T cell receptor for identifying KRAS mutation and encoding sequence thereof

Abstract

Provided in the present invention are a specific T cell receptor targeting a KRAS G12V mutated epitope peptide (the amino acid sequence as shown in SEQ ID NO: 2) and the anti-tumor use thereof. The specific T cell receptor consists of two peptide chains α and β chains, and can specifically bind to a VVGAVGVGK-HLA-A*11:01 complex. Further provided in the present invention are an antigen-binding fragment of the specific T cell receptor, a nucleic acid sequence encoding the T cell receptor, a vector containing the nucleic acid sequence, an engineered cell expressing the T cell receptor, a composition containing the T cell receptor, and the use thereof. In addition, further provided in the present invention is a method for screening a KRAS G12V-specific T cell receptor. The specific T cell receptor and the antigen-binding fragment thereof provided by the present invention can be used as an immune effect activator to stimulate an immune response of the body, so as to generate the effects of resisting diseases such as tumors.

Description

A T cell receptor recognizing KRAS mutation and its coding sequence Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to an HLA-A*11:01 restricted T cell receptor (TCR) or an antigen-binding fragment thereof that can specifically recognize and bind to the tumor cell KRAS G12V mutant epitope peptide VVGAVGVGK. Background Art

[0002] The KRAS gene (GeneID: 3845) is a member of the RAS gene family, located on human chromosome 12 and widely expressed in various tissues, including the intestine and brain. The GTPase encoded by the KRAS gene is involved in regulating cell growth. When certain sites in the KRAS gene mutate, the GTPase encoded by it becomes persistently activated, causing abnormal cell proliferation and leading to cancer. Clinical studies have shown that KRAS gene mutations are highly prevalent in various malignancies, such as colorectal cancer (30%-50%), pancreatic cancer (~85%), and non-small cell lung cancer (15%-25%). More than 97% of KRAS mutations occur at codons 12 and 13 of the KRAS gene, such as G12C, G12V, G12D, G12R, and G13D (Julian Downward, 2002). KRAS mutations are also a driver of tumor resistance. Patients with KRAS mutations have an extremely low response to EGFR inhibitors, at only approximately 0-5% (Jackman DM et al., 2009). Compared with patients with wild-type KRAS tumors, patients with KRAS mutations have shorter progression-free and overall survival and are more likely to experience recurrence and metastasis after surgery.

[0003] In recent decades, structural biology studies of KRAS have shown that GTP binds to the KRAS protein with an extremely high affinity (pM level), making it extremely difficult to develop competitive small molecule inhibitors against the KRAS protein itself. Therefore, the development of KRAS inhibitors in recent years has mainly focused on interfering with KRAS modifications, such as by designing compounds to irreversibly covalently bind to the cysteine ​​residues of the KRAS G12C mutation, locking the KRAS G12C mutant in an inactive state, thereby inhibiting the activity of KRAS G12C. However, there are currently no successful cases of drug development for other KRAS mutants (such as G12V and G13D). Moreover, as an intracellular protein, KRAS cannot be an effective target for antibody or chimeric antigen receptor T cell therapy.

[0004] T cell receptor gene engineered T cell (TCR-T) therapy involves transducing tumor antigen-specific T cell receptor (TCR) genes into normal T cells, enabling them to recognize antigen epitopes expressed on the surface of target cells, such as peptide-histocompatibility complex (p-MHC). This enhances or re-enables the T cells' ability to recognize tumor antigens, allowing them to specifically target and kill tumor cells. TCR-T therapy targets both tumor-associated antigens (TAAs) and tumor-specific antigens (TSAs). Because TAs are normal antigens in the body and are only highly expressed in tumor tissue, TCR-T therapies targeting TAs have poor therapeutic efficacy and high safety risks. However, TCR-T therapies targeting TSAs do not face these two issues. Currently, TSA targets primarily target cancer-driving gene mutations, such as KRAS G12V. These mutations create novel amino acid sequences in cancer cells, and TCR-T therapies developed with these mutations offer higher tumor specificity and a stronger immune response. In addition, whether such mutations can form effective TCR-T targets depends on the Major histocompatibility complex (MHC) that performs antigen presentation function, whose encoding gene is the human leukocyte antigen gene (HLA). Therefore, TSA targets are strictly HLA-restricted. Currently, most TCR-T therapies targeting TSA targets are restricted to HLA-A*02:01, which is a high HLA subtype in European and American populations. However, there are fewer effective TCR sequences for other more frequent HLA subtypes in Asian populations, including HLA-A*11:01 and HLA-A*24:02. Therefore, there is a need in this field to isolate TCRs that specifically bind to HLA-A*11:01 / KRAS G12V, and to transduce T cells with this TCR to construct engineered T cells targeting KRAS G12V, so that they can play a role in cellular immunotherapy.

[0005] Currently, the methods for obtaining TCRs mainly include screening from humanized HLA animal models, healthy human peripheral blood, or tumor-infiltrating lymphocytes (TILs) of tumor patients. However, for the method of screening TCRs from humanized HLA animal models, due to interspecies differences, the TCR diversity between humans and mice is very different, and it is very likely that the corresponding TCR does not exist in the animal, resulting in low screening efficiency. Even if an effective TCR sequence is obtained, it should be considered that non-human TCRs have not been screened by the human immune system. When used in humans, they may cause non-specific immune reactions, which will affect efficacy and pose safety risks. For the method of screening TCRs from healthy human peripheral blood, since the immune system of healthy people has not been exposed to tumor mutations, the low mutation antigen state of the body results in an extremely low percentage of TCRs targeting the antigen in the peripheral blood (as low as one in 100,000 or one in a million). Therefore, the positive rate of antigen-specific T cell expansion obtained by in vitro stimulation of a limited total amount of peripheral blood lymphocytes is low. In addition, the effective TCRs screened from T cells derived from the peripheral blood of healthy people through a single in vitro system have not been screened in vivo and may be screened out by the body's own immune system after being infused back into the body, and their effectiveness is not guaranteed. For the method of screening TCRs from TILs of tumor patients, since the number of TILs obtained from tumor tissue at one time is small, the in vitro culture and expansion of TILs is time-consuming and labor-intensive and has bias. Long-term in vitro TILs culture can lead to a large number of bystander T cells and reduce the percentage of target antigen-specific T cells. After the TILs are cultured, the affinity of the TCRs obtained by screening using T cell activation markers such as 4-1BB / OX40 / CD107a to bind to the target p-MHC is difficult to guarantee. Compared with the TCRs obtained by the method of screening with highly specific p-MHC polymers, their affinity is often poor and requires subsequent verification and affinity optimization through a large amount of screening work. Moreover, the same mutant antigen may be presented by different HLAs of tumor patients, so the effective TCR screened from TILs may not be the high-frequency HLA-restricted type in the population, and the population that can benefit from it is limited, which becomes an obstacle to later clinical development. Summary of the Invention

[0006] Therefore, to address the above-mentioned issues, the present invention provides a T cell receptor or antigen-binding fragment thereof that can specifically recognize and bind to the VVGAVGVGK-HLA-A*11:01 complex, cells and pharmaceutical compositions comprising the TCR, nucleic acids encoding the TCR, vectors and host cells for preparing the TCR, and methods for using the TCR to treat subjects. The TCR provided by the present invention is HLA-A*11:01-restricted, which is the HLA subtype that is predominant in the Asia-Pacific population, and thus can benefit more patients in the Asia-Pacific region.

[0007] In one aspect, the present invention provides an isolated T cell receptor or an antigen-binding fragment thereof, which can specifically recognize and bind to the VVGAVGVGK-HLA-A*11:01 complex, wherein the T cell receptor or the antigen-binding fragment thereof comprises an α chain variable region and a β chain variable region, the α chain variable region comprises the α chain complementary determining region CDR3 as shown in SEQ ID NO:5 or a variant having 1 or 2 amino acid residue changes with the sequence, and the β chain variable region comprises the β chain complementary determining region CDR3 as shown in SEQ ID NO:8 or a variant having 1 or 2 amino acid residue changes with the sequence.

[0008] In one embodiment, the α chain variable region comprises the α chain complementary determining regions CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 3, 4, and 5, respectively, or variants thereof having 1 or 2 amino acid residue changes compared to said sequences, and the β chain variable region comprises the β chain complementary determining regions CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 6, 7, and 8, respectively, or variants thereof having 1 or 2 amino acid residue changes compared to said sequences.

[0009] The amino acid sequence of TCR can be embedded into any suitable framework structure to prepare an effective chimeric TCR. As long as the framework structure is compatible with the CDR region of the TCR of the present invention, those skilled in the art can design or synthesize TCR molecules with corresponding functions based on the CDR region disclosed in the present invention. Therefore, the TCR molecule of the present invention refers to a TCR molecule comprising the above-mentioned α chain and β chain CDR region sequences and any suitable framework structure. In one embodiment, the T cell receptor or antigen-binding fragment thereof comprises an α chain variable region as shown in SEQ ID NO: 9, or an amino acid sequence having at least 90% sequence identity with the sequence; and a β chain variable region as shown in SEQ ID NO: 11, or an amino acid sequence having at least 90% sequence identity with the sequence.

[0010] In one embodiment, the T cell receptor or antigen binding fragment thereof further comprises a constant region.

[0011] The TCR of the present invention is a hybrid TCR comprising sequences derived from more than one species. For example, studies have shown that TCRs containing mouse TCR constant regions are more effectively expressed in human T cells than TCRs containing human TCR constant regions. Therefore, the TCR of the present invention may comprise a human variable region and a human T cell receptor constant region or a mouse T cell receptor constant region. In a preferred embodiment, the α chain and β chain constant regions of the TCR of the present invention are mouse T cell receptor constant regions, and those skilled in the art know or can obtain the mouse constant region amino acid sequence by consulting relevant books or the public database of IMGT (International Immunogenetics Information System).

[0012] In one embodiment, the T cell receptor or antigen-binding fragment thereof has an alpha chain as shown in SEQ ID NO: 17, or an amino acid sequence having at least 90% sequence identity to said sequence; and a beta chain as shown in SEQ ID NO: 18, or an amino acid sequence having at least 90% sequence identity to said sequence.

[0013] In one embodiment, the α chain and / or β chain of the T cell receptor or its antigen-binding fragment further comprises a signal peptide sequence, for example, a signal peptide sequence is comprised at the N-terminus of the α chain and / or β chain, preferably a signal peptide sequence is comprised at the N-terminus of the α chain and β chain.

