Kras mutant-specific t cell receptors and uses thereof

Abstract

The present invention relates to the field of immunology and tumor therapy. In particular, the present invention relates to T cell receptors (TCRs) specifically recognizing RAS G12V mutants, fusion proteins comprising said TCRs, conjugates comprising said TCRs or fusion proteins, and their use for the prevention or treatment of tumors having a RAS G12V mutation, such as a KRAS G12V, HRAS G12V and / or NRAS G12V mutation.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to international patent application PCT / CN2025 / 101160, filed on June 16, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to the field of protein engineering. Specifically, this invention relates to a RAS mutant-specific T cell receptor, a fusion protein comprising the receptor, and methods for preparing and using the same. Background Technology

[0004] RAS is one of the most widespread and common oncogenes in human cancers. The protein encoded by the RAS gene is a small GTPase, and most RAS mutations lead to loss of GTPase activity and constitutive activation of RAS. Studies have shown that approximately one-third of human cancers involve mutations in RAS genes (KRAS, NRAS, and HRAS). These hotspot mutations promote the development of human cancers, and their location and mutation type exhibit a tumor type-dependent distribution. The protein encoded by the KRAS gene (KRAS proto-oncogene) has GTPase activity and is an important gene involved in cell proliferation and cell cycle regulation. KRAS gene mutations occur in 20% of human solid tumors, such as pancreatic cancer, colorectal cancer, and lung cancer, with the proportion of patients with KRAS gene mutations reaching 20-90%. KRAS gene mutations typically occur at the G12, G13, and Q61 positions, with major mutants including G12D, G12V, G13D, and Q61H, among which G12V mutations account for 20-40% of KRAS gene mutations. Public databases (such as TCGA and The Cancer Genome Altas) indicate that approximately 4-5% of human solid tumors carry the G12V gene mutation. Based on this, it is estimated that 4.5 million new cancer patients are diagnosed in China each year (WHO), of whom approximately 200,000 carry the KRAS G12V mutation. Compared to KRAS wild-type patients, patients with KRAS mutations do not benefit from existing therapies, and many drugs are only effective for KRAS wild-type patients. For example, metastatic colorectal cancer patients with KRAS gene mutations do not achieve progression-free survival benefit when treated with PD-1 antibodies (T. Andre, NEJM, 2020). In particular, cancer patients with KRAS G12V gene mutations have shorter overall survival and progression-free survival compared to other patients (Robert P Jones, BJC, 2017; Jacob E. Till, Nature Communications, 2024). To date, no drugs specifically targeting the KRAS G12V mutation have been approved for marketing.

[0005] T cell receptors (TCRs) are naturally expressed on the surface of T lymphocytes. They typically form a dimer (e.g., TCRαβ) with CD3 to form a complex (TCR / CD3 complex), recognizing the pMHC complex (peptide and major histocompatibility complex) on the surface of antigen-presenting cells. TCRs are generated by rearrangements of the human TCR V, D, and J genes. After negative and positive selection in the thymus, only TCR clones that recognize the pMHC complex and have low affinity for their own antigen pMHC complex are released into the peripheral blood. During pathogen infection, cancer, or autoimmune diseases, pathogen-associated peptides are displayed in the human HLA system and recognized by the TCR, activating T cells and destroying pathogen-infected or tumorigenic cells. Since 2016, multiple studies have found that KRAS gene mutations can be presented in the human HLA system and recognized by the TCR (Rami Yossef, JCI insight, 2018; Gal Cafri, Nature communications, 2019; QiAi, Front. Immunol, 2023). KRAS G12V can specifically present on HLA-DP genes, such as HLA-DPB1*03:01. Studies have shown that HLA-DP molecules present antigens more like HLA-I molecules, which is a spontaneous presentation (Yamashita, Nat Commun, 2017). The KRAS G12V mutant protein can simultaneously produce antigenic peptides that bind to HLA-DP via the proteasome / TAP or lysosome pathway. The resulting pMHC complex is expressed on the surface of tumor cells and induces the production of specific TCRs (QiAi, Front. Immunol, 2023). Multiple studies have found that MHC-II is expressed in human solid tumors; for example, 77.7% of pancreatic cancer samples express MHC-II, and over 50% of pancreatic cancer samples show high expression (Renato B. Baleeiro, OncoImmunology, 2022). Simultaneously, approximately 30-40% of pancreatic cancers have KRAS G12V gene mutations, therefore pancreatic cancer cells can present a pHLA complex formed by a KRAS G12V mutant peptide and HLA-DP. Utilizing KRAS G12V mutation-specific TCRs, TCR gene-modified T cell therapy (TCR-T) or soluble TCR drugs (sTCR) can be developed for the treatment of human diseases.

[0006] Soluble TCRs refer to proteins that have been modified from membrane proteins into stable proteins in solution using protein engineering and recombinant expression technologies. Several technological platforms have the potential to modify TCRs into soluble proteins, such as ImmTAC technology (Immune-mobilizing monoclonal TCR against cancer). This technology uses *E. coli* to express TCRα and β chains separately, and then uses interchain disulfide bonds to link the TCRα and β chains in vitro to form a biologically functional dimer (Joanne Oates, Mol Immunol, 2015). The TCR drug Tebentafusp, manufactured using ImmTAC technology, has been approved for marketing for the treatment of uveal melanoma. Its structure does not contain a half-life extension and only includes the TCR and effector domain CD3scFv, resulting in a shorter human half-life. Furthermore, its manufacturing process is relatively complex (US20120225481).

[0007] Therefore, there is a need to develop a new soluble TCR protein drug to provide patients with new treatment options. Summary of the Invention

[0008] This invention provides a novel soluble TCR fusion protein drug that significantly prolongs the drug's half-life through fusion with the Fc domain of human immunoglobulin. After TCR germline gene screening and optimization, the candidate soluble TCR fusion protein exhibits good thermostability and can be produced as a soluble TCR αβ dimer using a eukaryotic expression system, avoiding the in vitro dimer production process and simplifying the manufacturing process. Simultaneously, this soluble TCR fusion protein drug can induce and activate T cells to recognize and kill tumors with RAS mutations (e.g., KRAS G12V, HRAS G12V, and / or NRAS G12V mutations) and HLA-DP positivity, including but not limited to pancreatic cancer, colorectal cancer, lung cancer, and bladder cancer.

[0009] In some aspects, the present invention provides an isolated T-cell receptor (TCR) or an antigen-binding fragment thereof capable of specifically recognizing mutants or fragments of RAS proteins (e.g., KRAS, HRAS, or NRAS proteins) with G12V substitution, said TCR or antigen-binding fragment comprising an α-chain variable region (Vα) and a β-chain variable region (Vβ), wherein: said Vα comprises CDR1α, CDR2α, and CDR3α, and said Vβ comprises CDR1β, CDR2β, and CDR3β; preferably, said TCR is a soluble TCR. The antigenic peptide of the RAS protein mutant with G12V substitution can be presented by MHC molecules, and the TCR or antigen-binding fragment disclosed herein can specifically recognize and bind said antigenic peptide presented by MHC molecules.

[0010] In some embodiments, CDR1α comprises an amino acid sequence having at least 95% identity with the sequence shown in SEQ ID NO: 17, CDR2α comprises an amino acid sequence having at least 95% identity with the sequence shown in any one of SEQ ID NO: 18-22, and CDR3α comprises an amino acid sequence having at least 95% identity with the sequence shown in SEQ ID NO: 23.

[0011] In some embodiments, CDR1α comprises an amino acid sequence having a substitution, insertion, or deletion of up to two amino acids relative to the sequence shown in SEQ ID NO: 17, CDR2α comprises an amino acid sequence having a substitution, insertion, or deletion of up to two amino acids relative to the sequence shown in any one of SEQ ID NO: 18-22, and CDR3α comprises an amino acid sequence having a substitution, insertion, or deletion of up to two amino acids relative to the sequence shown in SEQ ID NO: 23.

[0012] In some embodiments, CDR1β comprises an amino acid sequence having at least 95% identity with the sequence shown in SEQ ID NO: 24 or 25, CDR2β comprises an amino acid sequence having at least 95% identity with the sequence shown in any one of SEQ ID NO: 26-28, and CDR3β comprises an amino acid sequence having at least 95% identity with the sequence shown in any one of SEQ ID NO: 29-35.

[0013] In some embodiments, CDR1β comprises an amino acid sequence having a substitution, insertion, or deletion of up to two amino acids relative to the sequence shown in SEQ ID NO: 24 or 25, CDR2β comprises an amino acid sequence having a substitution, insertion, or deletion of up to two amino acids relative to the sequence shown in any one of SEQ ID NO: 26-28, and CDR3β comprises an amino acid sequence having a substitution, insertion, or deletion of up to two amino acids relative to the sequence shown in any one of SEQ ID NO: 29-35.

[0014] In some embodiments, CDR1α comprises the amino acid sequence shown in SEQ ID NO: 17, CDR2α comprises the amino acid sequence shown in any one of SEQ ID NO: 18-22, and CDR3α comprises the amino acid sequence shown in SEQ ID NO: 23.

[0015] In some embodiments, the CDR1β, the CDR2β, and the CDR3β have sequences selected from: (i) the CDR1β comprises the amino acid sequence shown in SEQ ID NO: 24, the CDR2β comprises the amino acid sequence shown in SEQ ID NO: 26, and the CDR3β comprises the amino acid sequence shown in any one of SEQ ID NO: 29-35; (ii) the CDR1β comprises the amino acid sequence shown in SEQ ID NO: 25, the CDR2β comprises the amino acid sequence shown in SEQ ID NO: 27, and the CDR3β comprises the amino acid sequence shown in SEQ ID NO: 29; or (iii) the CDR1β comprises the amino acid sequence shown in SEQ ID NO: 24, the CDR2β comprises the amino acid sequence shown in SEQ ID NO: 28, and the CDR3β comprises the amino acid sequence shown in SEQ ID NO: 29.

[0016] In some embodiments, Vα comprises a sequence having at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with any of the sequences shown in SEQ ID NO: 36-40; and / or, Vβ comprises a sequence having at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with any of the sequences shown in SEQ ID NO: 41-49.

[0017] In some embodiments, Vα comprises the sequence shown in any one of SEQ ID NO: 36-40, and Vβ comprises the sequence shown in SEQ ID NO: 41; or, Vα comprises the sequence shown in SEQ ID NO: 36, and Vβ comprises the sequence shown in any one of SEQ ID NO: 42-49.

[0018] In some embodiments, the TCR or its antigen-binding fragment is capable of specifically recognizing the RAS G12V antigenic peptide or a pMHC complex containing the antigenic peptide. Preferably, the antigenic peptide is presented by an MHC class II molecule, for example, HLA-DP. Preferably, the HLA-DP is selected from the group consisting of HLA-DPA1*02:01, HLA-DPA1*02:02, HLA-DPA1*01:03, and HLA-DPB1*03:01, HLA-DPB1*14:01, and HLA-DPB1*104:01, more preferably HLA-DPB1*03:01. In some embodiments, the RAS G12V antigenic peptide may be a KRAS G12V antigenic peptide, an HRAS G12V antigenic peptide, and / or a NRAS G12V antigenic peptide. In some embodiments, the RAS G12V antigenic peptide comprises the sequence shown in SEQ ID NO: 1.

[0019] In some embodiments, the TCR or its antigen fragment further comprises an α-chain constant region (Cα) and a β-chain constant region (Cβ). Optionally, the Cα and the Cβ can interact via interchain disulfide bonds. Optionally, the Cα has the sequence shown in SEQ ID NO: 50, and the Cβ has the sequence shown in SEQ ID NO: 51.

[0020] In some embodiments, the α chain comprises an amino acid sequence having at least 85%, 90%, 95%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 54-58; and / or, the β chain comprises an amino acid sequence having at least 85%, 90%, 95%, 99%, or 100% identity with the sequence shown in any one of SEQ ID NO: 59-65 and 92-93.

[0021] In some embodiments, the α chain comprises the amino acid sequence shown in any one of SEQ ID NO: 54-58, and the β chain comprises the amino acid sequence shown in SEQ ID NO: 59; or, the α chain comprises the amino acid sequence shown in SEQ ID NO: 54, and the β chain comprises the amino acid sequences shown in any one of SEQ ID NO: 60-65 and 92-93.

[0022] In some aspects, the present invention provides a fusion protein comprising a TCR or its antigen-binding fragment disclosed herein and an immunoglobulin Fc moiety; wherein the C-terminus of the α-chain and / or β-chain of the TCR or its antigen-binding fragment is operatively linked to the immunoglobulin Fc moiety, the immunoglobulin Fc moiety comprising a first Fc domain (Fc1) and a second Fc domain (Fc2). The first Fc domain and the second Fc domain may be on the same polypeptide chain. Preferably, the first Fc domain and the second Fc domain are operatively linked, for example, through a peptide linker. Alternatively, the first Fc domain and the second Fc domain may be on different polypeptide chains.

[0023] In some embodiments, the immunoglobulin Fc domain is the human immunoglobulin Fc domain or a variant thereof, such as the Fc domain of human IgG1, IgG2, IgG3, or IgG4 or a variant thereof. Preferably, the immunoglobulin Fc domain is the Fc domain of human IgG1 or a variant thereof.

[0024] In some embodiments, the fusion protein further comprises an effector domain operatively linked to the N-terminus of an α-chain variable region (Vα) and / or a β-chain variable region (Vβ). Optionally, the effector domain may be a peptide or protein capable of activating effector cells, which may be selected from T lymphocytes, NK cells, dendritic cells, and macrophages. Preferably, the effector domain is an antibody or its antigen-binding moiety, and the antibody may be an anti-CD3 antibody (e.g., UCHT1, SP34, OKT3), an anti-FcRn antibody, an anti-PD1 antibody, an anti-LAG3 antibody, or an anti-CD28 antibody. Preferably, the effector domain is an anti-CD3 antibody or its antigen-binding moiety, such as CD3 scFv, which may have an amino acid sequence as shown in SEQ ID NO: 52.

[0025] In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain includes the following domain operably linked: Vα-Cα-Fc1-Fc2, and the second polypeptide chain includes the following domain operably linked: an effector domain -Vβ-Cβ. Preferably, a non-natural interchain disulfide bond exists between Cβ and Cα. Preferably, the operably linked structure can be a direct link or a link via a peptide linker, which can be a flexible peptide linker commonly used in the art, such as the GS series linker. In some embodiments, the C-terminus of Fc1 is operably linked to the N-terminus of Fc2 via a peptide linker, for example, the CH3 region of Fc1 is operably linked to the CH2 region or hinge region of Fc2.

