Antigen binding proteins targeting krass mutations and uses thereof

Abstract

The present invention relates to antigen binding proteins comprising a T cell receptor variable region and targeting a KRAS G12V mutant epitope. The present invention also provides fusion proteins, conjugates or compositions comprising the antigen binding proteins, nucleic acid molecules encoding the antigen binding proteins or fusion proteins, vectors comprising the nucleic acid molecules, recombinant cells comprising the nucleic acid molecules or vectors, recombinant cells expressing the antigen binding proteins or fusion proteins, methods of making the antigen binding proteins, fusion proteins or recombinant cells, and therapeutic uses thereof.

Description

Technical Field

[0001] This invention belongs to the field of immunology, specifically relating to an antigen-binding protein that specifically targets the KRAS G12V mutant epitope and its therapeutic uses. Background Technology

[0002] The development of human tumors is often accompanied by driver mutations. Among them, mutations in the RAS gene family (including KRAS, NRAS, and HRAS) are widespread in various types of solid tumors, especially showing a high frequency in pancreatic cancer, colorectal cancer, and non-small cell lung cancer. KRAS (Kirsten rat sarcoma viral oncogene homolog) mutations account for more than 80% of all RAS mutations, making them one of the most common oncogenic mutations. KRAS mutations typically lead to the persistent activation of its protein products, continuously driving cell proliferation and inhibiting apoptosis, thereby promoting tumor development and progression.

[0003] Among numerous KRAS mutations, KRAS G12V (where glycine at position 12 is replaced by valine) is a clinically significant hotspot mutation, typically associated with poorer prognosis, increased tumor invasiveness, and resistance to conventional therapies such as chemotherapy or EGFR-targeted therapy. Therefore, KRAS G12V mutation is a key molecular target for which effective targeting strategies urgently need to be developed.

[0004] Because KRAS proteins are primarily located intracellularly, they are difficult to target directly using traditional antibodies or small molecule drugs. Therefore, in recent years, researchers have turned to activating specific T cell-mediated immune responses to eliminate tumor cells expressing mutated KRAS. Mutated KRAS fragments can be processed intracellularly and presented on the surface of tumor cells as peptide-major histocompatibility complex (pMHC), where they are recognized by specific T cells. This mechanism provides a theoretical basis for developing T cell receptor (TCR) therapies targeting KRAS mutations.

[0005] Currently, there is a lack of candidate TCR molecules with good affinity and specificity for the KRAS G12V target. Therefore, there is an urgent need to develop TCR molecules that can specifically recognize KRAS G12V mutations and exhibit good anti-tumor activity in the HLA-restricted background, in order to expand the target range of tumor immunotherapy and meet unmet clinical treatment needs. Summary of the Invention

[0006] This invention provides an antigen-binding protein comprising a variable region of a T-cell receptor (TCR) that can specifically recognize and bind with high affinity the VVGAVGVGK (SEQ ID NO: 2)-MHC complex, particularly the VVGAVGVGK (SEQ ID NO: 2)-HLA-A complex. 11 complexes, such as VVGAVGVGK (SEQ ID NO: 2)-HLA-A The 11:01 complex, and the antigen-binding protein can also effectively mediate the cytotoxic effects of immune effector cells on target cells expressing the KRAS G12V mutation. This invention provides a safer and more effective solution for developing immunotherapies against tumors with the KRAS G12V mutation, and has significant clinical value.

[0007] In one aspect, the present invention provides an antigen-binding protein comprising a TCR α-chain variable region and a TCR β-chain variable region, wherein the antigen-binding protein specifically recognizes and binds to the VVGAVGVGK (SEQ ID NO: 2)-MHC complex, wherein the TCR α-chain variable region comprises CDR3 with the amino acid sequence as shown in SEQ ID NO: 5 or a functional variant having one or two amino acid residue changes from the sequence, and the TCR β-chain variable region comprises CDR3 with the amino acid sequence as shown in SEQ ID NO: 8 or a functional variant having one or two amino acid residue changes from the sequence.

[0008] In another aspect, the present invention provides an antigen-binding protein comprising a TCR α-chain variable region and a TCR β-chain variable region, wherein the antigen-binding protein specifically recognizes and binds to the VVGAVGVGK (SEQ ID NO: 2)-MHC complex, wherein the TCR α-chain variable region comprises CDR3 with an amino acid sequence as shown in SEQ ID NO: 11 or a functional variant having one or two amino acid residue changes from the sequence, and the TCR β-chain variable region comprises CDR3 with an amino acid sequence as shown in SEQ ID NO: 14 or a functional variant having one or two amino acid residue changes from the sequence.

[0009] As used herein, the term "antigen-binding protein" refers to any protein or polypeptide capable of specifically recognizing and binding to a target antigen. The antigen-binding proteins described herein include TCR-derived CDRs, particularly proteins or polypeptides comprising at least one TCR α-chain CDR3 (CDR3α) and / or at least one TCR β-chain CDR3 (CDR3β) as defined herein, and capable of specifically targeting the KRASG12V mutant epitope. The antigen-binding proteins described herein may take many different forms as discussed herein. In some embodiments, the antigen-binding proteins described herein further comprise at least one TCR-derived CDR1α, CDR2α, CDR1β, CDR2β, α-chain variable region, β-chain variable region, α-chain and / or β-chain, or combinations thereof, as defined herein, and optionally one or more additional domains optionally fused directly or indirectly to the aforementioned TCR structural fragments. Furthermore, the invention also includes fragments of the antigen-binding protein, meaning portions of the antigen-binding protein that retain the ability to bind to the target antigen.

[0010] In some embodiments, the variable region of the TCR α chain comprises amino acid sequences such as CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, or functional variants having one or two amino acid residue changes from the sequences, and the variable region of the TCR β chain comprises amino acid sequences such as CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, or functional variants having one or two amino acid residue changes from the sequences.

[0011] In other embodiments, the variable region of the TCR α chain comprises amino acid sequences such as CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, or functional variants having one or two amino acid residue changes from the sequences, and the variable region of the TCR β chain comprises amino acid sequences such as CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, or functional variants having one or two amino acid residue changes from the sequences.

[0012] Unless otherwise stated, all CDRs described herein are defined according to the IMGT numbering scheme (Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; imgt.org / IMGTindex / V-QUEST.php). In some embodiments, the CDR3 amino acid sequence described herein may further include one or more junction amino acids, such as an N-terminal cysteine ​​and / or a C-terminal phenylalanine.

[0013] In some embodiments, the VVGAVGVGK (SEQ ID NO: 2)-MHC complex is VVGAVGVGK (SEQ ID NO: 2)-HLA-A. 11 complexes, such as VVGAVGVGK (SEQ ID NO: 2)-HLA-A 11:01 complex.

[0014] The amino acid sequences of the CDRs disclosed herein can be embedded into any suitable framework structure to prepare chimeric TCRs. As long as the framework structure is compatible with the CDRs from which the TCRs described herein are derived, those skilled in the art can design or synthesize TCR molecules with corresponding functions based on the CDRs disclosed herein. Therefore, in some embodiments, the antigen-binding protein described herein can be a TCR molecule comprising the above-described α-chain and / or β-chain CDR sequences and any suitable framework structure.

[0015] In some embodiments, the TCR α chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with said sequence, and / or the TCR β chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 17 or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with said sequence.

[0016] In other embodiments, the TCR α chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 19 or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with said sequence, and / or the TCR β chain variable region comprises an amino acid sequence as shown in SEQ ID NO: 21 or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with said sequence.

[0017] In some embodiments, the antigen-binding protein further comprises a TCR constant region or a fragment thereof.

[0018] The antigen-binding protein disclosed herein can be a heterozygous TCR containing sequences derived from more than one species. For example, studies have shown that TCRs containing a murine TCR constant region are expressed more efficiently in human T cells than TCRs containing a human TCR constant region. Therefore, the TCR constant region described herein can be a human TCR constant region or a murine TCR constant region. In a preferred embodiment, the TCR constant region described herein is a murine TCR constant region. Those skilled in the art will know or can obtain the amino acid sequence of the wild-type human or murine TCR constant region by consulting relevant books or the publicly available database of IMGT (International Immunogenetic Information System).

[0019] In some implementations, the TCR constant region includes a TCR α chain constant region and / or a TCR β chain constant region.

[0020] In some embodiments, the TCR α chain constant region contains an amino acid sequence as shown in SEQ ID NO: 23 or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with said sequence, and / or the TCR β chain constant region contains an amino acid sequence as shown in SEQ ID NO: 25 or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with said sequence.

[0021] To enhance proper pairing of the α and β chains and reduce mismatches with the endogenous TCR, the TCR α chain constant region and / or the TCR β chain constant region may also contain other mutations not present in the natural TCR. A non-limiting example includes the substitution of cysteine ​​residues at position 48 of the TCR α chain constant region and position 57 of the TCR β chain constant region according to the IMGT number to form a disulfide bond between the TCR α chain constant region and the β chain constant domain, thereby promoting proper pairing of the TCR α and β chains.

[0022] In some embodiments, the antigen-binding protein is a T-cell receptor or its antigen-binding fragment.

[0023] The antigen-binding proteins disclosed herein can be used in any TCR structural form. For example, in some embodiments, the TCR α-chain variable region and the TCR β-chain variable region are present in a single polypeptide chain. For example, the antigen-binding protein described herein can be a single-chain TCR (scTCR) comprising a TCR α-chain variable region and a TCR β-chain variable region linked by a peptide linker. Such scTCRs may include TCR α-chain variable regions and TCR β-chain variable regions, each linked to a TCR constant region. Alternatively, scTCRs may also include TCR α-chain variable regions and / or TCR β-chain variable regions not linked to a TCR constant region. Exemplary scTCRs are described in PCT Publications WO 2003 / 020763, WO 2004 / 033685, and WO 2011 / 044186. As described in the aforementioned patent documents, those skilled in the art can readily construct single-chain TCR molecules comprising at least one TCR source, such as CDR1α, CDR2α, CDR1β, CDR2β, α-chain variable region, β-chain variable region, α-chain and / or β-chain, or combinations thereof. It should be noted that the peptide linker can be any peptide chain suitable for linking Vα and Vβ.

[0024] In other embodiments, the TCR α-chain variable region and the TCR β-chain variable region are located in different polypeptide chains. For example, the antigen-binding protein described herein may comprise two polypeptide chains (e.g., an α-chain and a β-chain). In some embodiments, these chains have been engineered to each have cysteine ​​residues capable of forming interchain disulfide bonds. Thus, in some embodiments, the TCR disclosed herein comprises 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.

[0025] In some preferred embodiments, the antigen-binding protein is an α / β heterodimer TCR containing a TCR α chain comprising a variable region of a TCR α chain and a constant region of a TCR α chain, and / or a TCR β chain comprising a variable region of a TCR β chain and a constant region of a TCR β chain. The TCR α chain contains an amino acid sequence as shown in SEQ ID NO: 27 or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 28, and / or the TCR β chain contains an amino acid sequence as shown in SEQ ID NO: 28 or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with the sequence shown in SEQ ID NO: 28.

[0026] In some other preferred embodiments, the antigen-binding protein is an α / β heterodimer TCR containing a TCR α chain comprising a TCR α chain variable region and a TCR α chain constant region, and / or a TCR β chain comprising a TCR β chain variable region and a TCR β chain constant region, wherein the TCR α chain contains an amino acid sequence as shown in SEQ ID NO: 33 or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with said sequence, and / or the TCR β chain contains an amino acid sequence as shown in SEQ ID NO: 34 or an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity with said sequence.