[0014] In this context, the term "signal peptide" refers to a guide sequence located at the N-terminus of a nascent protein after translation, which guides the nascent protein to a designated expression site. A non-limiting example of a signal peptide is the α chain signal peptide sequence (MKRILGALLGLLSAQVCCVR) as shown in amino acids 1-20 of SEQ ID NO: 19, or the β chain signal peptide sequence (MGSRLLCWVLLCLLGAGPV) as shown in amino acids 1-19 of SEQ ID NO: 20. Those skilled in the art know or can obtain the signal peptide amino acid sequence by consulting relevant books or public databases.

[0015] In one embodiment, the T cell receptor or antigen-binding fragment thereof is soluble or membrane-bound.

[0016] In one embodiment, the T cell receptor can be used in any TCR configuration, such as a full-length T cell receptor, a soluble T cell receptor, or a single-chain T cell receptor.

[0017] In one embodiment, the TCR can be a full-length TCR comprising a full-length α chain and a full-length β chain.

[0018] In one embodiment, the TCR is a soluble TCR lacking one or more transmembrane regions and / or cytoplasmic regions. In one embodiment, a soluble TCR is produced by fusing the extracellular domain of the TCR of the invention to other protein domains (e.g., maltose binding protein, thioredoxin, human constant kappa domain, or leucine zipper).

[0019] In one embodiment, the TCR of the present invention may also be a single-chain TCR (scTCR), which includes Vα and Vβ connected by a peptide linker. Such scTCRs may include Vα and Vβ, each of which is connected to a TCR constant region. Alternatively, scTCRs may include Vα and Vβ, wherein Vα, Vβ, or both Vα and Vβ are not connected to a TCR constant region. Exemplary scTCRs are described in PCT Publications Nos. WO 2003 / 020763, WO 2004 / 033685, and WO 2011 / 044186, each of which is incorporated herein by reference in its entirety.

[0020] In one embodiment, the TCR of the present invention may include two polypeptide chains (e.g., α chain and β chain), wherein the chains have been engineered to each have a cysteine ​​residue that can form an interchain disulfide bond. Therefore, the TCR of the present invention may include two polypeptide chains connected by an engineered disulfide bond. Exemplary TCRs with engineered disulfide bonds are described in U.S. Patents Nos. 8,361,794 and 8,906,383, each of which is incorporated herein by reference in its entirety.

[0021] In another aspect, the present invention can also be provided in the form of a multivalent T cell receptor complex comprising at least two T cell receptor molecules, at least one of which is a T cell receptor or an antigen-binding fragment thereof as described above. The TCR complex of the present invention can be used to track or target cells presenting specific antigens in vitro or in vivo, and can also be used to generate intermediates for other multivalent TCR complexes with such applications.

[0022] In another aspect, the present invention provides a conjugate comprising the aforementioned isolated T cell receptor or antigen-binding fragment thereof or multivalent T cell receptor complex and an effector conjugated thereto.

[0023] 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 a novel activity to the molecule. In one embodiment, the effector is a biologically active compound or polypeptide (e.g., a compound or polypeptide that has an effect on cells targeted by a TCR), or a detectable marker.

[0024] In this context, the term "conjugation" refers to any method known in the art for functionally linking protein domains, including but not limited to: recombinant 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 one embodiment, conjugation to the effector can be performed by chemical or recombinant means, wherein the chemical means is the formation of a covalent bond between two molecules to form one molecule.

[0025] In one embodiment, the effector can be a therapeutic moiety. A therapeutic moiety refers to a compound or polypeptide that can be used as a therapeutic agent. The conjugate utilizes the targeting properties of the TCR so that the therapeutic moiety produces a therapeutic effect on the cells targeted by the TCR.

[0026] In one embodiment, the therapeutic moiety is selected from an immunostimulatory agent, such as an immunostimulatory cytokine or an immunostimulatory antibody. In certain exemplary embodiments, the immunostimulatory cytokine is 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, for example, including full-length proteins, active fragments or mutants thereof. For example, IL-2 refers to a polypeptide having IL-2 activity, which can be full-length IL-2, an active fragment or mutant of IL-2. In certain exemplary embodiments, the immunostimulatory antibody is 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 one embodiment, the immunostimulatory agent is selected from anti-CD3 antibodies, anti-CD28 antibodies, IL-2, IL-15, or any combination thereof.

[0027] In one embodiment, the therapeutic moiety is selected from a cytotoxic agent. As used herein, the cytotoxic agent includes any agent that is detrimental to cells (eg, kills cells).

[0028] In one embodiment, the cytotoxic agent is selected from an alkylating agent, a microtubule inhibitor or mitotic inhibitor, an antitumor antibiotic, an antimetabolite, a topoisomerase inhibitor, a tyrosine kinase inhibitor, a radionuclide agent, and any combination thereof.

[0029] In one embodiment, the effector is capable of increasing the solubility of the TCR. In one embodiment, the effector is selected from various parts of the heavy chain or light chain constant region of various subclass immunoglobulins (e.g., IgG, IgM, IgA, IgE). In one embodiment, the effector is selected from the constant region of a human immunoglobulin, such as a heavy chain constant region or a light chain constant region.

[0030] In one embodiment, the effector is selected from a detectable label. The detectable label of the present invention can be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such labels are well known in the art, and examples thereof include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C 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 ester compounds), magnetic beads, calorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g., streptavidin) modified with the above-mentioned 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 counter, 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 action of the enzyme on the substrate, and calorimetric labels are detected by simple visual coloring markers. In certain exemplary embodiments, the detectable label is selected from an enzyme, a radionuclide, a fluorescent dye, a luminescent substance (such as a chemiluminescent substance), or biotin.

[0031] In one embodiment, the TCR or antigen binding fragment thereof of the present invention is optionally conjugated to an effector via a linker (e.g., a peptide linker). In one embodiment, the effector is connected to the N-terminus or C-terminus of the TCR or antigen binding fragment thereof of the present invention.

[0032] In one embodiment, when the effector is a peptide or protein, the conjugate is preferably a fusion protein.

[0033] In another aspect, the present invention also provides a fusion protein comprising the aforementioned isolated T cell receptor or antigen-binding fragment thereof or multivalent T cell receptor complex and a peptide or protein expressed fused therewith.

[0034] In one embodiment, the TCR or its antigen binding fragment of the present invention is optionally fused to another peptide or protein via a connector (e.g., a peptide linker). In one embodiment, the peptide or protein is connected to the N-terminus or C-terminus of the TCR or its antigen binding fragment of the present invention.

[0035] In one embodiment, the peptide or protein can be selected from the various effectors that are peptides or proteins described above.

[0036] In one embodiment, the peptide or protein is selected from a therapeutic peptide or protein, an immunoglobulin constant region (eg, a human immunoglobulin constant region), a detectable protein label, or a protein tag.

[0037] In one embodiment, the protein tag is selected from His, Flag, GST, MBP, HA or Myc, etc. Those skilled in the art know how to select a suitable protein tag according to the desired purpose (eg, purification, detection or tracing).

[0038] In one embodiment, the peptide or protein is an anti-CD3 antibody, which is expressed as a fusion with the C-terminus or N-terminus of the α or β chain of the T cell receptor or antigen-binding fragment thereof.

[0039] In another aspect, the present invention provides an isolated nucleic acid molecule comprising a nucleic acid sequence encoding the aforementioned isolated T cell receptor or antigen-binding fragment thereof, or multivalent T cell receptor complex, or fusion protein.

[0040] The nucleotide sequence of the nucleic acid molecule of the present invention may be single-stranded or double-stranded, the nucleic acid molecule may be RNA or DNA, and may or may not contain introns.

[0041] In one embodiment, the nucleotide sequence does not contain introns but is capable of encoding a polypeptide of the present invention.

[0042] In one embodiment, the nucleic acid sequence is a nucleic acid sequence that has been codon-optimized for a host cell. It will be understood by those skilled in the art that different cells differ in the use of specific codons, and the codons in the sequence can be altered to increase expression levels depending on the type of cell. Codon usage tables for mammalian cells and a variety of other organisms are well known to those skilled in the art.

[0043] Those skilled in the art will appreciate that, due to the degeneracy of the genetic code, different nucleotide sequences can encode the same polypeptide. Therefore, the nucleic acid sequence encoding the TCR of the present invention can be identical to the nucleic acid sequence shown in Table 1 of the present invention or a degenerate variant.

[0044] In a preferred embodiment, the isolated nucleic acid molecule comprises a first nucleic acid sequence encoding the T cell receptor α chain and a second nucleic acid sequence encoding the T cell receptor β chain, wherein the first nucleic acid sequence comprises a nucleic acid sequence encoding the T cell receptor α chain variable region as shown in SEQ ID NO: 10 or its complementary sequence, and a nucleic acid sequence encoding the murine T cell receptor α chain constant region as shown in SEQ ID NO: 14 or its complementary sequence, more preferably, the first nucleic acid sequence comprises a nucleic acid sequence encoding the T cell receptor α chain as shown in SEQ ID NO: 21 or its complementary sequence; the second nucleic acid sequence comprises a nucleic acid sequence encoding the T cell receptor β chain variable region as shown in SEQ ID NO: 12 or its complementary sequence, and a nucleic acid sequence encoding the murine T cell receptor β chain constant region as shown in SEQ ID NO: 16 or its complementary sequence, more preferably, the second nucleic acid sequence comprises a nucleic acid sequence encoding the T cell receptor β chain as shown in SEQ ID NO: 22 or its complementary sequence; the first nucleic acid sequence and the second nucleic acid sequence are optionally linked by a nucleic acid sequence encoding a self-cleaving peptide, non-limiting examples of which include P2A, E2A, F2A or T2A.

[0045] In another aspect, the present invention provides a vector comprising the above-mentioned nucleic acid molecule, wherein the nucleic acid molecule is operably linked to an expression control sequence.

[0046] In one embodiment, the vector is a viral vector.

[0047] In one embodiment, the vector is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector or a baculoviral vector.

[0048] In another aspect, the present invention provides an engineered cell comprising the aforementioned nucleic acid molecule, or vector, or expressing the aforementioned isolated T cell receptor or antigen-binding fragment thereof, or multivalent T cell receptor complex or fusion protein.

[0049] In one embodiment, the engineered cells may be selected from immune cells, and the immune cells may be selected from lymphocytes.

[0050] In one embodiment, the immune cell is selected from T cells, tumor infiltrating lymphocytes (TIL), natural killer (NK) cells, natural killer T (NKT) cells, or any combination thereof. Exemplary immune cells that can be used to express the TCR of the present invention include PBMC, TIL and / or T cells.