[0026] In some embodiments, Fc1 comprises the sequence shown in SEQ ID NO: 94, and Fc2 comprises the sequence shown in SEQ ID NO: 95; or, Fc1 comprises the sequence shown in SEQ ID NO: 95, and Fc2 comprises the sequence shown in SEQ ID NO: 94. Preferably, Fc1 and Fc2 are linked by a peptide linker (e.g., a GS linker); preferably, a non-natural disulfide bond is formed between Fc1 and Fc2. In a specific embodiment, Fc1-Fc2 may comprise the sequence shown in SEQ ID NO: 53.

[0027] In some embodiments, the first polypeptide chain comprises an amino acid sequence having at least 95%, at least 99%, or 100% sequence identity with an amino acid sequence as shown in any one of SEQ ID NO: 3-7; and / or, the second polypeptide chain comprises an amino acid sequence having at least 95%, at least 99%, or 100% sequence identity with an amino acid sequence as shown in any one of SEQ ID NO: 8-16.

[0028] In some embodiments, the first polypeptide chain comprises an amino acid sequence as shown in any one of SEQ ID NO: 3-7, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 8; or, the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 3, and the second polypeptide chain comprises an amino acid sequence as shown in any one of SEQ ID NO: 9-16.

[0029] In some embodiments, the fusion protein is capable of specifically recognizing the RAS G12V antigenic peptide or a pMHC complex containing the antigenic peptide. Preferably, the antigenic peptide is presented by an MHC-II molecule. Preferably, the MHC-II molecule is HLA-DP, which may be selected from the group consisting of HLA-DPA1*02:01, HLA-DPA1*02:02, HLA-DPA1*01:03, and HLA-DPB1*03:01, HLA-DPB1*14:01, and HLA-DPB1*104:01, more preferably HLA-DPB1*03:01. In some embodiments, the RAS G12V antigenic peptide may be a KRAS G12V antigenic peptide, an HRAS G12V antigenic peptide, and / or a NRAS G12V antigenic peptide. In some embodiments, the RAS G12V antigenic peptide comprises the sequence shown in SEQ ID NO: 1.

[0030] In some embodiments, the EC50 of the fusion protein binding to the antigenic peptide or the pMHC complex containing the antigenic peptide is about 1 nM or less, about 100 pM or less, about 90 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less, or about 20 pM or less.

[0031] In some aspects, the present invention provides a conjugate comprising a TCR or its antigen-binding fragment disclosed herein, or a fusion protein disclosed herein, conjugated to an effector moiety, said effector moiety being selected from cytotoxic agents, nucleic acids (e.g., RNA), or radionuclides. Preferably, the cytotoxic agent is selected from alkylating agents, microtubule inhibitors, anticancer antibiotics, or antimetabolites.

[0032] In one aspect, the present invention provides a nucleic acid molecule comprising a nucleotide sequence encoding a TCR or an antigen-binding fragment thereof disclosed herein, or comprising a nucleotide sequence encoding a fusion protein disclosed herein.

[0033] In one aspect, the present invention provides a vector comprising a nucleic acid molecule disclosed herein, or a nucleotide sequence encoding a TCR or an antigen-binding fragment thereof disclosed herein, or a nucleotide sequence encoding a fusion protein disclosed herein.

[0034] In one aspect, the present invention provides a host cell comprising the nucleic acid molecules or vectors disclosed herein. The host cell may be a eukaryotic cell, such as a CHO cell or a 293 cell line.

[0035] In one aspect, the present invention provides a method for preparing the TCR or antigen-binding fragment thereof or fusion protein disclosed herein, comprising: culturing a host cell containing a nucleic acid molecule or vector disclosed herein under conditions allowing protein expression, and recovering the TCR or antigen-binding fragment thereof or fusion protein from the cultured host cell culture.

[0036] In some aspects, the present invention provides a pharmaceutical composition comprising a TCR or an antigen-binding fragment thereof disclosed herein, a fusion protein disclosed herein, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding said TCR or its antigen-binding fragment or fusion protein, and a pharmaceutically acceptable carrier and / or excipient. Preferably, the pharmaceutical composition further comprises additional therapeutic agents, such as antitumor agents or immune enhancers.

[0037] In some embodiments, the antitumor agent may be selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, radiosensitizers, antiangiogenic agents, cytokines, and immune checkpoint inhibitors (e.g., PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG-3 antibodies, or TIM3 antibodies).

[0038] In some embodiments, the immune enhancer may be selected from immunostimulatory antibodies (e.g., anti-CD3 antibody, anti-CD28 antibody, anti-CD40L (CD154) antibody, anti-41BB (CD137) antibody, anti-OX40 antibody, anti-GITR antibody or any combination thereof) or immunostimulatory cytokines (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFN-γ, IL-10, TGF-β, GM-CSF or any combination thereof).

[0039] In some aspects, the present invention relates to the use of the TCR or antigen-binding fragment thereof disclosed herein, the fusion protein disclosed herein, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or fusion protein thereof, or the conjugate disclosed herein, or the pharmaceutical composition disclosed herein in the preparation of a medicament for inducing an immune response in a subject against a tumor having a RAS G12V (e.g., KRAS G12V, HRAS G12V and / or NRAS G12V) mutation, and / or for the prevention or treatment in a subject of a tumor having a RAS G12V (e.g., KRAS G12V, HRAS G12V and / or NRAS G12V) mutation. The tumor with RAS G12V mutation can be selected from pancreatic cancer, pancreatic ductal adenocarcinoma, appendiceal adenocarcinoma, small intestinal adenocarcinoma, non-small cell lung cancer, colorectal cancer, cholangiocarcinoma (e.g., extrahepatic cholangiocarcinoma, intrahepatic cholangiocarcinoma), endometrial cancer, gastric cancer, breast cancer, prostate cancer, ovarian cancer, bladder cancer (e.g., transitional cell carcinoma of the bladder), oral cancer, thyroid cancer, and tumors of unknown primary origin. Preferably, the subject is a human. Preferably, the subject is HLA-DPB1*03:01, HLA-DPB1*14:01, or HLA-DPB1*104:01 positive. The TCR or its antigen-binding fragment, fusion protein, nucleic acid molecule, or carrier or host cell, or pharmaceutical composition can be administered in combination with other therapeutic agents, for example, simultaneously, separately, or sequentially; the other therapeutic agents can be immunostimulants or antitumor agents.

[0040] In some aspects, the present invention also relates to a method of treating a tumor with a RAS G12V (e.g., KRAS G12V, HRAS G12V, and / or NRAS G12V) mutation in a subject, comprising administering to the subject a therapeutically effective amount of a TCR or antigen-binding fragment thereof disclosed herein, a fusion protein disclosed herein, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding said TCR or antigen-binding fragment thereof or fusion protein, or a conjugate disclosed herein, or a pharmaceutical composition disclosed herein. The tumor with the RAS G12V mutation may be selected from pancreatic cancer, pancreatic ductal adenocarcinoma, appendiceal adenocarcinoma, small intestinal adenocarcinoma, non-small cell lung cancer, colorectal cancer, cholangiocarcinoma (e.g., extrahepatic cholangiocarcinoma, intrahepatic cholangiocarcinoma), endometrial cancer, gastric cancer, breast cancer, prostate cancer, ovarian cancer, bladder cancer (e.g., transitional cell carcinoma of the bladder), oral cancer, thyroid cancer, and tumors of unknown primary origin. Preferably, the subject is a mammal, such as a human. Preferably, the subject is HLA-DPB1*03:01, HLA-DPB1*14:01, or HLA-DPB1*104:01 positive. The TCR or its antigen-binding fragment, fusion protein, nucleic acid molecule, vector, host cell, or pharmaceutical composition can be administered in combination with other therapeutic agents, such as simultaneously, separately, or sequentially; the other therapeutic agents can be immunostimulants or antitumor agents.

[0041] In some aspects, the present invention relates to the TCR or antigen-binding fragment thereof disclosed herein, the fusion protein disclosed herein, nucleic acid molecules or vectors or host cells comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or fusion protein thereof, or conjugates disclosed herein, or pharmaceutical compositions disclosed herein for use as pharmaceuticals. The TCR or antigen-binding fragment thereof, the fusion protein disclosed herein, the nucleic acid molecules or vectors or host cells comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or fusion protein thereof, or conjugates disclosed herein, or pharmaceutical compositions disclosed herein may be used in subjects to induce an immune response against tumors with RAS G12V (e.g., KRAS G12V, HRAS G12V, and / or NRASG12V) mutations, and / or to prevent or treat tumors with RAS G12V (e.g., KRASG12V, HRAS G12V, and / or NRASG12V) mutations in subjects. Attached Figure Description

[0042] Figure 1The diagram shows a schematic structure of a TCR-CD3 polypeptide complex or fusion protein (Monovalent TCR-CD3scFv and hIgG1 fusion) according to some embodiments of the present invention, comprising a first chain with the structure Vα-Cα-Fc1-Fc2 and a second chain with the structure scFv-Vβ-Cβ. Cα is connected to Fc1-Fc2, scFv to Vβ, and Fc1 to Fc2 via linkers. Furthermore, one pair of interchain disulfide bonds may exist between Cα and Cβ, and three pairs of disulfide bonds may exist between Fc1 and Fc2. The dashed line indicates the linker between Fc1 and Fc2.

[0043] Figure 2 The results of stimulating Jurkat-NFAT-luc cells with different concentrations (10000, 1000, 100, 10, 1, 0.1 pM) of the TCR-CD3 molecule of the present invention are shown, with the biological activity of the TCR-CD3 molecule indicated by the luciferase chemiluminescence intensity.

[0044] Figure 3 The release of IFNγ from the TCR-CD3 molecules of the present invention at different concentrations (10000, 1000, 100, 10, 1, 0.1 pM) after co-culturing with CFPAC-DP223 cells (overexpressing HLA-DPA1*0202 / DPB1*0301), human PBMC cells, and pWT or pG12V peptides is shown.

[0045] Figure 4 The results show the killing effects of different concentrations (10000, 1000, 100, 10, 1, 0.1 pM) of the TCR-CD3 molecule of the present invention on non-target cells CFPAC-DP225-Luc (overexpressing HLA-DPA1*0202 / DPB1*0501) or target cells CFPAC-DP223-Luc (overexpressing HLA-DPA1*0202 / DPB1*0301) in the presence of pG12V and PBMC cells.

[0046] Figure 5The specific IFN-γ release of the present invention's TCR-CD3 molecules at different concentrations (25000, 5000, 1000, 200, 40, 8, 1.6, and 0 pM) was demonstrated in co-culture with negative control cells A549-DP223 (KRASG12S, HLA DPA1*02:02 / DPB1*03:01), CFPAC1-DP225 (KRASG12V, HLA DPA1*02:02 / DPB1*05:01), and two target cells CFPAC1-DP223 (KRASG12V, HLA-DPA1*02:02 / DPB1*03:01) and YAPC-DP223 (KRASG12V, HLA-DPA1*02:02 / DPB1*03:01) and human PBMC cells.

[0047] Figure 6 The invention’s TCR-CD3 molecules at different concentrations (25,000, 5,000, 1,000, 200, 40, 8, 1.6 and 0 pM) were shown to induce specific IFN-γ release when co-cultured with YAPC cells and human PBMC cells overexpressing different HLA-DP combinations.

[0048] Figure 7 The antitumor activity of the TCR-CD3 molecule of the present invention was demonstrated by co-culturing tumor cells and human PBMC cells that were positive for KRASG12V or HRASG12V and overexpressed target HLA (e.g., HLA-DPB1*03:01 and DPB1*14:01).

[0049] Figure 8 The in vivo antitumor activity of the TCR-CD3 molecule of the present invention was demonstrated. After treatment with the TCR-CD3 molecule of the present invention, tumor growth was significantly inhibited and tumor remission was induced compared with the control group. Detailed Implementation

[0050] In this invention, unless otherwise stated, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. All patents, patent applications, and other publications cited herein are incorporated herein by reference in their entirety. Furthermore, the terms and laboratory procedures related to immunology, molecular biology, biochemistry, nucleic acid chemistry, cell and tissue culture, etc., used herein are widely used terms and routine procedures in their respective fields. If any definition presented herein conflicts with a definition presented in a patent, patent application, or other publication incorporated herein by reference, the definition presented herein shall prevail.

[0051] As used herein, the term "separated" refers to a state obtained artificially from the natural state. A "separated" substance or component may exist in nature, or may be separated due to changes in its natural environment, separation from its natural environment, or both. For example, an unseparated polynucleotide or polypeptide may naturally exist within a living organism; the same polynucleotide or peptide isolated from this natural state with high purity is called a separated polynucleotide or polypeptide. The term "separated" does not exclude the presence of mixed artificial or synthetic substances, nor does it exclude other impurities that do not affect the activity of the separated substance.

[0052] As used in this article, the term "KRAS" refers to the kirsten rat sarcomaviral oncogene (KRAS), a proto-oncogene belonging to the RAS gene family. The human genome contains three RAS genes: HRAS (Gene ID: 3265), NRAS (Gene ID: 4893), and KRAS (Gene ID: 3845), which were the first oncogenes identified in human tumors. These three RAS genes share high sequence homology (>90%) and encode the HRAS, NRAS, and KRAS proteins, respectively. Ras proteins are primarily located on the inner side of the cell membrane, transmitting extracellular signals such as epidermal growth factor into the cell, and are widely involved in cell growth, differentiation, and tumorigenesis and development. In approximately one-third of human cancers, a single point mutation in RAS (e.g., mutations in G12, G13, or Q61 residues) causes it to preferentially bind to GTP, remaining in an activated state and leading to tumorigenesis and development. The KRAS protein encoded by the KRAS gene possesses GTPase activity, acting as a molecular switch between GTP and GDP binding and participating in numerous signaling pathways regulating cell proliferation, differentiation, and apoptosis, such as the MAPK and PI3K signaling pathways. KRAS gene mutations typically occur at the G12, G13, and Q61 positions, with major mutants including G12D, G12V, G13D, and Q61H, among which the G12V mutation accounts for 20-40% of KRAS gene mutations. The sequence of the KRAS protein encoded by the KRAS gene is well-known to those skilled in the art and can be found in various public databases. For example, the KRAS protein sequence can be found in NCBI:NP_001356715.1.

