; more preferably, the TCR is formed by linking the α chain and the β chain through a linker peptide chain SEQ ID NO.40, and the amino acid sequence of the TCR is SEQ ID NO.38.

[0073] In another preferred embodiment of the invention, the amino acids on the Vα and Vβ domains outside the antigen-binding CDR ring of the TCR provided by the present invention are replaceable. Simple variable region modifications are performed at a distance from the antigen-binding ring (Thomas, S. et al., Nat Commun, 2019, 10, 4451). Therefore, the TCR of the present invention can have amino acid substitutions made on residues in its α and β chain variable regions exposed to solvents, hydrophobic cores, Vα-Vβ interfaces, Vα-Cα, or Vβ-Cβ interfaces to increase TCR expression and improve effector function. Preferably, the amino acid residues are replaced by one or more sites selected from: amino acids at positions 5, 8, 19, 20, 24, 39, 50, 55, 66, 86, or 96 of TRAV; and amino acids at positions 9, 10, or 43 of TRBV.

[0074] In another preferred embodiment of the invention, a new artificial disulfide bond can be introduced between Thr48 in the α-chain constant region and Ser57 in the β-chain constant region (achieved by replacing these residues with cysteine. The original natural disulfide bonds in the TCR linker peptide can be retained in situ or removed). Therefore, the TCR provided by the present invention can contain an artificial disulfide bond formed by cysteine ​​introduced between the residues in its α and β-chain constant regions. It should be noted that the TCR provided by the present invention can contain a TRAC constant region sequence and a TRBC1 or TRBC2 constant region sequence, with or without the artificial disulfide bond introduced above. A set of natural disulfide bonds exists between the Cα and Cβ chains in the juxta-membrane region of the natural TCR; these are referred to as "natural interchain disulfide bonds" in this invention. The interchain covalent disulfide bonds artificially introduced in this invention, whose positions differ from those of the natural interchain disulfide bonds, are referred to as "artificial interchain disulfide bonds." Preferably, the cysteine ​​residue of the artificial disulfide bond is replaced by one or more 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.

[0075] Introducing artificial interchain disulfide bonds between the variable region of the TCR α chain and the constant region of the TCR β chain can improve the stability of the TCR. Therefore, the TCR α chain variable region and the constant region of the TCR β chain provided by the present invention may also contain artificial interchain disulfide bonds. In the present invention, the amino acid sequence positions of the variable regions TRAV and TRBV are numbered according to the position numbers listed in IMGT. Specifically, the cysteine ​​residues that form artificial interchain disulfide bonds between the TCR α chain variable region and the constant region of the TCR are replaced by: 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.

[0076] In another embodiment, the cysteine ​​residue of the artificial disulfide bond is further substituted with one or more sites selected from the following: amino acid at position 48, or 49, or 50 of the TRAV; amino acid at position 17, or 18, or 19 of the linking peptide between the α-chain variable region and the β-chain variable region.

[0077] Furthermore, the TCR provided by this invention can also be a heterozygous TCR comprising sequences derived from more than one species. The TCR of this invention may comprise a heterozygous TCR consisting of a variable region from humans and a constant region from mice.

[0078] Transducing human T cells with the double-stranded TCR (molecule) of the present invention (e.g., α- and β-chain molecules comprising the amino acid sequences given in SEQ ID NO. 10 and NO. 14) or a chimeric TCR (molecule) comprising the specific CDR described above can yield antigen-specific CTLs; similarly, transducing single-stranded TCRs can also be used to yield antigen-specific CTLs, and single-stranded TCRs have the advantage of not pairing with endogenous TCRs. Single-stranded TCRs can also be prepared as soluble TCRs in a manner similar to antibodies. In soluble TCRs, single-stranded TCRs do not contain transmembrane regions.

[0079] It should be understood that the amino acid names in this article are represented by internationally accepted single or three English letters. The correspondence between the single and three English letters in 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).

[0080] The fourth aspect of the invention provides a nucleic acid molecule encoding the TCR (molecule) or a portion thereof of the first and second aspects of the invention, said portion being one or more CDRs, variable regions of α and / or β chains, and α and / or β chains.

