Novel anti-tumor effector T cell subsets

Abstract

This invention discloses a novel CD8 + T cells and CD4 + T cell subsets. The CD8... + T cell subsets include: subsets characteristically expressing GZMK and PDCD1; subsets characteristically expressing GZMK, GPR183, IL7R, and ZNF683; subsets characteristically expressing GZMK and CXCL13; subsets characteristically expressing GZMK and ZNF683; and subsets expressing at least one of the following KIR genes: KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL3. The CD4+... + T cell subsets include: subsets that characteristically express FOXP3, TNFRSF9, and CTLA4; and subsets that characteristically express FOXP3 and BACH1.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically, to a novel subset of effector T cells for anti-tumor activity. Background Technology

[0002] Immune checkpoint blockade (ICB), particularly targeting CD8 expression + The inhibitory receptor programmed cell death 1 (PD-1) on T cells is one of the most successful immunotherapies for treating a wide range of advanced human cancers. However, the vast majority of patients with different types of cancer fail to benefit from this treatment. Combining ICBs with other therapies has improved the situation of poor response to anti-PD-1 monotherapy in some cancer patients. In particular, the combination of ICBs with anti-angiogenic therapy has improved efficacy in a variety of cancers, including hepatocellular carcinoma (HCC) and biliary tract cancer.

[0003] Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide, and chronic hepatitis B virus (HBV) infection is the primary causative factor of HCC in China. However, treatment options for HCC are limited, and the clinical success rate of immunotherapy is low. The combination of anti-PD-1 inhibitors with lenvatinib (a multi-target tyrosine kinase inhibitor that simultaneously targets the vascular endothelial growth factor receptor) significantly improved the response rate in HCC patients from 17% with anti-PD-1 monotherapy to 45%. However, the immune mechanisms underlying response and tolerance in this combination therapy remain unclear, hindering further efficacy improvements and the development of more successful treatments.

[0004] CD8 +T cells are the primary target of PD-1 blockade and the main force in killing tumor cells. T cells possess a specific receptor molecule, the T cell receptor (TCR), expressed on their cell surface, which can recognize and distinguish between self-antigens and exogenous antigens. Through antigen receptor responses, they transmit intracellular signals, promote cell proliferation, and initiate various immune responses, such as enhancing the production of inflammatory cytokines and chemokines. The TCR recognizes and binds to the major histocompatibility complex (MHC; in humans, it is called human leukocyte antigen, HLA) expressed by antigen-presenting cells, as well as antigenic peptides, forming a peptide-MHC (pMHC)-TCR complex. This complex distinguishes between self-antigens and non-self-antigens and recognizes the antigenic peptide. The TCR is a heterodimeric receptor molecule composed of two TCR polypeptide chains. The TCR gene consists of multiple regions encoded by different parts of the genome, including the V (variable region), J (connector region), D (variable region), and C (constant region). During T cell differentiation, these gene segments undergo gene rearrangement in various combinations. α- and γ-chain TCRs express genes composed of VJCs, while β- and δ-chain TCRs express genes composed of VDJCs. Most T cells express αβ-TCRs, and a minority express γδ-TCRs with specific functions. α- and β-chain TCR molecules form complexes with multiple CD3 molecules (CD3ζ, CD3ε, CD3γ, and CD3δ chains). The region where TCR molecules directly bind to the pMHC complex surface consists of three complementary regions (CDRs) within the V region: CDR1, CDR2, and CDR3. CDR3 specifically includes a portion of the V region, a portion of the J region, and a VDJ region formed by random sequences, forming the most diverse antigen recognition site. After TCRs recognize the pMHC complex, they activate intracellular signaling and initiate multiple T-cell immune responses.

[0005] Tumor antigens are divided into two main categories: tumor-specific antigens (TSA) and tumor-associated antigens (TAA). TSA possesses high tumor specificity and originates from viral antigens, gene coding sequence mutations or rearrangements, and antigens encoded by carcinoembryonic genes, possessing the potential to elicit an immune response. Tumor cells and other antigen-presenting cells use MHC I to present antigenic peptides derived from tumor-specific antigens to CD8+ T cells. The TCRs of CD8+ T cells specifically bind to the MHC I-antigen peptide complex, recognizing target cells and synthesizing IL-2. These cells then proliferate and differentiate into cytotoxic T lymphocytes (CTLs), which have a specific killing effect on tumor cells, thereby killing them. CD8+ T cells are not a homogeneous population but rather comprise subsets with different functional states. Studying the composition of the tumor-infiltrating T cell population and elucidating the functional states and interrelationships of each subset is crucial for providing new insights into T-cell-based immunotherapy.

[0006] On the other hand, early diagnosis, early treatment, and prognosis of tumors are crucial for improving treatment outcomes and patient survival rates. Identifying reliable tumor markers using molecular biology techniques is essential for predicting clinical outcomes, targeted therapy, and evaluating treatment efficacy. Currently discovered tumor prognostic markers mainly include tumor suppressor genes, oncogenes, cell cycle regulators, apoptosis regulators, invasion and metastasis-related markers, and telomerases. However, molecular markers suitable for monitoring and assessing tumor immune status are still limited. Understanding the interaction between the immune system and cancerous tissue is crucial for developing and improving immunotherapies, and also provides a means of assessing and monitoring the body's tumor immune status. However, the types, differentiation, and suppression pathways of tumor-infiltrating lymphocytes (TILs) infiltrating cancerous tissue are not yet fully understood. Currently, it is known that TILs contain populations of exhausted T cells, dysfunctional T cells, or senescent T cells.

[0007] Given the complexity of human immune cells, especially T cell types, there is a need for a more precise understanding of the classification, differentiation process, and functional genes of tumor-associated immune cells. This would provide more potential immune targets for the development of tumor immunotherapy drugs, as well as molecular markers that can be used for tumor prognosis.

[0008] In recent years, the rapidly developing single-cell sequencing technology has provided strong support for the study of gene expression differences in both population cells and single cells. Currently, several single-cell transcriptome deep sequencing methods have been developed, including SRT-Seq (single-cell tagged reverse transcription sequencing), Smart-Seq and Smart-Seq2, Cell-Seq (cell expression by linear amplification and sequencing), and PMA-Seq (Phi29-mRNA amplification and sequencing), as well as a variety of matching bioinformatics analysis methods. Summary of the Invention

[0009] The present invention aims to at least partially solve one of the technical problems in the related art.

[0010] The inventors of this invention studied HBV patients receiving neoadjuvant therapy with anti-PD-1 combined with lenvatinib. + T cells from HCC patients were analyzed using single-cell RNA sequencing (scRNA-seq), single-cell TCR sequencing (scTCR-seq), and flow cytometry to detect HBV. + This invention was completed by studying the single-cell gene expression profile of T cells from HCC patients, isolating and characterizing T cell subsets that reflect the body's tumor immune status, and further identifying new characteristic genes expressed by these cell subsets and the relationship between these characteristic genes and tumor prognosis.

[0011] Therefore, this invention is broadly directed to a series of markers, methods, compounds, compositions, and articles thereof that can be used to identify or characterize, and optionally to isolate, classify, separate, or enrich T cell subsets associated with tumor immunity. The T cells include CD8+. + T cells and CD4 + T cells.

[0012] More specifically, the inventors of this application have discovered a series of markers that can be used independently or collectively to accurately identify, sort, enrich, and / or characterize T cell subsets derived from tumors. Using selected biochemical techniques, by associating the markers of this invention with T cells in tumor tissue, it is possible to enrich, isolate, or purify T cell subsets that reflect the tumor immune status.

[0013] The markers disclosed in this invention can identify or characterize T cell subsets from tumors, constituting a universal characterization of these tumor immune cells, and can be used for the elucidation of therapeutic targets and the screening of drug compounds. Furthermore, they can be used in both clinical and non-clinical settings for the diagnosis, prognosis, classification, monitoring, and management of cancer patients, as well as for providing related kits or other manufactured products.

[0014] Based on this, in one aspect, the present invention proposes a CD8 + T cell subsets and CD4 + T cell subsets. According to an embodiment of the present invention, the CD8... + T cell subsets include the characteristic genes GZMK and PDCD1. According to an embodiment of the present invention, the CD8... + T cell subsets include: characteristic genes GZMK, GPR183, IL7R, and ZNF683. According to an embodiment of the present invention, the CD8... + T cell subsets include the characteristic genes GZMK and CXCL13. According to an embodiment of the present invention, the CD8... + T cell subsets include the characteristic genes GZMK and ZNF683. According to an embodiment of the present invention, the CD8... + T cell subsets include those expressing at least one of KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL3. According to an embodiment of the invention, the CD4... + T cell subsets include the characteristic genes FOXP3, TNFRSF9, and CTLA4. According to an embodiment of the present invention, the CD4... + T cell subsets include the characteristic genes FOXP3 and BACH1.

[0015] In another aspect, the present invention provides an antigenic epitope peptide. According to embodiments of the present invention, the amino acid sequence of the antigenic epitope peptide includes at least one of the amino acid sequences shown in SEQ ID NO:249-258; or at least one of the amino acid sequences having 90% sequence identity with it.

[0016] In another aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the aforementioned antigenic epitope peptide.

[0017] In another aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule.

[0018] In another aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises carrying the aforementioned nucleic acid molecule or the aforementioned expression vector; or expressing the aforementioned antigenic epitope peptide.

[0019] In another aspect, the present invention provides an antigen. According to embodiments of the present invention, the antigen comprises the aforementioned antigenic epitope polypeptide.

[0020] In another aspect, the present invention provides the use of the aforementioned antigenic epitope peptide, the aforementioned nucleic acid molecule, the aforementioned expression vector, and the aforementioned recombinant cells in the preparation of a drug for the prevention of hepatitis B virus infection or the treatment of hepatitis B virus-related diseases.

[0021] In another aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises the aforementioned antigenic epitope polypeptide.

[0022] In another aspect, the present invention provides the use of the aforementioned antigenic epitope peptide, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned pharmaceutical composition in screening TCRs that specifically recognize hepatitis B virus.

[0023] In another aspect, the present invention provides a separate TCR. According to an embodiment of the present invention, the isolated TCR comprises: a TCRα chain variable region and / or a TCRβ chain variable region, wherein the TCRα chain variable region comprises a CDR3 sequence selected from at least one of the following: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 and SEQ ID NO:72, or an amino acid sequence thereof in a conserved modified form; the TCRβ chain variable region comprises a CDR3 sequence selected from at least one of the following: SEQ ID NO:75, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 and SEQ ID NO:72, or an amino acid sequence thereof. The amino acid sequences of SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:141 and SEQ ID NO:144, or their conservatively modified forms.

[0024] In another aspect, the present invention provides a nucleic acid molecule. According to embodiments of the present invention, the nucleic acid molecule encodes the aforementioned isolated TCR.

[0025] In another aspect, the present invention provides an expression vector. According to an embodiment of the present invention, the expression vector carries the aforementioned nucleic acid molecule.

[0026] In another aspect, the present invention provides a recombinant cell. According to embodiments of the present invention, the recombinant cell comprises carrying the aforementioned nucleic acid molecule or the aforementioned expression vector; or expressing the aforementioned isolated TCR.

[0027] In another aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises the aforementioned isolated TCR, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell.

[0028] In another aspect, the present invention provides the use of the aforementioned isolated TCR, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of hepatitis B virus infection and / or related diseases caused by hepatitis B virus.

[0029] In another aspect, the present invention provides a method for preventing and / or treating hepatitis B virus infection and / or related diseases caused by hepatitis B virus. According to embodiments of the invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned pharmaceutical composition.

[0030] In another aspect, the present invention provides a method for screening the aforementioned isolated TCRs. According to an embodiment of the present invention, the method comprises: (i) forming an HLA-antigen epitope peptide complex by combining HLA and an antigen epitope peptide, and treating the HLA-antigen epitope peptide complex with T cells to screen for target T cells that specifically react with the HLA-antigen epitope peptide complex; and (ii) analyzing the TCR sequence and characteristic genes of the specifically reacting target T cells to screen for isolated TCRs.

[0031] In another aspect, the present invention provides a method for screening the aforementioned antigenic epitope peptides. According to an embodiment of the present invention, the method includes: forming an HLA-antigenic epitope peptide complex with HLA and the antigenic epitope peptide to be screened; and treating the HLA-antigenic epitope peptide with T cells to screen for the antigenic epitope peptide.

[0032] In another aspect, the present invention provides a T cell expressing the aforementioned isolated TCR or carrying the aforementioned nucleic acid molecule.

[0033] In another aspect, the present invention provides a method for screening the aforementioned T cells. According to an embodiment of the present invention, the method includes forming an HLA-antigen epitope peptide complex by combining HLA and an antigen epitope peptide, and then treating the HLA-antigen epitope peptide complex with T cells to screen for T cells that specifically respond to the HLA-antigen epitope peptide complex.

[0034] In another aspect, the present invention provides a method for identifying T cells with HBV antigen specificity. According to an embodiment of the present invention, the method includes (1) obtaining gene expression information in the T cells to be identified by staining and / or sequencing; (2) determining whether the T cells to be identified have HBV antigen specificity based on the aforementioned characteristic genes; and (3) analyzing and obtaining T cells with HBV antigen specificity from the T cells to be identified.

[0035] In another aspect, the present invention provides a method for identifying TCRs with HBV antigen specificity. According to an embodiment of the present invention, the method includes (1) obtaining gene expression information in T cells to be identified by staining and / or sequencing; (2) determining whether the T cells to be identified have HBV antigen specificity based on the aforementioned characteristic genes; and (3) analyzing and obtaining the TCR sequence of the T cells with HBV antigen specificity in the T cells to be identified.

[0036] In another aspect, the present invention provides a method for activating the aforementioned T cell subsets. According to embodiments of the present invention, the method includes co-culturing the aforementioned T cell subsets with genetically engineered artificial antigen-presenting cells expressing co-stimulatory molecules, or administering exogenous cytokines to activate the T cell subsets.

[0037] In another aspect, the present invention provides a method for inhibiting the aforementioned T cell subsets. According to embodiments of the present invention, the method includes identifying the T cell subsets using proteins specifically highly expressed by the aforementioned T cell subsets, targeting specific chemokines or receptors on their surface to block their infiltration, or using small molecule inhibitors to interfere with the regulation of downstream signal transduction by key intracellular signaling molecules, thereby inhibiting the T cell subsets.

[0038] In another aspect, the present invention provides a method for inducing the aforementioned T cell subsets. According to embodiments of the present invention, the method includes adding an antigenic epitope peptide or exogenous cytokine to an in vitro co-culture system with antigen-presenting cells to induce the generation of the aforementioned T cell subsets.

[0039] In another aspect, the present invention provides a method for amplifying the aforementioned T cell subsets. According to an embodiment of the present invention, the method includes: identifying and enriching the T cell subsets using the aforementioned characteristic genes, and adding antibodies or exogenous cytokines to promote the proliferation of the T cell subsets.

[0040] In another aspect, the present invention provides a method for in vitro expansion of antigen-specific T cells. According to an embodiment of the present invention, the method includes (1) isolating T cells from peripheral blood of a patient or donor, or from tumor tissue of a patient, based on the aforementioned characteristic genes; (2) culturing the T cells in vitro, and adding at least one of an antibody, cytokines, an antigen epitope peptide-MHC complex, and antigen-presenting cells to promote the expansion of the T cells; (3) detecting the tumor-killing function of the expanded T cells; wherein the antigen-specific T cells are the T cells identified by the aforementioned method.

[0041] In another aspect, the present invention provides the use of the aforementioned T cells in the preparation of drugs for treating and / or preventing cancer.