[0053] As used herein, the term "KRAS G12V mutant" refers to a KRAS mutant in which the 12th amino acid residue Gly is mutated to Val. In some embodiments, the amino acid sequence of the antigenic peptide of the KRAS G12V mutant is shown in SEQ ID NO: 1. Those skilled in the art will understand that the "12" in G12V indicates the position of the twelfth amino acid residue, starting from the first amino acid residue at the N-terminus of the KRAS protein amino acid sequence, and the first amino acid residue M at the N-terminus of the KRAS G12V antigenic peptide sequence is typically removed and not represented.

[0054] The HRAS and NRAS proteins share more than 84% sequence identity with the KRAS protein, wherein the amino acid sequences of at least positions 1 to 70 (e.g., positions 2 to 14) of the HRAS, NRAS, and KRAS proteins are identical. Exemplary amino acid sequences of the HRAS and NRAS proteins can be found in NP_001123914.1 and NP_002515.1, respectively. Similar to the KRAS protein, the HRAS and NRAS proteins also exhibit mutations at the 12th amino acid residue Gly, including a Gly mutation to Val (i.e., the G12V mutation). Therefore, the RAS G12V mutation described herein encompasses the KRAS G12V mutation, the HRAS G12V mutation, and / or the NRAS G12V mutation. In some embodiments, the amino acid sequence of the antigenic peptide of the HRAS G12V mutant and the NRAS G12V mutant comprises the sequence shown in SEQ ID NO: 1. NRAS G12V mutations have been found in diseases or conditions such as chronic myelomonocytic leukemia (CMML). Diseases or conditions associated with NRAS G12V mutations include, but are not limited to, salivary gland cancer, thyroid cancer, oral cancer, and bladder cancer.

[0055] As used herein, the terms “T cell receptor” and “TCR” are used interchangeably and refer to molecules that include a CDR or variable region from αβ or γδ T cell receptors. αβ TCRs are the major TCRs (95%) found on MHC-restricted T cells. The term TCR includes, but is not limited to, full-length TCRs, antigen-binding fragments of TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs containing variable regions linked by flexible linkers, and TCR chains linked by engineered disulfide bonds.

[0056] As used herein, the term "TCR variable region" refers to a portion of a mature TCR polypeptide chain (e.g., the TCRα chain or β chain) that is not encoded by the TRAC gene of the TCRα chain, the TRBC1 or TRBC2 gene of the TCRβ chain, the TRDC gene of the TCRδ chain, or the TRGC1 or TRGC2 gene of the TCRγ chain. In some embodiments, the TCR variable region of the TCRα chain encompasses all amino acids of the mature TCRα chain polypeptide encoded by the TRAV gene and / or the TRAJ gene, and the TCR variable region of the TCRβ chain encompasses all amino acids of the mature TCRβ chain polypeptide encoded by the TRBV gene, the TRBD gene, and / or the TRBJ gene (see, for example, Tcell receptor Factsbook, (2001), LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, which is incorporated herein by reference in its entirety). The TCR variable region typically includes frame regions (FRs) 1, 2, 3, and 4 and complementarity-determining regions (CDRs) 1, 2, and 3. In this document, the terms "α-chain variable region" and "Vα" are used interchangeably and refer to the variable region of the TCRα chain. The terms "β-chain variable region" and "Vβ" are used interchangeably and refer to the variable region of the TCRβ chain.

[0057] As used herein, the term “CDR” or “complementarity-determining region” in relation to TCR refers to a non-adjacent antigen-binding site found within a variable region of the TCR chain (e.g., α or β chain). These regions have been described in LeFranc, (1999), The Immunologist, 7:132-136; LeFranc et al., (1999), Nucleic Acids Res, 27:209-212; LeFranc, (2001), T cell receptor Facts book, Academic Press, ISBN 0-12-441352-8; LeFranc et al., (2003), Dev Comp Immunol, 27(1):55-77; and Kabat et al., (1991), Sequences of protein of immunological interest, all of which are incorporated herein by reference in their entirety. CDRs can be delimited according to various numbering systems well known in the art, such as the IMGT numbering system or the Kabat numbering system.

[0058] As used herein, the term “FR” or “frame region” in relation to TCR refers to those amino acid residues in the variable region of the TCR chain (e.g., the α chain or β chain) other than the CDR as defined above.

[0059] As used herein, the term "constant region" in relation to the TCR refers to a portion of the TCR encoded by the TRAC gene (for the TCRα chain), the TRBC1 or TRBC2 gene (for the TCRβ chain), the TRDC gene (for the TCRδ chain), or the TRGC1 or TRGC2 gene (for the TCRγ chain), optionally lacking all or a portion of the transmembrane region and / or all or a portion of the cytoplasmic region. In some embodiments, the TCR constant region lacks both the transmembrane and cytoplasmic regions.

[0060] As used herein, the term "antigen-binding portion" in relation to a TCR refers to any part or fragment of the TCR that retains the biological activity of the TCR (parental TCR) as part of the TCR. This biological activity may include the ability to specifically bind to the same antigen (e.g., the KRAS G12V mutant) or MHC-antigen complex bound to the parent TCR.

[0061] As used herein, the term "antigen-presenting cell" or "APC" refers to any cell capable of presenting peptide fragments of proteins associated with major histocompatibility complex (MHC) molecules on its cell surface. Such cells are well known to those skilled in the art and include, but are not limited to, dendritic cells, monocytes, macrophages, lymphoblastic cell lines (e.g., B lymphoblastoid cells, B-LCL), and the like.

[0062] As used herein, the terms "major histocompatibility complex" and "MHC" are used interchangeably. MHC is a tightly linked group of genes that determine tissue homology and are closely related to the immune response, primarily comprising MHC class I and MHC class II molecules. "MHC class I molecules" are dimers composed of the MHC class I α heavy chain and β2 microglobulin; T cells expressing CD8 molecules react with MHC class I molecules. "MHC class II molecules" are dimers composed of the MHC class II α chain and MHC class II β chain; T cells expressing CD4 molecules react with MHC class II molecules. In humans, the MHC is called the human leukocyte antigen (HLA) complex.

[0063] As used herein, the terms “MHC-peptide complex,” “MHC-peptide complex,” or “pMHC complex” are used interchangeably to refer to an MHC molecule (MHC class I or MHC class II) containing a peptide in a recognized MHC peptide-binding pocket. In some cases, the MHC molecule may be a membrane-bound protein expressed on the cell surface. In other cases, the MHC molecule may be a soluble protein lacking a transmembrane region or a cytoplasmic region.

[0064] In the context of TCRs, the terms "specific binding" or "specific recognition" refer to the ability of a TCR to preferentially bind to a specific antigen (e.g., a specific peptide or a specific peptide-MHC complex combination). Typically, a TCR that specifically binds to an antigen does not bind to or binds to other antigens with lower affinity. In some embodiments, the TCRs disclosed herein or their antigen-binding fragments specifically bind to the KRAS G12V mutant. In some embodiments, the TCRs disclosed herein or their antigen-binding fragments specifically bind to the sequence shown in SEQ ID NO: 1. In some embodiments, the TCRs disclosed herein or their antigen-binding fragments specifically bind to an MHC complex containing an antigenic peptide of the KRAS G12V mutant.

[0065] As used herein, the term "epitope" refers to a localized region of an antigen (e.g., a peptide or peptide-MHC complex) that a TCR can bind to, also known as an antigenic determinant. TCR-bound epitopes can be determined by, for example, NMR spectroscopy, X-ray diffraction crystallography, ELISA assays, hydrogen / deuterium exchange mass spectrometry (e.g., liquid chromatography-electrospray ionization mass spectrometry), flow cytometry analysis, mutagenesis mapping (e.g., site-directed mutagenesis mapping), and / or structural modeling. In some embodiments, the antigen is a peptide-MHC complex or a peptide presented by an MHC molecule.

[0066] As used herein, the term "identity" refers to the matching of sequences between two polypeptides or two nucleic acids. When a position in two compared sequences is occupied by the same base or nucleotide monomer subunit (e.g., a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. The "percentage identity" between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions compared × 100. For example, if six out of ten positions in two sequences match, then the two sequences are 60% identical. Typically, two sequences are compared to produce the maximum identity. Such comparisons can be performed using methods well known to those skilled in the art, for example, conveniently performed using computer programs such as the Align program (DNAstar, Inc.) Needleman et al. (1970) J. Mol. Biol. 48: 443-453.

[0067] As used herein, the term "effect domain" in relation to the fusion protein of the present invention refers to a peptide or protein that has the function of binding to effector cells, specifically, that is capable of binding to effector molecules on the surface of effector cells. The effector cells include, but are not limited to, T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells. In some embodiments, the effector domain is an antibody or its antigen-binding portion.

[0068] As used herein, the terms "antibody" and "its antigen-binding portion" refer to at least the smallest portion of an antibody capable of binding to a specified antigen targeted by the antibody, such as, in the case of a typical antibody produced by B cells, at least some complementarity-determining regions (CDRs) of the variable domain (VH) of the heavy chain and the variable domain (VL) of the light chain. The antigen-binding function of an antibody can be achieved by a fragment of the full-length antibody. The VH and VL regions include hypervariable regions called complementarity-determining regions (CDRs) and more conserved regions inserted therebetween called framework regions (FRs). The variable regions of the heavy and light chains contain binding domains that interact with the antigen. An antibody or its antigen-binding portion can be or is derived from polyclonal antibodies, monoclonal antibodies, human antibodies, humanized antibodies, or chimeric antibodies, single-chain antibodies, or epitope-binding fragments.

[0069] Examples of binding fragments covered in the term "antigen-binding moiety" of an antibody include (i) Fab fragments (fragments cleaved by papain) or similar monovalent fragments consisting of VL, VH, LC, and CH1 domains; (ii) F(ab')2 fragments (fragments cleaved by pepsin) or similar bivalent fragments consisting of two Fab fragments linked by disulfide bonds through a hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of VH domains (Ward et al., (1989) Nature 341:544-546); (vi) separate complementarity-determining regions (CDRs); and (vii) combinations of two or more separate CDRs, which may optionally be linked by synthetic linkers. Furthermore, although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be linked using recombination methods via a synthetic linker that allows them to be prepared as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

[0070] As used herein, antibodies or their antigen-binding portions also include "single-domain antibodies," examples of which include heavy chain antibodies, naturally occurring antibodies lacking light chains, single-domain antibodies derived from conventional four-chain antibodies, and engineered or recombinant single-domain antibodies. The variable heavy chain of a single-domain antibody lacking a light chain is referred to as a "VHH" or "nanobody." Similar to the traditional VH domain, a VHH contains four free radicals (FRs) and three core radicals (CDRs).

[0071] As used herein, the term "Fc" has the same meaning as when used for antibodies, referring to the antibody portion containing the second (CH2) and third (CH3) constant regions of the first heavy chain, which are bound to the second and third constant regions of the second heavy chain via disulfide bonds. The Fc region may contain all or part of the hinge region. The Fc region of an antibody is responsible for various effector functions, such as ADCC and CDC, but typically does not play a role in antigen binding. In this document, the term "Fc" includes wild-type Fc, Fc variants, and grafted Fc.

[0072] The term "operably linked" refers to the juxtaposition of two or more target biological sequences, with or without spacers or linkers, in a manner that allows them to function in the intended manner. When used for peptides, the intention is that the peptide sequences are linked in a manner that allows the linked product to have the expected biological function. For example, a TCR or its antigen-binding fragment can be operably linked to an Fc region to provide a stable product with antigen-binding activity. Through "operably linked," a TCR or its antigen-binding fragment can be directly linked to an Fc region, provided both portions function properly, or more preferably, the TCR or its antigen-binding fragment can be indirectly linked to an Fc region via a linker sequence (e.g., a hinge region). The term can also be used for polynucleotides. For example, when a polynucleotide encoding a peptide is operably linked to a regulatory sequence (e.g., a promoter, enhancer, silencer sequence, etc.), this means that the polynucleotide sequences are linked in a manner that allows the peptide to be expressed regulatedly from that polynucleotide.

[0073] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; bacteriophages; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0074] As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including but not limited to: cellular organisms such as *Escherichia coli* or *Bacillus subtilis*; fungal cells such as yeast cells or *Aspergillus*; insect cells such as S2 *Drosophila* cells or Sf9 cells; animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells, or human cells; and immune cells (such as T lymphocytes, NK cells, monocytes, macrophages, or dendritic cells). Host cells can include single cells or cell populations.

[0075] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, which is well known in the art and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining agents, absorption delaying agents, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. Osmotic pressure maintaining agents include, but are not limited to, sugars, NaCl, and their analogues. Delayed absorption agents include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Stabilizers have the meaning commonly understood by those skilled in the art as being able to stabilize the desired properties of the active ingredient in a pharmaceutical product, including but not limited to monosodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or their degradation products (such as lactalbumin hydrolysate). In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions). In some exemplary embodiments, such sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solutions (e.g., 0.9% (w / v) NaCl), glucose solutions (e.g., 5% glucose), solutions containing surfactants (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffer solutions), Ringer's solutions, and any combination thereof.

[0076] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, condition, or symptom (e.g., a tumor) in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical outcome. For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, alleviating symptoms, reducing the extent of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or alleviating the progression of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. Furthermore, "treatment" can also refer to prolonged survival compared to the expected survival (if no treatment had been received).

[0077] As used herein, the term "effective amount" means an amount sufficient to achieve, or at least partially achieve, the desired effect. For example, an effective amount for preventing disease (e.g., cancer) means an amount sufficient to prevent, stop, or delay the onset of disease (e.g., cancer); an effective amount for treating disease means an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is entirely within the capabilities of those skilled in the art. For example, an effective amount for therapeutic purposes will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general characteristics such as age, weight, and sex, the manner of administration of the drug, and any other concurrent treatments.

[0078] As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In some embodiments, the term "subject" refers to a living organism in which an immune response can be elicited. In some embodiments, the subject (e.g., a human) has a RAS G12V (e.g., KRAS G12V, HRAS G12V, and / or NRAS G12V) mutation-positive tumor, or is at risk of having such a disease.