[0051] In one embodiment, the T cells are selected from: αβT cells, γδT cells, iPSC-induced T cells, CD8+ 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, for example, constant natural killer T cells. In one embodiment, the immune cells comprise CD4+ T cells. It will be understood by those skilled in the art that immune cells can also include progenitor cells (precursor cells) of immune cells, wherein the progenitor cells can be induced in vivo or in vitro to differentiate into mature immune cells.

[0052] In another aspect, the present invention provides a method for preparing the aforementioned isolated T cell receptor or its antigen-binding fragment, or multivalent T cell receptor complex, or fusion protein, which comprises culturing the aforementioned engineered cells under conditions that allow protein expression, and recovering the T cell receptor or its antigen-binding fragment, or multivalent T cell receptor complex, or fusion protein from the cultured engineered cell culture.

[0053] Those skilled in the art will appreciate that the T cell receptors or antigen-binding fragments thereof, or multivalent T cell receptor complexes, or fusion proteins of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology. For example, DNA molecules encoding them can be obtained by chemical synthesis or PCR amplification; the resulting DNA molecules can be inserted into expression vectors and transfected into engineered cells; and the transfected engineered cells can then be cultured under specific conditions to express the T cell receptors or antigen-binding fragments thereof, or multivalent T cell receptor complexes, or fusion proteins of the present invention.

[0054] In another aspect, the present invention provides a method for preparing the aforementioned engineered cells, comprising: providing primary cells from a subject; introducing the aforementioned nucleic acid molecule or vector into the primary cells, wherein the nucleic acid molecule or vector comprises a nucleic acid sequence encoding the aforementioned isolated T cell receptor or its antigen-binding fragment, or multivalent T cell receptor complex, or fusion protein.

[0055] In one embodiment, the primary cells can be selected from the above-mentioned immune cells.

[0056] In one embodiment, the primary cells can be self / autologous ("self") or non-self ("non-self," e.g., allogeneic). "Self" refers to cells from the same subject; "allogeneic" refers to cells of the same species that are genetically different from the comparison cells.

[0057] In one embodiment, the method for introducing the nucleic acid molecule or vector into the cell is a conventional technical means in the art, non-limiting examples of which include calcium phosphate transfection, DEAE-dextran-mediated transfection, microinjection, electroporation, TALEN method, ZFN method, non-viral vector-mediated transfection (e.g., liposome) or viral vector-mediated transfection (e.g., lentiviral infection, retroviral infection, adenoviral infection), and other physical, chemical or biological means for transfer into host cells, such as transposon technology, CRISPR-Cas9 and other technologies.

[0058] In one embodiment, after introducing the nucleic acid molecule or vector into the cells, the method further comprises: expanding the obtained cells.

[0059] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned isolated T cell receptor or its antigen-binding fragment, or multivalent T cell receptor complex, or conjugate, or fusion protein, or nucleic acid molecule, or vector, or engineered cell; and a pharmaceutically acceptable carrier and / or excipient.

[0060] In one embodiment, the pharmaceutical composition further comprises an additional therapeutic agent, such as an anti-tumor agent or an immune-enhancing agent.

[0061] In one embodiment, the anti-tumor agent is selected from alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, antibodies specifically targeting tumor cells, and immune checkpoint inhibitors.

[0062] In one embodiment, the immune enhancer is selected from an immunostimulatory antibody or an immunostimulatory cytokine.

[0063] In another aspect, the present invention provides use of the aforementioned isolated T cell receptor or antigen-binding fragment thereof, or multivalent T cell receptor complex, or conjugate, or fusion protein, or nucleic acid molecule, or vector, or engineered cell, or pharmaceutical composition in the preparation of a medicament for inducing an immune response against a tumor having a KRAS G12V mutation in a subject, and / or preventing or treating a tumor having a KRAS G12V mutation in a subject.

[0064] In another aspect, the present invention provides a method for inducing an immune response against a tumor harboring a KRAS G12V mutation in a subject, and / or preventing or treating a tumor harboring a KRAS G12V mutation in a subject, comprising administering to the subject an effective amount of the aforementioned isolated T cell receptor or antigen-binding fragment thereof, or multivalent T cell receptor complex, or conjugate, or fusion protein, or nucleic acid molecule, or vector, or engineered cell, or pharmaceutical composition.

[0065] In one embodiment, the tumor having the KRAS G12V mutation is selected from acute lymphoblastic cancer, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, brain cancer, glioma, nasopharyngeal cancer, 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 intestine 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.

[0066] In one embodiment, the subject is HLA-A*11:01 positive.

[0067] In one embodiment, the isolated T cell receptor or antigen-binding fragment thereof, or multivalent T cell receptor complex, or conjugate, or fusion protein, or nucleic acid molecule, or vector, or engineered cell, or pharmaceutical composition can also be used in combination with another therapeutic agent or therapy, wherein the other therapeutic agent can be selected from anti-tumor agents or immunopotentiators, and the other therapy can be selected from surgery, chemotherapy, radiotherapy, targeted therapy, immunotherapy, hormone therapy, gene therapy or palliative care.

[0068] The isolated T cell receptor or its antigen-binding fragment, or multivalent T cell receptor complex, or conjugate, or fusion protein, or nucleic acid molecule, or vector, or engineered cell, or pharmaceutical composition of the present invention can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, lozenges, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the intended mode of administration and therapeutic use. The medicine of the present invention should be sterile and stable under production and storage conditions. A preferred dosage form is an injection. Such an injection can be a sterile injection solution. For example, a sterile injectable solution can be prepared by the following method: incorporating the necessary dose of the TCR or its antigen-binding fragment, conjugate, fusion protein, modified immune cell of the present invention, or a pharmaceutical composition comprising the same in an appropriate solvent, and optionally, simultaneously incorporating other desired ingredients (including but not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, isotonic agents, preservatives, diluents, or any combination thereof), followed by filtration sterilization. In addition, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze drying) for easy storage and use. Such sterile lyophilized powders can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% (w / v) NaCl), glucose solution (e.g., 5% glucose), a solution containing a surfactant (e.g., 0.01% polysorbate 20), a pH buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0069] The isolated T cell receptor or its antigen binding fragment, or multivalent T cell receptor complex, or conjugate, or fusion protein, or nucleic acid molecule, or vector, or engineered cell, or pharmaceutical composition of the present invention can be administered by any suitable method known in the art, including but not limited to, oral, oral, sublingual, ophthalmic, topical, parenteral, rectal, intrathecal, intracytoplasmic reticulum, inguinal, intravesical, topical (e.g., powder, ointment or drops), or nasal route. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous injection or push injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). It will be understood by the skilled person that the route and / or mode of administration will vary depending on the intended purpose. In one embodiment, the isolated T cell receptor or its antigen binding fragment, or multivalent T cell receptor complex, or conjugate, or fusion protein, or nucleic acid molecule, or vector, or engineered cell, or pharmaceutical composition of the present invention is administered by intravenous injection or push injection.

[0070] In another aspect, the present invention also provides a method for screening TCR, comprising the following steps:

[0071] 1) Obtain peripheral blood samples from tumor patients before and after treatment with mRNA vaccines or peptide vaccines targeting tumor-specific antigens;

[0072] 2) isolating peripheral blood mononuclear cells (PBMCs) from the peripheral blood sample after treatment with the mRNA vaccine or polypeptide vaccine in step 1), performing DC cell differentiation on the PBMCs, and stimulating the PBMCs with antigen epitope peptides corresponding to the tumor-specific antigen to promote the proliferation of antigen-specific T cells in the PBMCs;

[0073] 3) staining the antigen-specific T cells using a peptide-MHC complex tetramer of the antigen epitope peptide, and obtaining the antigen-specific T cell receptor clones by flow cytometry sorting;

[0074] 4) Single-cell sequencing to obtain the clonal frequency of antigen-specific TCR clones, the nucleic acid sequence information of their α and β chains, the αβ chain pairing information, and the CDR3 sequence information;

[0075] 5) Isolating PBMCs from peripheral blood samples before and after treatment with the mRNA vaccine or polypeptide vaccine described in step 1), extracting total RNA from the peripheral blood mononuclear cells, and performing T cell receptor bulk RNA sequencing, combined with bioinformatics analysis to quantitatively detect T cell clonal diversity in the CDR3 region;

[0076] 6) Analyze the percentage of T cell receptor clones that increase in peripheral blood before and after treatment with mRNA or peptide vaccines, and determine their CDR3 sequences;

[0077] 7) Comparing the CDR3 sequence obtained in step 6) with the CDR3 sequence obtained in step 4), the TCR present in both step 4) and step 6) is the target TCR and can be used for further functional screening;

[0078] Wherein, the steps 2)-4) and steps 5)-6) can be interchanged or performed simultaneously, and the tumor patient in step 1) meets the following conditions:

[0079] a) contains specific HLA alleles;

[0080] b) carrying tumor-specific antigens;

[0081] c) The patient is evaluated as having a clinical partial response (PR) or complete response (CR) after receiving treatment with an mRNA vaccine or polypeptide vaccine targeting the tumor-specific antigen.

[0082] In one embodiment, the increase in percentage content in step 6) means that the T cell receptor clone is not present in the peripheral blood sample of the patient before receiving the vaccine treatment, but is present in the peripheral blood sample of the patient after receiving the vaccine treatment, or the percentage content in the peripheral blood sample of the patient after receiving the vaccine treatment is ten times or more than the percentage content in the peripheral blood sample of the patient before receiving the vaccine treatment.

[0083] In this scenario, vaccine efficacy was evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1), which included complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). The term "complete response" or "CR" refers to the complete disappearance of all target lesions and the reduction of all pathological lymph nodes to normal size (short axis <10 mm); the term "partial response" or "PR" refers to a decrease of ≥30% in the sum of the diameters of all measurable target lesions from baseline, with the short diameter used for the sum of target nodules and the longest diameter used for the sum of all other target lesions; the term "progressive disease" or "PD" refers to a relative increase of at least 20% in the sum of the target lesion diameters, using the minimum sum of all target lesion diameters measured throughout the entire study as a reference (if the baseline measurement is the smallest, the baseline value is used as a reference). In addition, the absolute value of the sum of the diameters must increase by at least 5 mm (the appearance of one or more new lesions is also considered progressive disease); the term "stable disease" or "SD" refers to a decrease in the target lesions that does not reach the level of PR or the level of increase that does not reach the level of PD, but is somewhere in between. The minimum sum of the diameters can be used as a reference for research.