[0081] The nucleotide sequence encoding the CDR region of the α chain of the TCR (molecule) of the first aspect of this invention is as follows:

[0082] CDR1 is contained in the nucleotide sequence listed in SEQ ID NO.16; CDR2 is contained in the nucleotide sequence listed in SEQ ID NO.17; and CDR3 is contained in the nucleotide sequence listed in SEQ ID NO.18.

[0083] The nucleotide sequence encoding the CDR region of the β chain of the TCR (molecule) of the first aspect of this invention is as follows:

[0084] CDR1, which contains the nucleotide sequence listed in SEQ ID NO.19; CDR2, which contains the nucleotide sequence listed in SEQ ID NO.20; and CDR3, which contains the nucleotide sequence listed in SEQ ID NO.21.

[0085] Therefore, the nucleotide sequences of nucleic acid molecules encoding the TCR α chain of the present invention include SEQ ID NO.16, SEQ ID NO.17 and SEQ ID NO.18, and / or the nucleotide sequences of nucleic acid molecules encoding the TCR β chain of the present invention include SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21.

[0086] The nucleotide sequence of the nucleic acid molecule of the present invention can be single-stranded or double-stranded, and the nucleic acid molecule 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 can encode 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 contains 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 contains 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 contains 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 contains 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 contains 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 contains SEQ ID NO. 37. Alternatively, the nucleotide sequence of the nucleic acid molecule of the present invention comprises the nucleotide sequence SEQ ID NO.11 encoding the TCR α chain and / or the nucleotide sequence SEQ ID NO.15 encoding the TCR β chain. 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.

[0087] Example 1: Obtaining KRAS G12V antigen short peptide-specific T cell clones

[0088] Peripheral blood mononuclear cells (PBMCs) from HLA-A11-negative healthy volunteers were stimulated with T2-A11 cells loaded with the KRAS G12V synthetic short peptide VVGAVGVGK (as shown in SEQ ID NO.1, synthesized by Jiangsu Genscript Biotech Co., Ltd.). (T2 cells were transfused with HLA-A*11:01 and named T2-A11 cells. These cells can be effectively loaded with exogenous peptides because they lack the antigen processing-related factor TAP). T cells in PBMCs that can recognize the HLA-A*11:01 / VVGAVGVGK complex were activated and expanded.

[0089] T cells specific to the KRAS G12V antigen short peptide VVGAVGVGK can be detected using PE-labeled HLA-A*11:01-VVGAVGVGK tetramer (MBL International). The expanded T cells were stained with tetramer-PE and anti-CD8-APC and analyzed by flow cytometry, confirming the acquisition of double-positive cells.

[0090] To obtain truly tumor-specific CTLs that are both HLA-A*11:01 restricted and capable of recognizing the antigenic peptide VVGAVGVGK, this invention employed a limiting dilution method to culture double-positive cells obtained from tetramer-PE and anti-CD8-APC staining into single clones (the limiting dilution method for single-clone isolation can be found in Gross A, et al. Int J Mol Sci. 2015 Jul 24;16(8):16897-919). After screening dozens of single clones, this invention obtained one tumor-specific T cell clone (named F194) that is both HLA-A*11:01 restricted and capable of recognizing the antigenic peptide VVGAVGVGK. The FACS data of this single-clone T cell after staining with CD8 and tetramer are shown in the following figure. Figure 1 As shown.

[0091] Example 2: Obtaining the TCR gene of a KRAS G12V antigen short peptide-specific T cell clone and constructing a vector.

[0092] Single-cell VDJ sequencing is a method that has been developed in recent years and is well-known in the field, and is described in detail in manuals such as 10×Genomics based on microfluidics and oil droplet encapsulation technology.

[0093] Specifically, gel beads containing barcodes and primers were encapsulated in oil droplets along with single cells of a T-cell clone screened in Example 1 that was specific to the antigen short peptide VVGAVGVGK and HLA-A*11:01 restricted. Within each oil droplet, the gel beads dissolved, cells lysed, and mRNA was released. This mRNA was then reverse transcribed to produce barcode-encoded cDNA for sequencing, followed by TCR library construction. The V(D)J sequence of the TCR was enriched using nested PCR primers designed in the C region of the TCR. The library was then sequenced using an 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 containing a CDR with the following amino acid sequence:

[0094] CDR1α-SEQ ID NO.2; CDR2α-SEQ ID NO.3; CDR3α-SEQ ID NO.4.