[0027] In some embodiments, the TCR α chain and / or TCR β chain described herein further comprises a signal peptide. For example, a signal peptide sequence is included at the N-terminus of the α chain and / or β chain, preferably at the N-terminus of both the α chain and the β chain.

[0028] In this context, the term "signal peptide" refers to a guide sequence located at the N-terminus of a post-translational nascent protein that guides the nascent protein to a designated expression site. The signal peptide may be removed from the nascent protein during or after localization or secretion. A nascent protein with a signal peptide is referred to herein as a "preprotein," and a nascent protein without the signal peptide is referred to herein as a "mature" protein or polypeptide. In any of the embodiments disclosed herein, the antigen-binding protein or fusion protein may comprise or be a mature protein, or may comprise or be a preprotein.

[0029] Non-limiting examples of signal peptides include the α-chain signal peptide sequence (METLLGVSLVILWLQLARVN) represented by amino acids 1-20 of SEQ ID NO: 29, or the α-chain signal peptide sequence (MWGAFLLYVSMKMGGTA) represented by amino acids 1-17 of SEQ ID NO: 35, or the β-chain signal peptide sequence (MGFRLLCCVAFCLLGAGPV) represented by amino acids 1-19 of SEQ ID NO: 30, or the β-chain signal peptide sequence (MGPQLLGYVVLCLLGAGPL) represented by amino acids 1-19 of SEQ ID NO: 36. Those skilled in the art will know or be able to obtain the amino acid sequences of signal peptides by consulting relevant books or public databases.

[0030] In some embodiments, the antigen-binding protein described herein may be provided in a membrane-bound form. For example, it may be provided as a naturally occurring full-length TCR. A naturally occurring TCR is a membrane protein that is anchored to the cell membrane via its transmembrane region. Therefore, in some embodiments, the antigen-binding protein described herein comprises a full-length TCR α chain and a full-length TCR β chain having an endogenous transmembrane region.

[0031] Just as immunoglobulins (antibodies) serve as antigen recognition molecules, TCRs can also be developed for diagnostic and therapeutic applications, requiring soluble TCR molecules. Therefore, in some embodiments, the antigen-binding proteins described herein can also be provided in a soluble form, for example, as soluble TCRs. Soluble TCR molecules do not include the transmembrane region of the full-length TCR (and optionally, also exclude the cytoplasmic region). Therefore, in some embodiments, the antigen-binding proteins described herein comprise TCR α chains and TCR β chains lacking the transmembrane and / or cytoplasmic regions. Preferably, such antigen-binding proteins may comprise (i) all or part of the TCR α chain except for its transmembrane and / or cytoplasmic region, and / or (ii) all or part of the TCR β chain except for its transmembrane and / or cytoplasmic region, wherein (i) and (ii) both contain variable domains and at least a portion of constant domains of the TCR chain, and optionally the TCR α and TCR β chains are stabilized by disulfide bonds or covalently linked by suitable peptide linkers. It should be noted that the peptide linker in this article can be any peptide chain suitable for linking the TCR α chain and β chain.

[0032] Soluble TCRs have a wide range of applications. They can be used to study the interaction between TCRs and pMHC, as a diagnostic tool for detecting infections, or as biomarkers for autoimmune diseases. Similarly, soluble TCRs can be used to deliver therapeutic agents (such as cytotoxic compounds or immunostimulatory compounds) to cells that present specific antigens. Furthermore, soluble TCRs can be fused with other molecules (such as anti-CD3 antibodies) to redirect immune cells, thereby enabling immune cells to target cells that present specific antigens.

[0033] Therefore, in some embodiments, the antigen-binding protein further includes one or more antigen-binding sites that bind to different antigens or epitopes.

[0034] In some embodiments, the different antigens are immune cell surface antigens. Non-limiting examples of immune cell surface antigens are immune effector cell surface antigens, such as T cell surface antigens, B cell surface antigens, natural killer (NK) cell surface antigens, macrophage surface antigens, etc.

[0035] In some preferred embodiments, the immune cell surface antigen is a T cell surface antigen; more preferably, the T cell surface antigen is CD3.

[0036] In some implementations, the antigen-binding protein is isolated or purified.

[0037] As used herein, the term "isolated or purified" refers to an antigen-binding protein that has been identified, isolated, and / or recovered from its environment of origin, such that the "isolated or purified" antigen-binding protein is free from or substantially free from other contaminant components from its environment of origin that may interfere with its therapeutic or diagnostic use. Contaminant components may include enzymes, hormones, and other proteins or non-protein solutes. Therefore, an "isolated or purified" antigen-binding protein can be prepared by at least one purification step that removes or substantially removes these contaminant components.

[0038] In another aspect, the present invention provides a conjugate comprising the antigen-binding protein described herein and an effector conjugated thereto.

[0039] In this context, the term "effector" 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 is a biologically active compound or peptide (e.g., a compound or peptide that is effective for cells targeted by the antigen-binding protein described herein), a detectable label, or a pharmacokinetic modification.

[0040] 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 with effectors can be carried out chemically or recombinarily, wherein the chemical method involves forming a covalent bond between two molecules to form a single molecule.

[0041] In some embodiments, the effector 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 antigen-binding proteins to enable the therapeutic portion to produce a therapeutic effect on the cells targeted by the antigen-binding protein.

[0042] In some embodiments, the therapeutic component is selected from immune enhancers, such as immunostimulatory cytokines or immunostimulatory antibodies. In some exemplary embodiments, the immunostimulatory cytokines are selected from, for example, IL-2, IL-3, IL-12, IL-15, IL-18, IFN-γ, IL-10, TGF-β, GM-CSF, or any combination thereof. The various cytokines listed refer to polypeptides having the natural biological activity of the cytokine, including, for example, full-length proteins, their active fragments, or functional variants. 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 functional variant. In some exemplary embodiments, the immunostimulatory antibodies are selected from, for example, anti-CD3 antibodies, anti-CD28 antibodies, anti-CD40L (CD154) antibodies, anti-41BB (CD137) antibodies, anti-OX40 antibodies, anti-GITR antibodies, or any combination thereof. In some embodiments, the immune enhancer is selected from anti-CD3 antibodies, anti-CD28 antibodies, IL-2, IL-15, or any combination thereof.

[0043] 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).

[0044] 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.

[0045] In some embodiments, the effector can increase the solubility of the antigen-binding protein. In some embodiments, the effector is selected from various portions of the heavy or light chain constant regions of various subclasses of immunoglobulins (e.g., IgG, IgM, IgA, IgE). In some embodiments, the effector is selected from the constant regions of human immunoglobulins, such as the heavy chain constant region or the light chain constant region.

[0046] In some embodiments, the effector is selected from detectable markers. Detectable markers as described herein 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 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots, or cyanine dye derivatives (e.g., Cy7, Alexa 750)), luminescent substances (e.g., chemiluminescent substances, such as acridinium esters), magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) microbeads, and biotin for binding avidin (e.g., streptavidin) modified with the above-described labels. The detectable labels described above can be detected by methods known in the art. For example, radioactive labels can be detected using photographic film or a scintillation calculator, and fluorescent labels can be detected using a photodetector to detect emitted light. Enzyme labels are generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the enzyme's action on the substrate, and calorimetric labels are detected by simple visual coloring labels. In some exemplary embodiments, the detectable marker is selected from enzymes, radionuclides, fluorescent dyes, luminescent substances (such as chemiluminescent substances), or biotin.

[0047] In some embodiments, the effector is selected from pharmacokinetic modification moieties. These pharmacokinetic modification moieties include, but are not limited to, polyethylene glycol, PAS modification, albumin and albumin-binding domains, and / or immunoglobulin Fc regions. These pharmacokinetic modification moieties can be used to prolong the in vivo half-life of the antigen-binding proteins described herein.

[0048] In some embodiments, the antigen-binding protein described herein is optionally conjugated to an effector via a linker (e.g., a peptide linker or a chemical linker).

[0049] In some embodiments, when the effector is a peptide or protein, the conjugate is preferably a fusion protein.

[0050] Therefore, in another aspect, the present invention provides a fusion protein comprising the antigen-binding protein described herein and a peptide or protein expressed therewith in fusion.

[0051] In some embodiments, the antigen-binding protein described herein is optionally fused to another peptide or protein via a linker (e.g., a peptide linker).

[0052] In some embodiments, the peptide or protein may be selected from various effectors of peptides or proteins as described above, such as therapeutic peptides or proteins, immunoglobulin constant regions (e.g., human immunoglobulin constant regions), detectable protein labels, or protein tags.

[0053] In some implementations, the protein tag is selected from His, Flag, GST, MBP, HA, or Myc, etc., and those skilled in the art know how to select a suitable protein tag according to the desired purpose (e.g., purification, detection, or tracing).

[0054] In other embodiments, the peptide or protein may be an immunoglobulin variable region that can target and bind to immune cell surface antigens to redirect immune cells, thereby enabling immune cells to target cells that present specific antigens.

[0055] In some embodiments, non-limiting examples of the immune cell surface antigens are immune effector cell surface antigens, such as T cell surface antigens, B cell surface antigens, natural killer (NK) cell surface antigens, macrophage surface antigens, etc.

[0056] In some preferred embodiments, the immune cell surface antigen is a T cell surface antigen; more preferably, the T cell surface antigen is CD3.

[0057] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding an antigen-binding protein as described herein, or a nucleotide sequence encoding a fusion protein of the present invention.

[0058] "Nucleic acid molecule" generally refers to a polymer of DNA or RNA, which may be single-stranded or double-stranded, synthetic or derived from natural sources (e.g., isolated and / or purified), which may contain natural, non-natural or modified nucleotides, and which may contain natural, non-natural or modified internucleotide bonds, such as aminophosphate bonds or thiophosphate bonds, rather than phosphodiesters found between nucleotides of unmodified oligonucleotides. Preferably, the nucleic acids described herein are recombinant. As used herein, the term "recombinant" means (i) a molecule constructed outside a living cell by linking a natural or synthetic nucleic acid fragment to a nucleic acid molecule that can replicate in a living cell, or (ii) a molecule produced by replication of those described above (i). For the purposes of this document, replication may be in vitro or in vivo. Nucleic acids can be constructed based on chemical synthesis and / or enzymatic ligation reactions using procedures known in the art or commercially available (e.g., from Genscript, Thermo Fisher, etc.). See, for example, Sambrook et al., nucleic acids can be chemically synthesized using naturally occurring nucleotides or various modified nucleotides designed to increase the biological stability of the molecule or the physical stability of the double strands formed during hybridization (see, for example, Sambrook et al. 2001) (e.g., phosphate thioester derivatives and acridine-substituted nucleotides).

[0059] In some embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the variable region of the TCR α chain described herein and a second nucleotide sequence encoding the variable region of the TCR β chain.

[0060] In a preferred embodiment, the first nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID NO: 16 or its complementary sequence, or a nucleotide sequence or its complementary sequence having at least 90%, at least 95%, or at least 99% sequence identity with the sequence; the second nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID NO: 18 or its complementary sequence, or a nucleotide sequence or its complementary sequence having at least 90%, at least 95%, or at least 99% sequence identity with the sequence.

[0061] In a preferred embodiment, the first nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID NO: 20 or its complementary sequence, or a nucleotide sequence or its complementary sequence having at least 90%, at least 95%, or at least 99% sequence identity with the sequence; the second nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID NO: 22 or its complementary sequence, or a nucleotide sequence or its complementary sequence having at least 90%, at least 95%, or at least 99% sequence identity with the sequence.