[0042] In another aspect, the present invention provides a method for preparing T cells containing TCRs. According to an embodiment of the present invention, the method comprises (1) introducing a TCR α or TCR β gene into a viral or non-viral vector for gene expression; (2) creating a virus from a retroviral vector expressing the TCR α and TCR β genes; (3) independently and sequentially infecting lymphocytes collected from a patient with the virus carrying the TCR α and TCR β genes for transfection, or creating a gene expression retroviral vector including the TCR α and TCR β genes for one-time transformation of the two genes; introducing the DNA or mRNA of the TCR into T cells using electroporation or liposomes; (4) demonstrating that the TCR α / TCR β heterodimer is expressed on the surface of the T cells; wherein the TCR α or TCR β gene is derived from the aforementioned TCRs of the T cells, the aforementioned isolated TCRs, the TCRs screened by the aforementioned method, or the TCRs identified by the aforementioned method.

[0043] In another aspect, the present invention provides a method for adoptive cell therapy. According to an embodiment of the present invention, the method includes obtaining cells from a patient or donor, screening for specific T cell subsets based on the aforementioned characteristic genes, genetically modifying or not modifying the T cells, inducing or expanding the T cell subsets in vitro, and reinfusing the expanded T cell subsets into the patient for treatment; wherein the T cell subsets used for adoptive cell therapy are selected from the aforementioned T cells, T cells identified by the aforementioned method, or T cell subsets activated, expanded, or induced by the aforementioned method.

[0044] In another aspect, the present invention provides a method for a novel antitumor therapy by inhibiting or eliminating regulatory T cells. According to an embodiment of the present invention, the method includes identifying regulatory T cells using the aforementioned characteristic genes of T cells, screening and knocking out the identified regulatory T cells based on target genes, thereby inhibiting or eliminating the regulatory T cells; wherein the regulatory T cells include the aforementioned CD8+. + T cell subsets, the aforementioned CD4 + T cells and the aforementioned CD4 + At least one of the T cell subsets.

[0045] In another aspect, the present invention provides a method for preparing a cancer vaccine. According to an embodiment of the present invention, the method includes (1) synthesizing the aforementioned antigenic epitope peptide; (2) mixing the antigenic epitope peptide with an adjuvant or a carrier, or extracting monocytes from a patient, culturing them in vitro, and loading them with an antigen; (3) presenting the antigenic epitope peptide to antigen-specific T cells in vitro, and testing the cytotoxic activity of the antigen-specific T cells or the level of secreted IFN-γ and TNF-α cytokines to verify the immunogenicity of the antigenic epitope peptide; (4) verifying the immunogenicity in a tumor model or an immune humanization model; and (5) delivering it to the patient's tumor or lymph nodes.

[0046] In another aspect, the present invention provides a method for treating cancer using virus-specific T cells. According to an embodiment of the present invention, the method includes (1) preparing T cells using peripheral blood mononuclear cells or donor-derived blood; (2) stimulating the T cells with viral peptides, virus-infected target cells, or a vector encoding viral antigens; (3) expanding the corresponding virus-specific T cells under culture conditions containing supplemental cytokines; (4) ensuring, through in vitro killing experiments, that the expanded T cells have high specificity and low toxicity and can effectively kill target cells; (5) directly reinfusing the expanded T cells into the patient via intravenous injection, or reinfusing them into the patient after combining them with specific targeting technologies to enhance their specificity and function; wherein the T cells are derived from T cells identified by the aforementioned method.

[0047] In another aspect, the present invention provides a method for using bispecific or trispecific antibodies targeting T cells and other cells. According to embodiments of the present invention, the method includes selecting antigenic targets on T cells and other cells, designing and constructing antibodies, performing in vitro affinity testing and functional assays, and detecting efficacy, toxicity, and conducting clinical trials; wherein the T cells are derived from the aforementioned T cells, T cells identified by the aforementioned method, or T cells activated, expanded, or induced by the aforementioned method; the other cells include immune cells or non-immune cells, including: immune cells such as T cells, B cells, dendritic cells, macrophages, monocytes, NK cells, and ILC cells, or at least one of tumor cells, tumor-associated fibroblasts, endothelial cells, and epithelial cells.

[0048] In another aspect, the present invention provides a method for combination therapy. According to an embodiment of the present invention, the method comprises: a combination of the foregoing method with an immune checkpoint inhibitor or a small molecule drug; wherein the immune checkpoint inhibitor includes at least one of a CTLA-4 inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor; and the small molecule drug includes at least one of methotrexate, fluorouracil, and imatinib.

[0049] In another aspect, the present invention provides the use of the aforementioned T cell subsets in the treatment of autoimmune diseases. According to embodiments of the invention, this includes inhibiting or eliminating effector CD8+. + T cell subsets can reduce inflammation, increase the number or function of regulatory T cells to suppress excessive immune responses, or simultaneously target CD8. + Combined therapy of T cells and regulatory T cells.

[0050] In another aspect, the present invention provides a method for inhibiting the conversion of T cells into regulatory T cells. According to an embodiment of the present invention, the method includes: inhibiting CD8... + The expression of TGFB1 in T cells, or the inhibition of CD4 + Expression of BACH1 in T cells.

[0051] In another aspect, the present invention provides the use of an inhibitor in the preparation of a tumor therapeutic drug, said inhibitor inhibiting the expression of a target gene or a protein that inhibits the expression of a target gene, said target gene being selected from at least one of BACH1, TGFB1, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KLRC2, KLRC3, IKZF2, and TYROBP.

[0052] In another aspect, the present invention provides a method for enriching the aforementioned T cell subsets. According to an embodiment of the present invention, the method includes: contacting a population of immune cells infiltrating a tumor with a binding agent, said binding agent binding to at least one target gene or a protein or protein fragment expressed therein; and sorting the immune cells bound to said binding agent to enrich the T cell subsets; wherein the target gene is selected from the aforementioned genes.

[0053] In another aspect, the present invention provides a method for diagnosis or evaluation. According to an embodiment of the present invention, the method includes: (1) classifying a sample of immune cells infiltrating a tumor obtained from a subject as CD8. + T cells and CD4 + T cells; (2) the separated CD8 + T cells are contacted with at least one binding agent, which binds to a target gene or its expressed protein or protein fragment, wherein the target gene is selected from the aforementioned genes; (3) the separated CD4 + T cells are contacted with at least one binding agent, which binds to a target gene or its expressed protein or protein fragment, wherein the target gene is selected from the aforementioned genes.

[0054] In another aspect, the present invention provides a CD8 for diagnosis or monitoring. + A biomarker set for T cells. According to embodiments of the present invention, the biomarker set comprises the aforementioned genes or proteins or protein fragments expressed by the characteristic genes.

[0055] In another aspect, the present invention provides a CD4 for diagnosis or monitoring. + A biomarker set for T cells. According to embodiments of the present invention, the biomarker set comprises the aforementioned genes or proteins or protein fragments expressed by the characteristic genes.

[0056] In another aspect, the present invention provides a diagnostic reagent for diagnosis or monitoring. According to embodiments of the invention, the diagnostic reagent comprises a binder that binds to the aforementioned gene or its expressed protein or protein fragment.

[0057] In another aspect, the present invention provides a kit for diagnosing or monitoring tumor prognosis. According to embodiments of the invention, the kit comprises the aforementioned detection reagents.

[0058] In another aspect, the present invention provides a diagnostic reagent for diagnosis or monitoring. According to an embodiment of the invention, the kit comprises the aforementioned diagnostic reagent.

[0059] In another aspect, the present invention provides a method for identifying novel antitumor effector T cell subsets. According to embodiments of the present invention, the method includes: (1) Single-cell RNA sequencing (scRNA-seq), single-cell TCR sequencing (scTCR-seq), and flow cytometry analysis were performed on T cells of HBV+HCC patients who received neoadjuvant therapy with anti-PD-1 combined with lenvatinib. (2) Analyze the single-cell gene expression profile of T cells in cancer tissues and paired peripheral blood, and isolate and characterize T cell subsets that reflect the body's tumor immune status; (3) Determine the characteristic genes expressed by the T cell subsets and the relationship between these cell subsets and drug response.

[0060] In another aspect, the present invention provides a method for screening drugs. According to an embodiment of the present invention, the method includes: a) Mixing the test chemical substance with the characteristic gene or the protein it expresses, or mixing the test chemical substance with CD8 expressing the characteristic gene. + T cells or CD4 + T-cell mixing; b) Detect changes in the activity of the expressed protein, or whether the test chemical substance binds to the characteristic gene or the protein it expresses, or changes in the activity of the cell, or changes in the expression level of the characteristic gene in the cell; The characteristic gene is selected from at least one of the following genes: BACH1, TGFB1, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KLRC2, KLRC3, IKZF2, TYROBP, GZMK, MT1X, MT1E, MT2A, CCL4, XCL1, XCL2, GPR183, IL7R, ZNF683, CXCL13, CCL4L2, and NR4A2.

[0061] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0062] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 The CD8 variants in Example 1 of this invention have different characteristic genes and tissue distribution patterns. + T cell subsets and CD4 + T cell subsets. Figure 1A consists of 11 CD8s + UMAP distribution of T cell subsets Figure 1 B's heatmap shows 11 CD8s. + Characteristic gene expression of T cell subsets Figure 1 C is 9 CD4 + UMAP distribution of T cell subsets Figure 1 The heatmap for D shows 9 CD4s. + Characteristic gene expression of T cell subsets.

[0063] Figure 2 These are the characteristic genes and TCR features of the two T cell subsets, Tpex and cTem, belonging to the tumor Teff / Tem in Example 2 of this invention. Figure 2 A represents the distribution of Tpex and cTem cell subsets in the tumor Teff / Tem. Figure 2 B represents the expression level of gene CXCL13 in three T cell subsets (cTem, Tpex, tTex). Figure 2 C represents the exhaustion score in the three T cell subsets (cTem, Tpex, tTex), indicating its high or low level. Figure 2 The heatmap for D shows the expression levels of a series of genes related to T cell function in four T cell subsets (Temra, cTem, Tpex, tTex) within the tumor. Figure 2 E represents the enrichment of the transcriptomes of the three T cell subsets (cTem, Tpex, tTex) in cells under different mouse LCMV infection models (effective T cells on day 8 of Armstrong acute infection / memory T cells on day 30 of Armstrong acute infection / effector T cells on day 8 of Cl13 chronic infection / exhausted T cells on day 30 of Cl13 chronic infection). Figure 2 F represents the correlation between CD8+ in peripheral blood. + Distribution of tumor Tpex and cTem cells sharing TCR Figure 2 G represents the level of transformation with peripheral blood Temra cells, quantified using STARTRAC.

[0064] Figure 3 This describes the dynamic changes of tumor Teff / Tem cells in Example 3 of the present invention during combination therapy with anti-PD-1 and lenvatinib or anti-PD-1 monotherapy. Figure 3 A represents the CD8+ levels in tumors before and after treatment in responders and non-responders. + UMAP distribution of T cell subsets Figure 3 B represents the tumor Teff / Tem cell subset in response or non-responding tumor CD8 cells before and after combination therapy with anti-PD-1 and lenvatinib. + Percentage of T cells, Figure 3C represents the tumor Teff / Tem cell subset in response or non-responder CD8+ cells after anti-PD-1 monotherapy. + Percentage in T cells.

[0065] Figure 4 This describes the dynamic changes of tumor Tpex cells in Example 4 of the present invention during combination therapy with anti-PD-1 and lenvatinib or anti-PD-1 monotherapy. Figure 4 A represents the tumor Tpex cell subset in response or non-responding tumor CD8 cells before and after combination therapy with anti-PD-1 and lenvatinib. + Percentage of T cells, Figure 4 B represents a subset of tumor Tpex cells in response or non-responder CD8+ cells after anti-PD-1 monotherapy. + Percentage in T cells.

[0066] Figure 5 This describes the dynamic changes of tumor cTem cells in Example 5 of the present invention during combination therapy with anti-PD-1 and lenvatinib or anti-PD-1 monotherapy. Figure 5 A represents the tumor cTem cell subset in response or non-responding tumor CD8 cells before and after combination therapy with anti-PD-1 and lenvatinib. + Percentage of T cells, Figure 5 B represents a subset of tumor cTem cells in response to or non-responding tumor CD8 cells after anti-PD-1 monotherapy. + Percentage in T cells.

[0067] Figure 6 This describes the molecular mechanisms of combination therapy with anti-PD-1 and lenvatinib and anti-PD-1 monotherapy in Example 6 of this invention. Figure 6 A represents the level of tumor vascular normalization after combination therapy with anti-PD-1 and lenvatinib, and after anti-PD-1 monotherapy. Figure 6 B represents the levels of CD3 genes (CD3D, CD3E, CD3G) and CD8 in tumors of responders and non-responders after combination therapy with anti-PD-1 and lenvatinib, and anti-PD-1 monotherapy. + Average expression levels of Teff genes (CD8A, CD8B, PRF1, IFNG, GZMK).

[0068] Figure 7 These are the characteristic genes and TCR features of HBV-specific T cells in Example 7 of this invention. Figure 7 A describes the process of identifying and analyzing HBV-specific T cells using 31 HBV-peptide decanters with DNA barcodes. Figure 7 B represents the HBV-peptide decamer in CD8. + Flow cytometry staining of T cells Figure 7C represents a representative HBV-peptide decamer (code name HBVgp4_ag_core, SEQ ID NO: 250) on CD8. + T cells and CD4 + Distribution of binding cell number in T cells Figure 7 D represents the number of cells (horizontal axis) and the HBV peptides identified by DexTRAC in four representative HBV-specific clones. Figure 7 E represents the CD8+ content of HBV-specific T cells in different tissues (peripheral blood, tumor, adjacent normal tissue). + Percentage of T cell subsets Figure 7 F represents the CD8+ cells in tumor HBV-specific T cells. + The percentage of T cell subsets among all tumor-specific HBV T cells. Figure 7 G represents the percentage of HBV-specific T cells contained in tumor cTem and Tpex. Figure 7 H represents the clonal expansion levels of HBV-specific Temra and HBV-specific Teff / Tem, as well as other T cell subsets, quantified by STARTRAC. Figure 7 I represents the clonal expansion level and migration capacity of HBV-specific cTem cells and other cTem cells in responder tumors, quantified by STARTRAC. Figure 7 J represents differentially expressed characteristic genes in HBV-specific cTem cells and other cTem cells (red indicates high expression in HBV-specific cTem cells). Figure 7 K represents the HBV-specific T cell count, inferred from HBV scores, before and after combination therapy with anti-PD-1 and lenvatinib, in response or non-responding tumor CD8+ cells. + Percentage in T cells.

[0069] Figure 8 KIR in Embodiment 8 of the present invention + CD8 + Characteristic genes and TCR features of T cell subsets. Figure 8 A is KIR + CD8 + T cell characteristic genes in various CD8 、 Expression of T cell subsets Figure 8 B represents the tumor (C07) or peripheral blood (C06) KIR in this invention. + CD8 + T cells and KIR in autoimmune diseases + CD8 + T cells or KIR - CD8 + Pearson correlation coefficient of T cell transcriptome Figure 8 C represents the tumor KIR.+ CD8 + T cells and peripheral blood KIR + CD8 + Differentially expressed characteristic genes of T cells (blue indicates tumor KIR) + CD8 + (high expression of T cells) Figure 8 D represents the KIR2DL3 and KIR2DL4 genes of the KIR gene family from different sources (healthy human HC, multiple sclerosis MS, systemic lupus erythematosus SLE, tumor CO7, or peripheral blood CO6). + CD8 + Expression levels in T cells, Figure 8 E represents CD8 in peripheral blood. + KIR tumors that share T cells and TCR + CD8 + T cell distribution, Figure 8 F represents each CD8 quantized using STARTRAC. + T cell subsets and tumor KIR + CD8 + The level of T cell transformation.