[0079] T cell receptors

[0080] This invention provides an isolated T-cell receptor (TCR) or its antigen-binding fragment, capable of specifically recognizing KRAS protein mutants or their antigenic peptides, or pMHC complexes containing said antigenic peptides, or cells expressing said antigenic peptides. In some embodiments, the antigenic peptide is presented by an MHC class II molecule. In some embodiments, the MHC class II molecule is HLA-DP. In some embodiments, the HLA-DP is selected from HLA-DPA1*02:01, HLA-DPA1*02:02, HLA-DPA1*01:03, HLA-DPB1*03:01, HLA-DPB1*14:01, and HLA-DPB1*104:01, and combinations thereof. In some embodiments, the TCR or its antigen-binding fragment of the present invention may bind to DPA1*01:03 / DPB1*03:01 and DPA1*02:02 / DPB1*03:01, as well as DPB1*14:01 and DPB1*104:01.

[0081] Optionally, the TCR or its antigen-binding fragment is capable of specifically recognizing the KRAS G12V mutant, the TCR or its antigen-binding fragment comprising an α-chain variable region (Vα) and / or a β-chain variable region (Vβ), wherein, (a) the Vα comprises CDR1α, CDR2α, and CDR3α; and / or, (b) the Vβ comprises CDR1β, CDR2β, and CDR3β. Optionally, the TCR or its antigen-binding fragment further comprises an α-chain constant region (Cα) operatively linked to Vα and / or a β-chain constant region (Cβ) operatively linked to Vβ.

[0082] In some embodiments, CDR1α comprises an amino acid sequence having a substitution, insertion, or deletion of one or two amino acids relative to the sequence shown in SEQ ID NO: 17; CDR2α comprises an amino acid sequence having a substitution, insertion, or deletion of one or two amino acids relative to the sequence shown in any one of SEQ ID NO: 18-22; and CDR3α comprises an amino acid sequence having a substitution, insertion, or deletion of one or two amino acids relative to the sequence shown in SEQ ID NO: 23 or 101. Preferably, the substitution is a conserved amino acid substitution.

[0083] In some embodiments, the CDR2α has the sequence X1X2X3X4X5NLV (SEQ ID NO: 96), wherein X1 is selected from Y, A, K, or L; X2 is selected from I, P, A, G, or N; X3 is selected from T or L; X4 is selected from G, L, A, or R; and X5 is selected from D or E. In some embodiments, the CDR2α has the sequence shown in any one of SEQ ID NO: 18-22.

[0084] In some embodiments, CDR1β comprises an amino acid sequence having a substitution, insertion, or deletion of one or two amino acids relative to the sequence shown in SEQ ID NO: 24 or 25; CDR2β comprises an amino acid sequence having a substitution, insertion, or deletion of one or two amino acids relative to the sequence shown in any one of SEQ ID NO: 26-28; and CDR3β comprises an amino acid sequence having a substitution, insertion, or deletion of one or two amino acids relative to the sequence shown in any one of SEQ ID NO: 29-35. Preferably, the substitution is a conserved amino acid substitution.

[0085] In some embodiments, the CDR2β has the sequence SQX1X2X3X4 (SEQ ID NO: 97), wherein X1 is selected from I, L, or V; X2 is selected from V or M; X3 is selected from N or G; and X4 is selected from D, V, or H. In some embodiments, the CDR2β has the sequence shown in any one of SEQ ID NO: 26-28.

[0086] In some embodiments, the CDR3β has the sequence shown in ASSPGX1RX3X4SX6LH (SEQ ID NO: 98), wherein X1 is selected from Q, S, or T; X3 is selected from D or T; X4 is selected from N, F, Q, W, or T; and X6 is selected from P, Q, or A. In some embodiments, the CDR3β has the sequence shown in any one of SEQ ID NO: 29-35.

[0087] In some embodiments, the TCR or its antigen-binding fragment disclosed herein has a CDR1α comprising the sequence shown in SEQ ID NO: 17, a CDR2α comprising the sequence shown in any one of SEQ ID NO: 18-22, a CDR3α comprising the sequence shown in SEQ ID NO: 23; and / or, a CDR1β comprising the sequence shown in SEQ ID NO: 24, a CDR2β comprising the sequence shown in SEQ ID NO: 26, and a CDR3β comprising the sequence shown in SEQ ID NO: 29.

[0088] In some embodiments, the TCR or its antigen-binding fragment disclosed herein has a CDR1α comprising the sequence shown in SEQ ID NO: 17, a CDR2α comprising the sequence shown in SEQ ID NO: 22, and a CDR3α comprising the sequence shown in SEQ ID NO: 23; and / or, a CDR1β comprising the sequence shown in SEQ ID NO: 25, a CDR2β comprising the sequence shown in SEQ ID NO: 27, and a CDR3β comprising the sequence shown in SEQ ID NO: 29.

[0089] In some embodiments, the TCR or its antigen-binding fragment disclosed herein has a CDR1α comprising the sequence shown in SEQ ID NO: 17, a CDR2α comprising the sequence shown in SEQ ID NO: 22, and a CDR3α comprising the sequence shown in SEQ ID NO: 23; and / or, a CDR1β comprising the sequence shown in SEQ ID NO: 24, a CDR2β comprising the sequence shown in SEQ ID NO: 28, and a CDR3β comprising the sequence shown in SEQ ID NO: 29.

[0090] In some embodiments, the TCR or its antigen-binding fragment disclosed herein has a CDR1α comprising the sequence shown in SEQ ID NO: 17, a CDR2α comprising the sequence shown in SEQ ID NO: 22, a CDR3α comprising the sequence shown in SEQ ID NO: 23; and / or, a CDR1β comprising the sequence shown in SEQ ID NO: 24, a CDR2β comprising the sequence shown in SEQ ID NO: 26, and a CDR3β comprising the sequence shown in any one of SEQ ID NO: 30-35.

[0091] In some embodiments, Vα comprises a sequence having at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with any of the sequences shown in SEQ ID NO: 36-40, and Vβ comprises a sequence having at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with any of the sequences shown in SEQ ID NO: 41-49.

[0092] In some embodiments, the TCR of the present invention or its antigen-binding fragment comprises Vα having the sequence shown in any one of SEQ ID NO: 36-40 and / or Vβ having the sequence shown in any one of SEQ ID NO: 41-49. In some embodiments, the TCR of the present invention or its antigen-binding fragment comprises Vα having the sequence shown in any one of SEQ ID NO: 37-40 and Vβ having the sequence shown in SEQ ID NO: 41. In some embodiments, the TCR of the present invention or its antigen-binding fragment comprises Vα having the sequence shown in SEQ ID NO: 36 and Vβ having the sequence shown in any one of SEQ ID NO: 42-49.

[0093] In some embodiments, the TCR of the present invention or its antigen-binding fragment comprises CDR1α, CDR2α and CDR3α in Vα as shown in any one of SEQ ID NO: 37-40, and / or CDR1β, CDR2β and CDR3β in Vβ as shown in SEQ ID NO: 41.

[0094] In some embodiments, the TCR of the present invention or its antigen-binding fragment comprises CDR1α, CDR2α and CDR3α in Vα as shown in SEQ ID NO: 36, and / or CDR1β, CDR2β and CDR3β in Vβ as shown in any one of SEQ ID NO: 42-49.

[0095] In some embodiments, Vα comprises CDR1α as shown in SEQ ID NO: 17, CDR2α as shown in X1X2X3X4X5NLV (SEQ ID NO: 96), and CDR3α as shown in SEQ ID NO: 23, wherein X1 is selected from Y, A, K, or L; X2 is selected from I, P, A, G, or N; X3 is selected from T or L; X4 is selected from G, L, A, or R; and X5 is selected from D or E. In some embodiments, Vα comprises CDR1α as shown in SEQ ID NO: 17, CDR2α as shown in any one of SEQ ID NO: 18-22, and CDR3α as shown in SEQ ID NO: 23.

[0096] In some embodiments, the Vβ comprises CDR1β as shown in SEQ ID NO: 24, CDR2β as shown in SEQ ID NO: 26, and CDR3β as shown in ASSPGX1RX3X4SX6LH (SEQ ID NO: 98), wherein X1 is selected from Q, S, or T; X3 is selected from D or T; X4 is selected from N, F, Q, W, or T; and X6 is selected from P, Q, or A. In some embodiments, the Vβ comprises CDR1β as shown in SEQ ID NO: 24, CDR2β as shown in SEQ ID NO: 26, and CDR3β as shown in any one of SEQ ID NO: 29-35.

[0097] In some embodiments, the Vβ comprises CDR1β as shown in SEQ ID NO: 24, CDR2β as shown in SEQ ID NO: 28, and CDR3β as shown in SEQ ID NO: 29. In some embodiments, the Vβ comprises CDR1β as shown in SEQ ID NO: 25, CDR2β as shown in SEQ ID NO: 27, and CDR3β as shown in SEQ ID NO: 29.

[0098] In some implementations, the CDR described in any of the above implementations is defined according to the IMGT numbering system.

[0099] In some embodiments, the TCR or its antigen-binding fragment is capable of specifically recognizing the KRAS G12V mutant antigen or a fragment thereof (e.g., an antigenic peptide). In some embodiments, the TCR or its antigen-binding fragment is capable of specifically recognizing the antigenic peptide comprising SEQ ID NO: 1. In some embodiments, the antigenic peptides of the HRAS G12V mutant and the NRAS G12V mutant have the same amino acid sequence as the KRAS G12V mutant antigenic peptide. Therefore, in some embodiments, the TCR or its antigen-binding fragment is capable of specifically recognizing the HRAS G12V mutant or the NRAS G12V mutant antigenic peptide. In some embodiments, the antigenic peptide is presented by an MHC-II molecule. In some embodiments, the MHC-II molecule is HLA-DP. In some embodiments, the HLA-DP is a combination selected from HLA-DPA1*02:01, HLA-DPA1*02:02 and HLA-DPA1*01:03 and selected from HLA-DPB1*03:01, HLA-DPB1*14:01 and HLA-DPB1*104:01.

[0100] In some embodiments, the TCR is a soluble TCR lacking one or more transmembrane and / or cytoplasmic regions. In some embodiments, a soluble TCR is produced by fusing the extracellular domain of the TCR of the present invention with other protein domains (e.g., maltose-binding protein, thioredoxin, human immunoglobulin κ constant domain, or leucine zipper), see, for example, Løset GÅ et al., Front Oncol., 2015.1.12;4:378, which is incorporated herein by reference in its entirety.

[0101] In some embodiments, the TCR of the present invention may also be a single-chain TCR comprising Vα and Vβ linked via peptide linkers. Such a single-chain TCR may comprise Vα and Vβ, each linked to its own TCR constant region (TRC). Alternatively, a single-chain TCR may comprise Vα and Vβ, wherein Vα, Vβ, or both Vα and Vβ are not linked to the TCR constant region. Exemplary single-chain TCRs are described in PCT Publications WO 2003 / 020763, WO 2004 / 033685, and WO 2011 / 044186, each of which is incorporated herein by reference in its entirety.

[0102] In some embodiments, the TCR of the present invention may comprise two polypeptide chains (e.g., an α chain and a β chain), wherein said chains have been engineered to each have cysteine ​​residues capable of forming interchain disulfide bonds. Specifically, the α chain and β chain comprise variable regions (Vα and Vβ) and constant regions (Cα and Cβ), respectively, wherein said Cα and Cβ each have cysteine ​​residues capable of forming interchain disulfide bonds, and may also be engineered to have non-natural cysteine ​​residues capable of forming interchain disulfide bonds. Thus, the TCR of the present invention may comprise two polypeptide chains linked by engineered disulfide bonds. Exemplary TCRs with engineered disulfide bonds are described in U.S. Patent Nos. 8,361,794 and 8,906,383, each of which is incorporated herein by reference in its entirety.

[0103] Fusion proteins containing immunoglobulin Fc domains and / or effector domains

[0104] Furthermore, the present invention provides a fusion protein comprising an immunoglobulin Fc moiety and a TCR or its antigen-binding fragment disclosed herein, the fusion protein comprising a first polypeptide chain and a second polypeptide chain. In some embodiments, the first polypeptide chain comprises an α chain (Vα-Cα) of a TCR or its antigen-binding fragment, the second polypeptide chain comprises a β chain (Vβ-Cβ) of a TCR or its antigen-binding fragment, and the Fc moiety comprises a first Fc domain (Fc1) and a second Fc domain (Fc2), Fc1 and Fc2 being operatively linked to the C-terminus of the first and second polypeptide chains.

[0105] In some aspects, the present invention also provides a fusion protein comprising an immunoglobulin Fc moiety, an effector domain, and a TCR or antigen-binding fragment thereof disclosed herein, said fusion protein comprising a first polypeptide chain and a second polypeptide chain. In some embodiments, the first polypeptide chain comprises an α chain (Vα-Cα) of the TCR or antigen-binding fragment thereof, and the second polypeptide chain comprises a β chain (Vβ-Cβ) of the TCR or antigen-binding fragment thereof, optionally with the Fc moiety operably linked to the C-terminus of the first and / or second polypeptide chain, wherein the Fc moiety comprises a first Fc domain (Fc1) and a second Fc domain (Fc2). Further, the effector domain is linked to the N-terminus of the first and / or second polypeptide chain, or the effector domain is linked to the C-terminus of the first and / or second polypeptide chain. The first polypeptide chain and the second polypeptide chain can form a stable dimer through interchain disulfide bonds and / or non-covalent interactions. Fc1 and Fc2 can be operably linked on different chains or on the same chain. In some implementations, the CH3 region of Fc1 is operatively linked to the CH2 region or hinge region of Fc2 via a peptide linker.

[0106] The effector domain is preferably a peptide or protein with the function of binding to effector cells, including but not limited to T lymphocytes, NK cells, dendritic cells, and macrophages. When the effector domain of the fusion protein recognizes and binds to effector molecules located on the surface of effector cells, it leads to the activation of the effector cells. Preferably, the effector domain is an antibody or its antigen-binding moiety capable of binding to a specific antigen, and the antibody or its antigen-binding moiety may be in the form of Fab, Fab', F(ab')2, Fd, Fv, single-chain variable fragment (scFv), single-chain antibody, VHH, or vNAR. Preferably, the ratio of the number of TCRs or their antigen-binding fragments to the effector domains in the fusion protein disclosed herein is 1:1.