[0062] In some embodiments, the isolated nucleic acid molecule comprises a first nucleotide sequence encoding the TCR α chain described herein and a second nucleotide sequence encoding the TCR β chain.

[0063] In a preferred embodiment, the first nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID NO: 31 or its complementary sequence, or a nucleotide sequence or its complementary sequence having at least 90%, at least 95%, or at least 99% sequence identity with the sequence; the second nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID NO: 32 or its complementary sequence, or a nucleotide sequence or its complementary sequence having at least 90%, at least 95%, or at least 99% sequence identity with the sequence.

[0064] In a preferred embodiment, the first nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID NO: 37 or its complementary sequence, or a nucleotide sequence or its complementary sequence having at least 90%, at least 95%, or at least 99% sequence identity with the sequence; the second nucleotide sequence comprises a nucleotide sequence as shown in SEQ ID NO: 38 or its complementary sequence, or a nucleotide sequence or its complementary sequence having at least 90%, at least 95%, or at least 99% sequence identity with the sequence.

[0065] In some implementations, the first nucleotide sequence and the second nucleotide sequence are present in different, separate nucleic acid molecules.

[0066] In some embodiments, the first nucleotide sequence and the second nucleotide sequence exist in the same isolated nucleic acid molecule in any order. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are optionally linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, or T2A). In some embodiments, the self-cleaving peptide is P2A.

[0067] In some embodiments, the nucleotide sequence is a codon-optimized nucleotide sequence for the host cell. Those skilled in the art will understand that different cells utilize specific codons differently, and codons in the sequence can be changed to increase expression levels depending on the cell type. Codon selection tables for mammalian cells and many other organisms are well known to those skilled in the art.

[0068] Those skilled in the art will understand that, due to the degeneracy of the genetic code, different nucleotide sequences can encode the same polypeptide. Therefore, the nucleotide sequence encoding an antigen-binding protein can be the same as the nucleic acid sequences shown in Table 1 of this document or a degenerate variant.

[0069] In another aspect, the present invention provides a vector comprising the nucleic acid molecule of the present invention, wherein the nucleic acid molecule is operatively linked to an expression regulatory sequence.

[0070] A "vector" is any molecule or composition capable of carrying a nucleotide sequence into a suitable host cell, in which the synthesis of the encoded polypeptide can occur. Typically and preferably, a vector is a nucleic acid molecule engineered using recombinant DNA techniques known in the art to incorporate a desired nucleotide sequence (e.g., the nucleic acid molecule of the present invention). In some embodiments, the vector is a viral vector; preferably, the vector is a lentiviral vector, a retroviral vector, adenovirus vector, adeno-associated virus vector, baculovirus vector, or particles and / or vectors for gene therapy. The vector may contain a nucleic acid sequence that allows it to replicate in the host cell, such as an origin of replication. The vector may also include one or more selective marker genes and other genetic elements known to those skilled in the art, such as expression regulatory sequences, such as promoters, enhancers, terminators, etc. The vector is preferably an expression vector comprising a nucleic acid according to the present invention operatively linked to a sequence that allows expression of the nucleic acid. More preferably, the vector is a lentiviral vector.

[0071] In some embodiments, the vector comprises nucleotide sequences encoding the α-chain variable region and / or the β-chain variable region as described above, or the α-chain and / or β-chain thereof.

[0072] In some implementations, the first nucleotide sequence and the second nucleotide sequence are present in different vectors.

[0073] In some embodiments, the first nucleotide sequence and the second nucleotide sequence are present on the same vector in any order. In some embodiments, the first nucleotide sequence and the second nucleotide sequence are optionally linked by a nucleotide sequence encoding a self-cleaving peptide (e.g., P2A, E2A, F2A, or T2A). In some embodiments, the self-cleaving peptide is P2A.

[0074] In some embodiments, the vector contains a nucleotide sequence encoding the fusion protein as described above.

[0075] In another aspect, the present invention provides a recombinant cell comprising the nucleic acid molecules or vectors of the present invention, or expressing the antigen-binding proteins or fusion proteins described herein.

[0076] The recombinant cells provided by this invention include (i) “recombinant production cells” for expressing and producing soluble antigen-binding proteins or fusion proteins described herein and (ii) “recombinant effector cells” for expressing antigen-binding proteins or fusion proteins described herein and having effector functions. The “recombinant effector cells” are particularly suitable for therapeutic applications.

[0077] In some embodiments, the cells that can be used to generate the recombinant production cells may be bacteria, yeast, mammalian cells, or insect cells. The "recombinant production cells" used to express the antigen-binding protein or fusion protein described herein are preferably capable of expressing a large amount of the recombinant protein.

[0078] In other embodiments, the cells used to generate the recombinant effector cells are primary cells derived from the subject, which are processed to express the antigen-binding protein or fusion protein described herein. Preferably, such recombinant effector cells are capable of mediating effector functions through intracellular signal transduction after the antigen-binding protein or fusion protein binds to its specific target antigen.

[0079] In some preferred embodiments, the cells are immune cells.

[0080] In some preferred embodiments, the immune cells are lymphocytes.

[0081] In some preferred embodiments, the immune cells are selected from T cells, tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, natural killer T (NKT) cells, or any combination thereof. Exemplary immune cells that can be used to express the antigen-binding proteins or fusion proteins described herein include PBMCs, TILs, and / or T cells.

[0082] In some embodiments, the T cells are selected from: α / β T cells, γ / δ T cells, iPSC-induced T cells, CD8+ T cells, etc. + Cytotoxic T cells, CD4 + Cytotoxic T cells, CD4 + Helper T cells (e.g., Th1 or Th2 cells), CD4 / CD8 double-positive T cells, tumor-infiltrating T cells, thymocytes, memory T cells, natural killer T cells, such as constant-type natural killer T cells. In some embodiments, the immune cells contain CD4+. + T cells. Those skilled in the art will understand that immune cells may also include progenitor cells (precursor cells) of immune cells, wherein said progenitor cells can be induced in vivo or in vitro to differentiate into mature immune cells. Therefore, in some embodiments, immune cells include progenitor cells of immune cells, such as CD34-containing cells derived from umbilical cord blood, bone marrow, or flowing peripheral blood. + Hematopoietic stem cells (HSCs) within a cell population that differentiate into mature immune cells after being administered to a subject, or which can be induced to differentiate into mature immune cells in vitro.

[0083] In some preferred embodiments, the recombinant cells are recombinant T cells. In some embodiments, the recombinant T cells do not express endogenous TCRs.

[0084] In another aspect, the present invention provides a method for preparing the antigen-binding protein described herein or the fusion protein of the present invention, comprising: culturing the recombinant cells of the present invention under conditions that allow protein expression, and recovering the antigen-binding protein or fusion protein from the cultured recombinant cell culture.

[0085] Those skilled in the art will understand that the antigen-binding proteins or fusion proteins described herein can be prepared by various methods known in the art, such as through genetic engineering recombination techniques. For example, DNA molecules encoding them can be obtained through chemical synthesis or PCR amplification; the resulting DNA molecules can be inserted into an expression vector and transfected into engineered cells; then, the transfected engineered cells can be cultured under specific conditions to express the antigen-binding proteins or fusion proteins described herein. Once the antigen-binding proteins or fusion proteins described herein are expressed, they can be purified by any purification method known in the art, such as chromatography (e.g., ion exchange chromatography, such as hydroxyapatite chromatography; affinity chromatography, particularly protein A, protein G, or lectin affinity chromatography; large and small column chromatography), centrifugation, differential solubility chromatography, hydrophobic interaction chromatography, or any other standard technique for purifying proteins. Those skilled in the art will be able to readily select a suitable purification method based on the individual characteristics of the protein to be recovered.

[0086] In another aspect, the present invention provides a method for preparing the recombinant cells of the present invention, which includes introducing the nucleic acid molecules or vectors of the present invention into the cells.

[0087] In some implementations, the cells may be primary cells derived from the subject.

[0088] Primary cells can be obtained from a subject using any suitable method known in the art. In some embodiments, primary cells are obtained from blood collected from a subject using a variety of techniques known to those skilled in the art. In some embodiments, cells from a subject's circulating blood are obtained via therapeutic apheresis. Cells collected via therapeutic apheresis can be washed to remove the plasma fraction and placed in a suitable buffer (e.g., phosphate-buffered saline (PBS)) or culture medium for subsequent processing steps. The washing step can be performed using methods known to those skilled in the art, such as by using a semi-automatic "flow-through" centrifuge. After washing, the cells can be resuspended in various biocompatible buffers, such as, for example, Ca-free, Mg-free PBS, PlasmaLyte A, or other saline solutions with or without buffers. Alternatively, unwanted components in the therapeutic apheresis sample can be removed, and the cells can be directly resuspended in a culture medium. Therapeutic apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and removing monocytes, for example by Percoll gradient centrifugation or countercurrent centrifugation. Specific cell subpopulations can be further isolated using positive or negative selection techniques (e.g., antibody-coated beads, flow cytometry, etc.). In some embodiments, specific T cell subpopulations, such as CD3+, can be further isolated using positive or negative selection techniques (e.g., antibody-coated magnetic beads, flow cytometry, etc.). + CD28 + CD4 + CD8 + CD45RA + and CD45RO + T cells.

[0089] In some implementations, the primary cells may be the aforementioned immune cells.

[0090] In some implementations, the primary cells may be autologous or non-autologous (e.g., allogeneic). "Autologous" refers to cells from the same subject; "allogeneic" refers to cells from the same species that are genetically different from the comparison cells.

[0091] In some implementations, the methods for introducing the nucleic acid molecule or vector into cells are conventional techniques in the art. Non-limiting examples include calcium phosphate transfection, DEAE-glucan-mediated transfection, microinjection, electroporation, the TALEN method, the ZFN method, non-viral vector-mediated transfection (e.g., liposomes) or viral vector-mediated transfection (e.g., lentiviral infection, retroviral infection, adenovirus infection), and other physical, chemical, or biological methods for transfer into host cells, such as transposon technology, CRISPR-Cas9, etc.

[0092] In some embodiments, after introducing the nucleic acid molecule or vector into the cell, the method further includes: amplifying and / or activating the obtained cells.

[0093] In another aspect, the present invention provides a composition comprising the antigen-binding protein, conjugate, fusion protein, nucleic acid molecule, carrier and / or recombinant cell described herein; preferably, the composition further comprises a pharmaceutically acceptable carrier and / or excipient; more preferably, the composition further comprises an additional therapeutic agent.

[0094] In some embodiments, the carrier and / or excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesion agents, flow aids, preservatives, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, viscosity-improving agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers, and tension modifiers. Those skilled in the art know the importance of selecting suitable carriers and / or excipients to prepare the compositions desired herein. Exemplary carriers and / or excipients used in the compositions herein include saline, buffered saline, glucose, and water. Generally, the selection of suitable carriers and / or excipients depends particularly on the active molecule used, the disease to be treated, and the desired dosage form of the composition.

[0095] In some implementations, the additional therapeutic agent may be an antitumor agent or an immune enhancer.

[0096] In some embodiments, the antitumor agent is selected from alkylating agents, mitotic inhibitors, antitumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclides, 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).

[0097] 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, IFN-γ, IL-10, TGF-β, GM-CSF, or any combination thereof).