[0070] Figure 9 For KIR + CD8 + Dynamic changes of T cells in combination therapy with anti-PD-1 and lenvatinib or in anti-PD-1 monotherapy. Figure 9 A represents the tumor KIR. + CD8 + T cells and peripheral blood KIR + CD8 + T cell subsets before and after combination therapy with anti-PD-1 and lenvatinib in responding or non-responding tumor CD8 cells + Percentage of T cells, Figure 9 B represents the tumor KIR. + CD8 + T cells in responding or non-responding tumor CD8 cells after anti-PD-1 monotherapy + Percentage of T cells, Figure 9 C represents the tumor KIR in responders and non-responders before and after combined treatment in this invention. + CD8 + Pearson correlation coefficient of T cell transcriptome Figure 9 D represents the tumor KIR in responders after combination therapy. + CD8 + T-cell and non-responder tumor KIR + CD8 + Differentially characterized genes in T cells (red indicates non-responder tumor KIR) + CD8+ (high expression of T cells) Figure 9 E represents the gene TGFB1 in tumor KIR in responders and non-responders before and after combination therapy. + CD8 + T cell expression levels.

[0071] Figure 10 All CD4+ cells in tumors of responders and non-responders before and after combined treatment in Example 10 of this invention. + The proportion of activated Tregs and resting Tregs in T cells.

[0072] Figure 11 This describes the molecular mechanism by which activated Tregs are enriched in the tumors of non-responders after combined treatment in Example 11 of this invention. Figure 11 A represents differentially expressed genes in responder and non-responder tumor Treg cells after combination therapy (red indicates high expression in non-responder tumor Treg cells). Figure 11 B represents differentially expressed genes in responder and non-responder tumor Treg cells before combination therapy (red indicates high expression in non-responder tumor Treg cells). Figure 11 C represents the top 10 transcription factors in terms of regulatory activity within the activated Treg subset. Figure 11 D represents the regulatory network of three key transcription factors (BACH1, BATF, and AHR) in the activated Treg subset. Figure 11 E represents the expression level of gene BACH1 in Treg tumors of triple-negative breast cancer patients with PR and SD before anti-PD-L1 combined chemotherapy. Figure 11 F represents the survival curves of HCC patients with high tumor expression and low characteristic gene BACH1 in the TCGA dataset. Figure 11 G represents FOXP3 induced after CRISPR-Cas9 gene BACH1 or other genes were knocked out. + CD25high CD4 + The proportion of T cells. Detailed Implementation

[0073] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0074] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0075] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0076] To facilitate understanding of this invention, certain technical and scientific terms are specifically defined below. Unless explicitly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. The abbreviations for amino acid residues are the standard three-letter abbreviations used in the art to refer to one of the 20 commonly used L-amino acids. Embodiments of the invention are described in detail below. The embodiments described below are exemplary and are only used to explain the invention, and should not be construed as limiting the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0077] In this document, the terms “containing,” “comprising,” or “including” are open-ended expressions, meaning they include the contents specified in this invention but do not exclude other aspects.

[0078] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0079] In this document, the term "pharmaceutical composition" generally refers to a unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient (T cells) with a carrier constituting one or more accessory components. Typically, the composition is prepared by uniformly and sufficiently combining the active compound with a liquid carrier, a finely fragmented solid carrier, or both.

[0080] In this article, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0081] In this document, the term "pharmaceuticalally acceptable amount" or "pharmaceuticalally acceptable dose" refers to a dose suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio. For example, it may be "effective amount" or "effective dose," where "effective amount" or "effective dose" refers to an amount that is functional or active in humans and / or animals and is acceptable to them.

[0082] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, diluent, or other liquid excipient, etc., suitable for a particular target dosage form. The use of any conventional excipients, except those that are incompatible with the compounds of the present invention, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this invention.

[0083] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The T-cell or pharmaceutical composition of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the T-cell or pharmaceutical composition of the present invention is administered via intravenous injection.

[0084] In this document, the term "treatment" refers to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" encompasses diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in individuals susceptible to disease but not yet diagnosed with the disease; (b) suppression of disease, such as inhibiting disease progression; or (c) alleviating disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any medication that administers T cells or pharmaceutical compositions to an individual to treat, cure, alleviate, improve, reduce, or suppress the individual's disease, including but not limited to administering medications containing the T cells described herein to an individual in need.

[0085] In this paper, the terms “identity,” “homology,” or “similarity” are used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecular Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN procedure (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Institute)). Foundation, Washington, DC). Numerous algorithms exist for aligning sequences and determining sequence identity, including: the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; the similarity search method of Pearson et al. (1988) Proc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAST-2 (Altschul...). See, Meth.Enzym., 266:460-480 (1996); or GAP, BESTFIT, BLAST Altschul, etc., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, Mountain View, California.

[0086] Without substantially affecting antibody activity (retaining at least 80% or at least 90% of the activity), those skilled in the art can substitute, add, and / or delete one or more (e.g., 1, 2, or 3) amino acids in the sequences of this invention to obtain variants of the antibody or its antigen-binding fragment sequence. These are all considered to be included within the scope of protection of this invention. For example, amino acids with similar properties can be substituted in the variable region. The variant sequences of this invention can have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity (or homology) with the reference sequence. The sequence identity described in this invention can be measured using sequence analysis software, such as the computer program BLAST using default parameters, especially BLASTP or TBLASTN. The amino acid sequences mentioned in this invention are shown from the N-terminus to the C-terminus.

[0087] In this paper, the term "at least 80% homology" refers to at least 80% homology with each reference sequence, which may be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0088] In this paper, the term "at least 90% homology" means at least 90% homology with each reference sequence, which may be 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%.

[0089] In this document, the term "expression vector" generally refers to a nucleic acid molecule capable of self-replication within a suitable host, transferring the inserted nucleic acid molecule into and / or between cells or hosts. The expression vector may include expression vectors primarily for inserting DNA or RNA into cells, expression vectors primarily for replicating DNA or RNA, and expression vectors primarily for transcription and / or translation of DNA or RNA. The expression vector also includes vectors having a variety of the functions described above. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable cell or host. Typically, by culturing a suitable cell or host containing the vector, the vector can produce the desired expression product.

[0090] In this document, the term "recombinant cell" generally refers to a cell in which the genetic material of a host cell is modified or recombined using genetic engineering or cell fusion techniques to obtain a unique trait with stable inheritance. The term "host cell" refers to a prokaryotic or eukaryotic cell into which a recombinant vector can be introduced. The terms "transformed" or "transfected" as used herein refer to the introduction of nucleic acids (e.g., vectors) into cells using various techniques known in the art. Suitable host cells can be transformed or transfected with the DNA sequences of this invention and can be used for the expression and / or secretion of target proteins.

[0091] In this document, the terms "binding agent," "binding molecule," and "binding entity" are synonymous and can be used interchangeably. In the context of this invention, a binding agent binds to, recognizes, interacts with, reacts with, or otherwise associates with a selectable marker on the T cell subset. Exemplary binding agents may include, but are not limited to, antibodies or fragments thereof, antigens, aptamers, nucleic acids (e.g., DNA and RNA), proteins (e.g., receptors, enzymes, enzyme inhibitors, enzyme substrates, ligands), peptides, lectins, fatty acids or lipids, and polysaccharides. For example, in some embodiments of this invention, the binding agent comprises an antibody or fragment thereof, or a nucleic acid (e.g., DNA and RNA). In this document, the term "antibody" is used in the broadest sense and specifically encompasses synthetic antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, human antibodies, humanized antibodies, chimeric antibodies, primate-derived antibodies, Fab fragments, F (ab') fragments, single-chain FvFc (scFvFc), single-chain Fv (scFv), anti-idiotype (anti-Id) antibodies, and any other immunologically active antibody fragments, provided they exhibit the desired biological activity (i.e., label-associated or binding). In a broader sense, the antibodies of the present invention comprise immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules (i.e., molecules containing antigen-binding sites), wherein these fragments may or may not be fused to another immunoglobulin domain (including, but not limited to, the Fc region or fragments thereof). Furthermore, as outlined in more detail herein, the term antibody and various antibodies specifically include Fc variants or fragments thereof, including full-length antibodies and variant Fc-fusions containing an Fc region, which optionally contain at least one amino acid residue modification and are fused to an immunoglobulin's immunoactive fragment.

[0092] In this document, the term "diagnostic agent" refers to any molecule, compound, and / or substance used for the purpose of diagnosing a disease or disorder. In a preferred embodiment, the diagnostic agent should comprise a binding agent that binds to a reporter molecule. Other non-limiting examples of diagnostic agents include antibodies, antibody fragments, or other proteins, including those that bind to a detection reagent. The terms "detection reagent" or "reporter molecule" refer to any molecule, compound, and / or substance detectable by any methodology available to those skilled in the art, and non-limiting examples include dyes, fluorescent labels, gases, metals, or radioactive isotopes. In this document, the term "inhibition" refers to a reduction in the activity of a protein or cell compared to the absence of an inhibitor. In some embodiments, the term "inhibition" means a reduction in activity of at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95%. In other embodiments, inhibition means a reduction in activity of about 25% to about 50%, about 50% to about 75%, or about 75% to 100%. In some embodiments, inhibition means a reduction in activity of about 95% to 100%, for example, a reduction in activity of 95%, 96%, 97%, 98%, 99%, or 100%. Such a reduction can be measured using a variety of techniques known to those skilled in the art.

[0093] The terms "expression" and "gene expression" have the same meaning, referring to the process by which cells, during their life process, transcribe and translate the genetic information stored in the DNA sequence into biologically active protein molecules. The terms "increased expression" and "high expression" have the same meaning, referring to an increase in the number of gene transcription copies and / or increased translation compared to normal levels.

[0094] The term "prognosis" refers to predicting the possible course and outcome of a disease. It includes judging specific consequences of the disease (such as recovery, the appearance or disappearance of certain symptoms, signs and complications, and death), as well as providing time clues, such as predicting the likelihood of a certain outcome occurring within a certain period of time. In this article, the term "marker gene" is synonymous with "characteristic gene," referring to a gene in biological research that has a specific biological function or characteristic and can serve as a characteristic marker for a specific cell type, tissue, physiological state, or biological process.

[0095] In this article, the term "TCR" is synonymous with "isolated TCR" and refers to the T cell receptor, which is a structure on the surface of T cells that specifically recognizes antigens and plays a central role in the immune response.

[0096] In this paper, the term "TCR clonal type" refers to a specific type of T cell receptor (TCR) with unique nucleotide and amino acid sequences formed after gene rearrangement, reflecting the specificity and diversity of T cell recognition of antigens.

[0097] In this article, the term "CD8" + "T-cell clonal type" refers to CD8+ + T cells are a type of T lymphocyte that expresses the CD8 molecule, and CD8... + T-cell clonal types refer to distinct subsets of T cells with unique nucleotide sequences and functional characteristics, formed based on TCR gene rearrangements. Each CD8 cell type... + Each T cell clone has its own specific TCR, which can specifically recognize a specific antigenic epitope peptide presented by a major histocompatibility complex class I molecule (MHC-I) on the surface of antigen-presenting cells.

[0098] This invention proposes methods for identifying T cell subsets, peptides, TCRs, isolated nucleic acids, expression vectors, host cells, T cells, identifying antigen-specific T cells, identifying immunogenic peptides, identifying tumor and virus antigen-specific T cells, activating, inhibiting, inducing, and expanding T cell subsets, developing methods for in vitro expansion of antigen-specific T cells, applications, methods for preparing T cells containing the stated TCRs, developing adoptive cell therapy, developing novel anti-tumor therapies that inhibit or eliminate regulatory T cells, developing cancer vaccines, developing virus-specific T cell therapy for cancer, developing bispecific or trispecific antibodies targeting T cells and other cells, developing combination therapies, the application of T cell subsets in the treatment of autoimmune diseases, methods for inhibiting the transformation of T cells into regulatory T cells, the application of inhibitors in the preparation of tumor therapeutic drugs, methods for enriching T cell subsets, diagnostic or evaluation methods, and CD8 for diagnostic or monitoring purposes. + T cell biomarkers and CD4 for diagnosis or monitoring + T cell biomarker group.

[0099] In a first aspect, the present invention proposes seven T cells associated with the efficacy of anti-PD-1 combined with lenvatinib (or other anti-angiogenic drugs).

[0100] CD8 + T cell subsets According to embodiments of the present invention, a CD8 is proposed. + T cell subsets. According to an embodiment of the present invention, the CD8... + T cell subsets include those expressing the characteristic genes GZMK and PDCD1. According to an embodiment of the present invention, the CD8... +T cell subsets are effector or effector memory T cells. The inventors of this invention, through comparative studies, have for the first time discovered that the CD8... + T-cell subsets are positively correlated not only with the response to combination therapy with anti-PD-1 and lenfatinib, but also with the response to anti-PD-1 monotherapy.

[0101] According to an embodiment of the present invention, the CD8 + T cell subsets further express at least one of the characteristic genes MT1X, MT1E, MT2A, CCL4, XCL1, and XCL2. For example, CD8... + T cell subsets express the characteristic gene MT1X; or, the CD8... + T cell subsets express the characteristic genes MT1X and MT1E; or, the CD8+... + T cell subsets express characteristic genes MT1X, MT1E, and XCL1.

[0102] According to an optional embodiment of the present invention, the CD8 proposed in this invention + T cell subsets are GZMK + CD8 + T cells, GZMK + CD8 + T cells are positively correlated with good treatment response in cancer patients receiving neoadjuvant therapy with anti-PD-1 combined with lenvatinib, and they express the characteristic genes GZMK and PDCD1.

[0103] CD8 + T cell subsets According to embodiments of the present invention, a CD8 is proposed. + T cell subsets. According to an embodiment of the present invention, the CD8... + T cell subsets include those expressing the characteristic genes GZMK, GPR183, IL7R, and ZNF683. CD8 according to embodiments of the present invention... + T cell subsets, namely CD8 + T cell subsets are effector memory T cells with peripheral circulation characteristics. The inventors of this invention, through comparative studies, have for the first time discovered that the CD8... + T cell subsets specifically respond to combination therapy with anti-PD-1 and lenfatinib, rather than anti-PD-1 monotherapy.

[0104] According to an embodiment of the present invention, the CD8 + T cell subsets may further express at least one of the characteristic genes CXCR3, CXCR4, CD44, CCR7, LMNA, XCL1, and XCL2. For example, CD8... + T cell subsets express the characteristic gene CXCR3; or, the CD8...+ T cell subsets express characteristic genes CXCR3 and CCR7; or, the CD8... + T cell subsets express characteristic genes CXCR3, CCR7, and XCL1.

[0105] According to an optional embodiment of the present invention, the CD8 + The T cell subset is cTem cells, which are positively correlated with good treatment response in cancer patients receiving neoadjuvant therapy with anti-PD-1 combined with lenvatinib. They are a group of effector memory T cells that express the characteristic genes GZMK, GPR183, IL7R and ZNF683.

[0106] CD8 + T cell subsets According to embodiments of the present invention, a CD8 is proposed. + T cell subsets. According to an embodiment of the present invention, the CD8... + T cell subsets include those expressing the characteristic genes GZMK and CXCL13. CD8 according to embodiments of the present invention... + A subset of T cells, which is positively correlated with good treatment response in cancer patients receiving neoadjuvant therapy with anti-PD-1 combined with lenvatinib, is a group of progenitor-depleted T cells.

[0107] According to an embodiment of the present invention, the CD8 + T cell subsets may further express at least one of the characteristic genes CXCR4, DUSP4, DUSP1, IFNG, MT1X, MT1E, MT2A, XCL1, and XCL2. For example, the CD8+... + T cell subsets express the characteristic gene CXCR4; or, the CD8+ T cell subsets express the characteristic genes CXCR4 and DUSP4; or, the CD8+ T cell subsets express the characteristic genes CXCR4 and DUSP4. + T cell subsets express characteristic genes CXCR4, DUSP4, and DUSP1.