[0095] The β chain contains a CDR with the following amino acid sequence:

[0096] CDR1β-SEQ ID NO.5; CDR2β-SEQ ID NO.6; CDR3β-SEQ ID NO.7.

[0097] The TCRβ-2A-TCRα fragment was obtained by artificially synthesizing partial or full-length TCR α and TCR β chains using a Furin GSG linker to ligate the P2A sequence. This fragment was then processed using the standard method described in J Sambrook, David Russell, et al., Molecular Cloning: A Laboratory Manual, 2016, Third Edition. Not I+ EcoR The TCR-mRNA molecule is digested with enzyme I 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 containing the variable regions of the TCR α and TCR β chains shown in SEQ ID NO. 8 and SEQ ID NO. 12, as well as the amino acid sequence containing the single-stranded 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 containing the TCR α and β chains shown in SEQ ID NO. 34 and SEQ ID NO. 36. TCR-expressing mRNA can be obtained through in vitro transcription, 5' Cap1 capping, Pseudo-UTP(Ψ) substitution, 5-Methyl-CTP substitution, and 3' poly(A) tailing.

[0098] In order to enable the α and β chains of the TCR (molecule) of the present invention to form correct pairings more effectively during transduction, 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. The positions of the introduced cysteine ​​residues are Thr48 of TRAC and Ser57 of TRBC, respectively.

[0099] Example 3: Preparation of TCR-retrovirus

[0100] (1) Preparation of recombinant plasmid: The recombinant plasmid pMP71-TCR obtained in Example 2 was transformed into Stbl3 competent cells, spread evenly on LB solid medium plates containing 0.1% ampicillin by volume, and cultured at 37°C for 14 h. Then, a single colony was picked and cultured in LB liquid medium containing 0.1% ampicillin by volume at 37°C and 220 rpm / min for 14 h. The plasmid was extracted to obtain the pMP71-TCR recombinant plasmid.

[0101] (2) Packaging of recombinant plasmid: 293Vec-RD114 cells in logarithmic growth phase were used as packaging cells and seeded into 10 cm plates containing culture medium (DMEM medium containing 10% FBS by volume). When the cell density reached 80% of the surface 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 through a 0.45 μm filter membrane to remove cell debris, yielding a TCR-retrovirus suspension, which was stored at -80°C.

[0102] Example 4: Preparation of KRAS G12V-specific TCR-T cells and analysis of TCR expression

[0103] Peripheral blood was collected from healthy donors, lysed with erythrocyte lysis buffer (Solepro), and then sorted using CD8 magnetic beads (Miltenyi Biotec) to obtain CD8. + T cells. Adjust the cell density to 1×10⁶. 6 T cells were cultured at a density of 100 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 a -80°C freezer and slowly thawed at 4°C. 0.5 × 10⁶ cells / mL were then placed in each well of a 24-well plate pre-coated with RetroNectin (Takara). 6 CD8 + T cells were treated with 1.5 mL of viral supernatant and IL-2 (600 U / mL). The mixture was gently pipetted and centrifuged at 2200 rpm at 32°C for 90 minutes. The cells were then incubated at 37°C with 5% CO2. On day 4, the expression of KRAS G12V-TCR on T cells was detected using FACS. Figure 2 As shown, simulated transduced T cells could not be stained with KRAS G12V-tetramer, indicating that they lacked KRAS G12V specificity; while KRAS G12V-TCR transduced T cells could be stained with KRAS G12V-tetramer, 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 therein significantly proliferated.

[0104] Among them, the peripheral blood of healthy donors is the peripheral blood of hematopoietic stem cell transplant donors after mobilization.

[0105] Example 5: Detection of the function of KRAS G12V-specific TCR-T cells by intracellular immune factor staining.