[0107] In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain includes the following domains operably linked from the N-terminus to the C-terminus: Vα-Cα-Fc1-Fc2, and the second polypeptide chain includes the following domains operably linked from the N-terminus to the C-terminus: an effector domain-Vβ-Cβ or a Vβ-Cβ-effector domain. The first polypeptide chain and the second polypeptide chain are associated via interchain disulfide bonds (e.g., interchain disulfide bonds between Cα and Cβ) and / or non-covalent interactions. Preferably, Fc1 and Fc2 in the first polypeptide chain are linked by a flexible linker, allowing the two Fc sequences to form stable interactions as if they were on separate chains, through interchain disulfide bonds (e.g., hinge-to-hinge disulfide bonds).

[0108] As mentioned above, Vα and Vβ represent the variable regions of the α chain and β chain of the TCR or its antigen-binding fragment disclosed herein, respectively; Cα and Cβ represent the constant regions of the α chain and β chain of the TCR or its antigen-binding fragment disclosed herein, respectively; and Fc1 and Fc2 represent the first Fc domain and the second Fc domain of the immunoglobulin Fc region, respectively.

[0109] The operable connection (or denoted by "-") can be a direct connection between two parts, or a connection via a connector such as a peptide connector. The peptide connector can be any peptide connector conventionally used in the art, preferably a flexible connector, such as the GS series connectors. Furthermore, the TCR constant region (e.g., Cα or Cβ) can be connected to the first Fc domain or the second Fc domain via a hinge region.

[0110] The Fc portion of the fusion protein can be a wild-type Fc or an Fc variant. The wild-type Fc can be human IgG1, IgG2, IgG3, or IgG4 Fc. In some embodiments, the wild-type Fc is human IgG1-Fc. Compared to the wild-type Fc (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc), the Fc variant contains one or more amino acid residue modifications (e.g., substitution, insertion, and / or deletion). In some embodiments, compared to wild-type human IgG1 Fc, the Fc variant contains one or more amino acid residue modifications (e.g., substitution, insertion, and / or deletion). In some embodiments, compared to wild-type human IgG4 Fc, the Fc variant contains one or more amino acid residue modifications (e.g., substitution, insertion, and / or deletion). The first and second Fc domains can be associated with one or more disulfide bonds, and the sequences of Fc1 and Fc2 can be the same or different. In some embodiments, Fc1 and Fc2 are associated with one or more (e.g., one, two, or three) non-natural interchain disulfide bonds.

[0111] Numerous known mutations increase or decrease ADCC, ADCP, and CDC. One example of a human IgG1 heavy chain mutation is the LALA mutation, which blocks all effector functions—essentially those related to ADCC, ADCP, and CDC. The hIgG1 LALA sequence includes two mutations, L234A and L235A (EU numbers), which inhibit FcgR binding. When referring to residues in the constant region of the immunoglobulin heavy chain, the “EU numbering system” or “EU index” is commonly used (e.g., the EU index reported in Kabat et al., Sequences of Proteins of Immunological Interest (5th Ed.), USDept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242). “EU number in Kabat” or “EU index in Kabat” refers to the residue number of the human IgG1 EU antibody. Unless otherwise stated herein, residue numbers in the constant domain of the Fc region refer to residue numbers obtained through the EU numbering system.

[0112] The one or more amino acid modifications included in the Fc variant can alter binding to one or more FcγR receptors, alter binding to FcRn receptors, etc. In some embodiments, the Fc domain of the fusion protein contains one or more amino acid substitutions that improve pH-dependent binding to the neonatal Fc receptor (FcRn). This variant can have a prolonged pharmacokinetic half-life because it binds to FcRn at acidic pH, allowing it to escape degradation in lysosomes and then be transported and released from the cell. Methods for engineering antibodies and their antigen-binding fragments to improve binding affinity to FcRn are well known in the art; see, for example, Vaughn, D. et al., Structure, 6(1): 63-73, 1998; Kontermann, R. et al., Antibody Engineering, Volume 1, Chapter 27: Engineering of the Fc region for improved PK, published by Springer, 2010; Yeung, Y. et al., Cancer Research, 70: 3269-3277 (2010); and Hinton, P. et al., J. Immunology, 176: 346-356 (2006).

[0113] In some embodiments, the Fc portion of the fusion protein contains one or more amino acid substitutions that alter antibody-dependent cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC). Certain amino acid residues at the CH2 domain of the Fc may be substituted to provide reduced ADCC activity.

[0114] In some embodiments, the Fc moiety includes one or more amino acid modifications (e.g., substitutions) at the interface of the Fc region to facilitate and / or promote heterodimerization. For example, the first and second Fc domains of the Fc moiety are engineered to include a "knob-in-hole" structure to facilitate heterodimerization, which includes introducing a protrusion ("handle") into the first Fc domain and a cavity ("hole") into the second Fc domain, wherein the protrusion can be positioned within the cavity to facilitate the interaction between the first and second Fc domains to form a heterodimer or complex. Specifically, the Fc domain may include at least one "handle" (protrusion) and at least one "hole" (cavity), wherein the presence of the "handle" and the "hole" enhances the formation of the complex or heterodimer (see WO 2005 / 063816 for further details). In some embodiments, the Fc moiety disclosed herein includes first and second Fc domains, wherein each of the first and second Fc domains contains one or more mutations relating to wild-type human IgG1 Fc.

[0115] In some embodiments, the Fc domain is an IgG1 Fc variant containing a “FALA” mutation (i.e., F234A / L235A), thereby reducing binding to the Fc receptor or complement receptor. In some embodiments, the Fc variant contains mutations in L234A, L235A, and P329G. In some embodiments, the Fc variant comprises first and second Fc polypeptide chains, wherein the first Fc polypeptide contains a “handle” mutation and the second Fc polypeptide contains a “pore” mutation. In at least one embodiment, the “pore” mutation is Y349C, T366S, L368A, and / or Y407V, while the “handle” mutation is S354C and / or T366W, wherein S354C and Y349C can form a disulfide bond.

[0116] In some specific embodiments, Fc1 has the amino acid sequence shown in SEQ ID NO: 94, and Fc2 has the amino acid sequence shown in SEQ ID NO: 95. In some specific embodiments, there are three pairs of disulfide bonds (C212-C469, C215-C472, and C340-C592) between Fc1 and Fc2 in the fusion protein of the present invention.

[0117] In some embodiments, the effector domain includes a domain that specifically binds to CD3 (differentiation cluster 3) molecules, such as anti-CD3 antibodies (e.g., UCHT1, SP34, OKT3, etc.) or their antigen-binding portions. In some embodiments, the effector domain includes a domain that specifically binds to FcRn (CD16), such as anti-FcRn antibodies or their antigen-binding portions. In some embodiments, the effector domain includes a domain that specifically binds to PD1, such as anti-PD1 antibodies or their antigen-binding portions. In some embodiments, the effector domain includes a domain that specifically binds to LAG3, such as anti-LAG3 antibodies or their antigen-binding portions. In some embodiments, the effector domain includes a domain that specifically binds to CD28, such as anti-CD28 antibodies or their antigen-binding portions. In some embodiments, the effector domain is scFv (e.g., CD3 scFv). In a preferred embodiment, the effector domain has the amino acid sequence shown in SEQ ID NO: 52.

[0118] In some embodiments, the fusion protein comprising an Fc domain and an effector domain as described above comprises a first polypeptide chain having a structure as shown in Vα-Cα-Fc1-Fc2, and a second polypeptide chain having a structure as shown in CD3 scFv-Vβ-Cβ. The first polypeptide chain comprises an amino acid sequence having at least 95%, at least 99%, or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 3-7, and the second polypeptide chain comprises an amino acid sequence having at least 95%, at least 99%, or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 8-16.

[0119] In some embodiments, the first polypeptide chain comprises an amino acid sequence as shown in any one of SEQ ID NO: 4-7, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 8. In some embodiments, the first polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 3, and the second polypeptide chain comprises an amino acid sequence as shown in any one of SEQ ID NO: 9-16.

[0120] In some other embodiments, the fusion protein of the present invention may comprise a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain comprises an α chain (Vα-Cα) of a TCR or its antigen-binding fragment operably linked with a first Fc domain (Fc1) of the Fc moiety, the second polypeptide chain comprises an effector domain operably linked with a β chain (Vβ-Cβ) of a TCR or its antigen-binding fragment, and the third polypeptide chain comprises a second Fc domain (Fc2) of the Fc moiety. The first polypeptide chain and the second polypeptide chain, and the first polypeptide chain and the third polypeptide chain, form a stable protein complex through interchain disulfide bonds and / or non-covalent interactions. Preferably, Fc1 and Fc2 comprise a "handle" mutation and a "pore" mutation, respectively. In at least one embodiment, the "pore" mutation is Y349C, T366S, L368A, and / or Y407V, while the "handle" mutation is S354C and / or T366W, wherein S354C and Y349C can form a disulfide bond.

[0121] In some other embodiments, the fusion protein of the present invention may comprise a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises an α chain (Vα-Cα) of a TCR or an antigen-binding fragment thereof operably linked with a first Fc domain (Fc1) of the Fc portion, i.e., Vα-Cα-Fc1, and the second polypeptide chain comprises an effector domain, a β chain (Vβ-Cβ) of a TCR or an antigen-binding fragment thereof, and a second Fc domain (Fc2) of the Fc portion, i.e., effector domain-Vβ-Cβ-Fc2.

[0122] The fusion protein is capable of specifically recognizing and binding with high affinity to KRAS mutant proteins (e.g., KRASG12V mutants) or their antigenic peptides or pMHC complexes containing said antigenic peptides. In some embodiments, the fusion protein is capable of specifically recognizing and binding to antigenic peptides of RAS G12V mutants (e.g., KRAS G12V, HRASG12V, and / or NRAS G12V mutants) as shown in SEQ ID NO: 1 or pMHC complexes containing said antigenic peptides. In some embodiments, the fusion protein containing both an Fc domain and an effector domain can also recruit and induce the activation of effector cells, for example, the activation of T cells.

[0123] In some embodiments, T cell activation can be measured using any suitable indicator known in the art. Non-limiting examples of such suitable indicators include increased levels of cytokine (e.g., IL-2, IFNγ, etc.) secretion, proliferative activity, and / or increased expression levels of activation markers (e.g., CD25, CD69, CD107a, etc.). In some embodiments, T cell activation also includes the induction of apoptosis or death in a second cell (e.g., a tumor cell) displaying (e.g., displaying in an MHC-II background) antigenic peptide by the T cells. In some embodiments, the fusion protein disclosed herein is capable of inducing effector cell activation, recognizing tumor cells expressing a pMHC complex, releasing effector cytokines (e.g., IFNγ), and inducing tumor cell lysis; wherein the pMHC complex is a pMHC complex containing an antigenic peptide of a KRAS mutant protein.

[0124] The high affinity of the fusion protein can be characterized in a manner well known to those skilled in the art. For example, the EC50 of the fusion protein disclosed herein binding to a KRAS mutant protein (e.g., the KRAS G12V mutant) or its antigenic peptide or a pMHC complex containing the antigenic peptide is about 1 nM or less, about 900 pM or less, about 800 pM or less, about 700 pM or less, about 600 pM or less, about 500 pM or less, about 400 pM or less, about 300 pM or less, about 200 pM or less, about 100 pM or less, or about 90 pM or less, or about 80 pM or less, or about 70 pM or less, or about 60 pM or less, or about 50 pM or less, or about 40 pM or less, or about 30 pM or less, or about 20 pM or less.

[0125] Conjugate

[0126] The present invention provides a conjugate comprising the TCR described herein or its antigen-binding fragment or fusion protein and an effector moiety conjugated thereto.

[0127] In this context, the term "effective moiety" refers to a component or functional group that can modulate (e.g., increase or decrease) the natural activity of a molecule to which it is attached, or confer novel activity to that molecule. In some embodiments, the effector moiety is a biologically active compound (e.g., a compound that has an effect on cells targeted by TCR) or a detectable label.

[0128] In this context, the term "conjugation" refers to any method known in the art for connecting functional protein domains, including but not limited to: recombination fusion with or without a linker, intein-mediated fusion, non-covalent binding, and covalent bonding, such as disulfide bonding, peptide bonding, hydrogen bonding, electrostatic bonding, and conformational bonding, such as biotin-avidin binding. In some embodiments, conjugation to the effector moiety can be carried out chemically or recombinarily, wherein the chemical method involves forming a covalent bond between two molecules to form a single molecule.

[0129] In some embodiments, the effector portion may be a therapeutic portion. A therapeutic portion refers to a compound or peptide that can be used as a therapeutic agent. The conjugate utilizes the targeting properties of TCRs to enable the therapeutic portion to produce a therapeutic effect on the cells targeted by the TCRs.

[0130] In some embodiments, the therapeutic component is selected from immune enhancers, such as immunostimulatory cytokines or immunostimulatory antibodies and their functional fragments. In some exemplary embodiments, the immunostimulatory cytokines are selected from, for example, IL-2, IL-3, IL-12, IL-15, IL-18, IL-10, IFN-γ, TGF-β, GM-CSF, or any combination thereof. The various cytokines listed refer to polypeptides having the natural biological activity of that cytokine, including, for example, full-length proteins, their active fragments, or mutants. For example, IL-2 refers to a polypeptide having IL-2 activity, which can be full-length IL-2, an active fragment of IL-2, or a mutant.

[0131] In some embodiments, the treatment component is selected from cytotoxic agents. In this document, the cytotoxic agents include any agent that is harmful to cells (e.g., kills cells).

[0132] In some embodiments, the cytotoxic agent is selected from alkylating agents, microtubule inhibitors or mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, and any combination thereof.

[0133] In some embodiments, the effector portion is selected from detectable markers. The detectable markers described in this invention can be any substance detectable by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electrical, optical, or chemical means. Such markers are well known in the art, and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.) and radionuclides (e.g.,...). 3 H, 125 I, 35 S, 14 C or 32 P), fluorescent labels (e.g., fluorescein, rhodamine, dansyl, phycoerythrin, or Texas red), chromophore moieties, digoxigenin, biotin / avidin, etc. The detectable labels described above can be detected by methods known in the art.

[0134] In some embodiments, the TCR of the present invention, or its antigen-binding fragment or fusion protein, is optionally conjugated to an effector portion via a linker (e.g., a peptide linker). In some embodiments, the effector portion is linked to the N-terminus or C-terminus of the TCR of the present invention, or its antigen-binding fragment, or the fusion protein of the present invention.

[0135] Preparation of TCR and fusion protein

[0136] The TCR or TCR-containing fusion protein of the present invention can be prepared by various methods known in the art, such as by genetic engineering recombination techniques. For example, DNA molecules encoding them can be obtained by chemical synthesis or PCR amplification; the resulting DNA molecules can be inserted into an expression vector and then transfected into host cells; then, the transfected host cells can be cultured under specific conditions and the TCR or TCR-containing fusion protein of the present invention can be expressed.