[0108] According to embodiments of the present invention, a CD8 is proposed. + T cell subsets. According to an embodiment of the present invention, the CD8... + The T cell subset was CXCL13. + CD8 + Tpex cells, a population of progenitor exhausted T cells that are positively correlated with good treatment response in cancer patients receiving neoadjuvant therapy with anti-PD-1 combined with lenvatinib, express the characteristic genes GZMK and CXCL13.

[0109] According to an embodiment of the present invention, the CD8 +There is a transformation relationship between T cell subsets and terminally exhausted T cells.

[0110] According to an embodiment of the present invention, the CD8 + T cell subsets are progenitor cells of exhausted T cells capable of differentiating into terminally exhausted T cells. The inventors of this invention, through comparative studies, have for the first time discovered that the CD8... + T cell subsets respond simultaneously to both anti-PD-1 combined therapy with lenfatinib and anti-PD-1 monotherapy.

[0111] CD8 + T cell subsets According to embodiments of the present invention, a CD8 is proposed. + T cell subsets. According to an embodiment of the present invention, the CD8... + T cell subsets include those expressing the characteristic genes GZMK and ZNF683.

[0112] According to embodiments of the present invention, a CD8 is proposed. + T cell subsets. According to an embodiment of the present invention, the CD8... + T cell subsets are HBV antigen-specific CD8 + T cells express the characteristic genes GZMK and ZNF683.

[0113] According to an embodiment of the present invention, the CD8 + T cell subsets may further express one or more of the characteristic genes CCL4L2, NR4A2, and CCL4. For example, the CD8... + T cell subsets express the characteristic gene CCL4L2; or, the CD8... + T cell subsets express characteristic genes CCL4L2 and NR4A2; or, the CD8+... + T cell subsets express characteristic genes CCL4L2, NR4A2, and CCL4.

[0114] According to an embodiment of the present invention, the CD8 + T cell subsets have the ability to specifically recognize HBV antigens.

[0115] CD8 + T cell subsets According to embodiments of the present invention, a CD8 is proposed. + T cell subsets. According to an embodiment of the present invention, the CD8... + T cell subsets include those expressing at least one of KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL3. According to embodiments of the present invention, the CD8... +T cell subsets are a group of T cells with potential regulatory functions. The inventors of this invention, through comparative studies, have discovered for the first time that the CD8... + T cell subsets are specifically associated with resistance to combination therapy with anti-PD-1 and lenfatinib, but not with resistance to anti-PD-1 monotherapy.

[0116] According to an embodiment of the present invention, the CD8 + T cell subsets may further express at least one of the characteristic genes KLRC2, KLRC3, IKZF2, TYROBP, XCL1, and XCL2. For example, the CD8+... + T cell subsets express the characteristic gene KLRC2; or, the CD8... + T cell subsets express characteristic genes KLRC2 and KLRC3; or, the CD8+... + T cell subsets express characteristic genes KLRC2, KLRC3, and IKZF2.

[0117] According to an optional embodiment of the present invention, a CD8 is proposed. + T cell subsets. According to an embodiment of the present invention, the CD8+ T cell subset is KIR. + CD8 + T cells, a population of cells that are positively correlated with treatment resistance in cancer patients receiving neoadjuvant therapy with anti-PD-1 combined with lenvatinib, express at least one of the KIR gene family members KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL3.

[0118] CD4 + T cell subsets According to embodiments of the present invention, a CD4 is proposed. + T cell subsets. According to an embodiment of the present invention, the CD4... + T cell subsets include those expressing the characteristic genes FOXP3, TNFRSF9, and CTLA4. According to an embodiment of the present invention, the CD4... + T cell subsets are a group of T cells with regulatory functions. The inventors of this invention, through comparative studies, have for the first time discovered that the CD4... + T-cell subsets are associated with resistance to combination therapy of anti-PD-1 and lenfatinib.

[0119] According to an embodiment of the present invention, the CD4 + T cell subsets may further express at least one of the characteristic genes CTSC, IKZF2, STAM, and DUSP4. For example, CD4. + T cell subsets express the characteristic gene CTSC; or, the CD4+ T cell subsets express the characteristic genes CTSC and IKZF2; or, the CD4+ gene. + T cell subsets express characteristic genes CTSC, IKZF2, and STAM; or, CD4 + T cell subsets express characteristic genes CTSC, IKZF2, STAM, and DUSP4.

[0120] According to embodiments of the present invention, a CD4 is proposed. + T cell subsets. According to an optional embodiment of the invention, the CD4... + The T cell subset is tumor-resident Treg cells. According to embodiments of the present invention, these tumor-resident Treg cells are positively correlated with treatment resistance in cancer patients receiving neoadjuvant therapy with anti-PD-1 combined with lenvatinib, and they express the characteristic genes FOXP3, TNFRSF9, and CTLA4.

[0121] CD4 + T cell subsets According to embodiments of the present invention, a CD4 is proposed. + T cell subsets. According to an embodiment of the present invention, the CD4... + T cell subsets include those expressing the characteristic genes FOXP3 and BACH1. According to an embodiment of the present invention, the CD4... + T cell subsets are a group of T cells with regulatory functions. The inventors of this invention, through comparative studies, have for the first time discovered that the CD4... + T-cell subsets were enriched in pre-treatment tumors of cancer patients who did not respond to anti-PD-1 combined with lenvatinib combination therapy. The CD4... + The higher the proportion of T cell subsets infiltrating the tumor before treatment, the weaker the ability of the infiltrating T cells to kill tumor cells, and the worse the patient's response to treatment; the CD4... + The more types of genes expressed by T cell subsets among the six genes BACH1, REL, ICOS, AHR, CREM, and NFE2L2, the more accurate the diagnosis of the patient's tumor immune status and the more accurate the assessment of the patient's response to treatment.

[0122] According to an embodiment of the present invention, the CD4 + T cell subsets may further express at least one of the characteristic genes REL, ICOS, AHR, CREM, and NFE2L2. For example, CD4. + T cell subsets express the characteristic gene REL; or, the CD4 + T cell subsets express characteristic genes REL and ICOS; or, CD4 +T cell subsets express characteristic genes REL, ICOS, and AHR.

[0123] According to embodiments of the present invention, a CD4 is proposed. + T cell subsets. According to an optional embodiment of the invention, the CD4... + The T cell subset is BACH1 + Treg cells express the characteristic genes FOXP3 and BACH1.

[0124] In this invention, all seven T cell subsets mentioned above originate from tumors. In one specific embodiment of this invention, the tumor is HBV-positive hepatocellular carcinoma.

[0125] Antigenic epitope peptide In another aspect, the present invention provides an antigenic epitope peptide. According to embodiments of the present invention, the antigenic epitope peptide comprises at least one of the amino acid sequences shown in SEQ ID NO: 249-258; or at least one of the amino acid sequences having 90% sequence identity with it. The inventors used cells expressing the antigenic epitope peptide to stimulate T cells in vitro to obtain TCRs that specifically recognize hepatitis B virus. This TCR exhibits cross-reactivity and can recognize multiple antigenic epitope peptides.

[0126] Nucleic acid molecules, expression vectors and recombinant cells In another aspect, the present invention provides a nucleic acid molecule. According to embodiments of the invention, the nucleic acid molecule encodes the aforementioned antigenic epitope peptide.

[0127] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0128] In another aspect, the present invention provides an expression vector. According to embodiments of the invention, the vector carries the aforementioned nucleic acid molecule. When linking the nucleic acid molecule to the expression vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself, or they can be exogenous, i.e., not derived from the expression vector itself. Naturally, the connection between the nucleic acid molecule and the control elements only needs to be operably established.

[0129] In this article, "operable ligation" refers to ligating a foreign gene into a vector so that the control elements within the expression vector, such as transcriptional and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Of course, the polynucleotides encoding the antibody heavy and light chains can be inserted independently into different vectors, but it is more common to insert them into the same vector. Commonly used vectors include plasmids, phages, etc.

[0130] According to an embodiment of the present invention, the expression vector is a eukaryotic expression vector.

[0131] According to an embodiment of the present invention, the expression vector is a lentiviral vector.

[0132] In another aspect, the present invention provides a recombinant cell. According to embodiments of the invention, it comprises: carrying the aforementioned nucleic acid molecule or the aforementioned expression vector; or expressing the aforementioned antigenic epitope peptide. The expression vector can be introduced into a host cell to construct a recombinant cell expressing the aforementioned antigenic epitope peptide, the recombinant cell expressing the antigenic epitope peptide.

[0133] According to an embodiment of the present invention, the recombinant cells are obtained by introducing the aforementioned expression vector into host cells.

[0134] According to an embodiment of the present invention, the recombinant cells are eukaryotic cells.

[0135] antigen In another aspect of the invention, an antigen is provided. According to an embodiment of the invention, the antigen comprises the aforementioned antigenic epitope polypeptide. Recombinant cells expressing the above-mentioned antigen are used to stimulate T cells in vitro to obtain a TCR that specifically recognizes hepatitis B virus. This TCR has cross-reactivity, can recognize multiple antigenic epitope peptides and their mutants, and can rapidly respond to changes in hepatitis B virus mutant strains.

[0136] Pharmaceutical Composition In another aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, the pharmaceutical composition comprises the aforementioned antigenic epitope polypeptide. The pharmaceutical composition according to the invention can prevent infection with novel hepatitis B virus (HBV) or treat related diseases caused by HBV, such as HBV vaccines, which can induce a HBV-specific T-cell immune response, exhibiting good immunogenicity and immune efficacy.

[0137] According to embodiments of the present invention, the pharmaceutical composition further includes a carrier, wherein the antigenic epitope polypeptide is linked to the carrier.

[0138] According to an embodiment of the present invention, the pharmaceutical composition is selected from HBV vaccines.

[0139] According to an embodiment of the present invention, the HBV vaccine is selected from polypeptide vaccines.

[0140] use In another aspect of the invention, the invention provides the use of the aforementioned antigenic epitope peptide, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell in the preparation of a drug for the prevention of novel hepatitis B virus infection or the treatment of related diseases caused by novel hepatitis B virus.

[0141] According to an embodiment of the present invention, the drug is selected from HBV vaccines.

[0142] According to an embodiment of the present invention, the HBV vaccine is selected from polypeptide vaccines.

[0143] In another aspect, the present invention proposes the use of the aforementioned antigenic epitope peptide, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cell in screening TCRs that specifically recognize novel hepatitis B virus. Recombinant cells expressing the aforementioned antigenic epitope peptide are used to stimulate T cells in vitro to obtain TCRs that specifically recognize hepatitis B virus. These TCRs exhibit cross-reactivity, can recognize multiple antigenic epitope peptides and their mutants, and can rapidly respond to changes in hepatitis B virus mutant strains.

[0144] A method for screening antigenic epitope peptides In another aspect, the present invention provides a method for screening antigenic epitope peptides. According to an embodiment of the invention, the method includes: forming an HLA-antigen epitope peptide complex by combining HLA and the antigen epitope peptide to be screened; and treating the HLA-antigen epitope peptide with T cells to screen for the antigen epitope peptide. It should be noted that the human leukocyte antigen system (HLA) is the name of the gene complex encoding the human major histocompatibility complex (MHC) and includes HLA class I antigens (A, B, and C) and HLA class II antigens (DP, DQ, and DR). HLA alleles A, B, and C are presented primarily from intracellular proteins, such as peptides of proteins expressed intracellularly.

[0145] According to an optional embodiment of the present invention, the screening process is achieved by combining a complex containing the antigen epitope peptide sequence to be screened with single-cell sequencing technology, comprising the following steps: (1) determining the amino acid sequences of the candidate HLA and the antigen epitope peptide to be screened; (2) synthesizing the determined HLA and the antigen epitope peptide to be screened, and forming a complex in vitro; (3) reacting the HLA-antigen epitope peptide complex with T cells; (4) screening for HLA-antigen epitope peptide complexes and corresponding polypeptide sequences that can specifically bind to T cells by single-cell sequencing, thereby screening for the aforementioned antigen-specific antigen epitope peptides. In the present invention, the amino acid sequences of antigen-specific antigen epitope peptides are as shown in at least one of SEQ ID NO: 249~258.

[0146] According to embodiments of the present invention, the candidate HLA-selectable antigenic epitope peptide is selected from at least one of viruses, tumor neoantigens, or tumor-associated antigens. According to embodiments of the present invention, the amino acid sequence of the candidate HLA-selectable antigenic epitope peptide can be determined based on a score calculated using an HLA-binding peptide prediction algorithm. For example, at least one of BIMAS, SYFPEITHI, RANKPEP, or NetMHC can be used to determine the candidate HLA-selectable antigenic epitope peptide.

[0147] In this article, the term "T cell receptor (TCR)" refers to a molecule found on the surface of T cells that is responsible for recognizing antigens that bind to MHC molecules. Naturally occurring TCR heterodimers consist of alpha (α) and beta (β) chains in approximately 95% of T cells, while approximately 5% of T cells have TCRs composed of gamma (γ) and delta (δ) chains. During antigen processing, antigens are degraded intracellularly and then carried to the cell surface by major histocompatibility complex (MHC) molecules. T cells can recognize this antigen peptide-MHC complex on the surface of antigen-presenting cells. The binding of the TCR to the antigen peptide-MHC complex leads to T lymphocyte activation, and the TCR is expressed on T lymphocytes through a series of biochemical reactions mediated by related enzymes, co-receptors, and specialized helper molecules.

[0148] MHC molecules can be either class I or class II MHC molecules. This complex can be present on antigen-presenting cells, such as dendritic cells, B cells, or any other cell type (e.g., K562 cells). The human leukocyte antigen system (HLA) is the name for the gene complex that encodes the human major histocompatibility complex (MHC) and includes HLA class I antigens (A, B, and C) and HLA class II antigens (DP, DQ, and DR). HLA alleles A, B, and C are primarily presented from intracellular proteins, such as peptides of proteins expressed intracellularly. During T cell development in vivo, certain T cells and their expressed TCRs recognize only peptides presented by certain types of MHC molecules (i.e., MHC molecules encoded by specific HLA alleles), a phenomenon known as HLA restriction. For example, one HLA allele is HLA*A-11:01, which is expressed in most Asian populations. Therefore, it is advantageous to bind TCRs that bind to antigenic epitope peptides presented by MHC encoded by the HLA*A-11:01 cell line (i.e., HLA*A-11:01-restricted TCRs) because immunotherapies utilizing such TCRs would be suitable for treating most Asian populations.

[0149] Both the TCRα and TCRβ chains are members of the immunoglobulin superfamily and possess an N-terminal immunoglobulin (Ig) variable domain (V), an Ig constant domain (C), a transmembrane / cell transmembrane region, and a short C-terminal cytoplasmic tail. The variable domain (V) of both the TCRα and TCRβ chains contains three highly variable or complementarity-determining regions (CDRs). For example, the TCRα or β chain contains CDR1, CDR2, and CDR3 in an amino-to-carboxyl-terminal sequence. Typically, the antigenic peptide in the TCR that binds to the major histocompatibility complex (MHC) primarily binds to complementarity-determining region 3 (CDR3). CDR3 is generated during somatic rearrangement events, and the insertion / deletion of random nucleotides at rearrangement loci in each TCR chain gene significantly increases the diversity of the highly variable CDR3 sequence. Therefore, T cell receptor diversity is concentrated on CDR3.

[0150] Separated TCR Based on this, another aspect of the present invention proposes a separate TCR. According to an embodiment of the present invention, a separate TCR is proposed. According to an embodiment of the present invention, the isolated TCR comprises: a TCRα chain variable region and / or a TCRβ chain variable region, wherein the TCRα chain variable region comprises a CDR3 sequence selected from at least one of the following: SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 and SEQ ID NO:72, or an amino acid sequence thereof in a conserved modified form; the TCRβ chain variable region comprises a CDR3 sequence selected from at least one of the following: SEQ ID NO:75, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 and SEQ ID NO:72, or an amino acid sequence thereof. The amino acid sequences of SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:141 and SEQ ID NO:144, or their conserved modified forms.