[0098] In some embodiments, the antigen-binding proteins, conjugates, fusion proteins, nucleic acid molecules, carriers and / or recombinant cells described herein, along with the additional therapeutic agents, may be provided as separate components or as mixed components in the composition.

[0099] In another aspect, the present invention provides a method for inducing an immune response against a tumor with a KRAS G12V mutation in a subject, and / or for preventing or treating a tumor with a KRAS G12V mutation in a subject, the method comprising administering to the subject a therapeutically effective amount of the antigen-binding protein, conjugate, fusion protein, nucleic acid molecule, vector, recombinant cell and / or composition described herein.

[0100] In another aspect, the present invention provides the use of the antigen-binding proteins, conjugates, fusion proteins, nucleic acid molecules, vectors, recombinant cells and / or compositions described herein for inducing an immune response against tumors with KRAS G12V mutations in subjects, and / or for preventing or treating tumors with KRAS G12V mutations in subjects.

[0101] In some implementations, the recombinant cells are autologous or allogeneic to the subject.

[0102] In some embodiments, the tumor with the KRAS G12V mutation is selected from tumors caused by KRAS mutations. Preferably, the cancer is selected from solid tumors and hematologic malignancies. More preferably, the tumor is selected from acute lymphoblastic cancer, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, brain cancer, glioma, nasopharyngeal carcinoma, eye cancer, oral cancer, cervical cancer, esophageal cancer, liver cancer, intrahepatic bile duct cancer, gallbladder cancer, lung cancer, bone cancer, breast cancer, gastrointestinal tumors, colon cancer, small bowel cancer, colorectal cancer, rectal cancer, stomach cancer, skin cancer, melanoma, multiple myeloma, cervical cancer, endometrial cancer, uterine cancer, ovarian cancer, ureteral cancer, bladder cancer, penile cancer, testicular cancer, pancreatic cancer, prostate cancer, kidney cancer, soft tissue cancer, and thyroid cancer; preferably, the cancer is selected from lung cancer, pancreatic cancer, colorectal cancer, endometrial cancer, ovarian cancer, and prostate cancer.

[0103] In some implementations, the subject has HLA-A 11 alleles; preferably, the patient has HLA-A. 11:01 allele.

[0104] In some implementations, the method further includes the administration of additional therapeutic agents.

[0105] In some implementations, the additional therapeutic agent may be the aforementioned antitumor agent or immune enhancer.

[0106] In some implementations, the additional therapeutic agent may be administered simultaneously, separately, or sequentially.

[0107] In some implementations, the method further includes combination therapy with other treatment options.

[0108] In some implementations, the additional treatment options may be selected from surgery, chemotherapy, radiation therapy, targeted therapy, immunotherapy, hormone therapy, gene therapy, or palliative care.

[0109] In some implementations, the additional treatment may be administered simultaneously, separately, or sequentially.

[0110] The antigen-binding proteins, conjugates, fusion proteins, nucleic acid molecules, carriers, recombinant cells, and / or compositions described herein 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, intravesical, topical (e.g., powder, ointment, or drops), or nasal routes. However, for many therapeutic uses, the preferred route of administration is parenteral (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 antigen-binding proteins, conjugates, fusion proteins, nucleic acid molecules, carriers, recombinant cells, and / or compositions described herein are administered by intravenous injection or bolus.

[0111] Those skilled in the art can determine the exact dosage of the applied antigen-binding protein, conjugate, fusion protein, nucleic acid molecule, carrier, recombinant cell, and / or composition using known techniques. An appropriate dosage is sufficient to elicit, for example, a therapeutic or prophylactic response in the subject within a reasonable timeframe. In some embodiments, the dosage of the applied antigen-binding protein, conjugate, fusion protein, nucleic acid molecule, carrier, recombinant cell, and / or composition can be determined empirically in subjects who have received one or more doses. To assess efficacy, biomarkers of cancer cell status in the subject can be tracked. These include directly measuring cancer cell proliferation and cell death using FACS or other imaging techniques; assessing health improvements using such measurements; or measuring improvements in quality of life or extended survival using recognized assays. It will be apparent to those skilled in the art that the dosage will vary depending on the treatment objective (e.g., remission / maintenance versus acute disease exacerbation), route of administration, timing and frequency of administration, formulation, age, weight, overall health status, sex, diet, disease severity, concomitant drug use, sensitivity to response, and tolerance / responsiveness to treatment. The dosage used for administration can be adjusted based on several parameters, particularly the method of administration, the relevant pathology, or alternatively, the desired duration of treatment.

[0112] In some implementations, it can be in about 10 2 To about 10 10 Cells / kg body weight, approximately 10 5 To about 10 9 Cells / kg body weight, approximately 10 5 To about 10 8 Cells / kg body weight, approximately 10 5 To about 10 7 Cells / kg body weight, approximately 10 7 To about 10 9 Cells / kg body weight or 107 To about 10 8 A dose of cells per kilogram of body weight is administered of the recombinant cells described herein or compositions containing the recombinant cells described herein, including all integer values ​​within these ranges.

[0113] In some embodiments, the antigen-binding proteins, conjugates, fusion proteins, nucleic acid molecules and / or carriers described herein, or compositions comprising the antigen-binding proteins, conjugates, fusion proteins, nucleic acid molecules and / or carriers described herein, may be administered at doses of about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1 mg / kg to about 5 mg / kg, about 0.01 mg / kg to about 1 mg / kg or about 0.1 mg / kg to about 1 mg / kg, including all integer values ​​within these ranges.

[0114] It should be recognized that treatment may require a single or multiple administrations of a therapeutically effective dose of the antigen-binding proteins, conjugates, fusion proteins, nucleic acid molecules, vectors, recombinant cells, and / or compositions described herein. For example, depending on the formulation, half-life, and clearance rate of a particular composition, some compositions may be administered every 3 to 4 days, weekly, every two weeks, or once a month.

[0115] In another aspect, the present invention provides the use of the antigen-binding proteins, conjugates, fusion proteins, nucleic acid molecules, vectors, recombinant cells and / or compositions described herein in the preparation of medicaments for inducing an immune response in a subject against a tumor having a KRAS G12V mutation, and / or for preventing or treating a tumor having a KRAS G12V mutation in a subject.

[0116] The antigen-binding proteins, conjugates, fusion proteins, nucleic acid molecules, carriers, recombinant cells, and / or compositions described herein 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 injectable 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 medicines described herein should be sterile and stable under the conditions of manufacture and storage. A preferred dosage form is an injection. Such injections can be sterile injectable solutions. For example, sterile injectable solutions can be prepared by incorporating the required dose of the antigen-binding proteins, conjugates, fusion proteins, nucleic acid molecules, carriers, recombinant cells, and / or compositions described herein into a suitable solvent, and optionally, simultaneously 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. In addition, 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 prior to 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), solution containing surfactant (e.g., 0.01% polysorbate 20), pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof. Typically, the antigen-binding proteins, conjugates, fusion proteins, nucleic acid molecules, carriers, recombinant cells, and / or compositions described herein can be provided in unit dosage forms, usually in sealed containers, and may be provided as part of a kit. Such kits typically (but not necessarily) include instructions for use, and may also include multiple of the said unit dosage forms.

[0117] In another aspect, the present invention provides a method for detecting the presence of KRAS G12V mutation in a sample, the method comprising contacting the sample with the antigen-binding protein, conjugate, fusion protein and / or recombinant cells described herein.

[0118] In another aspect, the present invention provides the use of the antigen-binding proteins, conjugates, fusion proteins and / or recombinant cells described herein for detecting the presence of KRAS G12V mutations in samples.

[0119] In another aspect, the present invention provides the use of the antigen-binding proteins, conjugates, fusion proteins and / or recombinant cells described herein in the preparation of reagents or kits for detecting KRAS G12V mutations in samples.

[0120] In some embodiments, the sample may be a blood sample, urine sample, tissue sample, or cell sample derived from the subject. In some embodiments, the method is performed in vitro.

[0121] In some embodiments, the method includes (i) contacting the sample with the conjugate described herein, wherein the conjugate contains a detectable marker; and (ii) detecting the presence of a KRAS G12V mutation in the sample by detecting the detectable marker. Attached Figure Description

[0122] The invention is described below by way of example only with reference to the accompanying drawings. It should be emphasized that the details shown are merely illustrative and represent preferred embodiments of the invention to describe the principles and concepts of the invention. Those skilled in the art will readily recognize how to practice the invention in various forms as described with reference to the accompanying drawings.

[0123] Figure 1 : Lentiviral shuttle vector structure that can express the target TCR.

[0124] Figure 2 Flow cytometry was used to detect the positive rate and antigen specificity of J3-TCR-T cells.

[0125] Figure 3 Flow cytometry was used to detect the positive rate and antigen specificity of J4-TCR-T cells.

[0126] Figure 4 : Dose-dependent activation of J3-TCR-T cells in response to antigenic peptide stimulation.

[0127] Figure 5 : Dose-dependent activation of J4-TCR-T cells in response to antigenic peptide stimulation.

[0128] Figure 6 The ELISpot assay was used to detect the specific IFN-γ secretion of J3-TCR-T and J4-TCR-T cells on co-cultured target cells.

[0129] Figure 7 : Detection of cytotoxicity of J3-TCR-T cells against target cells.

[0130] Figure 8 : Detection of cytotoxicity of J4-TCR-T cells to target cells.

[0131] Figure 9 The in vivo tumor-inhibiting effects of J3-TCR-T and J4-TCR-T cells on tumor-bearing mice were assessed using an unpaired two-tailed Student's T test to evaluate the difference in tumor volume between groups at the experimental endpoint. Detailed Implementation

[0132] Before describing the invention in more detail, definitions of certain terms used herein may aid in understanding. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Furthermore, the procedures used herein, such as immunology, molecular biology, biochemistry, nucleic acid chemistry, and cell culture, are all standard procedures widely used in their respective fields. Additionally, to better understand the invention, definitions and explanations of relevant terms are provided below.

[0133] Unless the context clearly indicates otherwise, as used herein, an entity qualifier without a specific quantity shall refer to one or more of the entity, and the singular forms “a,” “an,” and “the / said” include plural references. Thus, the terms “a,” “an,” “one or more,” and “at least one” without a specific quantity are used interchangeably herein.

[0134] As used herein, the term “and / or” when used to connect two or more options should be understood to mean any one of the options or any two or more of the options.

[0135] As used herein, the terms “comprising” or “including” mean to include the stated elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms “comprising” or “including” are used, unless otherwise specified, they also cover situations consisting of the stated elements, integers, or steps. For example, when referring to “comprising” a variable region of a particular sequence, it is also intended to cover the TCR variable region consisting of that particular sequence.

[0136] As used herein, the term "RAS" refers to a proto-oncogene whose encoded RAS protein possesses GTPase activity and participates in numerous signaling pathways regulating cell proliferation, differentiation, and apoptosis, such as the MAPK, PI3K, and STAT signaling pathways. The human genome contains three RAS genes: HRAS (GeneID: 3265), NRAS (GeneID: 4893), and KRAS (GeneID: 3845), which share high sequence homology (>90%). RAS gene mutations are a driving factor in cancer development, and are the most frequent proto-oncogene mutations. The sequences of the RAS proteins encoded by RAS genes are well-known to those skilled in the art and can be found in various public databases. For example, the sequence of the KRAS protein can be found in NCBI:NP_004976.2, the sequence of the NRAS protein in NCBI:NP_002515.1, and the sequence of the HRAS protein in NCBI:NP_001123914.1.