[0084] Compared to TCR sequences targeting HLA-A*11:01 / KRAS-G12V screened by existing systems, TCR sequences isolated from the peripheral blood of patients with effective mRNA immune stimulation and clinical treatment have higher safety, stronger specificity and affinity, and precise killing function. In addition, HLA-A*11:01 restriction can cover a wider range of Asian populations. The present invention provides T cell receptors or antigen-binding fragments thereof that specifically recognize the KRAS G12V mutant, nucleic acid molecules encoding the TCRs, vectors containing the nucleic acid molecules, engineered cells expressing the TCRs, and the use of the TCRs and engineered cells in treating tumors harboring the KRAS G12V mutation. The TCRs provided by the present invention can induce an immune response against tumors harboring the KRAS G12V mutation and thereby treat the aforementioned tumors in a subject. In addition, the TCRs provided by the present invention are HLA-A*11:01 restricted, which is the HLA subtype that is overwhelmingly predominant in the Asia-Pacific population, thus benefiting more patients in the Asia-Pacific region. In addition, this HLA restriction is also widely distributed in European, American, and Oceanian populations, thus having broad application prospects. Therefore, the present invention provides a new T cell-based immunotherapy for the treatment of KRAS G12V mutation-positive tumors, which has significant clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The present invention is described below with reference to the accompanying drawings by way of example only. It should be emphasized that the details shown are merely exemplary and illustrative of preferred embodiments of the present invention, and are intended to illustrate the principles and concepts of the present invention. Those skilled in the art will readily appreciate how to practice the present invention in various forms as described with reference to the accompanying drawings.

[0086] Figure 1: Flow cytometric analysis of antigen-specific T cells isolated from PBMCs of patients treated with KRAS G12V mRNA vaccine and whose tumors significantly shrank.

[0087] Figure 2: Single-cell sequencing detects high clonal frequency TCRs.

[0088] Figure 3: Number of monoclonal T cells in subjects before and after vaccination.

[0089] Figure 4: Lentiviral shuttle vector construct expressing target TCR.

[0090] Figure 5: Flow cytometry was used to detect whether the target TCR was correctly expressed in Jurkat cells.

[0091] Figure 6: Flow cytometry analysis of in vitro stimulation of Jurkat cells expressing antigen-specific TCR with KRAS WT peptide or KRAS G12V-9 peptide.

[0092] Figure 7: Detection of M5-TCR expression in engineered donor-derived T cells.

[0093] Figure 8: ELISpot assay to detect IFN-γ release.

[0094] Figure 9: ELISpot counts of IFN-γ formed in the co-culture system.

[0095] Figure 10: In vitro cytotoxicity assay of M5-TCR-T.

[0096] Figure 11: The inhibitory effect of M5-TCR-T on tumors in mice. DETAILED DESCRIPTION

[0097] Before elaborating on the present disclosure in more detail, it may be helpful to provide definitions of certain terms used herein. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those of ordinary skill in the art. Furthermore, the procedures used herein in immunology, molecular biology, biochemistry, nucleic acid chemistry, cell culture, and the like are conventional procedures widely used in the corresponding fields. In addition, to facilitate a better understanding of the present disclosure, definitions and explanations of relevant terms are provided below.

[0098] An entity that is not limited to a specific number should refer to one or more (species) of that entity; for example, "multivalent T cell receptor complex" should be understood to mean one or more (species) T cell receptor complex. Similarly, the terms "one or more" and "at least one" are used interchangeably herein without specific quantitative definitions.

[0099] As used herein, the term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 10% less than the specified numerical value and an upper limit that is 10% greater than the specified numerical value.

[0100] As used herein, the term "and / or," when used to link two or more alternatives, should be understood to mean any one of the alternatives or any two or more of the alternatives.

[0101] As used herein, the terms "comprising" or "including" are intended to include the recited 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 indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to a variable region "comprising" a specific sequence, it is intended to encompass a TCR variable region consisting of that specific sequence.

[0102] As used herein, the term "RAS" refers to a proto-oncogene whose encoded RAS protein has GTPase activity and participates in numerous signaling pathways regulating cell proliferation, differentiation, and apoptosis, such as MAPK, PI3K, and STAT signaling pathways. Human genes contain three RAS genes, namely HRAS (GeneID: 3265), NRAS (GeneID: 4893), and KRAS (GeneID: 3845). The three RAS genes have a high degree of sequence homology (>90%). RAS gene mutations are driving factors for cancer development, and RAS gene mutations are the most frequent proto-oncogene mutations. The sequences of 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 KRAS protein can be found in NCBI: NP_001356715.1, the sequence of NRAS protein can be found in NCBI: NP_002515.1, and the sequence of HRAS protein can be found in NCBI: NP_001123914.1.

[0103] As used herein, the term "KRAS" refers to the Kirsten rat sarcoma viral oncogene homolog. KRAS is an important member of the RAS protein family. KRAS is regulated upstream by the epidermal growth factor receptor EGFR family. EGFR signals can activate SOS proteins, thereby regulating the activation of KRAS. The inactivation and activation state transitions of KRAS proteins in cells are determined by the molecules they bind to. The guanine nucleotide exchange factor GEF catalyzes KARS to bind to GTP, activating KRAS; and the GTPase activating protein GAP can promote the hydrolysis of GTP bound to KRAS to GDP, thereby leading to KRAS inactivation. Activated KRAS regulates its downstream signaling pathways such as MAPK and PI3K, which are related to functions such as cell proliferation and cell migration. KRAS mutations cause it to continuously bind to GTP, maintaining an activated state, resulting in continuous activation of downstream signaling pathways, thereby promoting tumorigenesis.

[0104] As used herein, the terms "KRAS G12V," "G12V," or "G12V mutation" are used interchangeably to refer to a KRAS mutant in which the amino acid residue glycine at position 12 is mutated to valine, which can be specifically detected by the immune system of an organism.

[0105] As used herein, the terms "histocompatibility complex" and "MHC" are used interchangeably to refer to a group of tightly linked genes that determine whether transplanted tissues are identical, are closely related to immune responses, and primarily include MHC-I and MHC-II molecules. "MHC-I" refers to a dimer of the MHC class I α chain and the β2 microglobulin chain, while "MHC-II" refers to a dimer of the MHC class II α chain and the MHC class II β chain. The human MHC is called the human leukocyte antigen (HLA) complex.

[0106] As used herein, the term "peptide-MHC complex" or "p-MHC complex" or "p-MHC" refers to an MHC molecule (MHC class I or MHC class II) that contains a peptide bound to the art-recognized MHC peptide binding pocket. In some cases, the MHC molecule can be a membrane-bound protein expressed on the cell surface. In other cases, the MHC molecule can be a soluble protein that lacks a transmembrane region or a cytoplasmic region.

[0107] As used herein, the term "antigen" is any molecule that can be specifically detected by an organism's immune system.

[0108] As used herein, the term "tumor-associated antigen" or "TAA" refers to an antigen that is differentially expressed in tumor cells (eg, has significantly increased expression on tumor cells) compared to normal cells.

[0109] As used herein, the term "tumor-specific antigen" or "TSA" refers to an antigen that is unique to tumor cells and is not present in normal tissues and cells.

[0110] As used herein, the term "epitope" refers to a local region of an antigen (e.g., a peptide or peptide-MHC complex) to which a TCR can bind. In certain embodiments, the antigen is a peptide-MHC complex or a peptide presented by an MHC molecule.

[0111] As used herein, the terms "T cell receptor" and "TCR" are used interchangeably to refer to proteins on the surface of T cells that are responsible for the specific recognition of antigenic peptides bound to MHC (major histocompatibility complex). The T cell receptor or TCR is a T cell surface receptor that specifically recognizes specific antigenic peptides presented on the major histocompatibility complex (MHC). In the immune system, the binding of antigen-specific TCR to the p-MHC complex triggers direct physical contact between T cells and antigen-presenting cells (APCs), and then other molecules on the cell surfaces of both T cells and APCs can also interact, which causes a series of subsequent cell signaling and other physiological reactions, thereby allowing T cells with different antigenic specificities to exert immune effects on target cells. There are 4 TCR genes in the human genome: two encoding light chain TCRs: TRA genes encoding TCRα and TRG genes encoding TCRγ; two encoding heavy chain TCRs: TRB genes encoding TCRβ and TRD genes encoding TCRδ. Heavy chain TCR and light chain TCR form heterodimers to form a complete TCR. There are two types of TCR in humans: TCRα / β and TCRγ / δ, of which 95% of T cells express TCRα / β, called αβT cells; 5% of T cells express TCRγ / δ, called γ / δT cells. This ratio changes during individual development and in diseased states (such as leukemia), and also varies 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 TCR variable regions attached by flexible linkers, TCR chains connected by engineered disulfide bonds, etc., as long as the TCR retains its ability to recognize antigen targets.

[0112] As used herein, the term "variable region" or "variable domain" refers to the domain of the α chain or β chain (or the γ chain and δ chain for γδTCR) of the TCR that is involved in antigen recognition and binding. The variable domains of the α chain and β chain of the natural TCR (Vα and Vβ, respectively) generally have similar structures, with each domain comprising four generally conserved framework regions (FRs) and three complementarity determining regions (CDRs).

[0113] In TCR, the framework region separates the CDRs, and the CDRs are located between the framework regions (ie, in the primary structure). The Vα domain is encoded by two separate DNA fragments, the variable gene segment and the connecting gene segment (VJ). The Vβ domain is encoded by three independent DNA fragments (variable gene segment, diversity gene segment and connecting gene segment (VDJ)). Human TCR V, D and J alleles, 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. In addition, the Vα or Vβ domain can be used to separate the TCR that binds to a specific antigen from the TCR that binds to the antigen to screen the library of complementary Vα or Vβ domains, respectively.

[0114] 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 TCR α and β chains, which generally confer antigen specificity and / or binding affinity and are separated from each other by framework sequences in the primary structure. In some cases, framework amino acids may also contribute to binding, for example, they may also contact antigens or antigen-containing molecules. Typically, there are three CDRs in each variable region, of which CDR3 is considered to be the main CDR responsible for recognizing antigens, and its sequence is highly variable; CDR1 and CDR2 mainly or in some cases only interact with MHC, and their sequences are relatively conserved. The variable domain sequence can be aligned with a numbering scheme (e.g., Kabat numbering, Chothia numbering, International Immunogenetics Information System (IMGT) and Aho Antibody Database), and the equivalent residue positions can be annotated and different molecules can be compared using the Antigen Receptor Numbering and Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15: 298-300).