[0106] The artificially synthesized KRAS G12V antigenic peptide VVGAVGVGK and control peptides (unmutated wild-type WT antigenic peptide VVVGAGGVGK and mutated KRAS G12D antigenic peptide VVVGADGVGK) were incubated with T2-A11 cells at 37°C and 5% CO2 for 2 hours (the concentrations of KRAS G12V peptides in the experimental groups were 10 μM, 1 μM, 100 nM, 10 nM, 1 nM and 100 pM, and the concentration of peptides in the control group was consistent with the highest concentration of 10 μM in the experimental group). Unbound antigenic peptides and control peptides were washed to remove them, and then the cells were collected to obtain T2-A11 cells loaded with antigenic peptides and control peptides.

[0107] 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 96-well plates at 37°C and 5% CO2. The concentrations of both T cells and target cells were 0.4 × 10⁻⁶. 6 Each cell was filled with T cells and BFA (BFA is used to retain immune factors in T cells and prevent them from being released so that they can be monitored by FACS staining) to a final concentration of 1.5 μg / mL.

[0108] Cells were collected after 24 hours of co-culture. The cell surface was first stained with anti-CD3 / CD8-APC, and then intracellular immune factors were stained using the Fix and Perm kit (Invitrogen) according to the manufacturer's instructions. The production of various immune factors in the stained cells was detected by FACS.

[0109] 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 could be as low as the picomolar (pM) level. These results indicate that the high-affinity TCR obtained in this invention can specifically recognize the picomolar (pM) level of the KRAS G12V antigen peptide VVGAVGVGK. T cells transduced with the TCR of this 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.

[0110] Example 6: Preparation of K562 / THP-1 / PANC-1 / H460-A11-WT-GFP and K562 / THP-1 / PANC-1 / H460-A11-G12V-GFP in target cells

[0111] HLA-A*11:01-CD34-puro, KRAS WT-GFP, and KRASG12V-GFP can be obtained using artificial gene synthesis methods. A11 and CD34 are linked via a P2A sequence, CD34 and puro are linked via a T2A sequence, and KRAS WT / G12V and GFP are linked via a T2A sequence. Recombinant plasmids pMP71-HLA-A*11:01-CD34-puro, pMP71-KRAS WT-GFP, and pMP71-KRAS G12V-GFP can be obtained using the same method as in Example 2. Using the same methods as in Examples 3 and 4, HLA-A*11:01-CD34-puro, KRAS WT-GFP, and KRAS G12V-GFP were transfected into leukemia cell lines K562 and THP-1, lung cancer cell line H460, and pancreatic cancer cell line PANC-1 to obtain the positive target cells K562 / THP-1 / PANC-1 / H460-A11-G12V-GFP and control target cells K562 / THP-1 / PANC-1 / H460-A11-WT-GFP required by this invention. The HLA-A11-CD34 staining and GFP expression of these target cells are shown in [the table below]. Figure 4 and Figure 5 .

[0112] 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 allows it to be recognized by the KRAS G12V-TCR-T of this 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, expressing HLA-A*11:01 and unmutated wild-type KRAS, but without the G12V mutation, were used as control target cells.

[0113] Example 7: Killing effect of KRAS G12V-TCR-T cells on target cells

[0114] Using the K562 / THP-1 / PANC-1-A11-G12V-GFP constructed in Example 6 as positive target cells and K562 / THP-1 / PANC-1-A11-WT-GFP as negative control target cells, 1×10⁻⁶ cells were added to each well of 100 μL RPMI 1640 medium in a U-bottom 96-well plate. 4 Positive target cells and control target cells (K562 lineage / THP-1 lineage), and 1×10⁶ cells were added to each well of 100 μL DMEM medium in a flat-bottomed 96-well plate. 4 Positive target cells and control target cells (PANC-1 lineage) were then mixed and cultured with 100 μL of nonspecific T cells (mock) and KRAS G12V-TCR-T cells at different effector-to-target ratios (E:T).

[0115] Twenty-four hours later, for the K562 and THP-1 lines, all cells from each well were aspirated into flow cytometry tubes for flow cytometry analysis to detect changes in the GFP positivity rate of positive and control target cells. For the PANC-1 line, the culture supernatant was discarded, and the changes in the OD values ​​of positive and control target cells were detected using sulfonylrhodamine B (SRB) colorimetric analysis. Changes in the GFP positivity rate and SRB OD values ​​of target cells indicate whether target cells have been killed.