[0137] As used in this article, the term "KRAS" refers to the Kirsten rat sarcoma viral oncogene homolog. KRAS is an important member of the RAS protein family. Upstream of KRAS, it is regulated by the epidermal growth factor receptor (EGFR) family. EGFR signaling can activate SOS protein, thereby regulating KRAS activation. The inactivation and activation transitions of intracellular KRAS proteins are determined by the molecules they bind to. Guanine nucleotide exchange factor (GEF) catalyzes the binding of KARS to GTP, activating KRAS; while GTPase activator protein (GAP) promotes the hydrolysis of GTP bound to KRAS to GDP, leading to KRAS inactivation. Activated KRAS regulates downstream signaling pathways such as MAPK and PI3K, which are related to cell proliferation and migration. KRAS mutations result in persistent GTP binding, maintaining an activated state and leading to continued activation of downstream signaling pathways, thereby promoting tumorigenesis.

[0138] As used herein, the terms “KRAS G12V”, “G12V”, or “G12V mutation” are used interchangeably to refer to a KRAS variant in which the 12th amino acid residue glycine is mutated to valine, which can be specifically detected by the organism’s immune system.

[0139] As used herein, the terms "histiocompatibility complex" and "MHC" are used interchangeably, referring to a group of tightly linked genes that determine whether transplanted tissues are identical and are closely related to the immune response. These primarily include MHC class I and MHC class II molecules. "MHC class I molecules" refer to dimers of the MHC class I α chain and β2-microglobulin chain, while "MHC class II molecules" refer to dimers of the MHC class II α chain and MHC class II β chain. In humans, the MHC is called the human leukocyte antigen (HLA) complex.

[0140] As used herein, the terms "peptide-MHC complex," "p-MHC complex," or "p-MHC" refer to an MHC molecule (MHC class I or MHC class II) that contains a peptide bound in a peptide-binding pocket recognized in the art. 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.

[0141] As used in this article, the term "antigen" is any molecule that can be specifically detected by an organism's immune system.

[0142] 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. In some embodiments, the antigen is a peptide-MHC complex or a peptide presented by an MHC molecule.

[0143] As used herein, the term "T cell receptor" or "TCR" refers to the receptor on the surface of T cells that specifically recognizes antigenic peptides presented by the major histocompatibility complex (MHC). In the immune system, the specific binding of TCRs to the p-MHC complex leads to direct physical contact between T cells and antigen-presenting cells (APCs), which in turn promotes interactions between other molecules on the surface of both T cells and APCs, triggering a series of subsequent cell signaling and other physiological responses, ultimately guiding T cells with different antigen specificities to exert an immune effect on target cells. There are four TCR genes in the human genome: two encoding light chain TCRs: the TRA gene encodes TCRα, and the TRG gene encodes TCRγ; and two encoding heavy chain TCRs: the TRB gene encodes TCRβ, and the TRD gene encodes TCRδ. Heavy chain TCRs and light chain TCRs form heterodimers to constitute the complete TCR. In humans, there are two types of TCRs: TCRα / β and TCRγ / δ. 95% of T cells express TCR α / β, and are called α / β T cells; 5% of T cells express TCR γ / δ, and are called γ / δ T cells. This ratio varies during individual development and in disease states (e.g., leukemia), and also differs between species. Examples of TCRs include, but are 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, as long as the TCR retains its ability to recognize antigen targets.

[0144] As used herein, the term "variable region" or "variable domain" refers to a domain of the α-chain or β-chain (or γ-chain and δ-chain for γ / δ TCR) of a TCR that participates in antigen recognition and binding. The variable domains (Vα and Vβ, respectively) of the α-chain and β-chain of a natural TCR typically have similar structures, with each domain containing four generally conserved framework regions (FRs) and three complementarity-determining regions (CDRs).

[0145] In a TCR, the framework separates the CDRs, and the CDRs are located between the framework regions (i.e., in the primary structure). The Vα domain is encoded by two separate DNA segments, a variable gene segment and a linker gene segment (VJ). The Vβ domain is encoded by three independent DNA segments (a variable gene segment, a variable gene segment, and a linker gene segment (VDJ)). The V, D, and J alleles of the human TCR, including their nucleotides and encoded amino acid sequences, are known in the art. A single Vα or Vβ domain may be sufficient to confer antigen-binding specificity. Furthermore, TCRs binding to specific antigens can be isolated from antigen-binding TCRs using either the Vα or Vβ domains to screen libraries of complementary Vα or Vβ domains, respectively.

[0146] As used herein, the terms “complementarity-determining region” and “CDR” are known in the art to refer to the amino acid sequences in the variable regions of the TCR α and β chains that typically confer antigen specificity and / or binding affinity and are separated from each other in the primary structure by a framework sequence. In some cases, framework amino acids may also facilitate binding, for example, by also contacting the antigen or antigen-containing molecules. Typically, there are three CDRs in each variable region, with CDR3 considered the major CDR responsible for antigen recognition and having a highly variable sequence; CDR1 and CDR2 interact primarily or, in some cases, only with the MHC and have relatively conserved sequences.

[0147] The TCR variable domain sequence can be aligned with a numbering scheme (such as IMGT, Kabat, or Chothia) that allows for the annotation of equivalent residue positions and comparison of different molecules using software tools such as ANACRCI (2016, Bioinformatics 15:298-300). This numbering scheme provides a standardized partitioning of the frame region and CDRs within the TCR variable domain. Unless otherwise stated, the CDRs described herein are defined according to the IMGT numbering scheme (Lefranc et al., Dev.Comp.Immunol. 27:55, 2003; imgt.org / IMGTindex / V-QUEST.php). In some embodiments, the CDR3 amino acid sequence described herein may further include one or more junction amino acids.

[0148] As used herein, the term "constant region" refers to a portion of the TCR encoded by the TRAC gene (for the TCR α chain), the TRBC1 or TRBC2 gene (for the TCR β chain), the TRDC gene (for the TCR δ chain), or the TRGC1 or TRGC2 gene (for the TCR γ chain), optionally lacking all or part of the transmembrane region and / or all or part of the cytoplasmic region. In some embodiments, the TCR constant region lacks both the transmembrane and cytoplasmic regions. The TCR constant region does not contain amino acids encoded by the TRAV, TRAJ, TRBV, TRBD, TRBJ, TRDV, TRDD, TRDJ, TRGV, or TRGJ genes (see, for example, T Cell Receptor Information Manual, (2001), LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, which is incorporated herein by reference in its entirety). In the context of TCR, the terms "extracellular region" refer to one or more portions of the TCR chain located outside the cell, "transmembrane region" refer to one or more portions of the TCR chain embedded in the cell's plasma membrane, and "cytoplasmic region" refer to one or more portions of the TCR chain located in the cell's cytoplasm.

[0149] As used herein, the term "full-length TCR" refers to a TCR comprising a dimer of a first polypeptide chain and a second polypeptide chain, each of which includes a TCR variable region and a TCR constant region comprising a TCR transmembrane region and a TCR cytoplasmic region. In some embodiments, a full-length TCR comprises one or two unmodified TCR chains, such as an unmodified TCR α chain or a TCR β chain. In some embodiments, a full-length TCR comprises one or two modified TCR chains, such as chimeric TCR chains and / or TCR chains containing one or more amino acid substitutions, insertions, or deletions relative to unmodified TCR chains. In some embodiments, a full-length TCR comprises a mature full-length TCR α chain and a mature full-length TCR β chain.

[0150] As used herein, the term "antigen-binding fragment" refers to any portion or fragment of a TCR that retains the biological activity of the TCR (maternal 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 epitope) or p-MHC complex bound by the maternal TCR.

[0151] As used herein, the terms "specific binding" or "specific recognition" refer to a non-random binding reaction between two molecules, such as the reaction between a TCR and its targeted antigen (e.g., a specific peptide or a specific peptide-MHC complex combination). Typically, TCRs that specifically bind to antigens do not bind to or bind to other antigens with lower affinity. For example, antigen-specific TCRs bind to target antigens at a Ka of at least 2, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 times the association constant (Ka) compared to other non-specific antigens. In some embodiments, the antigen-binding protein disclosed herein specifically binds to the KRAS G12V mutant epitope. In some embodiments, the antigen-binding protein disclosed herein binds to VVGAVGVGK-HLA-A 11:01 Complex specifically binds.

[0152] As used herein, the term "linker" refers to one or more amino acid residues inserted between domains or between a domain and a reagent, designed to provide sufficient mobility for a domain or element (e.g., a domain of the antigen-binding protein of the present invention) to fold correctly to form an antigen-binding site. Linkers can be inserted at the amino acid sequence level at transitions between variable domains or between a variable domain and a constant domain (or other domains). Transitions between domains can be identified because the approximate sizes of antibody domains and TCR domains are well known to those skilled in the art. Linker sequences are typically flexible because they consist primarily of amino acids (e.g., glycine, alanine, and serine) and do not have large side chains that could limit flexibility. The usable or optimal length of the linker sequence can be readily determined.

[0153] As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions. Immune cells typically include cells that play a role in the immune response and often have a hematopoietic origin. The term "effector function" refers to the specialized function of an immune cell, such as the function or response that enhances or promotes the immune attack on target cells (e.g., killing of target cells, or inhibiting their growth or proliferation). For example, the effector function of T cells may be, for instance, cytolytic activity or an activity that assists or includes the secretion of cytokines. Examples of immune cells include T cells (e.g., α / β T cells and γ / δ T cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages, among others.

[0154] As used herein, the term "T cell" refers to an immune system cell that matures in the thymus and produces a TCR. T cells can be naive (not exposed to antigens; with increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO compared to TCM), memory T cells (TM) (cells that have experienced antigens for a long time and survive), or effector T cells (cytotoxic T cells that have experienced antigens). TM can be further divided into central memory T cells (TCM, with increased expression of CD62L, CCR7, CD28, CD127, CD45RO, and CD95, and decreased expression of CD54RA compared to naive T cells) and effector memory T cells (TEM, with decreased expression of CD62L, CCR7, CD28, and CD45RA, and increased expression of CD127 compared to naive T cells or TCM).

[0155] As used herein, the term "cytotoxic agent" includes any agent that is harmful to cells (e.g., kills cells), such as chemotherapy drugs, bacterial toxins, plant toxins, or radioactive isotopes.

[0156] As used herein, the term "nucleic acid" refers to a nucleotide chain of any length and includes both DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that can be incorporated into the chain by DNA or RNA polymerases.

[0157] As used herein, the term "vector" refers to a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing said genes or sequences in the host cell. Vectors can be introduced into host cells through transformation, transduction, or transfection. Examples of vectors include, but are not limited to, viral vectors, plasmids, granules, or phage vectors. Vectors may contain nucleic acid sequences, such as replication initiation regions, that allow the gene or sequence of interest to replicate in the host cell. Vectors may also contain one or more optional marker genes and other genetic elements known to those skilled in the art. Vectors are preferably expression vectors containing nucleic acids as described herein, said nucleic acids being operatively linked to sequences that allow said nucleic acid expression.

[0158] As used herein, the term “operationally linked” refers to a functional link between a nucleic acid expression regulatory sequence (e.g., but not limited to, promoter sequences, transcription termination sequences, etc.) and a nucleic acid sequence encoding a target protein (e.g., a coding sequence or an open reading frame), such that the transcription and translation of the nucleic acid sequence are controlled and regulated by the expression regulatory sequence.