[0115] As used herein, the term "constant region" refers to a portion of a TCR encoded by a TRAC gene (for TCR α chain), TRBC1 or TRBC2 gene (for TCR β chain), TRDC gene (for TCR δ chain) or TRGC1 or TRGC2 gene (for TCR γ chain), optionally lacking all or part of the transmembrane region and / or all or part of the cytoplasmic region. In certain embodiments, the TCR constant region lacks the transmembrane region and the cytoplasmic region. The TCR constant region does not include amino acids encoded by TRAV, TRAJ, TRBV, TRBD, TRBJ, TRDV, TRDD, TRDJ, TRGV or TRGJ genes (see, e.g., T Cell Receptor Databook, (2001), LeFranc and LeFranc, Academic Press, ISBN 0-12-441352-8, which is incorporated herein by reference in its entirety). In the context of TCRs, the term "extracellular" refers to the portion(s) of the TCR chain that are located outside the cell, "transmembrane" refers to the portion(s) of the TCR chain that are embedded in the plasma membrane of a cell, and "cytoplasmic" refers to the portion(s) of the TCR chain that are located in the cytoplasm of a cell.

[0116] 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 comprises a TCR variable region and a TCR constant region comprising a TCR transmembrane region and a TCR cytoplasmic region. In certain embodiments, a full-length TCR comprises a mature full-length TCR α chain and a mature full-length TCR β chain. In certain embodiments, a full-length TCR comprises a mature full-length TCR γ chain and a mature full-length TCR δ chain.

[0117] 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 (parent TCR) as part of the TCR. The biological activity may include the ability to specifically bind to the same antigen (e.g., KRAS G12V mutant) or p-MHC complex that the parent TCR binds.

[0118] As used herein, the term "specific binding" or "specific recognition" refers to a non-random binding reaction between two molecules, such as a reaction between a TCR and an antigen to which it is directed (e.g., a specific peptide or a specific peptide-MHC complex combination). Typically, a TCR that specifically binds to an antigen does not bind to or binds to other antigens with a lower affinity. For example, an antigen-specific TCR binds to a target antigen with 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 nonspecific antigens. In certain embodiments, the TCR disclosed herein, or its antigen-binding fragment, specifically binds to a KRAS G12V mutant. In certain embodiments, the TCR disclosed herein, or its antigen-binding fragment, specifically binds to a VVGAVGVGK-HLA-A*11:01 complex.

[0119] As used herein, the term "immune cell" refers to any cell of the immune system with one or more effector functions. Immune cells typically include cells that play a role in immune responses, and they generally have hematopoietic origins. The term "effector function" refers to the specialized functions of immune cells, such as enhancing or promoting the function or reaction of an immune attack on a target cell (such as killing of a target cell, or inhibiting its growth or proliferation). For example, the effector function of a T cell, for example, can be cytolytic activity or auxiliary or activity including the secretion of cytokines. Examples of immune cells include T cells (such as α / βT cells and γ / δT cells), B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived macrophages.

[0120] As used herein, the term "T cell" is a kind of immune system cell that matures in the thymus and produces TCR. T cells can be naive (not exposed to antigens; Compared with TCM, the expression of CD62L, CCR7, CD28, CD3, CD127 and CD45RA increases, while the expression of CD45RO decreases), memory T cells (TM) (experienced antigen and long-survival cells) or effector T cells (experienced antigen cytotoxic T cells). TM can be further divided into central memory T cells (TCM, compared with original T cells, CD62L, CCR7, CD28, CD127, CD45RO and CD95 expression increases, and CD54RA expression decreases) and effector memory T cells (TEM, compared with original T cells or TCM, CD62L, CCR7, CD28, CD45RA expression decreases, while CD127 expression increases).

[0121] As used herein, the term "hematopoietic progenitor cell" is a cell derived from hematopoietic stem cells (HSC) or fetal tissue that is capable of further differentiation into mature cell types (e.g., cells of the T cell lineage). In certain embodiments, CD24 lo Lin - CD117 + Hematopoietic progenitor cells are useful. As defined herein, hematopoietic progenitor cells can include embryonic stem cells that are capable of further differentiating into cells of the T cell lineage. Hematopoietic progenitor cells can be derived from a variety of animals, including humans, mice, rats, or other mammals. "Thymocyte progenitor cells" or "thymocytes" are hematopoietic progenitor cells that reside in the thymus.

[0122] As used herein, the term "cytotoxic agent" includes any agent that is detrimental to (eg, kills) cells, such as a chemotherapeutic drug, a bacterial toxin, a plant toxin, or a radioactive isotope.

[0123] As used herein, the term "nucleic acid" refers to a chain of nucleotides of any length, and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate capable of being incorporated into a chain by DNA or RNA polymerase.

[0124] As used herein, the term "vector" refers to a construct that is capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the gene or sequence in the host cell. The vector can be introduced into a host cell by transformation, transduction, or transfection. Examples of vectors include, but are not limited to, viral vectors, plasmids, cosmids, or phage vectors. The vector can comprise a nucleic acid sequence that allows the gene or sequence of interest to replicate in the host cell, such as an origin of replication. The vector can also comprise one or more selectable marker genes and other genetic elements well known to those skilled in the art. The vector is preferably an expression vector comprising a nucleic acid according to the present invention, and the nucleic acid is operably connected to a sequence that allows the expression of the nucleic acid.

[0125] As used herein, the term "operably linked" refers to a functional connection between a nucleic acid expression regulatory sequence (for example, but not limited to, a promoter sequence, a transcription termination sequence, etc.) and a nucleic acid sequence encoding a target protein (for example, a coding sequence or an open reading frame), so that the transcription and translation of the nucleic acid sequence are controlled and regulated by the expression regulatory sequence.

[0126] As used herein, the term "engineered cell" refers to a cell into which an exogenous nucleic acid has been introduced, including the progeny of such cells. Engineered cells include "transfected cells," which include the primary transfected cell and its progeny, regardless of the number of passages. Progeny may not be identical to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny having the same function or biological activity as the cell screened or selected in the initial transfected cell are included herein.

[0127] As used herein, the term "isolated" means having been separated or purified from components with which it naturally accompanies it (eg, nucleic acids, proteins, or other naturally occurring biological or organic molecules).

[0128] As used herein, the term "identity" refers to the degree to which polynucleotide or polypeptide segments are identical in an alignment of sequences (e.g., nucleotide sequences or amino acid sequences). An alignment of sequences is generated by manually aligning two sequences, e.g., a sequence shown as provided herein and another sequence as a reference, to produce the highest number of matching elements, e.g., individual nucleotides or amino acids, while allowing gaps to be introduced into either sequence. The "identity score" of a sequence aligned with a reference sequence is the number of matching elements, divided by the full length of the reference sequence, excluding gaps introduced into the reference sequence by the alignment process.

[0129] As used herein, the term "variant" in the context of a polypeptide (including polypeptides) also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by the introduction of amino acid residue substitutions, deletions or additions. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, for example, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection / 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, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived. For example, compared to the TCR provided by the present invention, those skilled in the art know that it is possible to produce TCRs that incorporate minimal changes in their constant and / or variable regions but do not change the affinity of interaction with the VVGAVGVGK-HLA-A*11:01 complex, and the scope of the present invention includes such insignificant variants, and TCRs containing one or more conservative substitutions also constitute part of the present invention.

[0130] As used herein, the term "conservative substitution" means an amino acid substitution that does not adversely affect or change the expected properties of the protein / polypeptide comprising the amino acid sequence. For example, conservative substitutions can be introduced by 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 of residues physically or functionally similar to corresponding amino acid residues (e.g., having similar size, shape, charge, chemical properties, including the ability to form covalent bonds or hydrogen bonds, etc.). 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, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace the corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative amino acid substitutions are well known in the art (see, e.g., 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).

[0131] As used herein, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. Also, in the present invention, 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.

[0132] As used herein, "pharmaceutical composition" may refer to a composition for use in treating a disease or in vitro cell culture experiments. When used in the treatment of a disease, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of combining the active ingredient with an excipient that constitutes one or more adjunct ingredients. Typically, the composition is prepared by uniformly and thoroughly combining the active compound with a liquid excipient, a finely divided solid excipient, or both.

[0133] As used herein, the term "pharmaceutically acceptable" means that the substance or composition must be chemically and / or toxicologically compatible with the other ingredients of the formulation and / or the mammal to be treated therewith. Preferably, the "pharmaceutically acceptable" herein means approved by federal regulatory agencies or national governments or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopeia for use in animals, particularly humans.

[0134] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the particular target dosage form. Except to the extent that any conventional excipient is incompatible with the compounds of the present invention, for example, by producing any adverse biological effect or interacting in a deleterious manner with any other component of the pharmaceutically acceptable composition, their use is also contemplated by the present invention.

[0135] 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., cows, pigs, horses, sheep, goats, etc.), and experimental animals (e.g., rats, mice, guinea pigs, etc.). Typically, the subject is a human in need of treatment.

[0136] As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease or disorder or symptom (e.g., a tumor) in a subject. The term "treatment" refers to a method used to refer to obtaining a desired pharmacological and / or physiological effect. The effect can be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or disorder in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the development of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0137] As used herein, the term "effective amount" refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, a prophylactic amount is an amount sufficient to prevent, arrest, or delay the onset of the disease; a therapeutic amount is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and any other concurrently administered therapies.

[0138] The present invention is described in further detail below in conjunction with the examples, which are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. Modifications or improvements made without departing from the present invention are intended to fall within the scope of protection of the present invention. The experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications. In the following examples, unless otherwise specified, all cell lines used were purchased from the American Type Culture Collection (ATCC).

[0139] The present invention relates to sequences as shown in the following table:

[0140] Table 1:

[0141] Example 1. Obtaining peripheral blood mononuclear cells (PBMC) from tumor patients

[0142] 1.1 Identify cancer patients for blood sampling. Patients must meet the following criteria:

[0143] 1) The tumor patient's HLA genotype contains at least one HLA-A*11:01 allele;

[0144] 2) tumors harboring KRAS-G12V mutations;

[0145] 3) Tumor patients received KRAS-G12V mRNA or peptide vaccines and their tumors were significantly reduced.