[0151] It should be noted that, in this document, "conservatively modified amino acid sequences" refers to amino acid modifications that do not significantly affect or alter the binding properties of the antibody containing that amino acid sequence. These modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into the antibodies of this invention using standard techniques such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions involve replacing an amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been identified in the art. These families include amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid), amino acids with uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), amino acids with nonpolar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), amino acids with β-branched side chains (such as threonine, valine, and isoleucine), and amino acids with aromatic side chains (such as tyrosine, phenylalanine, tryptophan, and histidine). Therefore, one or more amino acid residues in the CDR region of the antibody of the present invention can be replaced by other amino acid residues from the same side chain family, and the retained function of the modified antibody can be tested using the functional assay methods described herein. Preferably, the number of conservative modifications does not exceed one or two.

[0152] According to embodiments of the present invention, the TCRα chain variable region further comprises an amino acid sequence selected from at least one of the following: SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 and SEQ ID NO:70, or a conserved modified form thereof.

[0153] According to embodiments of the present invention, the TCRα chain variable region further comprises an amino acid sequence selected from at least one of the following: SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 and SEQ ID NO:71, or a conserved modified form thereof.

[0154] According to embodiments of the present invention, the TCRβ chain variable region further comprises an amino acid sequence selected from at least one of the following: SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:130, SEQ ID NO:133, SEQ ID NO:136, SEQ ID NO:139 and SEQ ID NO:142, or a conserved modified form thereof.

[0155] According to embodiments of the present invention, the TCRβ chain variable region further comprises a CDR2 sequence selected from at least one of the following: SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:113, SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:122, SEQ ID NO:125, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:134, SEQ ID NO:137, SEQ ID NO:140 and SEQ ID NO:143, or an amino acid sequence thereof with a conservative modification thereof.

[0156] According to embodiments of the present invention, the variable region of the TCRα chain comprises: CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 1-3 or amino acid sequences having at least 90% homology therewith; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 4-6 or amino acid sequences having at least 90% homology therewith; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 7-9 or amino acid sequences having at least 90% homology therewith; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 10-12 or amino acid sequences having at least 90% homology therewith; and CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 13-15 or amino acid sequences having at least 90% homology therewith. The following sequences are listed: CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 16-18 or amino acid sequences having at least 90% homology with them; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 19-21 or amino acid sequences having at least 90% homology with them; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 22-24 or amino acid sequences having at least 90% homology with them; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 25-27 or amino acid sequences having at least 90% homology with them; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 28-30 or amino acid sequences having at least 90% homology with them; and CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 19-21; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 19-21; CDR2, ... The amino acid sequences 31-33 or amino acid sequences having at least 90% homology with them, representing CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 34-36 or amino acid sequences having at least 90% homology with them, representing CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 37-39 or amino acid sequences having at least 90% homology with them, representing CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 40-42 or amino acid sequences having at least 90% homology with them, representing CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 43-45 or amino acid sequences having at least 90% homology with them, representing CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 46-48 or amino acid sequences having at least 90% homology with them, representing CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: The CDR1, CDR2, and CDR3 sequences shown in amino acid sequences 49-51 or amino acid sequences having at least 90% homology with them; the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 52-54 or amino acid sequences having at least 90% homology with them; the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 55-57 or amino acid sequences having at least 90% homology with them; and the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 58-60 or amino acid sequences having at least 90% homology with them.The CDR1, CDR2, and CDR3 sequences respectively shown in the amino acid sequences of SEQ ID NO: 61-63 or amino acid sequences having at least 90% homology therewith; the CDR1, CDR2, and CDR3 sequences respectively shown in the amino acid sequences of SEQ ID NO: 64-66 or amino acid sequences having at least 90% homology therewith; the CDR1, CDR2, and CDR3 sequences respectively shown in the amino acid sequences of SEQ ID NO: 67-69 or amino acid sequences having at least 90% homology therewith; or the CDR1, CDR2, and CDR3 sequences respectively shown in the amino acid sequences of SEQ ID NO: 70-72 or amino acid sequences having at least 90% homology therewith.

[0157] According to embodiments of the present invention, the variable region of the TCRα chain comprises: CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 1-3, respectively; CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 4-6, respectively; CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 7-9, respectively; CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 10-12, respectively; CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 13-15, respectively; CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 16-18, respectively; CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 19-21, respectively; CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 22-24, respectively; and CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 19-21 ... The CDR1, CDR2, and CDR3 sequences shown in amino acid sequences 25-27; the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 28-30; the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 31-33; the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 34-36; the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 37-39; the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 40-42; the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 43-45; the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 46-48; and the sequences shown in amino acid sequences SEQ ID NO: 28-39. The CDR1, CDR2, and CDR3 sequences shown in amino acid sequences 49-51; the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 52-54; the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 55-57; the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 58-60; and the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 61-63.The CDR1, CDR2, and CDR3 sequences respectively shown in the amino acid sequences of SEQ ID NO: 64-66; the CDR1, CDR2, and CDR3 sequences respectively shown in the amino acid sequences of SEQ ID NO: 67-69; or the CDR1, CDR2, and CDR3 sequences respectively shown in the amino acid sequences of SEQ ID NO: 70-72.

[0158] According to embodiments of the present invention, the variable region of the TCRβ chain comprises: CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 73-75 or amino acid sequences having at least 90% homology therewith; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 76-78 or amino acid sequences having at least 90% homology therewith; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 79-81 or amino acid sequences having at least 90% homology therewith; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 82-84 or amino acid sequences having at least 90% homology therewith; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 85-87 or amino acid sequences having at least 90% homology therewith; and CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 76-78 or amino acid sequences having at least 90% homology therewith; and CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 79-81 ... The following sequences are included: CDR1, CDR2, and CDR3 sequences with amino acid sequences of 88-90 or amino acid sequences having at least 90% homology; CDR1, CDR2, and CDR3 sequences with amino acid sequences of SEQ ID NO: 91-93 or amino acid sequences having at least 90% homology; CDR1, CDR2, and CDR3 sequences with amino acid sequences of SEQ ID NO: 94-96 or amino acid sequences having at least 90% homology; CDR1, CDR2, and CDR3 sequences with amino acid sequences of SEQ ID NO: 97-99 or amino acid sequences having at least 90% homology; CDR1, CDR2, and CDR3 sequences with amino acid sequences of SEQ ID NO: 100-102 or amino acid sequences having at least 90% homology; and CDR1, CDR2, and CDR3 sequences with amino acid sequences of SEQ ID NO: 103-105 or amino acid sequences having at least 90% homology. The following sequences are listed: CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 106-108 or amino acid sequences having at least 90% homology with them; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 109-111 or amino acid sequences having at least 90% homology with them; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 112-114 or amino acid sequences having at least 90% homology with them; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 115-117 or amino acid sequences having at least 90% homology with them; CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 118-120 or amino acid sequences having at least 90% homology with them; and CDR1, CDR2, and CDR3 sequences as shown in SEQ ID NO: 109-111. The amino acid sequences 121-123 or amino acid sequences having at least 90% homology with them, representing CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 124-126 or amino acid sequences having at least 90% homology with them, representing CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 127-129 or amino acid sequences having at least 90% homology with them, representing CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 130-132 or amino acid sequences having at least 90% homology with them, representing CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 133-135 or amino acid sequences having at least 90% homology with them, representing CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: The CDR1, CDR2, and CDR3 sequences shown in amino acid sequences 136-138 or amino acid sequences having at least 90% homology with them; the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 139-141 or amino acid sequences having at least 90% homology with them; or the CDR1, CDR2, and CDR3 sequences shown in amino acid sequences SEQ ID NO: 142-144 or amino acid sequences having at least 90% homology with them.

[0159] According to embodiments of the present invention, the variable region of the TCRβ chain includes: CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 73-75; CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 76-78; CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 79-81; and CDR1, CDR2, and CDR3 sequences as shown in the amino acid sequences of SEQ ID NO: 82-84. The following sequences are listed: CDR1, CDR2, and CDR3, as shown in the amino acid sequences of SEQ ID NO: 85-87; CDR1, CDR2, and CDR3, as shown in the amino acid sequences of SEQ ID NO: 88-90; CDR1, CDR2, and CDR3, as shown in the amino acid sequences of SEQ ID NO: 91-93; CDR1, CDR2, and CDR3, as shown in the amino acid sequences of SEQ ID NO: 94-96; CDR1, CDR2, and CDR3, as shown in the amino acid sequences of SEQ ID NO: 97-99; CDR1, CDR2, and CDR3, as shown in the amino acid sequences of SEQ ID NO: 100-102; CDR1, CDR2, and CDR3, as shown in the amino acid sequences of SEQ ID NO: 103-105; CDR1, CDR2, and CDR3, as shown in the amino acid sequences of SEQ ID NO: 106-108; and CDR1, CDR2, and CDR3, as shown in the amino acid sequences of SEQ ID NO: 85-87; and CDR2, CDR3, as shown in the amino acid sequences of SEQ ID NO: 88-90; CDR1, CDR2, and CDR3, as shown in the amino acid sequences of SEQ ID NO: 81-93; CDR1, CDR2, and CDR3, as shown in the amino acid sequences of SEQ ID NO: 84-96; CDR1, CDR2, and CDR3, as shown in the amino acid sequences of SEQ ID NO: 85-87; and CDR3, as shown in the amino acid sequences of SEQ ID NO: 85-87; and CDR2, CDR3, as shown in the amino acid sequences of SEQ ID NO: 85-87; and CDR3, as shown in the amino acid sequences of SEQ ID NO: The amino acid sequences 109-111 show the CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 112-114 show the CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 115-117 show the CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 118-120 show the CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 121-123 show the CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 124-126 show the CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 127-129 show the CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 130-132 show the CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 109-111 show the CDR1, CDR2, and CDR3 sequences; the amino acid sequences SEQ ID NO: 119-110 ... The CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO: 133-135; the CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO: 136-138; the CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO: 139-141; or the CDR1, CDR2, and CDR3 sequences shown in the amino acid sequences of SEQ ID NO: 142-144.

[0160] According to embodiments of the present invention, the isolated TCR has a TCRα chain variable region having an amino acid sequence as shown in SEQ ID NO:145 or having at least 90% homology therewith, and a TCRβ chain variable region having an amino acid sequence as shown in SEQ ID NO:169 or having at least 90% homology therewith; the isolated TCR has a TCRα chain variable region having an amino acid sequence as shown in SEQ ID NO:146 or having at least 90% homology therewith, and a TCRβ chain variable region having an amino acid sequence as shown in SEQ ID NO:170 or having at least 90% homology therewith; the isolated TCR has a TCRα chain variable region having an amino acid sequence as shown in SEQ ID NO:147 or having at least 90% homology therewith, and a TCRβ chain variable region having an amino acid sequence as shown in SEQ ID NO:171 or having at least 90% homology therewith; the isolated TCR has a TCRα chain variable region having an amino acid sequence as shown in SEQ ID NO:148 or having at least 90% homology therewith, and a TCRβ chain variable region having an amino acid sequence as shown in SEQ ID NO:149 ... The isolated TCR has a TCRβ chain variable region as shown in SEQ ID NO:172 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:149 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:173 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:150 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:174 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:151 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:175 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:172; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:151 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:175 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:172; The TCR has a variable region of the TCRα chain as shown in NO:152 or having at least 90% homology with the amino acid sequence thereon and a variable region of the TCRβ chain as shown in SEQ ID NO:176 or having at least 90% homology with the amino acid sequence thereon; the isolated TCR has a variable region of the TCRα chain as shown in SEQ ID NO:153 or having at least 90% homology with the amino acid sequence thereon and a variable region of the TCRβ chain as shown in SEQ ID NO:177 or having at least 90% homology with the amino acid sequence thereon;The isolated TCR has a TCRα chain variable region with an amino acid sequence as shown in SEQ ID NO:154 or having at least 90% homology therewith, and a TCRβ chain variable region with an amino acid sequence as shown in SEQ ID NO:178 or having at least 90% homology therewith; the isolated TCR has a TCRα chain variable region with an amino acid sequence as shown in SEQ ID NO:155 or having at least 90% homology therewith, and a TCRβ chain variable region with an amino acid sequence as shown in SEQ ID NO:179 or having at least 90% homology therewith; the isolated TCR has a TCRα chain variable region with an amino acid sequence as shown in SEQ ID NO:156 or having at least 90% homology therewith, and a TCRβ chain variable region with an amino acid sequence as shown in SEQ ID NO:180 or having at least 90% homology therewith; the isolated TCR has a TCRα chain variable region with an amino acid sequence as shown in SEQ ID NO:157 or having at least 90% homology therewith, and a TCRβ chain variable region with an amino acid sequence as shown in SEQ ID NO:158 or having at least 90% homology therewith, and a TCRβ chain variable region with an amino acid sequence as shown in SEQ ID NO:159 ... The isolated TCR has a TCRβ chain variable region as shown in SEQ ID NO:181 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:158 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:182 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:159 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:183 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:160 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:184 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:181; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:158 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:181 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:181 or having at least 90% homology with the amino acid sequence shown in SEQ ID NO:184; the isolated TCR has a TCRβ chain variable region as shown in SEQ ID NO:181 or having at least 90% homology with the amino acid sequence ... The isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:161 or having at least 90% homology with the amino acid sequence therein, and a TCRβ chain variable region as shown in SEQ ID NO:185 or having at least 90% homology with the amino acid sequence therein; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:162 or having at least 90% homology with the amino acid sequence therein, and a TCRβ chain variable region as shown in SEQ ID NO:186 or having at least 90% homology with the amino acid sequence therein; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:163 or having at least 90% homology with the amino acid sequence therein, and a TCRβ chain variable region as shown in SEQ ID NO:187 or having at least 90% homology with the amino acid sequence therein.The isolated TCR has a TCRα chain variable region with an amino acid sequence as shown in SEQ ID NO:164 or having at least 90% homology therewith, and a TCRβ chain variable region with an amino acid sequence as shown in SEQ ID NO:188 or having at least 90% homology therewith; the isolated TCR has a TCRα chain variable region with an amino acid sequence as shown in SEQ ID NO:165 or having at least 90% homology therewith, and a TCRβ chain variable region with an amino acid sequence as shown in SEQ ID NO:189 or having at least 90% homology therewith; the isolated TCR has a TCRα chain variable region with an amino acid sequence as shown in SEQ ID NO:166 or having at least 90% homology therewith, and a TCRβ chain variable region with an amino acid sequence as shown in SEQ ID NO:190 or having at least 90% homology therewith; the isolated TCR has a TCRα chain variable region with an amino acid sequence as shown in SEQ ID NO:167 or having at least 90% homology therewith, and a TCRβ chain variable region with an amino acid sequence as shown in SEQ ID NO:188 or having at least 90% homology therewith. The TCR β chain variable region is shown in SEQ ID NO:191 or has at least 90% homology with the amino acid sequence thereon; or the isolated TCR has a TCR α chain variable region shown in SEQ ID NO:168 or has at least 90% homology with the amino acid sequence thereon and a TCR β chain variable region shown in SEQ ID NO:192 or has at least 90% homology with the amino acid sequence thereon.