[0159] As used herein, the term "expression regulatory sequence" refers to a nucleic acid sequence necessary for the expression of a coding sequence of an operatively linked (hetero)polynucleotide in a specific host organism or host cell. Therefore, the expression regulatory sequence includes both transcriptional and translational regulatory sequences. Typically, the regulatory sequences required for the expression of heteropolynucleotide sequences in prokaryotes include a promoter, an optional operon sequence, and a ribosome binding site; in eukaryotes, a promoter, a polyadenylation signal, an enhancer, and an optional splicing signal are generally required. Furthermore, specific initiation and secretion signals can be introduced into the vector to allow the peptide of interest to be secreted into the culture medium.

[0160] As used herein, the terms “engineered cell” or “recombinant cell” refer to cells in which exogenous nucleic acids have been introduced, including the progeny of these cells. Engineered cells or recombinant cells include “transfected cells,” which include primary transfected cells and their progeny, regardless of passage number. Progeny cells may not be identical to parent cells in terms of nucleic acid content, but may contain mutations. This article includes mutant progeny that have the same function or biological activity as cells screened or selected in the initially transfected cells.

[0161] As used herein, the term "sequence identity" refers to the degree to which polynucleotide or polypeptide segments have the same amino acid residues at the same positions in a sequence alignment (e.g., nucleotide or amino acid sequence), and is typically expressed as a percentage. Methods for evaluating the degree of sequence identity between polynucleotides or polypeptides are known to those skilled in the art. For example, amino acid sequence identity is typically measured using sequence analysis software. For instance, the BLAST procedure of the NCBI database can be used to determine identity. For determining sequence consistency, see, for example: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

[0162] As used herein, the term "functional variant" refers to an antigen-binding protein that has at least or significantly similar sequence identity to the parent antigen-binding protein and retains the biological activity of the parent antigen-binding protein, such as those containing conserved amino acid substituents. Functional variants encompass, for example, variants of the antigen-binding protein or fragments thereof described herein that retain a similar, equal, or greater degree of target antigen recognition ability than the parent antigen-binding protein. Compared to the parent antigen-binding protein, for example, the amino acid sequence of the functional variant may have, for example, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity with the parent antigen-binding protein.

[0163] For example, a functional variant may comprise the amino acid sequence of a parent antigen-binding protein having at least one conserved amino acid substituent. Alternatively or additionally, the functional variant may comprise the amino acid sequence of a parent antigen-binding protein having at least one non-conserved amino acid substituent. In this case, the non-conserved amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. Preferably, the non-conserved amino acid substitution enhances the biological activity of the functional variant, thereby increasing the biological activity of the functional variant relative to the parent antigen-binding protein.

[0164] The modified TCRs, peptides, and antigen-binding proteins (including functional parts and functional variants) described herein may have any length, i.e., may contain any number of amino acids, provided that the modified TCRs, peptides, or proteins (or their functional parts or functional variants) retain their biological activity, such as the ability to bind specifically to antigens, detect diseased cells in the host, or treat or prevent disease in the host.

[0165] The antigen-binding proteins described herein (including functional portions and functional variants) may contain synthetic amino acids that replace one or more natural amino acids. Such synthetic amino acids are known in the art.

[0166] In some cases, the term "functional variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to the polypeptide or peptide). For example, but not limited to, polypeptides can be modified, such as by glycosylation, acetylation, polyethylene glycolation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, linking to cellular ligands or other proteins, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, and the metabolic synthesis of tunicamycin.

[0167] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or alter the intended properties of a protein / peptide containing an amino acid sequence. For example, conservative substitutions can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions include substitutions of amino acid residues with amino acid residues having similar side chains, such as substitutions with residues that are physically or functionally similar to the corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent or hydrogen bonds). Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid and glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), β-branched side chains (e.g., threonine, valine, and isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, and histidine). Therefore, it is preferable to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conserved amino acid substitutions are well known in the art (see, for example, Brummell et al., Biochem. 32:1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10):879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94:412-417 (1997), which are incorporated herein by reference).

[0168] As used herein, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Furthermore, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0169] As used herein, "composition" can refer to something used for the treatment of a disease or for in vitro cell culture experiments. When used for the treatment of a disease, the term "composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients that constitute one or more adjunct components. Typically, compositions are prepared by uniformly and adequately combining the active compound with liquid excipients, finely chopped solid excipients, or both.

[0170] As used herein, the term "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by a federal regulatory agency or national government, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0171] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" can include any solvent, solid excipient, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. The use of any conventional excipients, except for those incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0172] As used herein, the terms “subject” and “patient” are used interchangeably and refer to mammals in need of treatment, such as pets (e.g., dogs, cats, etc.), livestock (e.g., cattle, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). Typically, a subject is a human being in need of treatment.

[0173] As used herein, the term “prevention” means a method implemented to prevent or delay the occurrence of a disease or condition or symptom (e.g., a tumor) in a subject. The term “treatment” means used to refer to achieving a desired pharmacological and / or physiological effect. Such effect may be preventative in terms of complete or partial prevention of the disease or its symptoms, and / or therapeutic in terms of partial or complete cure of the disease and / or adverse effects caused by the disease. As used herein, “treatment” covers diseases in mammals, particularly humans, including: (a) prevention of the occurrence of a disease or condition in individuals susceptible to the disease but not yet diagnosed with it; (b) inhibition of the disease, such as blocking disease progression; or (c) relief of the disease, such as reducing symptoms associated with the disease. As used herein, “treatment” covers any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual’s disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0174] 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 disease prevention means an amount sufficient to prevent, stop, or delay the onset of said disease; an effective amount for disease treatment 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 other concurrent treatments, etc.

[0175] sequence list

[0176] The sequences involved in this article are shown in the table below:

[0177] Table 1:

[0178]

[0179]

[0180]

[0181]

[0182]

[0183]

[0184] Example

[0185] The present invention will be further described in detail below with reference to embodiments. It should be understood that these embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Modifications or improvements made without departing from the basis of the present invention are all within the protection scope of the present invention. The following embodiments are provided by way of example and are not intended to limit the invention. These embodiments are not intended to represent all or only the experiments conducted below.

[0186] Unless otherwise stated, the experimental methods used in the following examples are conventional methods, and the experimental materials and reagents used are commercially available.

[0187] Example 1. Screening for KRAS G12V-specific TCRs

[0188] Peripheral blood samples were obtained from cancer patients who met the following criteria: 1) HLA genotype was HLA-A 1) Type 11:01; 2) Carrying KRAS-G12V mutation in tumor tissue; 3) Significant tumor shrinkage (PR) after the patient receives KRAS-G12V mRNA or peptide vaccine treatment.

[0189] After obtaining peripheral blood from patients meeting the above criteria, peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation using Ficoll-Paque (Cytiva, 17144003) lymphocyte separation medium. The target T cells in the patient's PBMCs were then stimulated in vitro with the KRASG12V mutant epitope peptide VVGAVGVGK (SEQ ID NO: 2) to expand, thereby enriching antigen-specific T cells.

[0190] Stimulated PBMCs were harvested and stained with PE-labeled tetramer HLA-A. 11:01 / KRAS G12V (G12V-9mer-tetramer) and control tetramer staining reagent HLA-A 11:01 / KRAS WT (WT-tetramer) and HLA-A 11:01 / KRAS G12V (G12V-10mer-tetramer) (both purchased from Beijing Bomei Biotechnology Co., Ltd.) was used to stain PBMCs (staining method followed the reagent instructions), and the HLA-A inhibitors were obtained by flow cytometry. 11:01 / KRAS G12V (9mer) specific positive T cells. The sorted positive T cells were subjected to 10x Genomics single-cell sequencing to obtain a series of candidate TCR clones.

[0191] Simultaneously, bulk RNA sequencing was performed on PBMC samples from patients before and after mRNA or peptide vaccine treatment, and TCRs with significantly increased clonal frequency before and after mRNA or peptide vaccine treatment were selected as candidate TCR clones.

[0192] Through comparison and experimental verification of various candidate TCR clones, two HLA-A receptors that specifically recognize KRAS G12V were finally obtained. The 11:01 restricted TCRs were named J3-TCR and J4-TCR, respectively, and their TCR α chain and β chain genotypes are shown in Table 2.

[0193] Table 2. α-chain and β-chain genotypes of J3-TCR and J4-TCR

[0194]

[0195] Sequencing revealed that the amino acid sequences of the α-chain variable region and their encoding nucleotide sequences of the J3-TCR are shown in SEQ ID NO:15 and SEQ ID NO:16, respectively, and the amino acid sequences of the β-chain variable region and their encoding nucleotide sequences are shown in SEQ ID NO:17 and SEQ ID NO:18, respectively. Specifically, the amino acid sequences of the α-chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively, and the amino acid sequences of the β-chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, respectively.

[0196] The amino acid sequences of the α-chain variable region of J4-TCR and their encoding nucleotide sequences are shown in SEQ ID NO: 19 and SEQ ID NO: 20, respectively; the amino acid sequences of the β-chain variable region and their encoding nucleotide sequences are shown in SEQ ID NO: 21 and SEQ ID NO: 22, respectively; wherein, the amino acid sequences of the α-chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively; and the amino acid sequences of the β-chain variable regions CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14, respectively.

[0197] Example 2. Construction of TCR gene and vector for HLA-A*11:01 / KRAS G12V specific T cell clones

[0198] like Figure 1As shown, following the TRBV-MusBC-P2A-TRAV-MusAC sequence, the α-chain variable region and β-chain variable region of J3-TCR and J4-TCR were fused with the mouse constant region for expression. TRBV represents the β-chain variable region of the TCR, MusBC represents the mouse TCR β-chain constant region (its amino acid and nucleotide sequences are shown in SEQ ID NO: 25 and SEQ ID NO: 26, respectively), the amino acid and nucleotide sequences of the J3-TCR β-chain are shown in SEQ ID NO: 30 and SEQ ID NO: 32, respectively, and the amino acid and nucleotide sequences of the J4-TCR β-chain are shown in SEQ ID NO: 36 and SEQ ID NO: 38, respectively; TRAV represents the α-chain variable region of the TCR, MusAC represents the mouse TCR α-chain constant region (its amino acid and nucleotide sequences are shown in SEQ ID NO: 23 and SEQ ID NO: 24, respectively), and the amino acid and nucleotide sequences of the J3-TCR α-chain are shown in SEQ ID NO: 29 and SEQ ID NO: 20, respectively. As shown in NO: 31, the amino acid and nucleotide sequences of the J4-TCR α chain are shown in SEQ ID NO: 35 and SEQ ID NO: 37, respectively; P2A is a self-cleaving peptide (its amino acid and nucleotide sequences are shown in SEQ ID NO: 39 and SEQ ID NO: 40, respectively). The above TRBV-MusBC-P2A-TRAV-MusAC sequence (wherein, the J3-TCR amino acid sequence and its encoding nucleotide sequence are shown in SEQ ID NO: 41 and SEQ ID NO: 42, respectively, and the J4-TCR amino acid sequence and its encoding nucleotide sequence are shown in SEQ ID NO: 43 and SEQ ID NO: 44, respectively) was cloned into the lentiviral shuttle vector pLvx-EF1a to form a lentiviral vector capable of expressing J3-TCR and J4-TCR.