[0146] 1.2 Isolation of peripheral blood mononuclear cells (PBMC) to stimulate target T cell proliferation in vitro

[0147] Peripheral blood of tumor patients was diluted with PBS buffer (purchased from Gibco) at a volume ratio of 1:1. The diluted whole blood and lymphocyte separation solution Ficoll-Paque (Cytiva, 17144003) were mixed at a volume ratio of 4:3 and centrifuged at 400×g for 40 min. After the centrifugation, the buffy coat layer was aspirated with a Pasteur tube to obtain peripheral blood mononuclear cells (PBMCs). After washing PBMCs twice with PBS buffer, PBMCs were isolated at a concentration of 1×10 6 The cells were inoculated at a density of 1:1 / mL into a dedicated culture medium and cultured at 37°C / 5% CO2 for 24 h. The dedicated culture medium was RPMI 1640 medium containing 5% (v / v) inactivated human AB serum, 100 ng / ml GM-CSF, 100 ng / ml IL-4 and 100 ng / ml Flt3-L.

[0148] On the second day, the induced PBMCs were resuspended and centrifuged, and the cells were resuspended in stimulation medium and plated at 10 per well in a 96-well plate. 5 The cells were divided into three groups at a cell density of 10 wells: a WT peptide stimulation group (10 wells), a KRAS G12V mutant epitope peptide stimulation group (10 wells), and a blank control group (5 wells). Peptides were added to the corresponding groups at a final concentration of 1 mM, mixed thoroughly, and the cell suspension was added to a U-shaped 96-well plate at a rate of 200 μL / well and incubated in a 37°C / 5% CO2 incubator for 24 hours. The stimulation medium consisted of RPMI 1640 medium containing 5% (v / v) inactivated human AB serum, 100 ng / ml GM-CSF, 100 ng / ml IL-4, 100 ng / ml Flt3-L, 10 ng / ml IL-6, 10 ng / ml IL-1b, 10 ng / ml TNFα, 100 ng / ml PGE2, and 50 ng / mL LPS.

[0149] On day 3, 100 μL of culture medium was aspirated from the 96-well plate and 100 μL of 2× feeding medium was added in a half-well medium exchange. The cells were then cultured for another 48 hours in a 37°C / 5% CO2 incubator. The 2× feeding medium consisted of RPMI 1640 supplemented with 5% (v / v) inactivated human AB serum, 10 ng / ml IL-2, 10 ng / ml IL-7, and 10 ng / ml IL-15.

[0150] On the fifth day, 100 μL of the culture medium in the 96-well plate was aspirated and discarded, and 100 μL of 2× feeding medium was added by half-medium replacement. The cells were placed in a 37° C. / 5% CO 2 incubator and cultured for a further 72 hours.

[0151] On day 8, 100 μL of the culture medium in the 96-well plate was aspirated and replaced with 100 μL of safe culture medium using a half-fluid exchange. The cells were then incubated in a 37°C / 5% CO2 incubator for another 24 hours. The complete culture medium was RPMI 1640 supplemented with 5% (v / v) inactivated human AB serum.

[0152] On day 9, resuspend the cells from the same group in the 96-well plate and transfer them to a 1.5 mL centrifuge tube. Centrifuge at 1500 rpm for 5 minutes. Discard the supernatant and use the cell pellet for subsequent tetramer staining.

[0153] Example 2. Obtaining HLA-A*11:01 / KRAS G12V-specific T cell clones and single-cell sequencing

[0154] The PE-labeled tetramer staining reagent used in this example is HLA-A*11:01 / KRAS G12V (G12V-9mer-tetramer). This tetramer can mark HLA-A*11:01 / KRAS G12V-specific T cells as positive cells. The control tetramer staining reagents are HLA-A*11:01 / KRAS WT (WT-tetramer) and HLA-A*11:01 / KRAS G12V (G12V-10mer-tetramer). The above three reagents were purchased from Beijing Borma Biotechnology Co., Ltd. Stimulated PBMCs were harvested and stained with the tetramers described above (staining methods were performed according to the reagent instructions). Positive T cells were then flow cytometry sorted. As shown in Figure 1, patients who received the KRAS G12V mRNA vaccine and were assessed as having a partial response (30% tumor reduction) showed a certain proportion of KRAS G12V-specific T cells (marked by red boxes) within their PBMCs. These T cells were labeled by the G12V-9mer-tetramer but not by the WT-tetramer or G12V-10mer-tetramer, indicating that these T cells were highly specific for HLA-A*11:01 / KRAS G12V. These positive cells were flow cytometry sorted, the supernatant removed by centrifugation, and resuspended in serum-free DMEM for single-cell sequencing on 10x Genomics. After sequencing, the top 10 double-positive TCR clones with the highest clonal frequency were selected as candidate TCRs (Figure 2). Among them, the fifth T cell clone was identified through sequencing and multiple experimental verification and was determined to be an HLA-A*11:01-Kras-G12V antigen-specific TCR, named M5-TCR. Its TCRα and β chain genotypes are shown in Table 2.

[0155] Table 2. Genotypes of the α and β chains of M5-TCR

[0156] After sequencing, the sequence information of the α chain and β chain of the M5-TCR are as follows: the amino acid sequence and nucleotide sequence of the TCRα chain variable region are SEQ ID NOs: 9 and 10, respectively; the amino acid sequence and nucleotide sequence of the TCRβ chain variable region are SEQ ID NOs: 11 and 12, respectively.

[0157] The α chain of M5-TCR was identified to contain CDRs with the following amino acid sequence:

[0158] αCDR1-DSVNN (SEQ ID NO: 3)

[0159] αCDR2-IPSGT (SEQ ID NO: 4)

[0160] αCDR3-CAAVLSMEYGNKLVF (SEQ ID NO:5)

[0161] The beta chain contains a CDR with the following amino acid sequence:

[0162] βCDR1-SGHRS (SEQ ID NO: 6)

[0163] βCDR2-YFSETQ (SEQ ID NO: 7)

[0164] βCDR3-CASTWTGLPEETQYF (SEQ ID NO:8).

[0165] Example 3. T cell receptor sequencing of peripheral blood of patients receiving tumor vaccine

[0166] PBMCs from vaccine recipients were isolated according to the method in Example 1. RNA was extracted from the PBMCs using a kit (RNeasy PowerLyzer Tissue & Cells Kit, QIAGEN). RNA samples were quality-checked using a nucleic acid quality analyzer (2100 Bioanalyzer System, Agilent Technologies). Samples that passed the quality check were subsequently used for cDNA library construction. For specific library construction procedures, refer to the product manual for the TruSeq RNA Exome Kit (Illumina). The cDNA library was sequenced using a HiSeq 2500 (Illumina) sequencing machine with a read length of 150 nucleotides at each end. The sequencing depth required a read count of 2.5 × 10 for each sample. 7. The raw sequencing data were statistically analyzed using Salmon v.0.12.0 and then aligned to the reference genome (GENCODE v.29). TCR was assembled using MiXCR v.3.0.125 software, and finally the different TCR clones were distinguished and counted by the CDR3 sequence of the TCRβ chain. As shown in Figure 3, the number of T cells of clone No. 5 proliferated more than 10 times after vaccination compared to before vaccination, and after comparison, this clone was identical to the M5-TCR clone sequence obtained by single-cell sequencing in Example 2, and was the same T cell clone. This data shows that clone No. 5 can proliferate in large quantities both in the patient's body and during in vitro culture, and it is very likely that it is a T cell that has an immune response to the VVGAVGVGK-HLA-A*11:01 antigen.

[0167] Example 4. Construction of TCR genes and vectors for HLA-A*11:01 / KRAS G12V-specific T cell clones

[0168] As shown in Figure 4, DNA encoding M5-TCR was synthesized according to the sequence TRAV-MusAC-P2A-TRBV-MusBC, wherein TRAV is the α chain variable region of M5-TCR (its amino acid sequence and nucleotide sequence are shown in SEQ ID NOs: 9 and 10, respectively), MusAC is the mouse TCR α chain constant region (its amino acid sequence and nucleotide sequence are shown in SEQ ID NOs: 13 and 14, respectively), and the amino acid sequence and nucleotide sequence of the α chain are shown in SEQ ID NOs: 19 and 21, respectively; TRBV is the M5-TCR β chain variable region (its amino acid sequence and nucleotide sequence are shown in SEQ ID NOs: 11 and 12, respectively), MusBC is the mouse TCR β chain constant region (its amino acid sequence and nucleotide sequence are shown in SEQ ID NOs: 15 and 16, respectively), and the amino acid sequence and nucleotide sequence of the β chain are shown in SEQ ID NOs: 20 and 22, respectively; P2A is a self-cleaving peptide (its amino acid sequence and nucleotide sequence are shown in SEQ ID NOs: 23 and 24, respectively). The TRAV-MusAC-P2A-TRBV-MusBC sequence (whose amino acid sequence and nucleotide sequence are shown in SEQ ID NOs: 25 and 26, respectively) was cloned into the lentiviral shuttle vector pLvx-EF1a-IRES-GFP to form the lentiviral vector pLvx-EF1a-TRAV-MusAC-P2A-TRBV-MusBC-IRES-GFP (abbreviated as M5-TCR) capable of expressing M5-TCR. At the same time, an empty pLvx-EF1a-IRES-GFP vector of this vector was used as a control group (abbreviated as pLvx-Ctrl).

[0169] Example 5. HLA-A*11:01 / KRAS G12V-specific T cell receptor lentiviral packaging

[0170] Lentivirus was prepared by express-mediated transient transfection of 293T cells, and lentivirus containing the desired TCR gene was packaged using a second-generation lentiviral packaging system. 293T cells were transfected with three plasmids (the lentiviral shuttle plasmid containing M5-TCR or pLvx-Ctrl described in Example 4, and two plasmids containing other components necessary for the construction of infectious but non-replicating lentiviral particles) using express-mediated transient transfection. For transfection, 2×10 cells were seeded in 15 cm culture dishes on day 0. 7293T cells were plated evenly on the culture dish to a confluency slightly above 50%. On day 1, plasmids were transfected to package the M5-TCR and pLvx-Ctrl control viruses. The expression plasmids were mixed with the packaging plasmid psPAX and the envelope plasmid pMD.2G. The following amounts were used for a 15 cm diameter culture dish: 20 μg:12 μg:8 μg. The transfection reagent Lipo2000 (Invitrogen, product number 11668019) was used at a 2:1 ratio of plasmid to transfection reagent. 80 μl was used per 15 cm dish. The specific operation is as follows: the expression plasmid and packaging plasmid are added to 2 mL of OPTI-MEM (Gibco, catalog number 31985-070) culture medium according to the above dosage, mixed evenly, and allowed to stand at room temperature for 5 minutes to form a DNA mixture; the corresponding amount of transfection reagent Lipo2000 is mixed evenly with 2 mL of OPTI-MEM culture medium, and allowed to stand at room temperature for 5 minutes. The DNA mixture and the transfection reagent mixture are mixed together and allowed to stand at room temperature for 30 minutes, and then added to 293T cells that have been converted into 12 ml of OPTI-MEM. The culture dish is gently shaken to mix the culture medium evenly, and then cultured at 37°C / 5% CO2 for 8 hours. The transfection medium is removed and replaced with DMEM (Gibco, product number C11995500bt) complete medium containing 10% fetal bovine serum, and cultured at 37°C / 5% CO2. On days 3 and 4, the culture supernatant containing the packaged lentivirus was collected and centrifuged at 3000 × g for 15 minutes to remove cell debris. The supernatant was then filtered through a 0.45 μm filter (Merck Millipore, product number SLGP033RB) and concentrated by centrifugation. The specific concentration method was performed according to the instructions of the Lentivirus Concentration Kit (Clonetech, product number 632200). Most of the supernatant was removed and the supernatant was finally concentrated to 1 ml. The aliquots were packaged and frozen at -80°C. The concentrated samples were subjected to lentiviral titer determination according to the instructions of the p24 ELISA kit (Takara, product number 631476).