[0161] According to embodiments of the present invention, the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:145 and a TCRβ chain variable region as shown in SEQ ID NO:169; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:146 and a TCRβ chain variable region as shown in SEQ ID NO:170; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:147 and a TCRβ chain variable region as shown in SEQ ID NO:171; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:148 and a TCRβ chain variable region as shown in SEQ ID NO:172; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:149 and a TCRβ chain variable region as shown in SEQ ID NO:173 ...5 and a TCRβ chain variable region as shown in SEQ ID NO:169; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:146 and a TCRβ chain variable region as shown in SEQ ID NO:170; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:147 and a TCRβ chain variable region as shown in SEQ ID NO:171; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:148 and a TCRβ chain variable region as shown in SEQ ID NO:172; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:149 and a TCRβ chain variable region as shown in SEQ ID NO:173; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:149 and a TCRβ chain variable The TCRα chain variable region of the amino acid sequence shown in NO:150 and the TCRβ chain variable region of the amino acid sequence shown in SEQ ID NO:174. The isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:151 and a TCRβ chain variable region as shown in SEQ ID NO:175; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:152 and a TCRβ chain variable region as shown in SEQ ID NO:176; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:153 and a TCRβ chain variable region as shown in SEQ ID NO:177; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:154 and a TCRβ chain variable region as shown in SEQ ID NO:178; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:155 and a TCRβ chain variable region as shown in SEQ ID NO:179 ...1; the isolated TCR has a TCRβ chain variable region as shown in SEQ ID NO:152; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:176; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:153 and a TCRβ chain variable region as shown in SEQ ID NO:177; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:154 and a TCRβ chain variable region as shown in SEQ ID NO:178; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:155 and a TCRβ chain variable region as shown in SEQ ID NO:179; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:154; the isolated TCR has a TCRβ chain variable region as shown in SEQ ID NO:175; the isolated TCR The isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:156 and a TCRβ chain variable region as shown in SEQ ID NO:180; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:157 and a TCRβ chain variable region as shown in SEQ ID NO:181; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:158 and a TCRβ chain variable region as shown in SEQ ID NO:182; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:159 and a TCRβ chain variable region as shown in SEQ ID NO:183; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:160 and a TCRβ chain variable region as shown in SEQ ID NO:184; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:161 and a TCRβ chain variable region as shown in SEQ ID NO:184. The TCR β chain variable region of the amino acid sequence shown in NO:185; the isolated TCR has a TCR α chain variable region of the amino acid sequence shown in SEQ ID NO:162 and a TCR β chain variable region of the amino acid sequence shown in SEQ ID NO:186; the isolated TCR has a TCR α chain variable region of the amino acid sequence shown in SEQ ID NO:163 and a TCR β chain variable region of the amino acid sequence shown in SEQ ID NO:187; The isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:164 and a TCRβ chain variable region as shown in SEQ ID NO:188; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:165 and a TCRβ chain variable region as shown in SEQ ID NO:189; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:166 and a TCRβ chain variable region as shown in SEQ ID NO:190; the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:167 and a TCRβ chain variable region as shown in SEQ ID NO:191; or the isolated TCR has a TCRα chain variable region as shown in SEQ ID NO:168 and a TCRβ chain variable region as shown in SEQ ID NO:192.

[0162] According to an embodiment of the present invention, the separated TCR further comprises: The constant regions of the TCRα chain and / or the constant regions of the TCRβ chain.

[0163] According to embodiments of the present invention, the TCRα chain constant region described herein includes TRAC*01, which is the most common TCRα chain constant region sequence, and its amino acid sequence is highly conserved among different individuals. The amino acid sequence of TRAC*01 is as follows: IQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS (SEQ ID NO: 259) According to an embodiment of the present invention, the TCRα chain constant region sequence is exemplified as follows: IQNPDPA VYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIPEDTFFPSX1X2X3X4CDVKLVEKSFETDTNLNFQNLLVIVLRILLLKVAGFNLLM TLRLWSS, wherein X1 is selected from either S or P, X2 is selected from either D or E, X3 is selected from either V or S, and X4 is selected from either P or S.

[0164] The TCR is a structure on the surface of T cells that specifically recognizes antigens. It typically consists of a heterodimer composed of α and β chains, and in rare cases, γ and δ chains. The constant region of the TCR α chain is located near the membrane end of the TCR α chain, i.e., on the side closest to the T cell membrane, and together with the variable region of the TCR α chain, forms the complete α chain. The variable region is responsible for recognizing specific antigens, while the constant region is relatively conserved across different T cells, and its amino acid sequence is relatively stable. Those skilled in the art will understand that the constant region of the TCR α chain mentioned herein includes, but is not limited to, the aforementioned sequence.

[0165] According to embodiments of the present invention, the constant region sequence of the TCR β chain in this document includes, but is not limited to, TRBC1*01 and TRBC2*01. The amino acid sequence of TRBC1*01 is as follows: DLNKVFPPEVAVFEPSEAEISHTQKATLVCLATGFFPDHVELSWWVNGKEVHSGVTDPPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCOVQFYGLSENDewTQDRAKPVTQIVSAEAWGRADCGFTVSYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDF (SEQ ID NO: 260). The TCR is typically a heterodimer composed of an α chain and a β chain linked by disulfide bonds. The constant region of the TCR β chain is located at the end of the β chain closest to the cell membrane, and together with the variable region, constitutes the complete β chain. It consists of a relatively stable amino acid sequence and exhibits high conservation among different T cells. Those skilled in the art will understand that the constant region of the TCR β chain in this document includes, but is not limited to, the aforementioned sequence.

[0166] According to an embodiment of the present invention, the constant region is a mouse constant region or a human constant region.

[0167] According to embodiments of the present invention, the isolated TCR is isolated, purified, or recombinant.

[0168] According to an embodiment of the present invention, the isolated TCR is human.

[0169] According to an embodiment of the present invention, the isolated TCR is monoclonal.

[0170] According to an embodiment of the present invention, the separated TCR is a single chain.

[0171] According to an embodiment of the present invention, the separated TCR comprises two chains.

[0172] According to an embodiment of the present invention, the isolated TCR is in a cell-bound form or a soluble form, preferably in a soluble form.

[0173] According to an embodiment of the present invention, the isolated TCR binds to the antigenic epitope peptide-HLA complex, preferably, the amino acid sequence of the antigenic epitope peptide is shown as at least one of SEQ ID NO:249~258.

[0174] Nucleic acid molecules, expression vectors, recombinant cells In the process of preparing or obtaining the isolated TCR, nucleic acid molecules expressing the isolated TCR can be linked with different vectors and then expressed in host cells to obtain recombinant cells expressing the isolated TCR.

[0175] Therefore, in another aspect of the invention, the invention also provides an isolated nucleic acid molecule. According to embodiments of the invention, the nucleic acid molecule encodes the aforementioned isolated TCR.

[0176] According to an embodiment of the present invention, the nucleic acid molecule is DNA.

[0177] In another aspect, the present invention also provides an expression vector. According to an embodiment of the present invention, the expression vector carries the isolated nucleic acid molecule described above. When the nucleic acid molecule is linked to the expression vector, the nucleic acid molecule can be directly or indirectly linked to control elements on the expression vector, as long as these control elements can control the translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the expression vector itself, or they can be exogenous, i.e., not derived from the expression vector itself. Naturally, the connection between the nucleic acid molecule and the control elements only needs to be operably established.

[0178] In this article, "operable ligation" refers to ligating a foreign gene into a vector so that the control elements within the expression vector, such as transcriptional and translational control sequences, can perform their intended functions of regulating the transcription and translation of the foreign gene. Of course, the polynucleotides encoding the antibody heavy and light chains can be inserted independently into different vectors, but it is more common to insert them into the same vector. Commonly used vectors include plasmids, phages, etc.

[0179] According to an embodiment of the present invention, the expression vector is a eukaryotic expression vector.

[0180] According to an embodiment of the present invention, the expression vector is a lentiviral vector.

[0181] In another aspect, the present invention also provides a recombinant cell. According to embodiments of the invention, the recombinant cell carries the aforementioned isolated nucleic acid molecule or the aforementioned expression vector; or expresses the aforementioned isolated TCR. The expression vector can be introduced into a host cell to construct a recombinant cell expressing the aforementioned isolated TCR, which is capable of specifically recognizing a predetermined antigenic peptide, and exhibits stronger recognition of antigenic organisms carrying said antigenic peptide.

[0182] According to an embodiment of the present invention, the recombinant cells are obtained by introducing the aforementioned expression vector into a host cell.

[0183] According to an embodiment of the present invention, the host cell includes lymphocytes.

[0184] For example, host cells include, but are not limited to, T cells and NK cells.

[0185] Those skilled in the art will understand that the features and advantages described above for the isolated TCR also apply to the nucleic acid molecule, expression vector, and recombinant cells, and will not be repeated here.

[0186] Pharmaceutical Composition In another aspect, the present invention provides a pharmaceutical composition. According to embodiments of the invention, the pharmaceutical composition comprises the aforementioned isolated TCR, the aforementioned nucleic acid molecule, the aforementioned expression vector, or the aforementioned recombinant cells. Through extensive experimentation, the inventors have found that the above-mentioned pharmaceutical composition can effectively prevent and / or treat hepatitis B virus infection and / or prevent and / or treat related diseases caused by hepatitis B virus.

[0187] According to embodiments of the present invention, pharmaceutically acceptable excipients are further included.

[0188] Those skilled in the art will understand that the features and advantages described above for the isolated TCR, nucleic acid molecules, expression vectors and recombinant cells also apply to this pharmaceutical composition, and will not be repeated here.

[0189] use In another aspect of the invention, the invention provides the use of the aforementioned isolated TCR, the aforementioned nucleic acid molecule, the aforementioned expression vector, the aforementioned recombinant cell, or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of hepatitis B virus infection and / or the prevention and / or treatment of related diseases caused by hepatitis B virus.

[0190] Those skilled in the art will understand that the features and advantages described above for isolated TCRs, nucleic acid molecules, expression vectors, recombinant cells and pharmaceutical compositions are equally applicable to this use, and will not be repeated here.

[0191] Methods for preventing and / or treating hepatitis B virus infection and / or related diseases caused by hepatitis B virus In another aspect, the present invention provides a method for preventing and / or treating hepatitis B virus infection and / or related diseases caused by hepatitis B virus. According to an embodiment of the invention, the method comprises administering to a subject a pharmaceutically acceptable amount of the aforementioned pharmaceutical composition. According to an embodiment of the invention, the method can effectively prevent and / or treat hepatitis B virus infection and / or related diseases caused by hepatitis B virus.

[0192] According to an embodiment of the present invention, the method is administered via intravenous injection.

[0193] Those skilled in the art will understand that the features and advantages described above for the pharmaceutical composition also apply to this method, and will not be repeated here.

[0194] Methods for screening TCR In another aspect, the present invention provides a method for screening TCRs. According to an embodiment of the present invention, the method includes: (i) forming an HLA-antigen epitope peptide complex by combining HLA and an antigen epitope peptide, and treating the HLA-antigen epitope peptide complex with T cells to screen for T cells that specifically respond to the HLA-antigen epitope peptide complex; (ii) analyzing the TCR sequence and characteristic genes of the T cells that specifically respond to the complex to screen for TCRs.

[0195] According to embodiments of the present invention, the antigenic epitope peptide can have multiple sources, such as sequences from viruses, tumor neoantigens, or tumor-associated antigens. The amino acid sequence of the candidate HLA-antigenic epitope peptide complex can be determined based on a score calculated using an HLA-binding antigenic epitope peptide prediction algorithm. For example, BIMAS, SYFPEITHI, RANKPEP, or NetMHC can be used to determine the candidate HLA-antigenic epitope peptide complex.

[0196] According to an embodiment of the present invention, the amino acid sequence of the antigenic epitope peptide is selected from at least one of SEQ ID NO:249~258.

[0197] According to an optional embodiment of the present invention, the screening process is achieved by combining a complex containing an antigen epitope peptide sequence and single-cell sequencing technology, including: (1) determining the amino acid sequences of candidate HLA and antigen epitope peptides; (2) synthesizing the determined HLA and antigen epitope peptides and forming a complex in vitro; (3) reacting the HLA-antigen epitope peptide complex with T cells; (4) identifying antigen-specific T cells that can specifically bind to the HLA-antigen epitope peptide complex by single-cell sequencing; and (5) analyzing the TCR sequence and characteristic genes of the identified antigen-specific T cells by bioinformatics analysis of single-cell data.

[0198] T cells In another aspect, the present invention provides a T cell. According to embodiments of the invention, the T cell expresses the aforementioned isolated TCR or carries the aforementioned nucleic acid molecules.

[0199] According to embodiments of the present invention, the T cells include at least one of a T cell population, a T cell line, or a recombinantly expressed T cell.

[0200] According to embodiments of the present invention, the T cell population, T cell line, recombinantly expressed T cells, or nucleic acid sequence encoding the TCR are useful in diagnosis or treatment. For diagnosis, cancer can be detected, or pathological conditions or prognosis can be predicted, by examining whether the aforementioned sequence is only found in cancer patients, whether the aforementioned sequence is more frequently observed in cancer patients, or whether the aforementioned sequence accumulates in cancerous tissues of cancer patients. For cancer treatment, T cell populations, T cell lines, or recombinantly expressed T cells possessing the aforementioned TCR can be utilized.

[0201] In one embodiment of the invention, the cancer includes liver cancer, particularly hepatocellular carcinoma.

[0202] Methods for screening T cells In another aspect, the present invention provides a method for screening T cells. According to an embodiment of the invention, the method includes: forming an HLA-antigen epitope peptide complex by combining HLA and an antigen epitope peptide; and treating T cells by contacting the HLA-antigen epitope peptide complex with T cells to screen for T cells that specifically respond to the HLA-antigen epitope peptide complex.

[0203] According to an optional embodiment of the present invention, the identification process is achieved by combining a complex containing an antigenic epitope peptide sequence and single-cell sequencing technology, including: (1) determining the amino acid sequences of candidate HLA and antigenic epitope peptide; (2) synthesizing the determined HLA and antigenic epitope peptide and forming a complex in vitro; (3) reacting the HLA-antigenic epitope peptide complex with T cells; and (4) identifying antigen-specific T cells that can specifically bind to the HLA-antigenic epitope peptide complex by single-cell sequencing.

[0204] According to embodiments of the present invention, the specific antigenic epitope peptide can have multiple sources, such as sequences from viruses, tumor neoantigens, or tumor-associated antigens. The amino acid sequence of the candidate HLA-peptide can be determined based on a score calculated using an HLA-binding antigenic epitope peptide prediction algorithm. For example, BIMAS, SYFPEITHI, RANKPEP, or NetMHC can be used to determine the candidate HLA-antigenic epitope peptide.

[0205] Methods for identifying HBV antigen-specific T cells In another aspect of the invention, a method for identifying T cells with HBV antigen specificity is provided. According to an embodiment of the invention, the method includes: (1) obtaining gene expression information in the T cells to be identified by staining and / or sequencing; (2) determining whether the T cells to be identified have HBV antigen specificity based on the characteristic genes described in the first aspect of the invention; and (3) analyzing and obtaining T cells with HBV antigen specificity from the T cells to be identified.

[0206] According to an optional embodiment of the present invention, the method includes: (1) obtaining gene expression information in the T cells to be identified by staining and / or sequencing; (2) determining whether the T cells to be identified have HBV antigen specificity based on the characteristic genes described in the first aspect of the present invention; and (3) analyzing and obtaining T cells with HBV antigen specificity from the T cells to be identified. According to an embodiment of the present invention, the staining method may include flow cytometry staining, immunofluorescence staining, MHC tetramers or multimers with fluorescent groups, etc.; the sequencing method includes high-throughput sequencing such as second-generation sequencing and third-generation sequencing, etc.

[0207] Methods for identifying TCRs with HBV antigen specificity In another aspect of the invention, a method for identifying TCRs with HBV antigen specificity is proposed. According to an embodiment of the invention, the method includes: (1) obtaining gene expression information in the T cells to be identified by staining and / or sequencing; (2) determining whether the T cells to be identified have HBV antigen specificity based on the characteristic genes described in the first aspect of the invention; and (3) analyzing and obtaining the TCR sequence of the T cells with HBV antigen specificity in the T cells to be identified.