[0199] Example 3. HLA-A*11:01 / KRAS G12V specific T cell receptor lentiviral packaging

[0200] Lentiviral cells were prepared via transient transfection of 293T cells, and lentiviruses containing the target TCR gene were packaged using a second-generation lentiviral packaging system. For transfection, 2 × 10⁶ cells were seeded in 15 cm culture dishes on day 0. 7293T cells were evenly distributed on a culture dish, with a confluence slightly higher than 50%. After 24 hours of cell seeding, the cells were transfected with plasmids. The lentiviral vector constructed in Example 2 was mixed with the packaging plasmid psPAX and the envelope plasmid pMD.2G. The amounts used for a 15 cm diameter culture dish were as follows: 20 μg: 12 μg: 8 μg. The ratio of transfection reagent Lipo2000 (Invitrogen, product number 11668019) to plasmid was 2:1, with 80 μL used per 15 cm dish. The specific procedure is as follows: Add the expression plasmid and packaging plasmid to 2 mL of OPTI-MEM medium (Gibco, catalog number 31985-070) according to the above dosage, mix well, and let stand at room temperature for 5 minutes to form a DNA mixture; take the corresponding amount of transfection reagent Lipo2000 and mix it with 2 mL of OPTI-MEM medium, let it stand at room temperature for 5 minutes, mix the DNA mixture and transfection reagent mixture together and let it stand at room temperature for 30 minutes, then add it to 293T cells that have been converted to 12 mL of OPTI-MEM, gently shake the culture dish to mix the medium evenly, and culture at 37℃ / 5% CO2 for 6 hours, remove the transfection medium, replace it with DMEM (Gibco, product number C11995500bt) complete medium containing 10% fetal bovine serum, and culture at 37℃ / 5% CO2. On days 3 and 4, the culture supernatant containing the packaged lentivirus was collected. The collected culture supernatant was centrifuged at 3000×g for 15 minutes to remove cell debris, and then filtered through a 0.45-micron filter (Merck Millipore, product number SLGP033RB). The supernatant was then concentrated by centrifugation according to the Lentiviral Concentration Kit (clonetech, product number 632200) instructions. Most of the supernatant was removed, and the final concentration was reduced to 1 mL. The concentrate was aliquoted and stored at -80°C. The lentivirus titer of the concentrated sample was determined according to the p24 ELISA (Takara, product number 631476) kit instructions.

[0201] Example 4. Construction of primary TCR-T cells expressing the target TCR

[0202] Peripheral blood lymphocytes were collected from healthy donors, and T cells were sorted and counted using magnetic beads. CD3 / CD28 antibody-conjugated magnetic beads (Gibco, 11132D) were added to the T cell culture system in equal amounts to the cell count to activate the T cells. After 24 hours of activation, lentivirus expressing the target TCR obtained in Example 3 was added, followed by centrifugation infection. The virus-infected T cells were cultured in vitro for 7 days to obtain a sufficient number of J3-TCR-T cells and J4-TCR-T cells, which were used as effector cells for subsequent experiments. Uninfected T cells (Mock T) served as control cells.

[0203] Example 5. HLA-A*11:01 / KRAS G12V tetramer staining to verify expression

[0204] The TCR expression positivity rate of T cells after viral infection in Example 4 was detected by flow cytometry. CD8 antibody (Biolegend, clone RPA-T8) and CD4 antibody (Biolegend, clone SK3) were used to target cytotoxic (CD8+) T cells. + ) and auxiliary (CD4) + These two main types of T cells were distinguished, and the expression positivity rates of J3-TCR and J4-TCR on the surface of these two types of T cells were detected using antibodies targeting the constant region of murine TCR. Simultaneously, the T cells expanded in Example 4 were labeled using the PE-labeled tetramer staining reagent (tetramer-PE) used in Example 1 to further detect the specificity of primary TCR-T cells.

[0205] Following the reagent instructions, G12V-9mer-tetramer-PE was thoroughly mixed with the expanded T cells from Example 4 and incubated on ice for 40 minutes. Then, APC-labeled anti-mouse TCR β constant region (mouse TCR-βC-BV605) antibody (Biolegend, H57-597) was added, and incubation on ice continued for 15 minutes. After washing the samples twice with PBS containing 2% FBS, double-positive T cells labeled with G12V-9mer-tetramer and mouse TCR-βC-BV605 were detected using a Cytek Aurora flow cytometer. Data analysis was performed using Cytek SpectroFlo software.

[0206] For J3-TCR-T, the flow cytometry analysis results ( Figure 2 This indicates that CD8+ in donor-derived T cells... + T cells and CD4 + The proportions of T cells were 33.6% and 52.3%, respectively. After engineering modification, HLA-A The positive rates of 11:01 / KRAS G12V-9mer tetramer were 38.66% and 30.59%, respectively, and no HLA-A was detected. 11:01 / KRAS G12WT-9mer and HLA-A 11:01 / KRASG12V-10mer tetramer positive cells.

[0207] For J4-TCR-T, the flow cytometry analysis results ( Figure 3 This indicates that CD8+ in donor-derived T cells... + T cells and CD4 + The proportions of T cells were 33.28% and 51.69%, respectively. After engineering modification, HLA-A The positive rates for 11:01 / KRAS G12V-9mer tetramer were 30.9% and 7.62%, respectively, and no HLA-A was detected. 11:01 / KRAS G12WT-9mer and HLA-A 11:01 / KRASG12V-10mer tetramer positive cells.

[0208] The above data demonstrate that, after engineered modification, most donor T cells stably express J3-TCR and J4-TCR, and these primary J3-TCR-T and J4-TCR-T cells effectively inhibit HLA-A expression. The 11:01 / KRAS G12V-9 peptide complex exhibits high specificity.

[0209] Example 6. Co-culture activation and IFN-γ release level detection

[0210] The following peptides for in vitro stimulation were custom-synthesized by Jiangsu Genscript Co., Ltd.: the short peptide VVGAGGVGK (SEQ ID NO: 1) (also referred to as "KRAS WT peptide") of the amino acid sequence from position 8 to position 16 of wild-type KRAS and the short peptide VVGAVGVGK (SEQ ID NO: 2) (also referred to as "KRAS G12V-9 peptide") of the amino acid sequence from position 8 to position 16 of KRAS G12V after the amino acid at position 12 of KRAS is mutated from G to V.

[0211] The KRAS G12V-9 peptide was resuspended in DMSO at an initial concentration of 1 mM, followed by serial dilutions to final concentrations of 10 μM, 1 μM, and 10 μM. -1 μM, 10 -2 μM, 10 -3 μM, 10 -4 μM and 10 -5μM KRAS G12V-9 peptide was used to pulse-stimulate the Panc1 human pancreatic cancer cell line (genotype HLA-A) in vitro. 11:01), and after being thoroughly mixed with the J3-TCR-T cells and J4-TCR-T cells obtained in Example 4, were added to a flat-bottomed 96-well plate, wherein the amount of Panc1 cells was 4 × 10⁻⁶. 4 The number of TCR-T cells was 2 × 10⁶ cells. 4 Cells were cultured for 24 hours. After culture, the IFN-γ secretion levels of J3-TCR-T cells and J4-TCR-T cells stimulated with different concentrations of KRAS G12V-9 peptide were detected by ELISA (BD, 555142).

[0212] like Figure 4 and Figure 5 The data show that as the concentration of KRAS G12V-9 peptide in the culture system gradually increases, the IFN-γ secretion level of TCR-T cells expressing the target TCR increases in a dose-dependent manner, indicating that HLA-A The 11:01 / KRAS G12V-9 peptide complex can specifically activate J3-TCR-T cells and J4-TCR-T cells.

[0213] Example 7. Detection of TCR-T cell-target cell interaction

[0214] KRAS G12V / HLA-A*11:01 Target Cell Preparation

[0215] The CDS sequence of the wild-type KRAS gene transcript (NCBI: NM_004985.5_191..757; SEQ ID NO: 45) and the CDS sequence of the KRAS gene transcript containing the G12V mutation (NCBI: NM_004985.5_c.35G>T(p.Gly12Val); SEQ ID NO: 46) were cloned into the lentiviral vector Phage-IRES-RFP to construct the eukaryotic recombinant expression plasmids Phage-IRES-KRASG12WT-RFP and Phage-IRES-KRASG12V-RFP. Lentiviral packaging was performed according to the method in Example 3, and stable expression of wild-type KRAS and human pancreatic cancer cell lines Panc1 (G12WT-Panc1 and G12V-Panc1) containing the KRAS G12V mutation were constructed via lentiviral infection. These cells naturally express HLA-A. The 11:01 molecule can present the KRAS mutant peptide VVGAVGVGK to the cell surface, forming VVGAVGVGK-HLA-A. The 11:01 complex. Therefore, G12V-Panc1 cells can be used as target cells in subsequent experiments, while G12WT-Panc1 cells can be used as control target cells.

[0216] Detection of TCR-T cell activation by target cells

[0217] The cell suspensions of G12WT-Panc1 control cells and G12V-Panc1 target cells were centrifuged at 200-250×g for 5 minutes at room temperature. The supernatant was removed, and the cells were resuspended in RPMI 1640 complete medium containing 1% FBS and counted. The two cell types were then diluted to 4×10⁻⁶ cells / mL. 5 Cells / mL, the prepared cell suspension was seeded into ELISpot 96-well plates (Mabtech, 3420-4HPT-10), 100 μL of cell suspension per well (4 × 10⁶ cells / mL). 4 (cells). Simultaneously, effector cells J3-TCR-T, J4-TCR-T, and control effector cells Mock T constructed in Example 4 were collected, and the three cell types were diluted to 1.6 × 10⁻⁶ cells. 6 Add 50 μL of cell suspension (8 × 10⁶ cells / mL) to each well. 4 (cells).

[0218] Each of the target cells was co-cultured with each of the effector cells. A positive control was set up, in which effector cells were co-cultured with CD3 antibody (Miltenyi Biotec, clone OKT3), and a negative control was set up, containing only effector cells. The IFN-γ secretion level of effector cells after co-culturing with target cells was detected.

[0219] Each experimental group was set up with two replicates. After incubation at 37℃ for 24 hours, the plate was washed, incubated with antibody, developed color, and terminated according to the operating procedures of the ELISpot Plus: HumanIFN-γ (HRP) (Mabtech, 3420-4HPT-2) product instructions. The 96-well plate was placed on an ELISA spot analyzer (AID, model ELR08IFL) and the corresponding parameters were set before detection.

[0220] The data from this experiment show that ( Figure 6 T cells derived from healthy donors, after being engineered into TCR-T cells, were able to secrete large amounts of IFN-γ upon stimulation by target cells, while unengineered cells could not secrete IFN-γ upon stimulation by target cells. This result indicates that both J3-TCR-T cells and J4-TCR-T cells can effectively recognize HLA-A... 11:01 KRAS G12V mutant antigen peptide presented by the molecule. Furthermore, control target cells expressing wild-type KRAS could not effectively activate J3-TCR-T cells and J4-TCR-T cells, indicating that J3-TCR-T cells and J4-TCR-T cells are not effective in distinguishing HLA-A mutants. The KRAS G12V mutant antigenic peptide presented by the 11:01 molecule exhibits high specificity for the corresponding wild-type antigenic peptide.

[0221] Example 8. Detection of the killing function of TCR-T cells against target cells

[0222] This embodiment assesses the cytotoxic function of TCR-T cells against target cells by detecting the release of lactate dehydrogenase (LDH) in the co-culture system. When cells are damaged, cell membrane permeability changes, and LDH is released from the cell into the extracellular space. This release is proportional to the number of lysed cells; therefore, the degree of cell damage can be reflected by quantifying LDH in the co-culture system. J3-TCR-T cells, J4-TCR-T cells, or Mock T cells (E) were co-cultured with G12WT-Panc1 or G12V-Panc1 target cells (T) at different effector cell / target cell ratios of 4:1, 2:1, and 1:1. After 24 hours of culture, the LDH in the supernatant of the culture system was quantified using an LDH detection kit (Dongren Chemical Technology, CK12).