[0171] Example 6. Construction of Jurkat cells expressing target TCR

[0172] Jurkat cells were revived and cultured in RPMI 1640 medium (Gibco, product number 11875101) supplemented with 10% FBS (Gibco, product number 10099141C) at 37°C / 5% CO2. Concentrated lentivirus expressing the M5-TCR-specific T cell receptor gene or pLvx-Ctrl control virus was added at an MOI of 2 and infected by centrifugation at 32°C and 200×g for 90 minutes. After infection, the lentiviral infection medium was removed and the cells were resuspended in RPMI 1640 medium supplemented with 10% FBS and 50 IU / ml IL-2 and 10 ng / ml IL-7. The cells were cultured at 37°C / 5% CO2 for 3 days. GFP-positive Jurkat cells, indicating lentiviral recombinant expression, were isolated by flow cytometry and expanded in culture for subsequent experiments.

[0173] Example 7. HLA-A*11:01 / KRAS G12V Tetramer Staining

[0174] In this experiment, the GFP-positive Jurkat cells sorted in Example 6 were labeled with the tetramer (tetramer-PE) with PE fluorescence used in Example 2 to detect the expression and specificity of the target TCR.

[0175] According to the reagent instructions, G12V-9mer-tetramer-PE was thoroughly mixed with the GFP-positive Jurkat cells sorted in Example 6 and incubated on ice for 40 minutes. APC-labeled anti-mouse TCRβ constant region (mouse TCR-βC-APC) antibody (Biolegend, H57-597) was then added and incubated on ice for another 15 minutes. The samples were washed twice with PBS containing 2% FBS, and double-positive Jurkat cells labeled with G12V-9mer-tetramer and mouse TCR-βC-APC were analyzed using a Cytek Aurora flow cytometer. Data were analyzed using Cytek SpectroFlo software. The data in Figure 5 demonstrate that the TCR can be stably expressed on the surface of Jurkat cells and that the recombinantly expressed TCR can recognize the HLA-A*11:01 / KRAS 9mer-tetramer with high specificity.

[0176] Example 8. Co-culture Activation

[0177] The following peptides for in vitro stimulation were custom-synthesized by Jiangsu GenScript Pharmaceuticals: a short peptide VVGAGGVGK (SEQ ID NO: 1) representing the amino acid sequence from positions 8 to 16 of wild-type KRAS (also referred to herein as the "KRAS WT peptide"), and a short peptide VVGAVGVGK (SEQ ID NO: 2) representing the amino acid sequence from positions 8 to 16 of KRAS G12V, where the amino acid at position 12 of KRAS is mutated from G to V (also referred to herein as the "KRAS G12V-9 peptide").

[0178] KRAS WT peptide and KRAS G12V-9 peptide were resuspended in DMSO and pulsed in vitro to a final concentration of 10 μM in the Panc1 human pancreatic cancer cell line (genotype: HLA-A*11:01). The cells were then thoroughly mixed with the GFP-positive Jurkat cells isolated in Example 6 and co-cultured for 24 hours. The mixed cells were collected and stained with G12V-9mer-tetramer and an antibody against CD69 (Biolegend, FN50), a T cell activation marker. The samples were washed twice with PBS containing 2% FBS and then detected using a Cytek Aurora flow cytometer for double-positive Jurkat cells labeled with G12V-tetramer-PE and CD69. Data were analyzed using Cytek SpectroFlo software. The data in Figure 6 show that HLA-A*11:01 of PANC1 cells can activate Jurkat cells expressing specific TCR only when loaded with KRAS G12V-9 peptide, and there is no activation effect when loaded with KRAS WT peptide. It also shows that the recombinantly expressed TCR can effectively transmit signals to the cell after recognizing the HLA-A*11:01 / KRAS G12V antigen, promote the activation of effector cells, and have physiological functions.

[0179] Example 9. Preparation of primary M5-TCR-T cells and detection of their interaction with target cells

[0180] Preparation of Kras G12V / HLA-A*1101 target cells

[0181] The CDS sequence of the wild-type Kras gene transcript (NCBI: NM_004985.5_191..757; SEQ ID NO: 27) 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:28) was cloned into the lentiviral vector Phage-IRES-RFP to construct eukaryotic recombinant expression plasmids Phage-IRES-KrasG12WT-RFP and Phage-IRES-KrasG12V-RFP. Lentiviral packaging was performed using the method in Example 5, and human pancreatic cancer cell line PANC1 cells (G12WT-Panc1 and G12V-Panc1) stably expressing wild-type Kras and containing the Kras-G12V mutation were constructed by lentiviral infection. The HLA-A*11:01 naturally expressed in these cells can present the Kras mutant peptide VVGAVGVGK to the cell surface to form an HLA-A*11:01-VVGAVGVGK complex. Therefore, G12V-Panc1 cells can be used as target cells in subsequent experiments, and G12WT-Panc1 can be used as control target cells.

[0182] M5-TCR-T cell preparation

[0183] Peripheral blood lymphocytes were collected from two healthy donors (D1 and D2), and T cells were sorted and counted using magnetic beads. CD3 / CD28 antibody-coupled magnetic beads (Gibco, 11132D) were added to the T cell culture system in an amount equal to the number of cells to activate the T cells. After 24 hours of activation, the M5-TCR-expressing lentivirus obtained in Example 5 was added and infected by centrifugation. The virus-infected T cells were cultured in vitro for 7 days to obtain a sufficient number of M5-TCR-T cells. These cells were used as effect cells for subsequent experiments, and uninfected T cells (Mock T) were used as control cells.

[0184] M5-TCR-T cell positive rate detection

[0185] The M5-TCR expression positivity of T cells after virus infection was detected by flow cytometry. Antibodies against CD8 and CD4 were used to distinguish the two main types of T cells: cytotoxic (CD8+) and helper (CD4+). Antibodies against the constant region of mouse TCR were used to detect the M5-TCR expression positivity of these two types of T cells. At the same time, the M5-TCR-T cells were stained with the Kras G12V / HLA-A*1101 tetramer used in Example 7 to further confirm the specificity of the primary TCR-T (Figure 7). The results showed that the proportions of CD8+ and CD4+ positive T cells in donor-derived T cells were 55.25% and 38.98%, respectively. After engineering modification, the Kras G12V / HLA-A*1101 tetramer-positive proportions were 49.04% and 50.7%, respectively, and there were no WT tetramer-positive cells. The above data indicate that after engineering modification, most donor T cells stably express M5-TCR, and this TCR is highly specific for Kras G12V / HLA-A*1101.

[0186] Detection of M5-TCR-T cell activation by target cells

[0187] The cell suspensions of G12WT-Panc1 and G12V-Panc1 target cells were centrifuged at room temperature at 200-250 × g for 5 minutes, the supernatant was removed, and the cells were resuspended in RPMI 1640 complete medium containing 1% FBS and counted. The cells were diluted to 4 × 10 5 The prepared cell suspension was plated onto a 96-well ELISpot plate (Mabtech, 3420-4HPT-10) with 100 μL of cell suspension (4×10 4 The above effector cells M5-TCR-T and its control cells Mock T were collected and diluted to 1.6×10 6 cells / mL, 50 μL cell suspension per well (4×10 4 The mixing method of the above cells for co-culture is shown in the following table:

[0188] Table 3:

[0189] Table 4:

[0190] Each of the above combinations was replicated twice. After incubation at 37°C for 24 h, the ELISpot Plus: Human IFN-γ (HRP) product instructions were followed for plate washing, antibody incubation, color development, and termination. The 96-well plate was placed on an enzyme-linked immunosorbent assay (AID, model ELR08IFL) and the corresponding parameters were set for detection.

[0191] The data from this experiment showed that T cells from two healthy donors that underwent TCR-T engineering could secrete large amounts of IFN-γ upon stimulation with target cells, but the unengineered cells could not be stimulated to secrete IFN-γ by target cells (Figures 8 and 9). This data indicates that the M5-TCR can effectively recognize the Kras G12V mutant antigen peptide presented by HLA-A*1101, and that control target cells expressing wild-type Kras cannot effectively activate the M5-TCR-T (Figures 8 and 9), indicating that the M5-TCR has a high degree of specificity for distinguishing between the Kras G12V mutant antigen peptide presented by HLA-A*1101 and the wild-type antigen peptide.

[0192] Example 10. Detection of the killing function of M5-TCR-T cells on target cells

[0193] This embodiment evaluates the killing function of M5-TCR-T on target cells by detecting the release of lactate dehydrogenase (LDH) in the co-culture system. When the cells are damaged, the permeability of the cell membrane changes, and LDH is released from the cell to the extracellular space. This release is proportional to the number of cells lysed, so the degree of cell damage can be reflected by detecting the quantification of LDH in the co-culture system. M5-TCR-T cells or Mock T cells (E) and G12WT-Panc1 or G12V-Panc1 target cells (T) are co-cultured according to 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 is quantified using an LDH detection kit (Dongren Chemical Technology, CK12). The results showed that only when M5-TCR-T cells were co-cultured with G12V-Panc1 target cells did they result in significant LDH release, and the amount of LDH released had a significant dose-response relationship with different effector cell / target cell ratios (Figure 10). M5-TCR-T cells only had a specific killing function against G12V-Panc1 target cells.