[0208] According to an optional embodiment of the present invention, the method includes: (1) obtaining gene expression information in the T cells to be identified by staining and / or sequencing; (2) determining whether the T cells to be identified have HBV antigen specificity based on the characteristic genes described in the first aspect of the present invention; and (3) analyzing and obtaining the TCR sequence of the T cells with HBV antigen specificity in the T cells to be identified. According to an embodiment of the present invention, the staining method may include flow cytometry staining, immunofluorescence staining, MHC tetramers or multimers with fluorescent groups, etc.; the sequencing method includes high-throughput sequencing such as second-generation sequencing and third-generation sequencing, etc.

[0209] As can be seen from the foregoing, the CD8 described in this invention +T cell subsets (cTem) are HBV antigen-specific and constitute a group of T cells with both tumor-specific and virus-specific characteristics. Therefore, this invention provides a method for identifying cTem cells using the characteristic genes described in the first aspect of this invention, thereby identifying HBV antigen-specific T cells and their TCR sequences.

[0210] A method for activating, inhibiting, inducing, or expanding T cell subsets In another aspect, the present invention provides a method for activating, inhibiting, inducing, or amplifying T cell subsets.

[0211] A method for activating the aforementioned T cell subsets In another aspect, the present invention provides a method for activating a T cell subset. According to an embodiment of the present invention, the method includes: co-culturing the aforementioned T cell subset with genetically engineered artificial antigen-presenting cells expressing co-stimulatory molecules, or administering exogenous cytokines to activate the T cell subset.

[0212] According to embodiments of the present invention, the T cell subsets are derived from the seven T cell subsets described in the first aspect of the present invention. The present invention provides potential target information for subsequent activation of these cells by identifying the gene expression profiles of these T cell subsets. According to embodiments of the present invention, cTem cells and KIR... + CD8 + T cells and BACH1 + Treg cells are used as an example.

[0213] According to an optional embodiment of the present invention, the present invention provides a method for activating cTem cells. According to an embodiment of the present invention, the method enhances the tumor-killing ability of cTem cells by co-culturing them with genetically engineered artificial antigen-presenting cells expressing co-stimulatory molecules, such as antigen-presenting cells expressing 4-1BBL (APCs, such as dendritic cells), thereby activating cTem cells by enhancing the co-stimulatory signal. In addition, exogenous cytokines or combinations thereof, such as IL-2, IL-7, IL-12, IL-15, or IL-18, are administered to synergistically stimulate cTem cells, promoting their rapid differentiation into highly efficient tumor-killing cells and releasing large amounts of anti-tumor factors.

[0214] According to an optional embodiment of the present invention, the present invention provides an activation of KIR. + CD8 + T-cell methods. According to embodiments of the present invention, the method, in addition to the addition of CD3 and CD28 antibodies, adds HLA ligand stimulation to activate the KIR receptor, thereby activating KIR.+ CD8 + T cells. For example, using APCs (dendritic cells or tumor cells) loaded with specific HLA-I molecules and KIR. + CD8 + T cell co-culture; or direct addition of recombinant HLA molecules and antigens (such as peptides); or direct activation of KIR cells via anti-KIR antibodies (such as anti-KIR2DS1, anti-KIR3DL1). + T cells.

[0215] According to an optional embodiment of the present invention, the present invention provides an activation method for BACH1. + A method using Treg cells. According to embodiments of the present invention, the method, in addition to the addition of CD3 and CD28 antibodies, administers IL-2 and TGF-β1 to promote the inhibitory function of Treg cells and maintain FOXP3 expression. This is achieved by activating the intracellular TGF-β1-Helios signaling pathway, upregulating the expression of immunosuppression-related genes, and enhancing their inhibitory function.

[0216] A method for suppressing the aforementioned T cell subsets In another aspect, the present invention provides a method for inhibiting T cell subsets. According to embodiments of the present invention, the method includes: identifying the T cell subsets using proteins specifically highly expressed by the aforementioned T cell subsets; targeting specific chemokines or receptors on their surface to block their infiltration; or using small molecule inhibitors to interfere with the regulation of downstream signal transduction by key intracellular signaling molecules, thereby inhibiting the T cell subsets.

[0217] According to embodiments of the present invention, the T cell subsets are derived from the seven T cell subsets described in the first aspect of the present invention. The present invention provides potential target information for subsequent inhibition of these cells by identifying the gene expression profiles of these T cells. According to embodiments of the present invention, cTem cells and KIR... + CD8 + T cells and BACH1 + Taking Treg cells as an example, we will now describe them in detail.

[0218] According to an optional embodiment of the present invention, the present invention provides a method for inhibiting cTem cells. According to an embodiment of the present invention, the method includes recognizing cTem cells with highly expressed cTem cell-specific proteins such as GZMK, GPR183, IL7R, and ZNF683 when the body experiences an excessive inflammatory response or when excessive activation of cTem cells in the tumor microenvironment triggers an immune-related adverse reaction, and targeting specific chemokines or receptors on the surface of cTem cells to block their infiltration, thereby alleviating the inflammatory response. For example, blocking cTem cell infiltration by targeting, but not limited to, chemokines XCL1 or XCL2, chemokine receptors CXCR3 or CXCR4 on their surface, thereby inhibiting the excessive immune activity of cTem cells. Simultaneously, small molecule inhibitors are used to interfere with the regulation of downstream signal transduction by key intracellular signaling molecules, reducing the proliferation and effector function of cTem cells.

[0219] According to an optional embodiment of the present invention, the present invention provides a method for suppressing KIR. + CD8 + A T-cell approach. According to embodiments of the invention, the method includes, based on KIR... + CD8 + T cells may play a detrimental role in certain tumors, autoimmune diseases, or transplant rejection; blocking HLA molecules can help alleviate KIR. + CD8 + The inhibitory effect of T cells. Therefore, soluble KIR molecules can be designed to competitively bind to HLA molecules on the surface of target cells, thus blocking KIR. + CD8 + T cell activation signals are blocked, inhibiting the cell's immunosuppressive function. Furthermore, according to the KIR described in the first aspect of the invention... + CD8 + T cells highly express TGFB1, KLRC2, KLRC3, and IKZF2 (Helios), therefore, targeting KIR can be used, but is not limited to. + CD8 + Specific antibodies or small molecule inhibitors targeting TGF-β1 on T cells can block TGF-β1 and Helios, or techniques such as RNA interference and CRISPR-Cas9 can be used to target and silence KLRC2 and KLRC3 or other KLR-related receptors. + CD8 + Genes related to T-cell suppression function should be prevented from exerting excessive suppression to maintain immune balance.

[0220] According to an optional embodiment of the present invention, the present invention provides a method for suppressing BACH1 + A method using Treg cells. According to embodiments of the invention, the method includes, in the treatment of tumors or autoimmune diseases, when BACH1...+ When Treg cell dysfunction leads to immune imbalance, BACH1 can be inhibited through, but not limited to, the following methods. + Treg cells weaken BACH1 + Immunosuppressive function of Treg cells, rebalancing the immune response: RNA interference, CRISPR-Cas9 and other technologies target and silence the BACH1 gene; small molecule inhibitors block genes related to the suppressive function of Treg cells, such as BACH1, REL or BATF; antibody blocking of BACH1 + Treg cells have co-inhibitory receptors on their surface, such as CTLA4, which limit their immunosuppressive function.

[0221] Methods for inducing the aforementioned T cell subsets In another aspect, the present invention provides a method for inducing T cell subsets. According to embodiments of the present invention, the method includes adding an antigenic epitope peptide or exogenous cytokine to an in vitro co-culture system with antigen-presenting cells to induce the generation of the T cell subsets.

[0222] According to embodiments of the present invention, the T cell subsets are derived from the seven T cell subsets described in the first aspect of the present invention. The present invention provides potential target information for subsequent induction of these cells by identifying the gene expression profiles of these T cell subsets. According to embodiments of the present invention, cTem cells and KIR... + CD8 + T cells and BACH1 + Taking Treg cells as an example, we will now describe them in detail.

[0223] According to an optional embodiment of the present invention, the present invention provides a method for inducing cTem cells. According to an embodiment of the present invention, the method includes: inducing cTem cells from naïve T cells; specifically, a tumor antigen peptide may be added to an in vitro co-culture system with antigen-presenting cells to guide the differentiation of naïve T cells into cTem cells with tumor-specific memory and effector functions. Additionally, exogenous cytokines or combinations thereof, such as IL-2, IL-12, IL-15, or IL-18, may be added to promote the proliferation and survival of cTem cells.

[0224] According to an optional embodiment of the present invention, the present invention provides an induced KIR + CD8 + A method for using T cells. According to embodiments of the invention, the method includes inducing KIR from naïve T cells. + CD8 +T cells can be induced to express TGF-β1 by adding specific cytokines such as IL-10 and IL-6 to the culture system; alternatively, recombinant TGF-β1 can be directly added to the culture medium to promote greater TGF-β1 expression through a feedback mechanism, thereby guiding cells towards KIR. + CD8 + T cell lineage differentiation.

[0225] According to an optional embodiment of the present invention, the present invention provides an induction method for BACH1. + A method using Treg cells. According to embodiments of the invention, the method includes BACH1... + Treg cells: from regular CD4 + T cells induce the production of BACH1 + Treg cells, when cultured in vitro with inducers such as IL-2 and TGF-β1, express the key transcription factor Foxp3, driving the cells to migrate towards the immunosuppressive BACH1 receptor. + Treg cell transformation. This is to specifically induce BACH1... + Treg cells can be used to regulate signaling pathways or the transcriptional environment based on traditional methods, such as using inhibitors to suppress the Nrf2 pathway to indirectly enhance BACH1 expression.

[0226] Methods for expanding the aforementioned T cell subsets In another aspect, the present invention provides a method for amplifying T cell subsets. According to an embodiment of the present invention, the method includes: identifying and enriching T cell subsets using the aforementioned characteristic genes, and adding antibodies or exogenous cytokines to promote the proliferation of T cell subsets.

[0227] According to embodiments of the present invention, the T cell subsets are derived from the seven T cell subsets described in the first aspect of the present invention. The present invention provides potential target information for subsequent expansion of these cells by identifying the gene expression profiles of these T cell subsets. According to embodiments of the present invention, cTem cells and KIR... + CD8 + T cells and BACH1 + Taking Treg cells as an example, we will now describe them in detail.

[0228] According to an optional embodiment of the present invention, the present invention provides a method for amplifying cTem cells. According to an embodiment of the present invention, the method includes identifying and enriching cTem cells using specific genes GZMK, GPR183, and IL7R; optimizing culture conditions based on amplification methods with the addition of CD3 and CD28 antibodies; and adding exogenous cytokines or combinations thereof, such as IL-2, IL-12, IL-15, or IL-18, to promote cTem cell proliferation.

[0229] According to an optional embodiment of the present invention, the present invention provides an amplified KIR... + CD8 + A T-cell approach. According to embodiments of the invention, the method includes targeting KIRs via specific gene KIRs. + CD8 + T cells were identified and enriched. Based on the amplification method involving the addition of CD3 and CD28 antibodies, culture conditions were optimized, and exogenous cytokines or combinations, such as IL-2, IL-12, IL-15, or IL-18, were administered to promote KIR. + CD8 + T cell proliferation.

[0230] According to an optional embodiment of the present invention, the present invention provides an amplification of BACH1 + A method using Treg cells. According to embodiments of the invention, the method includes targeting BACH1 cells with specific genes as described in the first aspect of the invention via the genes TNFRSF9 and CTLA4. + Treg cells were identified and enriched, and IL-2 and TGF-β1 were administered in addition to CD3 and CD28 antibodies to promote BACH1. + The expansion of Treg cells.

[0231] A method for in vitro expansion of antigen-specific T cells In another aspect of the present invention, the present invention provides a method for in vitro expansion of antigen-specific T cells. According to an embodiment of the present invention, the method includes (1) isolating T cells from peripheral blood of a patient or donor, or from tumor tissue of a patient, based on the aforementioned characteristic genes; (2) culturing the T cells in vitro, and adding at least one of an antibody, cytokines, an antigen epitope peptide-MHC complex, and antigen-presenting cells to promote the expansion of the T cells; (3) detecting the tumor-killing function of the expanded T cells; wherein the antigen-specific T cells are the T cells identified by the aforementioned method.

[0232] According to an optional embodiment of the present invention, the antigen-specific T cells of the present invention are derived from the cTem cells described in the sixth aspect of the present invention, and the in vitro expansion method may include the following steps: (1) Isolating cTem cells from the peripheral blood of a patient or donor or from the tumor tissue of a patient according to the characteristic gene described in the first aspect of the present invention; (2) In vitro culturing of the isolated cells, adding stimuli to promote cell expansion, such as anti-CD3 / CD28 antibody, cytokines such as IL-2, antigen epitope peptide-MHC complex, co-culturing with antigen-presenting cells (dendritic cells or B cells), co-culturing with tumor cells, etc.; (3) Detecting the tumor killing function of the expanded cells, such as tumor killing experiment, measuring the expression level of anti-tumor factors such as IFN-γ and TNF-α, etc.

[0233] Use of T cells in the preparation of drugs for treating and / or preventing cancer In another aspect, the present invention provides the use of T cells in the preparation of drugs for treating and / or preventing cancer.

[0234] According to embodiments of the present invention, this invention provides the use of the aforementioned T cells in the preparation of pharmaceuticals. The pharmaceuticals are used to treat or prevent cancer, for example, by using in vitro expanded T cells or TCR-T cells for adoptive cell transfer, and by synthesizing immunogenic antigen sequences for cancer vaccines. The T cells of this invention can effectively kill or inhibit tumor killing. The methods for activating, inhibiting, inducing, or expanding the aforementioned T cell subsets of this invention contribute to the development of novel anti-tumor immunotherapies for the treatment or prevention of cancer.

[0235] According to embodiments of the present invention, the cancer includes at least one of the following: liver cancer, stomach cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphoma, esophageal cancer, intestinal cancer, bone marrow cancer, nasal cancer, bone cancer, head and neck cancer, oral cancer, kidney cancer, and Kaposi's sarcoma.

[0236] Methods for preparing T cells containing TCR In another aspect of the invention, the invention provides a method for preparing T cells containing TCRs. According to an embodiment of the invention, the method comprises (1) introducing a TCR α or TCR β gene into a viral or non-viral vector for gene expression; (2) creating a virus from a retroviral vector expressing the TCR α and TCR β genes; (3) independently and sequentially infecting lymphocytes collected from a patient with the virus carrying the TCR α and TCR β genes for transfection, or creating a gene expression retroviral vector including the TCR α and TCR β genes for one-time transformation of the two genes; introducing the DNA or mRNA of the TCR into T cells using electroporation or liposomes; (4) demonstrating that the TCR α / TCR β heterodimer is expressed on the surface of the T cells; wherein the TCR α or TCR β gene is derived from the aforementioned TCRs of the T cells, the aforementioned isolated TCRs, the TCRs screened by the aforementioned method, or the TCRs identified by the aforementioned method.

[0237] In an optional embodiment of the present invention, the preparation method may include the following steps: (1) introducing the TCRα or TCRβ gene described above into a viral vector or non-viral vector for gene expression, such as a lentivirus or retrovirus, and a non-viral vector such as an electroporator or liposome; (2) for the introduction into a viral vector, creating a virus from a retroviral vector expressing the TCRα and TCRβ genes; (3) for the introduction into a viral vector, independently and sequentially infecting lymphocytes collected from a patient with the virus carrying the TCRα and TCRβ genes for transfection, or creating a gene expression retroviral vector including the TCRα and TCRβ genes to transform the two genes at once; for the introduction into a non-viral vector, introducing the DNA or mRNA of the TCR into T cells using an electroporator or liposome; (4) demonstrating that the TCRα / TCRβ heterodimer is expressed on the surface of T cells.