[0137] Therefore, in one aspect, the present invention provides a nucleic acid molecule comprising:

[0138] (i) The nucleotide sequence encoding the TCR of the present invention or its antigen-binding fragment or its α-chain variable region and / or β-chain variable region;

[0139] (ii) A nucleotide sequence encoding one or more polypeptide chains of the fusion protein of the present invention.

[0140] In some embodiments, the isolated nucleic acid molecule comprises a nucleotide sequence encoding an α-chain variable region of the TCR or its antigen-binding fragment disclosed herein and a nucleotide sequence encoding its β-chain variable region. The nucleotide sequences encoding the α-chain variable region and the nucleotide sequences encoding the β-chain variable region are present on different nucleic acid molecules.

[0141] In some embodiments, the nucleotide sequences encoding the α-chain variable region and the nucleotide sequences encoding the β-chain variable region are present on the same nucleic acid molecule in any order; in some embodiments, the nucleotide sequences encoding the α-chain variable region and the nucleotide sequences encoding the β-chain variable region are linked in any order by nucleotide sequences encoding self-cleaving peptides (e.g., P2A, E2A, F2A, or T2A).

[0142] In some embodiments, the isolated nucleic acid molecule comprises one or more nucleotide sequences encoding one or more polypeptide chains of the fusion protein disclosed herein. The one or more nucleotide sequences encoding one or more polypeptide chains are present on different nucleic acid molecules.

[0143] In some embodiments, the one or more nucleotide sequences encoding one or more polypeptide chains are present on the same nucleic acid molecule in any order; in some embodiments, the one or more nucleotide sequences encoding one or more polypeptide chains are linked in any order by nucleotide sequences encoding self-cleaving peptides (e.g., P2A, E2A, F2A, or T2A).

[0144] In one aspect, the present invention provides a vector (e.g., a cloning vector or an expression vector) comprising the nucleic acid molecules disclosed herein. In some embodiments, the vector of the present invention is, for example, a plasmid, a granulosome, a bacteriophage, etc. In some embodiments, the vector comprises a nucleotide sequence encoding a TCR as described above, or an antigen-binding fragment thereof, or an α-chain variable region thereof and / or a β-chain variable region thereof, or an α-chain and / or a β-chain thereof.

[0145] In some embodiments, the vector comprises a nucleotide sequence encoding the variable region of the TCRα chain and a nucleotide sequence encoding the variable region of the TCRβ chain.

[0146] In some embodiments, the vector comprises a nucleotide sequence encoding an α-chain of the TCR or an antigen-binding fragment thereof and a nucleotide sequence encoding a β-chain of the TCR or an antigen-binding fragment thereof.

[0147] In some embodiments, the nucleotide sequences encoding the TCRα chain variable region and the nucleotide sequences encoding the TCRβ chain variable region are present on different vectors. In some embodiments, the nucleotide sequences encoding the TCRα chain variable region and the nucleotide sequences encoding the TCRβ chain variable region are present on the same vector in any order. In some embodiments, the nucleotide sequences encoding the TCRα chain variable region and the nucleotide sequences encoding the TCRβ chain variable region are linked in any order by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A).

[0148] In some embodiments, the nucleotide sequences encoding the TCRα chain and the nucleotide sequences encoding the TCRβ chain are present on different vectors. In some embodiments, the nucleotide sequences encoding the TCRα chain and the nucleotide sequences encoding the TCRβ chain are present on the same vector in any order. In some embodiments, the nucleotide sequences encoding the TCRα chain and the nucleotide sequences encoding the TCRβ chain are linked in any order by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A).

[0149] In some embodiments, the vector contains a nucleotide sequence encoding the fusion protein as described above.

[0150] In some embodiments, the vector comprises one or more nucleotide sequences encoding one or more polypeptide chains of the fusion protein. In some embodiments, one or more nucleotide sequences encoding one or more polypeptide chains of the fusion protein are present on different vectors. In some embodiments, one or more nucleotide sequences encoding one or more polypeptide chains of the fusion protein are present on the same vector in any order; in some embodiments, one or more nucleotide sequences encoding one or more polypeptide chains of the fusion protein are linked in any order by nucleotide sequences encoding self-cleaving peptides (e.g., P2A, E2A, F2A, or T2A).

[0151] In one aspect, the present invention provides a host cell comprising the nucleic acid molecules or vectors disclosed herein. Such host cells include, but are not limited to, prokaryotic cells such as *Escherichia coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). In some embodiments, the host cell is a CHO cell or a 293 cell.

[0152] In another aspect, the present invention also provides a method for preparing the TCR or a fusion protein containing the TCR of the present invention, comprising culturing the host cells disclosed herein under conditions that allow protein expression, and recovering the TCR or the fusion protein containing the TCR from the cultured host cell culture.

[0153] TCR-based therapies

[0154] The TCR or fusion protein containing the present invention can be used in immunotherapy to kill tumors containing KRASG12V mutations, HRAS G12V mutations, and / or NRAS G12V mutations. Therefore, the present invention provides a pharmaceutical composition comprising: the TCR disclosed herein or an antigen-binding fragment thereof, a conjugate comprising the TCR or an antigen-binding fragment thereof, or a fusion protein comprising the TCR or an antigen-binding fragment thereof, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or an antigen-binding fragment thereof, or a conjugate or fusion protein thereof; and a pharmaceutically acceptable carrier and / or excipient.

[0155] In some embodiments, the pharmaceutical composition further comprises additional therapeutic agents, such as antitumor agents or immune enhancers. In some embodiments, the antitumor agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers (e.g., gemcitabine, 5-fluorouracil, taxane, cisplatin, etc.), antiangiogenic agents, cytokines (e.g., GM-CSF, IL-7, IL-12, IL-15, IL-18, IL-21, etc.), specific tumor cell-targeting antibodies (e.g., CD20 antibodies such as rituximab, Her2 antibodies such as trastuzumab, VEGF antibodies such as bevacizumab, EGFR antibodies such as cetuximab, etc.), and immune checkpoint inhibitors (e.g., PD-1 antibodies, PD-L1 antibodies, CTLA-4 antibodies, LAG-3 antibodies, or TIM3 antibodies).

[0156] In some embodiments, the immune enhancer is selected from immunostimulatory antibodies (e.g., anti-CD3 antibody, anti-CD28 antibody, anti-CD40L (CD154) antibody, anti-41BB (CD137) antibody, anti-OX40 antibody, anti-GITR antibody, or any combination thereof) or immunostimulatory cytokines (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFNγ, IL-10, TGF-β, GM-CSF, or any combination thereof).

[0157] In some embodiments, the TCR of the present invention or its antigen-binding fragment, conjugate or fusion protein, along with the additional therapeutic agent, may be provided as separate components or as mixed components in the pharmaceutical composition.

[0158] In another aspect, the present invention provides methods for inducing an immune response in a subject against tumors with RAS G12V mutations (e.g., KRAS G12V, HRAS G12V, and / or NRAS G12V mutations), and / or for preventing or treating tumors with RAS G12V mutations (e.g., KRAS G12V, HRAS G12V, and / or NRAS G12V mutations) in a subject, the methods comprising administering to a subject in need an effective amount of the TCR or antigen-binding fragment thereof disclosed herein, a conjugate comprising the TCR or antigen-binding fragment thereof, or a fusion protein comprising the TCR or antigen-binding fragment thereof, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or conjugate or fusion protein thereof, or the pharmaceutical composition thereof.

[0159] In some implementations, tumors with KRAS G12V mutations are selected from pancreatic cancer, pancreatic ductal adenocarcinoma, appendiceal adenocarcinoma, small intestinal adenocarcinoma, non-small cell lung cancer, colorectal cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, endometrial cancer, gastric cancer, breast cancer, prostate cancer, and tumors of unknown primary origin. In some implementations, tumors with HRAS G12V mutations may be selected from bladder cancer, thyroid cancer (e.g., follicular thyroid carcinoma), and oral cancer (e.g., oral squamous cell carcinoma).

[0160] In some embodiments, the subject is a mammal, such as a human. In some embodiments, the subject is HLA-DPA1*02:01 positive, HLA-DPA1*02:02 positive, or HLA-DPA1*01:03 positive, and / or HLA-DPB1*03:01 positive, HLA-DPB1*14:01 positive, or HLA-DPB1*104:01 positive.

[0161] In some embodiments, the TCR or its antigen-binding fragment disclosed herein, a conjugate comprising the TCR or its antigen-binding fragment, or a fusion protein comprising the TCR or its antigen-binding fragment, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or its antigen-binding fragment or conjugate or fusion protein, or the pharmaceutical composition may be administered in combination with other therapeutic agents (e.g., immune enhancers or antitumor agents). Therefore, in some embodiments, the method further includes administering other therapeutic agents (e.g., immune enhancers or antitumor agents) to the subject, such as simultaneously, separately, or sequentially.

[0162] In some embodiments, the TCR or its antigen-binding fragment disclosed herein, a conjugate comprising the TCR or its antigen-binding fragment, or a fusion protein comprising the TCR or its antigen-binding fragment, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or its antigen-binding fragment, conjugate, or fusion protein, or the pharmaceutical composition thereof, may be administered in combination with additional therapies, such as concurrently, separately, or sequentially. Such additional therapies may be any therapy known for use oncology, such as surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

[0163] The TCR or its antigen-binding fragment, conjugate, fusion protein, nucleic acid molecule or carrier or host cell comprising a nucleotide sequence encoding said TCR or its antigen-binding fragment or conjugate or fusion protein, or pharmaceutical compositions comprising them, of the present invention can be formulated into any dosage form known in the medical field, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalers, sprays, etc. Preferred dosage forms depend on the intended route of administration and therapeutic use. The pharmaceuticals of the present invention should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections may be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating an appropriate dose of the TCR of the present invention or its antigen-binding fragments, conjugates, fusion proteins, or pharmaceutical compositions comprising them into a suitable solvent, and optionally, by incorporating other desired components (including, but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by sterile filtration. Alternatively, sterile injectable solutions can be prepared as sterile lyophilized powders (e.g., by vacuum drying or freeze-drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), surfactant-containing solution (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0164] Therefore, in some exemplary embodiments, the pharmaceutical composition comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution). In some exemplary embodiments, such a sterile injectable liquid is selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0165] The TCR or its antigen-binding fragments, conjugates, fusion proteins, nucleic acid molecules or carriers or host cells comprising a nucleotide sequence encoding the TCR or its antigen-binding fragments, conjugates or fusion proteins, or pharmaceutical compositions comprising them of the present invention may be administered by any suitable method known in the art, including but not limited to oral, oral, sublingual, ocular, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum groove, groin, bladder, topical (e.g., powder, ointment or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration is parenteral administration (e.g., intravenous injection or bolus, subcutaneous injection, intraperitoneal injection, intramuscular injection). Those skilled in the art will understand that the route of administration and / or method will vary depending on the intended purpose. In some embodiments, the TCR or its antigen-binding fragments, conjugates, fusion proteins, nucleic acid molecules or carriers or host cells comprising a nucleotide sequence encoding the TCR or its antigen-binding fragments, conjugates or fusion proteins, or pharmaceutical compositions comprising them of the present invention are administered by intravenous injection or bolus.

[0166] The pharmaceutical composition may comprise a “therapeutic effective amount” or a “preventative effective amount” of the present invention’s TCR or its antigen-binding fragment, conjugate, fusion protein, nucleic acid molecule or vector comprising a nucleotide sequence encoding the TCR or its antigen-binding fragment or conjugate or fusion protein, or a host cell. In this context, a “therapeutic effective amount” is an amount capable of generating an immune response in a treated subject that reduces or inhibits the proliferation of tumor cells and / or eliminates tumor cells. A “preventative effective amount” refers to an amount capable of generating an immune response in a treated subject against target cells (e.g., tumor cells containing RAS mutations, such as KRAS, HRAS, or NRAS mutations) that prevents tumor formation in the subject or substantially reduces the subject’s chance of developing or continuing to develop tumors.

[0167] In some embodiments, the administration of a therapeutically effective amount of the TCR or its antigen-binding fragment disclosed herein, a conjugate comprising the TCR or its antigen-binding fragment, or a fusion protein comprising the TCR or its antigen-binding fragment, a nucleic acid molecule or carrier or host cell comprising a nucleotide sequence encoding the TCR or its antigen-binding fragment or conjugate or fusion protein, or the pharmaceutical composition to a subject may result in at least a portion of the tumor in the subject regressing or alleviating, and / or reducing or inhibiting the proliferation of tumor cells, or reducing the tumor volume. In some embodiments, the therapeutically effective amount of the TCR or its antigen-binding fragment disclosed herein, or a fusion protein comprising the TCR or its antigen-binding fragment, may be from about 100 mg / kg to about 0.01 mg / kg, for example, from about 100 mg / kg to about 50 mg / kg, from about 50 mg / kg to about 20 mg / kg, from about 20 mg / kg to about 10 mg / kg, from about 10 mg / kg to about 5 mg / kg, from about 5 mg / kg to about 1 mg / kg, from about 1 mg / kg to about 0.1 mg / kg, or from about 0.1 mg / kg to about 0.01 mg / kg. In some embodiments, the therapeutically effective amount of the TCR or its antigen-binding fragment disclosed herein, or a fusion protein comprising the TCR or its antigen-binding fragment, may be about 10 mg / kg, about 9 mg / kg, about 8 mg / kg, about 7 mg / kg, about 6 mg / kg, about 5 mg / kg, about 4 mg / kg, about 3 mg / kg, about 2 mg / kg, about 1 mg / kg, about 0.9 mg / kg, about 0.8 mg / kg, or about 0.7 mg / kg. g / kg, about 0.6 mg / kg, about 0.5 mg / kg, about 0.4 mg / kg, about 0.3 mg / kg, about 0.2 mg / kg, about 0.1 mg / kg, about 0.09 mg / kg, about 0.08 mg / kg, about 0.07 mg / kg, about 0.06 mg / kg, about 0.05 mg / kg, about 0.04 mg / kg, about 0.03 mg / kg, about 0.02 mg / kg, or about 0.01 mg / kg.