[0194] Example 11. Detection of the tumor-suppressing effect of M5-TCR-T cells in vivo

[0195] This example further demonstrates its tumor-killing function by detecting the inhibitory effect of M5-TCR-T on subcutaneous tumors in immunodeficient mice. scid Il2rg em1Smoc ) dorsal subcutaneously, with a target cell inoculation rate of 5×10 6 On the 30th day after tumor formation (when the tumor enters a rapid growth phase), the mice were divided into two groups according to the tumor growth status. M5-TCR-T cells or control Mock T cells were injected into the mice through the tail vein at a dose of 2×10 7 , and monitor changes in tumor volume. The data show that the tumor growth curves of the two groups of mice were very close before T cell inoculation, but on the 7th day after T cell inoculation, the tumor growth of mice inoculated with M5-TCR-T cells was significantly inhibited and continued to shrink. At the same time, the tumors of the control group mice inoculated with Mock T cells were not inhibited and continued to grow (Figure 11). The experimental data show that after the donor T cells are engineered to express M5-TCR, they can be effectively activated by the HLA-A*1101-Kras G12V mutant antigen peptide complex on the surface of tumor cells in mice, and exert a killing function, thereby inhibiting the occurrence and development of tumors. This further illustrates that the M5-TCR sequence provided by the present invention has very high clinical application potential.

[0196] The above embodiments are only provided for understanding the method and core concept of the present invention. It should be noted that, without departing from the principles of the present invention, a number of improvements and modifications may be made to the present invention by a person skilled in the art, and such improvements and modifications shall fall within the scope of protection of the claims of the present invention.

Claims

1. An isolated T cell receptor or antigen-binding fragment thereof, which can specifically recognize and bind to the VVGAVGVGK-HLA-A*11:01 complex, wherein: The T cell receptor or antigen-binding fragment thereof comprises an α chain variable region and a β chain variable region, wherein the α chain variable region comprises the α chain complementary determining region CDR3 as shown in SEQ ID NO: 5 or a variant thereof having 1 or 2 amino acid residue changes compared with the sequence, and the β chain variable region comprises the β chain complementary determining region CDR3 as shown in SEQ ID NO: 8 or a variant thereof having 1 or 2 amino acid residue changes compared with the sequence.

2. The T cell receptor or antigen-binding fragment thereof according to claim 1, wherein The α chain variable region comprises the α chain complementary determining regions CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 3, 4, and 5, respectively, or variants thereof having 1 or 2 amino acid residue changes compared with the sequences, and the β chain variable region comprises the β chain complementary determining regions CDR1, CDR2, and CDR3 as shown in SEQ ID NOs: 6, 7, and 8, respectively, or variants thereof having 1 or 2 amino acid residue changes compared with the sequences, respectively.

3. The T cell receptor or antigen-binding fragment thereof according to claim 1 or 2, comprising the α chain variable region as shown in SEQ ID NO: 9, or an amino acid sequence having at least 90% sequence identity to said sequence; and the β chain variable region as shown in SEQ ID NO: 11, or an amino acid sequence having at least 90% sequence identity to said sequence. 4 . The T cell receptor or antigen-binding fragment thereof according to claim 1 , further comprising a constant region. 5 . The T cell receptor or antigen-binding fragment thereof according to claim 4 , wherein the constant region is a human T cell receptor constant region or a murine T cell receptor constant region.

6. The T cell receptor or antigen-binding fragment thereof according to claim 4, which comprises an α chain as shown in SEQ ID NO: 17, or an amino acid sequence having at least 90% sequence identity with said sequence; and a β chain as shown in SEQ ID NO: 18, or an amino acid sequence having at least 90% sequence identity with said sequence. 7 . The T cell receptor or antigen-binding fragment thereof according to claim 1 , wherein the α chain and / or β chain further comprises a signal peptide.

8. The T cell receptor or antigen-binding fragment thereof according to claim 1, which is soluble or membrane-bound. 9 . The T cell receptor or antigen-binding fragment thereof according to claim 1 , wherein the T cell receptor is a full-length T cell receptor, a soluble T cell receptor or a single-chain T cell receptor.

10. A multivalent T cell receptor complex comprising at least two T cell receptor molecules, wherein at least one T cell receptor molecule is the T cell receptor or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. A conjugate comprising the isolated T cell receptor or antigen-binding fragment thereof according to any one of claims 1 to 9 or the multivalent T cell receptor complex according to claim 10 and an effector conjugated thereto, wherein the effector is selected from an antibody, an antibody fragment, a cytokine, a cytotoxic agent, an immunoglobulin constant region, a detectable label or any combination thereof, and the T cell receptor or antigen-binding fragment thereof and the effector have or do not have a linker.

12. A fusion protein comprising the isolated T cell receptor or antigen-binding fragment thereof according to any one of claims 1 to 9 or the multivalent T cell receptor complex according to claim 10 and a peptide or protein expressed fused therewith, wherein the peptide or protein is selected from an antibody, an antibody fragment, a cytokine, a cytotoxic protein, an immunoglobulin constant region, a detectable marker, a protein tag, or any combination thereof, and the T cell receptor or antigen-binding fragment thereof and the peptide or protein may or may not have a linker.

13. The fusion protein according to claim 12, wherein the antibody expressed by fusion with the T cell receptor or its antigen-binding fragment is an anti-CD3 antibody linked to the C-terminus or N-terminus of the α or β chain of the T cell receptor or its antigen-binding fragment.

14. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding the isolated T cell receptor or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multivalent T cell receptor complex according to claim 10, or the fusion protein according to claim 12 or 13. The nucleic acid molecule according to claim 14 , wherein the nucleic acid sequence is codon-optimized for a host cell.

16. A vector comprising the nucleic acid molecule according to claim 14 or 15, wherein the nucleic acid molecule is operably linked to an expression control sequence. The vector according to claim 16 , which is a viral vector. The vector according to claim 16 , which is a lentiviral vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector or a baculoviral vector.

19. An engineered cell comprising the nucleic acid molecule of claim 14 or 15, or comprising the vector of any one of claims 16-18, or expressing the isolated T cell receptor or antigen-binding fragment thereof of any one of claims 1-9, or the multivalent T cell receptor complex of claim 10, or the fusion protein of claim 12 or 13.

20. The engineered cell of claim 19, wherein the engineered cell is an immune cell.

21. The engineered cell of claim 20, wherein the immune cell is selected from hematopoietic progenitor cells, T cells, tumor infiltrating lymphocytes, natural killer cells, or any combination thereof.

22. The engineered cell of claim 21, wherein the T cell is a CD8+ T cell or a CD4+ T cell.

23. A method for preparing the isolated T cell receptor or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multivalent T cell receptor complex according to claim 10, or the fusion protein according to claim 12 or 13, comprising culturing the engineered cell according to any one of claims 19 to 22 under conditions that allow protein expression, and recovering the T cell receptor or antigen-binding fragment thereof, or multivalent T cell receptor complex, or fusion protein from the cultured engineered cell.

24. A method for preparing the engineered cell according to any one of claims 19 to 22, comprising: providing primary cells from a subject; The nucleic acid molecule of claim 14 or 15, or the vector of any one of claims 16 to 18, is introduced into the primary cells, wherein the nucleic acid molecule or vector comprises a nucleic acid sequence encoding the isolated T cell receptor or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multivalent T cell receptor complex according to claim 10, or the fusion protein according to claim 12 or 13.

25. A pharmaceutical composition comprising the isolated T cell receptor or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multivalent T cell receptor complex according to claim 10, or the conjugate according to claim 11, or the fusion protein according to claim 12 or 13, or the nucleic acid molecule according to claim 14 or 15, or the vector according to any one of claims 16 to 18, or the engineered cell according to any one of claims 19 to 22; and a pharmaceutically acceptable carrier and / or excipient.

26. Use of the isolated T cell receptor or antigen-binding fragment thereof according to any one of claims 1 to 9, or the multivalent T cell receptor complex according to claim 10, or the conjugate according to claim 11, or the fusion protein according to claim 12 or 13, or the nucleic acid molecule according to claim 14 or 15, or the vector according to any one of claims 16 to 18, or the engineered cell according to any one of claims 19 to 22, or the pharmaceutical composition according to claim 25 in the preparation of a medicament for inducing an immune response against a tumor having a KRAS G12V mutation in a subject, and / or preventing or treating a tumor having a KRAS G12V mutation in a subject.

27. The method of claim 26, wherein the tumor having the KRAS G12V mutation is selected from the group consisting of acute lymphoblastic cancer, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, brain cancer, glioma, nasopharyngeal cancer, 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 intestine 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.

28. The use according to claim 26, wherein the subject is HLA-A*11:01 positive.

29. The use according to claim 26, which can also be used in combination with another therapeutic agent.

30. A method for screening T cell receptors, comprising the following steps: 1) Obtain peripheral blood samples from tumor patients before and after treatment with mRNA vaccines or peptide vaccines targeting tumor-specific antigens; 2) isolating peripheral blood mononuclear cells from the peripheral blood sample after treatment with the mRNA vaccine or polypeptide vaccine in step 1), performing DC cell differentiation on the cells, and stimulating the peripheral blood mononuclear cells with antigenic epitope peptides corresponding to the tumor-specific antigen to promote the proliferation of antigen-specific T cells in the peripheral blood mononuclear cells; 3) staining the antigen-specific T cells using a peptide-MHC complex tetramer of the antigen epitope peptide, and obtaining the antigen-specific T cell receptor clones by flow cytometry sorting; 4) Single-cell sequencing to obtain the clonal frequency of antigen-specific T cell receptor clones and determine their CDR3 sequences; 5) isolating peripheral blood mononuclear cells from the peripheral blood samples before and after treatment with the mRNA vaccine or polypeptide vaccine described in step 1), extracting total RNA from the peripheral blood mononuclear cells, and performing bulk RNA sequencing to quantitatively detect the frequency of T cell receptor clones; 6) Analyze the percentage of T cell receptor clones that increase in peripheral blood before and after treatment with mRNA or peptide vaccines, and determine their CDR3 sequences; 7) comparing the CDR3 sequence obtained in step 6) with the CDR3 sequence obtained in step 4), and the T cell receptor present in both step 4) and step 6) is the target T cell receptor; Wherein, the steps 2)-4) and steps 5)-6) can be interchanged or performed simultaneously, and the tumor patient in step 1) meets the following conditions: a) contains specific HLA alleles; b) carrying tumor-specific antigens; c) The patient is evaluated as having clinical partial remission or complete remission after receiving treatment with an mRNA vaccine or polypeptide vaccine targeting the tumor-specific antigen.

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