[0116] Figure 6 The results showed that both control and positive target cells survived well when mock cells were added. However, when KRAS G12V-TCR-T cells were added, positive target cells were significantly killed, while control target cells still survived well. This indicates that the KRAS G12V-TCR-T cells of the present invention can selectively kill cells that simultaneously express HLA-A*11:01 and KRASG12V.

[0117] Example 8: ELISA detection of INF-γ secretion after co-culturing KRAS G12V-TCR-T cells with target cells

[0118] The K562 / PANC-1-A11-G12V-GFP constructed in Example 6 was used as a positive target cell, and the K562 / PANC-1-A11-WT-GFP was used as a negative control target cell. 1×10⁻⁶ cells were added to each well of 100 μL of RPMI 1640 medium in a U-bottom 96-well plate. 4 Positive target cells and control target cells (K562 lineage) were added to each well of a 96-well flat-bottom plate with 1×10⁻⁶ cells in 100 μL of DMEM medium. 4 Positive target cells and control target cells (PANC-1 lineage) were then mixed with 100 μL of nonspecific T cells (mock) and KRAS G12V-TCR-T cells at different effector-to-target ratios (E:T). After 24 hours, the supernatant from each well was aspirated for ELISA detection of INF-γ secretion.

[0119] like Figure 7 The results showed that, under different effector-to-target ratios, neither the control nor the positive target cell groups could stimulate mock cells to secrete IFN-γ. However, after co-culturing KRAS G12V-TCR-T cells with positive target cells, TCR-T cells could be stimulated to secrete a large amount of IFN-γ, while the control target cells could not stimulate KRAS G12V-TCR-T cells to produce immune factors, and the difference between the two was significant. This result further indicates that the KRAS G12V-TCR can specifically recognize the antigenic peptide VVGAVGVGK. After T cells transduced with this KRAS G12V-TCR recognize target cells, they can secrete IFN-γ, thereby killing the target cells. However, when encountering control target cells, they do not produce immune factors, thus avoiding unnecessary side effects.

[0120] As can be seen from the above, the T-cell receptor for the KRAS G12V antigen short peptide provided by this invention recognizes the KRAS G12V-HLA-A*11:01 complex with high affinity, and can recognize specific KRAS G12V antigen peptide concentrations down to the picomolar level, exhibiting a strong killing effect on tumor cells simultaneously expressing HLA-A*11:01 and KRAS G12V. The KRAS G12V-specific T cells of this invention can be used to treat KRAS G12V-related diseases presenting the KRAS G12V antigen short peptide VVGAVGVGK-HLA-A*11:01 complex.

[0121] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0122] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A T-cell receptor that recognizes a short peptide of the KRAS G12V antigen, characterized in that, The T-cell receptor is able to 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 α and β chains; The amino acid sequence of the α chain is as shown in SEQ ID NO.10, or is an amino acid sequence that has at least 90% sequence identity with it; The amino acid sequence of the β chain is as shown in SEQ ID NO.14, or is an amino acid sequence that has at least 90% sequence identity with it; The α chain includes a TCR α chain variable region, which includes three complementarity-determining regions CDR1α~CDR3α, and their amino acid sequences are shown in SEQ ID NO.2~SEQ ID NO.

4. The β chain includes a TCR β chain variable region, which contains three complementarity-determining regions CDR1β~CDR3β, and their amino acid sequences are shown in SEQ ID NO.5~SEQ ID NO.

7.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule includes a nucleotide sequence encoding the T cell receptor of claim 1 or its complementary sequence.

3. A vector or host cell, characterized in that, The vector or the host cell contains the nucleic acid molecule as described in claim 2.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the T-cell receptor of claim 1, the nucleic acid molecule of claim 2, or the carrier or host cell of claim 3, and a pharmaceutically acceptable carrier.

5. The use of the T-cell receptor of claim 1, the carrier or host cell of claim 3, or the pharmaceutical composition of claim 4 in the preparation of a medicament for treating tumors, characterized in that, The tumors are KRAS G12V positive and HLA-A*11:01 positive leukemia, lung cancer, and pancreatic cancer.

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