[0223] The results showed that ( Figure 7 and Figure 8 When only J3-TCR-T cells or J4-TCR-T cells were co-cultured with G12V-Panc1 target cells, significant LDH release was observed, and there was a significant dose-response relationship between the amount of LDH released and the effector cell / target cell ratio, indicating that both J3-TCR-T cells and J4-TCR-T cells have specific killing ability only against G12V-Panc1 target cells.

[0224] Example 9. Detection of the in vivo tumor-suppressing effect of TCR-T cells

[0225] This embodiment further demonstrates the tumor-killing function of J3-TCR-T cells and J4-TCR-T cells by detecting their inhibitory effects on subcutaneous tumors in immunodeficient mice. The G12V-Panc1 target cells constructed in Example 7 were inoculated into NSG mice (NOD.Cg-Prkdc...). scid Il2rg em1 / Smoc) Subcutaneous on the dorsal side, the target cell seeding amount per mouse was 5 × 10 6On day 30 after tumor formation (when the tumor enters a rapid growth phase), mice were grouped according to tumor growth. J3-TCR-T cells, J4-TCR-T cells, or control Mock T cells were injected into the mice via the tail vein. The cell injection dose per mouse was 2 × 10⁶ cells. 7 Individual cells. Monitor tumor volume changes after injection.

[0226] Data shows ( Figure 9 Before TCR-T cell inoculation, the tumor growth curves of the mice in each group were very similar. However, 7 days after TCR-T cell inoculation, tumor growth in mice treated with either T3-TCR-T cells or J4-TCR-T cells was significantly inhibited, and the tumor volume continued to shrink. Meanwhile, the tumors in the control group treated only with Mock T cells continued to grow. At the experimental endpoint, the tumor volume in the J3-TCR-T cell-injected group was significantly smaller than that in the Mock T cell-injected control group (P=1.19087×10⁻⁶). -11 The tumor volume in the experimental group injected with M2-TCR-T cells was significantly smaller than that in the control group injected with Mock T cells (P=4.39313×10⁻⁶). -11 This result indicates that donor T cells, after being engineered to express J3-TCR or J4-TCR, can be detected by HLA-A receptors on the surface of tumor cells in mice. The 11:01-KRAS G12V mutant antigen peptide complex is effectively activated and exerts its killing function, thereby inhibiting the occurrence and development of tumors. This further demonstrates that the J3-TCR and J4-TCR provided by this invention have extremely high clinical application potential.

[0227] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. An antigen-binding protein comprising a TCR α-chain variable region and a TCR β-chain variable region, wherein the antigen-binding protein specifically recognizes and binds to the VVGAVGVGK (SEQ ID NO: 2)-MHC complex, wherein, The variable region of the TCR α chain contains amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, respectively, and the variable region of the TCR β chain contains amino acid sequences CDR1, CDR2, and CDR3 as shown in SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, respectively.

2. The antigen-binding protein according to claim 1, wherein the VVGAVGVGK (SEQ ID NO: 2)-MHC complex is Complex.

3. The antigen-binding protein according to claim 2, wherein the VVGAVGVGK (SEQ ID NO: 2)-MHC complex is Complex.

4. The antigen-binding protein according to any one of claims 1-3, wherein, The TCR α chain variable region contains an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 15, and / or the TCR β chain variable region contains an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

17.

5. The antigen-binding protein according to any one of claims 1-3, wherein, The TCR α chain variable region contains an amino acid sequence as shown in SEQ ID NO: 15, and / or the TCR β chain variable region contains an amino acid sequence as shown in SEQ ID NO:

17.

6. The antigen-binding protein according to any one of claims 1-3, wherein the antigen-binding protein further comprises a TCR constant region or a fragment thereof.

7. The antigen-binding protein according to claim 6, wherein the TCR constant region is a human TCR constant region or a mouse TCR constant region.

8. The antigen-binding protein of claim 6, wherein the TCR constant region comprises a TCR α-chain constant region and / or a TCR β-chain constant region.

9. The antigen-binding protein according to claim 8, wherein, The TCR α chain constant region contains an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:23, and / or the TCR β chain constant region contains an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

25.

10. The antigen-binding protein according to claim 8, wherein, The TCR α chain constant region contains an amino acid sequence as shown in SEQ ID NO: 23, and / or the TCR β chain constant region contains an amino acid sequence as shown in SEQ ID NO:

25.

11. The antigen-binding protein according to any one of claims 1-3, wherein the antigen-binding protein is a T-cell receptor.

12. The antigen-binding protein according to any one of claims 1-3, wherein the antigen-binding protein is a soluble TCR lacking a transmembrane region and a cytoplasmic region.

13. The antigen-binding protein according to any one of claims 1-3, wherein the antigen-binding protein is a single-chain TCR containing a TCR variable region linked by a flexible linker.

14. The antigen-binding protein according to any one of claims 1-3, wherein the TCR α chain variable region and the TCR β chain variable region are present in a single polypeptide chain, or the TCR α chain variable region and the TCR β chain variable region are present in different polypeptide chains.

15. The antigen-binding protein of claim 14, wherein the antigen-binding protein is an α / β heterodimer TCR, comprising a TCR α chain including a TCR α chain variable region and a TCR α chain constant region, and / or a TCR β chain including a TCR β chain variable region and a TCR β chain constant region, wherein, The TCR α chain contains an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 27, and / or the TCR β chain contains an amino acid sequence that has at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

28.

16. The antigen-binding protein of claim 14, wherein the antigen-binding protein is an α / β heterodimer TCR, comprising a TCR α chain including a TCR α chain variable region and a TCR α chain constant region, and / or a TCR β chain including a TCR β chain variable region and a TCR β chain constant region, wherein, The TCR α chain contains the amino acid sequence shown in SEQ ID NO: 27, and / or the TCR β chain contains the amino acid sequence shown in SEQ ID NO:

28.

17. The antigen-binding protein of claim 15, wherein the TCR α chain and / or TCR β chain further comprises a signal peptide.

18. The antigen-binding protein according to any one of claims 1-3, wherein the antigen-binding protein is soluble or membrane-bound.

19. The antigen-binding protein according to any one of claims 1-3, wherein the antigen-binding protein further comprises one or more antigen-binding sites that bind different antigens or epitopes.

20. The antigen-binding protein of claim 19, wherein the different antigens are immune cell surface antigens.

21. The antigen-binding protein according to claim 20, wherein, The immune cell surface antigen is a T cell surface antigen.

22. The antigen-binding protein according to claim 21, wherein, The T cell surface antigen is CD3.

23. A fusion protein comprising the antigen-binding protein of any one of claims 1-22 and a peptide or protein expressed therewith in fusion, wherein the peptide or protein is a protein tag or a detectable protein marker.

24. An isolated nucleic acid molecule encoding the antigen-binding protein of any one of claims 1-22, or encoding the fusion protein of claim 23.

25. The nucleic acid molecule according to claim 24, wherein, The nucleic acid molecule in question is a codon-optimized nucleic acid molecule for the host cell.

26. A vector comprising the nucleic acid molecule of claim 24 or 25, wherein the nucleic acid molecule is operatively linked to an expression regulatory sequence.

27. The vector according to claim 26, wherein it is a viral vector.

28. The carrier according to claim 27, wherein, The vector is a lentiviral vector, a retroviral vector, adenovirus vector, adeno-associated virus vector, or baculovirus vector.

29. A recombinant cell comprising the nucleic acid molecule of claim 24 or 25 or the vector of any one of claims 26-28, or expressing the antigen-binding protein of any one of claims 1-22 or the fusion protein of claim 23.

30. The recombinant cell of claim 29, wherein the cell is a bacterium, yeast, mammalian cell or insect cell.

31. The recombinant cell of claim 29, wherein the recombinant cell is a primary cell derived from a subject.

32. The recombinant cell according to claim 31, wherein, The cells in question are immune cells.

33. The recombinant cell according to claim 32, wherein, The immune cells are selected from T cells, natural killer cells, or any combination thereof.

34. The recombinant cell according to claim 32, wherein, The immune cells are tumor-infiltrating lymphocytes.

35. The recombinant cell according to claim 33, wherein, The T cells are CD8. + T cells or CD4 + T cells.

36. A method for preparing the antigen-binding protein according to any one of claims 1-22 or the fusion protein according to claim 23, comprising: Under conditions that allow protein expression, the recombinant cells of any one of claims 29-35 are cultured, and the antigen-binding protein or fusion protein is recovered from the cultured recombinant cell culture.

37. A method for preparing recombinant cells according to any one of claims 29-35, comprising introducing a nucleic acid molecule according to claim 24 or 25 or a vector according to any one of claims 26-28 into in vitro or ex vivo cells.

38. A conjugate comprising the antigen-binding protein of any one of claims 1-22 and a detectable label conjugated thereto.

39. A composition comprising the antigen-binding protein of any one of claims 1-22, the fusion protein of claim 23, the nucleic acid molecule of claim 24 or 25, the vector of any one of claims 26-28, the recombinant cell of any one of claims 29-35, and / or the conjugate of claim 38.

40. The composition according to claim 39, wherein, The composition also contains a pharmaceutically acceptable carrier and / or excipient.

41. The composition according to claim 39, wherein, The composition also contains additional therapeutic agents.

42. Use of the antigen-binding protein of any one of claims 1-11 and 14-22, the nucleic acid molecule encoding the antigen-binding protein of any one of claims 1-11 and 14-22, the vector comprising the nucleic acid molecule encoding the antigen-binding protein of any one of claims 1-11 and 14-22, and / or the recombinant cell expressing the antigen-binding protein of any one of claims 1-11 and 14-22 in the preparation of a medicament for use in the prevention or treatment of tumors with KRAS G12V mutations in a subject, wherein the tumors with KRAS G12V mutations are selected from lung cancer, pancreatic cancer, and colorectal cancer.

43. Use in the preparation of a composition comprising the antigen-binding protein of any one of claims 1-11 and 14-22, a nucleic acid molecule encoding the antigen-binding protein of any one of claims 1-11 and 14-22, a vector comprising the nucleic acid molecule encoding the antigen-binding protein of any one of claims 1-11 and 14-22, and / or recombinant cells expressing the antigen-binding protein of any one of claims 1-11 and 14-22, of a composition comprising the antigen-binding protein of any one of claims 1-11 and 14-22, and / or recombinant cells expressing the antigen-binding protein of any one of claims 1-11 and 14-22, for the purpose of preventing or treating tumors with KRAS G12V mutations in a subject, wherein the tumors with KRAS G12V mutations are selected from lung cancer, pancreatic cancer, and colorectal cancer.

44. The use according to claim 42 or 43, wherein, The subject has Alleles.

45. The use according to claim 44, wherein, The subject has Alleles.

46. ​​The use according to claim 42 or 43, wherein, The drug is used in combination with other therapeutic agents.

47. Use of the antigen-binding protein of any one of claims 1-22, the fusion protein of claim 23, the recombinant cell of any one of claims 29-35, the conjugate of claim 38, and / or the composition of any one of claims 39-41 in the preparation of a reagent or kit for detecting the presence of a KRAS G12V mutation in a sample.

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