[0168] In some aspects, the present invention relates to the TCR or antigen-binding fragment thereof disclosed herein, conjugates comprising the TCR or antigen-binding fragment thereof, or fusion proteins comprising the TCR or antigen-binding fragment thereof, nucleic acid molecules or vectors or host cells comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or conjugates or fusion proteins, or the pharmaceutical compositions thereof, for inducing an immune response in a subject against tumors having RAS G12V mutations (e.g., KRAS G12V, HRAS G12V and / or NRAS G12V mutations), and / or for preventing or treating tumors having RAS G12V mutations (e.g., KRAS G12V, HRAS G12V and / or NRAS G12V mutations) in a subject.

[0169] In another aspect, the present invention provides the use of the TCR or antigen-binding fragment thereof disclosed herein, conjugates comprising the TCR or antigen-binding fragment thereof, or fusion proteins comprising the TCR or antigen-binding fragment thereof, nucleic acid molecules or vectors or host cells comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or conjugates or fusion proteins, or the pharmaceutical compositions thereof, in the preparation of a medicament for inducing an immune response in a subject against tumors having RASG12V mutations (e.g., KRAS G12V, HRAS G12V and / or NRAS G12V mutations), and / or for the prevention or treatment in a subject of tumors having RASG12V mutations (e.g., KRAS G12V, HRAS G12V and / or NRAS G12V mutations).

[0170] In another aspect, the present invention provides methods for inducing an immune response in a subject against tumors with RAS G12V mutations (e.g., KRAS G12V, HRAS G12V, and / or NRAS G12V mutations), and / or for preventing or treating tumors with RAS G12V mutations (e.g., KRAS G12V, HRAS G12V, and / or NRAS G12V mutations) in a subject, comprising administering to the subject a therapeutically effective amount of the TCR or antigen-binding fragment thereof disclosed herein, a conjugate comprising the TCR or antigen-binding fragment thereof, or a fusion protein comprising the TCR or antigen-binding fragment thereof, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or a conjugate or fusion protein thereof, or the pharmaceutical composition thereof. Beneficial effects of the present invention

[0171] This invention provides a soluble, high-affinity TCR fusion protein molecular structure and a production method thereof;

[0172] This invention provides a soluble, high-affinity TCR and TCR-CD3 fusion protein drug that specifically targets RAS G12V mutations (e.g., KRAS G12V, HRAS G12V, and / or NRASG12V mutations) and HLA-DP (DPB1*0301, DPB1*1401, and DPB1*10401) complexes, exhibiting high specificity, developability, good half-life, and in vivo antitumor activity.

[0173] The soluble high-affinity TCR and TCR-CD3 drugs provided by this invention can offer a new approach for the treatment of tumors with RAS G12V mutations (e.g., KRASG12V, HRAS G12V and / or NRAS G12V mutations).

[0174] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanatory of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope of protection intended by the present invention.

[0175] Example

[0176] Example 1. Obtaining high-affinity TCR mutants by phage display

[0177] The generated disulfide-linked TCR (dsTCR) mutant library was electroporated into TG1 *E. coli* and packaged into a phage library displaying dsTCR mutants according to well-known standard procedures. Positive panning was performed using biotinylated pMHCDPG12V (pMHC-biotin) coupled with Strepavidin-Dynabeads (SA-beads) as the antigen, with the concentration decreasing sequentially in each round of panning (e.g., 1 nM, 0.1 nM, 0.01 nM, 0.001 nM), and the number of washes and washing time increasing. The panned clones were then screened by ELISA and sequenced to obtain the pMHC-enriched dsTCR mutant sequences, as shown below.

[0178] Table A. CDR sequences of TCR

[0179] Example 2. Generation of monovalent dsTCR-CD3 fusion protein and pMHC binding assay

[0180] The sequences of the TCR clones (TRA and TRB strands) obtained from phage screening and sequencing are then processed as follows: Figure 1The TCR-CD3 fusion protein structure shown was inserted into a eukaryotic vector for transient recombinant expression (e.g., CHOK1 cell line). The CHO culture supernatant from days 9-10 of transient transformation was purified using a two-step chromatography method to remove TCR-CD3 and multimers (Protein A affinity chromatography and cation exchange chromatography). The sequences of the resulting dsTCR mutant fusion protein with CD3 are shown in Table 1.

[0181] First, the binding affinity between the TCR-CD3 fusion protein and pMHC was detected using SPR. pMHC-biotin was coupled to a Strepavidin chip, and the KD (kinetic density) of the TCR-CD3 molecule was determined using a Biacore 8K analyzer (concentration range 100 nM, 2-fold dilution, 5 concentrations). TCR-CD3 molecules were injected at 25 μl / min, with a binding time of 420 s and a dissociation time of 580 s. When the dissociation of TCR-CD3 molecules exceeded 10%, the next concentration determination was performed. The calculated KD results are as follows:

[0182] The above results indicate that three TCR-CD3 molecules (b246, b248, and b249) have a binding KD of less than 1 nM with pMHC, among which b248 exhibits the strongest binding affinity to pMHC. Since WT TCRs have extremely weak affinity for pMHC, molecule b215 was used as a control in this example and the examples below.

[0183] The ability of the generated TCR-CD3 fusion protein to bind to pMHC was then determined by ELISA, and the results are shown in Table 2.

[0184] Table 1. Sequence of TCR-CD3 fusion protein

[0185] Table 2. ELISA results showing the binding of TCR-CD3 fusion protein to pMHCWT and pMHCG12V.

[0186] The selectivity factor is the ratio of the EC50 of pMHCWT binding to the EC50 of pMHCG12V binding. ELISA binding assays showed that the above molecules can selectively bind to pMHCG12V, with the EC50 range of 20-100 pM for TCR-CD3 fusion proteins containing the mutant dsTCR (except b215) binding to pMHCG12V.

[0187] Example 3. Jurkat NFAT-luc reporter gene assay to detect TCR-CD3 biological activity

[0188] CFPAC-DP223 cells (overexpressing HLA-DPA1*0202 / DPB1*0301) were adjusted to a density of 4×10⁻⁶. 5 At a concentration of 50 μl / ml, 96-well plates were seeded with pG12V peptide (SEQ ID NO: 1) to a concentration of 1 μg / ml. Jurkat NFAT-luc reporter cells were added at a 1:1 ratio, followed by serially diluted TCR-CD3 molecules (final concentrations of 10000, 1000, 100, 10, 1, and 0.1 pM). After incubating the system overnight, 100 μl of ONE-GLO was added to each well, and the fluorescence signal intensity was read to calculate the biological activity of TCR-CD3 molecules. The activation activity of luciferase in Jurkat NFAT-luc cells by TCR-CD3 molecules was directly proportional to the chemiluminescence reading (NFAT-luc). The results are shown in Table 3 and [Table data missing]. Figure 2 As shown.

[0189] Table 3. Results of TCR-CD3 molecule activation of luciferase activity in Jurkat NFAT-luc cells

[0190] The results of reporter gene assays show that the TCR-CD3 fusion protein of the present invention can stimulate T cell activation, with an EC50 ranging from 48 to 122 pM, and has an enhanced ability to activate T cells compared to the control b215.

[0191] Example 4. IFNγ induction assay of TCR-CD3 biological activity

[0192] The density of CFPAC-DP223 cells (overexpressing HLA-DPA1*0202 / DPB1*0301) was adjusted to 2×10⁻⁶. 5 At a concentration of 100 μl / well, 100 μl of pWT (SEQ ID NO: 2) or pG12V peptide was seeded into each well of a 96-well plate, and the concentration was increased to 10 μg / ml. Human PBMC cells were added at a 1:5 ratio, and serially diluted TCR-CD3 (final concentrations of 10000, 1000, 100, 10, 1, and 0.1 pM) were added. The cells were co-cultured for 24 hours, and the release of specific IFNγ in the culture supernatant was measured. The results are shown in Table 4 and [Table data missing]. Figure 3 As shown.

[0193] Table 4. Results of TCR-CD3 induced IFNγ assay

[0194] The above results indicate that the TCR-CD3 molecule of the present invention significantly enhances the ability of human PBMCs to secrete IFNγ compared to b215, with an efficacy increase of 20-200 times.

[0195] Example 5. Detection of off-target cross-reactivity of TCR-CD3

[0196] The density of CAPAN1-DP223 cells (overexpressing HLA-DPA1*0202 / DPB1*0301) was adjusted to 2×10⁻⁶. 5 At a concentration of 100 μl / ml, 100 μl of TCR-CD3 molecules were seeded into each well of a 96-well plate. pWT, pG12V, and 27 sequence-similar peptides were added to a concentration of 10 μg / ml. Human PBMC cells were then added at a 1:5 ratio, followed by 1 nM TCR-CD3 molecules. The cells were co-cultured for 48 hours. The release of specific IFNγ in the culture supernatant (IFNγ MFI value) was detected using a CBA IFNγ Flexset (BD Bioscience) to determine potential off-target reactions of TCR-CD3 molecules. The results of IFNγ release detection are shown in Table 5.

[0197] Table 5. Off-target assay results of TCR-CD3 molecules (MFI by flow cytometry)

[0198] The above results indicate that, after affinity maturation, the TCR-CD3 molecule, at a peptide concentration of 10 μg / ml, cross-recognizes potential genes (defined as ≥3 times the PBMC background value), which are underlined. Further concentration gradient titration of the potentially binding peptides showed that the TCR-CD3 molecule significantly binds only at a peptide concentration of 10 μg / ml; below 10 μg / ml, the TCR-CD3 molecule binds only to KRASG12V. Therefore, the TCR-CD3 molecule of this invention exhibits excellent binding specificity to KRASG12V and does not bind to similar peptides under normal physiological conditions.

[0199] Example 6. Detection of the tumor cell killing ability of TCR-CD3

[0200] The densities of non-target cells CFPAC-DP225-Luc (overexpressing HLA-DPA1*0202 / DPB1*0501) and target cells CFPAC-DP223-Luc (overexpressing HLA-DPA1*0202 / DPB1*0301) were adjusted to 2×10⁻⁶. 5At a concentration of 100 μl / well, pG12V peptide was seeded into 96-well plates, and PBMC cells were added at an effector-to-target ratio of E:T = 10:1. Serially diluted TCR-CD3 (final concentrations of 10000, 1000, 100, 10, 1, and 0.1 pM) were added, and the cells were co-cultured for 72 hours. The killing effect on tumor cells was measured by adding ONE-Glo. Results are shown in Table 6. Figure 4 As shown.

[0201] Table 6. Results of TCR-CD3 molecule-specific killing assay

[0202] The above results indicate that even at a pG12V concentration of 10 μg / ml, TCR-CD3 maintains selective killing of target cells; at in vitro TCR-CD3 concentrations ≤100 pM, optimal selectivity is maintained, with a difference of 10 μg / ml from the effective concentration (EC50). 2 -10 4 Order of magnitude.

[0203] Example 7. Detection of TCR-CD3 molecule-specific IFN-γ induction activity

[0204] Two negative control cell lines, A549-DP223 (KRASG12S, overexpressing HLA-DPA1*02:02 / DPB1*03:01) and CFPAC1-DP225 (KRASG12V, overexpressing HLA-DPA1*02:02 / DPB1*05:01), and two target cell lines, CFPAC1-DP223 (KRASG12V, overexpressing HLA-DPA1*02:02 / DPB1*03:01) and YAPC-DP223 (KRASG12V, overexpressing HLA-DPA1*02:02 / DPB1*03:01), were co-cultured with human PBMC cells at an E:T ratio of 2:1 in IFN-γ. ELISPOT culture plates (MABTECH) were incubated with different concentrations of b248 molecules (25000, 5000, 1000, 200, 40, 8, 1.6 and 0 pM) for 24 hours, and the release of specific IFN-γ was detected by ELISPOT.

[0205] like Figure 5 As shown, the b248 molecule can specifically mediate the recognition of tumor cells by human PBMC cells that simultaneously express KRASG12V and target HLA (e.g., HLA-DPA1*02:02 / DPB1*03:01) and induce the release of IFN-γ.

[0206] Example 8. Detection of the effect of HLA-DP combination on the molecular biological activity of TCR-CD3

[0207] To confirm the effect of KRASG12V presented by various HLA-DP combinations on the biological activity of TCR-CD3 molecules (b248) (e.g., IFN-γ induction activity), YAPC cells were overexpressed with HLA-DP combinations to generate the following cell types (Table 7) for assay.

[0208] Table 7. Effects of YAPC cells expressing multiple HLA-DPs on TCR-CD3 biological activity

[0209] The above cells and negative control cells YAPC-DP225 were used to detect TCR-CD3-induced specific IFN-γ release according to the assay method in Example 7. The results are as follows: Figure 6 As shown, TCR-CD3 molecules can specifically recognize YAPC cells (KRASG12V+ cells) expressing HLA-DPB1*03:01 and DPB1*14:01 and induce the release of IFN-γ; the type of HLA-DPA1 allele has no significant effect on the biological activity of TCR-CD3 molecules.

[0210] Example 9. Detection of the tumor-killing activity of TCR-CD3 molecules

[0211] To determine TCR-CD3-mediated tumor-killing activity, target HLAs (e.g., HLA-DPB1*03:01 and DPB1*14:01) and Firefly Luciferase (Table 8) were overexpressed in tumor cells naturally expressing KRASG12V or HRASG12V as target tumor cells for TCR-CD3-mediated tumor killing. Human PBMC cells were cultured with the above cells at an E:T ratio of 10:1 for 72 hours, and TCR-CD3-mediated tumor killing was determined using the ONE-Glo Luciferase Assay (Promega).

[0212] Table 8. Tumor cell lines that are KRASG12V / HRASG12V positive and express the target HLA

[0213] Figure 7 The results show that TCR-CD3 molecules can recognize endogenously presented RASG12V and induce human PBMC cells to kill tumor cells with KRASG12V / HRASG12V and expressing the target HLA type.

[0214] Example 10. Detection of the in vivo antitumor effect of TCR-CD3 molecules

[0215] With 1×10 7 CFPAC1-DP223 and YAPC-DP223 tumor cells were subcutaneously inoculated into NPG mice (NOD.Cg-Prkdcscid Il2rgtm1 / Vst) at a dose of 1 cell per mouse. Inoculation continued until the tumor volume reached 50-100 mm². 3 At that time, human PBMC cells (5×10⁻⁶) were administered via tail vein. 6 Two hours later, TCR-CD3 molecules were administered via tail vein to control cells (PBS), control cells (TCE) (using ES516b1 as the control TCR-CD3 molecule, whose TCR domain specifically recognizes the HLA-B58-restricted TP53R282W mutation), and three doses of TCR-CD3 molecules (b248: 1 mg / kg, 0.1 mg / kg, and 0.01 mg / kg). The dosing frequency was Q3d (administered every 3 days) for 5 consecutive times. The in vivo antitumor effect of TCR-CD3 molecules was determined by detecting changes in tumor volume.

[0216] The results are as follows Figure 8 As shown, all three doses of TCR-CD3 could induce tumor growth inhibition or tumor remission in human PBMC cells, with the lowest effective dose being 0.01 mg / kg. In both animal models, all animals tolerated the treatment well, and no significant changes in body weight were observed.

[0217] Sequence Summary

[0218] Information on some of the sequences involved in this invention is provided in the table below. The first strand contains Vα-Cα-Fc-Fc, and the second strand contains scFv-Vβ-Cβ.

[0219] Those skilled in the art will further recognize that the invention can be embodied in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the invention discloses only exemplary embodiments thereunder, it should be understood that other variations are considered to be within the scope of the invention. Therefore, the invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims to indicate the scope and content of the invention.

Claims

1. An isolated T-cell receptor (TCR) or its antigen-binding fragment thereof, capable of specifically recognizing a RAS protein mutant with G12V substitution, said TCR or its antigen-binding fragment comprising an α-chain variable region (Vα) and a β-chain variable region (Vβ), wherein: (a) The Vα comprises CDR1α, CDR2α, and CDR3α; and (b) The Vβ includes CDR1β, CDR2β and CDR3β; Preferably, the TCR is a soluble TCR.

2. The TCR or its antigen-binding fragment according to claim 1, wherein: (i) The CDR1α comprises an amino acid sequence having at most two amino acid substitutions, insertions or deletions relative to the sequence shown in SEQ ID NO: 17; (ii) The CDR2α comprises an amino acid sequence having at most two amino acid substitutions, insertions or deletions relative to the sequence shown in any of SEQ ID NO: 18-22; (iii) The CDR3α comprises an amino acid sequence having at most two amino acid substitutions, insertions or deletions relative to the sequence shown in SEQ ID NO:

23.

3. The TCR or its antigen-binding fragment according to claim 1 or 2, wherein: (i) The CDR1β comprises an amino acid sequence having at most two amino acid substitutions, insertions or deletions relative to the sequence shown in SEQ ID NO: 24 or 25; (ii) The CDR2β comprises an amino acid sequence having at most two amino acid substitutions, insertions or deletions relative to the sequence shown in any of SEQ ID NO: 26-28; (iii) The CDR3β comprises an amino acid sequence having at most two amino acid substitutions, insertions or deletions relative to the sequence shown in any of SEQ ID NO: 29-35.

4. The TCR or its antigen-binding fragment according to any one of claims 1-3, wherein: The CDR1α contains the amino acid sequence shown in SEQ ID NO: 17, the CDR2α contains the amino acid sequence shown in any one of SEQ ID NO: 18-22, and the CDR3α contains the amino acid sequence shown in SEQ ID NO:

23.

5. The TCR or its antigen-binding fragment according to any one of claims 1-4, wherein: (i) The CDR1β contains the amino acid sequence shown in SEQ ID NO: 24, the CDR2β contains the amino acid sequence shown in SEQ ID NO: 26, and the CDR3β contains the amino acid sequence shown in any one of SEQ ID NO: 29-35; (ii) The CDR1β comprises the amino acid sequence shown in SEQ ID NO: 25, the CDR2β comprises the amino acid sequence shown in SEQ ID NO: 27, and the CDR3β comprises the amino acid sequence shown in SEQ ID NO: 29; or (iii) The CDR1β contains the amino acid sequence shown in SEQ ID NO: 24, the CDR2β contains the amino acid sequence shown in SEQ ID NO: 28, and the CDR3β contains the amino acid sequence shown in SEQ ID NO:

29.

6. The TCR or its antigen-binding fragment according to any one of claims 1-5, wherein: (a) The Vα comprises a sequence having at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with any of the sequences shown in SEQ ID NO: 36-40; and / or (b) The Vβ comprises a sequence having at least 85%, at least 90%, at least 95%, at least 99%, or 100% sequence identity with any of the sequences shown in SEQ ID NO: 41-49.

7. The TCR or its antigen-binding fragment according to any one of claims 1-6, wherein: (a) The Vα comprises the sequence shown in any one of SEQ ID NO: 36-40, and the Vβ comprises the sequence shown in SEQ ID NO: 41; or (b) The Vα comprises the sequence shown in SEQ ID NO: 36, and the Vβ comprises the sequence shown in any one of SEQ ID NO: 42-49.

8. The TCR or its antigen-binding fragment according to any one of claims 1-7, wherein the TCR or its antigen-binding fragment is capable of specifically recognizing the RAS G12V antigen peptide or a pMHC complex containing the antigen peptide; Preferably, the antigenic peptide is presented by MHC-II molecules; Preferably, the MHC-II molecule is HLA-DP; Preferably, the HLA-DP is selected from the group consisting of HLA-DPA1*02:01, HLA-DPA1*02:02, HLA-DPA1*01:03, HLA-DPB1*03:01, HLA-DPB1*14:01, and HLA-DPB1*104:01, and more preferably HLA-DPB1*03:

01.

9. The TCR of claim 8 or its antigen-binding fragment thereof, wherein the RAS G12V antigenic peptide is a KRAS G12V antigenic peptide, an HRAS G12V antigenic peptide and / or an NRAS G12V antigenic peptide, for example, the RAS G12V antigenic peptide comprises the sequence shown in SEQ ID NO:

1.

10. The TCR or its antigen fragment according to any one of claims 1-9, further comprising an α-chain constant region (Cα) and a β-chain constant region (Cβ). Optionally, the Cα and the Cβ interact via interchain disulfide bonds; Optionally, the Cα has a sequence as shown in SEQ ID NO: 50, and the Cβ has a sequence as shown in SEQ ID NO:

51.

11. The TCR or its antigen-binding fragment according to any one of claims 1-10, wherein: (a) The α chain comprises the amino acid sequence shown in any one of SEQ ID NO: 54-58; and / or (b) The β chain comprises the amino acid sequence shown in any one of SEQ ID NO: 59-65 and 92-93.

12. The TCR or its antigen-binding fragment according to claim 11, wherein: (a) The α chain comprises the amino acid sequence shown in any one of SEQ ID NO: 54-58, and the β chain comprises the amino acid sequence shown in SEQ ID NO: 59; or (b) The α chain comprises the amino acid sequence shown in SEQ ID NO: 54, and the β chain comprises the amino acid sequence shown in any one of SEQ ID NO: 60-65 and 92-93.

13. A fusion protein comprising any one of claims 1-12, a TCR or its antigen-binding fragment thereof, and an immunoglobulin Fc moiety; in, The C-terminus of the α-chain and / or β-chain of the TCR or its antigen-binding fragment is operatively linked to the Fc portion of the immunoglobulin, the Fc portion of which includes a first Fc domain (Fc1) and a second Fc domain (Fc2). Optionally, the first Fc structure domain and the second Fc structure domain are operatively connected.

14. The fusion protein of claim 13, wherein the immunoglobulin Fc domain is a human immunoglobulin Fc domain or a variant thereof, such as the Fc domain of human IgG1, IgG2, IgG3 or IgG4 or a variant thereof. Preferably, the immunoglobulin Fc domain is the Fc domain of human IgG1 or a variant thereof.

15. The fusion protein of claim 13 or 14, further comprising an effector domain operatively connected to the N-terminus of the α-chain variable region (Vα) and / or the β-chain variable region (Vβ); Optionally, the effector domain is a peptide or protein capable of activating effector cells, wherein the effector cells are selected from T lymphocytes, NK cells, dendritic cells, and macrophages; Preferably, the effector domain is an antibody or its antigen-binding portion, such as an anti-CD3 antibody or its antigen-binding portion, such as CD3 scFv; Preferably, the CD3 scFv has an amino acid sequence as shown in SEQ ID NO:

52.

16. The fusion protein of any one of claims 13-15, comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the following domains operably linked: Vα-Cα-Fc1-Fc2, and the second polypeptide chain comprises the following domains operably linked: effector domain -Vβ-Cβ. Preferably, there is a non-natural interchain disulfide bond between Cβ and Cα; Preferably, the operable connection is a direct connection or a connection via a peptide linker.

17. The fusion protein of claim 16, wherein Fc1 comprises the sequence shown in SEQ ID NO: 94, and Fc2 comprises the sequence shown in SEQ ID NO: 95, or, Fc1 comprises the sequence shown in SEQ ID NO: 95, and Fc2 comprises the sequence shown in SEQ ID NO: 94; Preferably, Fc1 and Fc2 are connected by a peptide linker; Preferably, one or more non-natural interchain disulfide bonds exist between Fc1 and Fc2; Preferably, Fc1-Fc2 comprises the sequence shown in SEQ ID NO:

53.

18. The fusion protein of claim 16 or 17, wherein: The first polypeptide chain comprises an amino acid sequence having at least 95%, at least 99%, or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 3-7, and / or The second polypeptide chain comprises an amino acid sequence having at least 95%, at least 99%, or 100% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 8-16.

19. The fusion protein according to any one of claims 16-18, wherein: (a) The first polypeptide chain comprises an amino acid sequence as shown in any one of SEQ ID NO: 3-7, and the second polypeptide chain comprises an amino acid sequence as shown in SEQ ID NO: 8; or (b) The first polypeptide chain contains an amino acid sequence as shown in SEQ ID NO: 3, and the second polypeptide chain contains an amino acid sequence as shown in any one of SEQ ID NO: 9-16.

20. The fusion protein of any one of claims 13-19, which is capable of specifically recognizing the RAS G12V antigenic peptide or a pMHC complex containing said antigenic peptide; Preferably, the antigenic peptide is presented by MHC-II molecules; Preferably, the MHC-II molecule is HLA-DP; Preferably, the HLA-DP is selected from the group consisting of HLA-DPA1*02:01, HLA-DPA1*02:02, HLA-DPA1*01:03, HLA-DPB1*03:01, HLA-DPB1*14:01, and HLA-DPB1*104:01, and more preferably HLA-DPB1*03:

01.

21. The fusion protein of claim 20, wherein the RAS G12V antigenic peptide is a KRAS G12V antigenic peptide, a HRASG12V antigenic peptide, and / or a NRAS G12V antigenic peptide, for example, the RAS G12V antigenic peptide comprises the sequence shown in SEQ ID NO:

1.

22. The fusion protein of claim 20 or 21, wherein the EC50 of the binding of the antigenic peptide or the pMHC complex containing the antigenic peptide is about 1 nM or less, about 100 pM or less, about 90 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less, or about 20 pM or less.

23. A conjugate comprising a TCR or an antigen-binding fragment thereof as described in any one of claims 1-12 or a fusion protein as described in any one of claims 13-22 conjugated to an effector moiety, said effector moiety being selected from cytotoxic agents, nucleic acids (e.g., RNA) or radionuclides; Preferably, the cytotoxic agent is selected from alkylating agents, microtubule inhibitors, anticancer antibiotics, or antimetabolites.

24. A nucleic acid molecule comprising a nucleotide sequence encoding a TCR or an antigen-binding fragment thereof as described in any one of claims 1-12, or comprising a nucleotide sequence encoding a fusion protein as described in any one of claims 13-22.

25. A carrier comprising the nucleic acid molecule of claim 24.

26. A host cell comprising the nucleic acid molecule of claim 24 or the vector of claim 25; Preferably, the host cell is a eukaryotic cell, such as a CHO cell or a 293 cell line.

27. A method for preparing the TCR or its antigen-binding fragment according to any one of claims 1-12 or the fusion protein according to any one of claims 13-22, comprising: The host cells of claim 26 are cultured under conditions that allow protein expression, and the TCR or its antigen-binding fragment or fusion protein is recovered from the cultured host cell culture.

28. A pharmaceutical composition comprising a TCR or an antigen-binding fragment thereof as claimed in any one of claims 1-12, a fusion protein as claimed in any one of claims 13-22, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or an antigen-binding fragment thereof or the fusion protein, and a pharmaceutically acceptable carrier and / or excipient; Preferably, the pharmaceutical composition further comprises additional therapeutic agents, such as antitumor agents or immune enhancers.

29. The pharmaceutical composition of claim 28, wherein the antitumor agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers, antiangiogenic agents, cytokines, and immune checkpoint inhibitors (e.g., PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG-3 antibody, or TIM3 antibody).

30. The pharmaceutical composition of claim 28, wherein the immune enhancer is selected from immunostimulatory antibodies (e.g., anti-CD3 antibody, anti-CD28 antibody, anti-CD40L (CD154) antibody, anti-41BB (CD137) antibody, anti-OX40 antibody, anti-GITR antibody, or any combination thereof) or immunostimulatory cytokines (e.g., IL-2, IL-3, IL-12, IL-15, IL-18, IFN-γ, IL-10, TGF-β, GM-CSF, or any combination thereof).

31. Use in the preparation of a medicament of any TCR or antigen-binding fragment thereof of any one of claims 1-12, a fusion protein of any one of claims 13-22, a nucleic acid molecule or vector or host cell comprising a nucleotide sequence encoding the TCR or antigen-binding fragment thereof or fusion protein thereof, or a conjugate of claim 23, or a pharmaceutical composition of any one of claims 28-30, wherein the medicament is used in a subject to induce an immune response against tumors having RAS G12V mutations (e.g., KRAS G12V, HRAS G12V and / or NRAS G12V mutations), and / or to prevent or treat tumors having RAS G12V mutations (e.g., KRAS G12V, HRAS G12V and / or NRAS G12V mutations) in a subject; Preferably, the tumor with RAS G12V mutation is selected from pancreatic cancer, non-small cell lung cancer, colon cancer, endometrial cancer, bile duct cancer, ovarian cancer, and bladder cancer; Preferably, the subject is a human being; Preferably, the subject is positive for HLA-DPB1*03:01, HLA-DPB1*14:01, or HLA-DPB1*104:01; Preferably, the TCR or its antigen-binding fragment, fusion protein, nucleic acid molecule or carrier or host cell, or pharmaceutical composition is administered in combination with another therapeutic agent, for example, simultaneously, separately or sequentially; preferably, the other therapeutic agent is an immunostimulant or an antitumor agent.

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