[0238] Adoptive cell therapy methods In another aspect, the present invention provides a method for adoptive cell therapy. According to an embodiment of the invention, the method includes obtaining cells from a patient or donor, screening specific T cell subsets based on the aforementioned characteristic genes, genetically modifying or not modifying the T cell subsets, inducing or expanding the T cell subsets in vitro, and reinfusing the expanded cells into the patient for treatment; wherein the T cell subsets used for adoptive cell therapy are derived from the aforementioned T cell subsets, T cells identified by the aforementioned method, or T cell subsets activated, expanded, or induced by the aforementioned method.

[0239] According to an embodiment of the present invention, the T cell subsets for adoptive cell therapy described in the present invention may be derived from: (1) the TCR-T cells or CAR-T cells prepared above, (2) the T cells described in the first aspect of the present invention, (3) the aforementioned T cells, and (4) the T cells obtained by activation, expansion or induction as described in the seventh aspect. According to an embodiment of the present invention, the specific steps of the adoptive cell therapy may include: (1) isolating tumor-infiltrating lymphocytes (TILs) from surgically removed tumor tissue of the patient, isolating monocytes (PBMCs) from peripheral blood, or collecting PBMCs from a donor; (2) using adherence method, magnetic bead sorting, flow cytometry, or screening for specific subsets of T cells using specific antigens; (3) genetically modifying the T cells, for example, by introducing specific TCRs, or without modification; (4) inducing or expanding the T cells in vitro, expanding the modified or unmodified T cells to the number required clinically; (5) performing functional testing and safety assessment on the expanded cells, such as cell killing activity, proliferation capacity, absence of microbial contamination, absence of off-target gene editing, etc.; and (6) directly reinfusing the cells into the patient via intravenous injection.

[0240] Novel anti-tumor therapies that inhibit or eliminate regulatory T cells In another aspect of the invention, a method for a novel antitumor therapy by inhibiting or eliminating regulatory T cells is provided. According to an embodiment of the invention, the method includes: identifying regulatory T cells using characteristic genes of the T cells described in the first aspect of the invention; screening and knocking out the identified regulatory T cells using target genes to inhibit or eliminate the regulatory T cells; wherein the regulatory T cells include the aforementioned CD8+. + T cell subsets, the aforementioned CD4 + T cells and the aforementioned CD4 + At least one of the T cell subsets.

[0241] According to embodiments of the present invention, the method may include developing novel antitumor therapies targeting regulatory T cells within tumors that inhibit the killing function of cytotoxic T cells, wherein the regulatory T cells may include KIR. + CD8 + T cells, CD4 cells expressing characteristic genes FOXP3 and TNFRSF9 in tumors + T cells or Bach1 + Treg cells, etc. According to embodiments of the present invention, by specifically inhibiting or eliminating these two cell groups, it is helpful to alleviate their inhibitory effect on the anti-tumor ability of cytotoxic T cells. The inhibition method may include the aforementioned inhibition steps, antibody-dependent cell-mediated cytotoxicity (ADCC) methods, or other inhibition methods to be developed. The development process may include the following steps: (1) Identifying KIRs through the characteristic genes described in the first aspect of the present invention.+ CD8 + T cells or FOXP3 + CD4 + Treg cells; (2) By further comparing the characteristic genes of this cell with those of other immune cell populations in the patient's tumor or peripheral blood, genes specifically highly expressed in this cell are screened as target genes; (3) By using gene editing technology, the effects of overexpression or knockout of the target gene in this cell on the differentiation, expansion or inhibition of the cell are studied in vitro or in animal models, thereby verifying the effectiveness of the target gene in anti-tumor therapy; for example, knocking out naïve CD8 in vitro. + This target gene in T cells was examined to verify its effect on KIR. + CD8 + The impact on T cell differentiation; or knockout of naïve CD4 in vitro. + This target gene in T cells was examined to verify its effect on FOXP3. + CD4 + The effect of Treg cell differentiation; or the tumor growth inhibition rate and the number and activity of tumor-infiltrating T cells in transgenic mouse tumor models in which the target gene in the cells has been knocked out under certain conditions.

[0242] Methods for preparing cancer vaccines In another aspect of the invention, the invention provides a method for preparing a cancer vaccine. According to an embodiment of the invention, the method includes: (1) synthesizing the aforementioned antigenic epitope peptide; (2) mixing the antigenic epitope peptide with an adjuvant or a carrier, or extracting monocytes from a patient, culturing them in vitro, and loading them with an antigen; (3) presenting the antigenic epitope peptide to antigen-specific T cells in vitro, and testing the killing activity of the antigen-specific T cells or the level of secreted IFN-γ and TNF-α cytokines to verify the immunogenicity of the antigenic epitope peptide; (4) verifying the immunogenicity in a tumor model or an immune humanization model; and (5) delivering it to the patient's tumor or lymph nodes.

[0243] In an optional embodiment of the present invention, the antigen of the cancer vaccine may be derived from the aforementioned epitope peptide. The synthesized therapeutic vaccine is used to activate the T-cell immune response in the tumor microenvironment or draining lymph nodes. The epitope peptide used for vaccine synthesis may have multiple sources, such as sequences of viruses, tumor neoantigens, or tumor-associated antigens. The delivery method includes dendritic cells, oncolytic viruses, liposomes, or nanoparticles. In an optional embodiment of the present invention, the cancer vaccine development method may include the following steps: (1) synthesizing the epitope peptide; (2) mixing it with an adjuvant or carrier, or extracting the patient's mononuclear cells, culturing them in vitro, and loading the antigen; (3) presenting the epitope peptide to antigen-specific T cells in vitro, and testing the killing activity of antigen-specific T cells or the level of secreted cytokines such as IFN-γ and TNF-α to verify the immunogenicity of the epitope peptide; (4) verifying the immunogenicity in a tumor model or an immune humanization model; (5) delivering it to the patient's tumor or lymph nodes.

[0244] Methods of treating cancer using virus-specific T cells In another aspect of the invention, the invention provides a method for treating cancer using virus-specific T cells. According to an embodiment of the invention, the method includes (1) preparing T cells using peripheral blood mononuclear cells or donor-derived blood; (2) stimulating the T cells with viral peptides, virus-infected target cells, or a vector encoding a viral antigen; (3) expanding the corresponding virus-specific T cells under culture conditions containing supplemental cytokines; (4) ensuring, through in vitro killing experiments, that the expanded T cells have high specificity and low toxicity and can effectively kill target cells; (5) directly reinfusing the expanded T cells into the patient via intravenous injection, or reinfusing them into the patient after combining them with specific targeting techniques to enhance their specificity and function; wherein the T cells are derived from T cells identified by the aforementioned method.

[0245] According to embodiments of the present invention, HBV-specific T cells can recognize HBV antigens expressed by liver cancer cells. Therefore, virus-specific T cells can recognize and kill tumor cells expressing viral antigens. By expanding the patient's own or donor's virus-specific T cells in vitro, the anti-tumor immune effect can be enhanced. The treatment method may include the following steps: (1) preparing T cells using peripheral blood mononuclear cells (PBMCs) or donor-derived blood; (2) stimulating T cells with viral peptides, virus-infected target cells, or vectors encoding viral antigens; (3) expanding the corresponding virus-specific T cells under culture conditions containing supplemental cytokines to ensure that their number is sufficient for treatment; (4) ensuring that the expanded T cells have high specificity and low toxicity and can effectively kill target cells by in vitro killing experiments; (5) directly reinfusing the expanded T cells into the patient via intravenous injection, or reinfusing them into the patient after combining them with specific targeting technologies (such as gene editing or CAR-T technology) to enhance their specificity and function.

[0246] According to embodiments of the present invention, the cancer may include virus-related cancer and non-virus-related cancer. Virus-related cancers include liver cancer infected with HBV and hepatitis C virus (HCV), cervical cancer or head and neck squamous cell carcinoma infected with human papillomavirus (HPV), nasopharyngeal carcinoma or lymphoma infected with Epstein-Barr virus (EBV), and Kaposi's sarcoma infected with Kaposi's sarcoma-associated herpesvirus (KSHV). For non-virus-related cancers, tumor cells can be infected with genetically modified oncolytic viruses (such as adenovirus), causing the infected tumor cells to release viral antigens carried by the oncolytic virus, guiding virus-specific T cells to attack the tumor, thereby enhancing the anti-tumor immune response to treat the cancer.

[0247] Methods using bispecific or trispecific antibodies targeting T cells and other cells In another aspect, the present invention provides a method for using bispecific or trispecific antibodies targeting T cells and other cells. According to embodiments of the present invention, the method includes: selecting antigenic targets on T cells and other cells; designing and constructing antibodies; performing in vitro affinity testing and functional assays; and detecting efficacy, toxicity, and conducting clinical trials. The T cells are derived from the aforementioned T cells, T cells identified by the aforementioned method, or T cells activated, expanded, or induced by the aforementioned method. The other cells include immune cells or non-immune cells, including: immune cells such as T cells, B cells, dendritic cells, macrophages, monocytes, NK cells, and ILC cells; or at least one of tumor cells, tumor-associated fibroblasts, endothelial cells, and epithelial cells.

[0248] According to embodiments of the present invention, the T cells described in the present invention may be derived from: (1) the TCR-T cells or CAR-T cells prepared above, (2) the T cells described in the first aspect of the present invention, (3) the aforementioned T cells, and (4) the aforementioned activated, expanded, or induced T cells. Other cells described in the present invention may include: immune cells such as the aforementioned T cells, B cells, dendritic cells, macrophages, monocytes, NK cells, and ILC cells, or non-immune cells such as tumor cells, tumor-associated fibroblasts, endothelial cells, and epithelial cells. Specific development steps may include: (1) selecting antigen targets on specific T cell subsets based on the characteristics of each T cell subset described in the first aspect of the present invention; (2) selecting antigen targets on other cells; (3) designing and constructing antibodies based on the selected targets; (4) evaluating the effectiveness of the antibody in recruiting and inducing T cells and its tumor-killing ability through in vitro affinity testing and functional tests, and detecting the efficacy and toxicity of the antibody in animal models; (5) verifying the safety, tolerability, and efficacy of the antibody through clinical trials.

[0249] A combination therapy method In another aspect, the present invention provides a method for combination therapy. According to embodiments of the present invention, the method includes a method for preparing T cells containing the aforementioned TCR, a method for adoptive cell therapy, a method for novel antitumor therapy that inhibits or eliminates regulatory T cells, a method for preparing a cancer vaccine, a method for treating cancer using virus-specific T cells, a method for combining a bispecific or trispecific antibody targeting T cells and other cells with an immune checkpoint inhibitor or a small molecule drug. The immune checkpoint inhibitor includes at least one of a CTLA-4 inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor; the small molecule drug includes at least one of a methotrexate, fluorouracil, and imatinib.

[0250] According to embodiments of the present invention, CTLA-4 inhibitors include ipilimumab.

[0251] According to embodiments of the present invention, PD-1 inhibitors include nivolumab and pembrolizumab.

[0252] According to embodiments of the present invention, PD-L1 inhibitors include atezolizumab and durvalumab.

[0253] The combined therapy described in this invention refers to the combination of various treatment methods developed for the T cell subsets described in this invention with one or more existing cancer treatment methods. The T cell subsets may include: (1) the TCR-T cells prepared above, (2) the T cells described in the first aspect of this invention, (3) the aforementioned T cells, and (4) the aforementioned activated, expanded, or induced T cells. The various developed treatment methods include the preparation of the aforementioned TCR-T cells, adoptive cell therapy, novel anti-tumor therapies that inhibit or eliminate Treg cells, cancer vaccines, virus-specific T cells, bispecific or trispecific antibodies targeting T cells and other cells, or similar techniques. The existing cancer treatment methods may include: immune checkpoint inhibitors, small molecule drugs, the preparation of TCR-T cells or CAR-T cells, adoptive cell therapy, cancer vaccines, bispecific or trispecific antibodies targeting T cells and other cells, or similar techniques.

[0254] According to embodiments of the present invention, the cancer includes at least one of the following: liver cancer, stomach cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphoma, esophageal cancer, intestinal cancer, bone marrow cancer, nasal cancer, bone cancer, head and neck cancer, oral cancer, kidney cancer, and Kaposi's sarcoma.

[0255] The Use of T Cell Subsets in the Treatment of Autoimmune Diseases In another aspect, the present invention provides the use of T cell subsets in the treatment of autoimmune diseases.

[0256] According to embodiments of the present invention, the present invention provides the use of the seven T cell subsets described in the first aspect in the treatment of autoimmune diseases. According to embodiments of the present invention, this includes: inhibiting or clearing effector CD8+. + T cell subsets can reduce inflammation, increase the number or function of regulatory T cells to suppress excessive immune responses, or simultaneously target CD8. + A combined therapy of T cells and regulatory T cells. Among them, the seven T cell subsets mentioned in the first aspect include GZMK. + CD8 + T cells, cTem cells, CXCL13 + CD8 + Tpex cells and HBV antigen-specific CD8 + T cells are effector CD8 cells. + T cells, KIR + CD8 + T cells, tumor-resident Treg cells, and BACHI1 +Treg cells are regulatory T cells. In autoimmune diseases such as rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis, organs and tissues such as joints, skin, and central nervous system are in a state of chronic inflammation, and abnormal immune cells are continuously activated and attack the body's own tissues.

[0257] The use of the T cell subsets described in this invention in autoimmune diseases may include: (1) the four effector CD8 cells mentioned above. + T cell subsets may destroy healthy tissue by targeting autoantigens. Therefore, inflammation can be alleviated by inhibiting or eliminating this cell subset to block the autoimmune response caused by its overactivation. The inhibition or elimination method may include the inhibition pathways described above or other pathways of the present invention. (2) In autoimmune diseases, the function or number of Treg cells may be suppressed, leading to the immune system attacking autoimmune tissues. By enhancing KIR + CD8 + T cells, tissue-resident Treg cells, or BACHI1 + The number or function of Treg cells can effectively suppress excessive immune responses and slow disease progression. The enhancement methods may include the aforementioned activation, induction, or expansion methods, as well as other methods that can enhance the number or function of the aforementioned regulatory T cells, such as reinfusion after expansion or enhancing Treg cell function through gene editing. (3) Simultaneously targeting the aforementioned CD8 + Combined therapy of T cells and regulatory T cells.

[0258] The autoimmune diseases described in this invention include at least one of the following: rheumatoid arthritis, systemic lupus erythematosus, systemic sclerosis, Sjögren's syndrome, antiphospholipid syndrome, multiple sclerosis, myasthenia gravis, Guillain-Barré syndrome, type 1 diabetes mellitus, Hashimoto's thyroiditis, Graves' disease (hyperthyroidism), Addison's disease (adrenal insufficiency), inflammatory bowel disease, autoimmune hepatitis, primary biliary cholangitis, vitiligo, psoriasis, scleroderma, idiopathic thrombocytopenic purpura, autoimmune hemolytic anemia, antiglomerular basement membrane disease, IgA nephropathy, inflammatory myopathy, Behçet's disease, and relapsing polychondritis.

[0259] Methods to inhibit the transformation of T cells into regulatory T cells In another aspect, the present invention provides a method for inhibiting the conversion of T cells into regulatory T cells. According to an embodiment of the present invention, the method includes inhibiting CD8+. + The expression of TGFB1 in T cells, or the inhibition of CD4 + Expression of BACH1 in T cells.

[0260] According to an embodiment of the present invention, the suppression of CD8 +The expression of TGFB1 in T cells, or the inhibition of CD4 + BACH1 expression in T cells occurs through at least one of the following mechanisms: gene silencing or gene editing, small molecule inhibitors, and antibody drugs.

[0261] According to embodiments of the present invention, the gene silencing or gene editing includes at least one of CRISPR-Cas9 virus and non-virus, TALEN, ZFN, shRNA, and siRNA.

[0262] According to an optional embodiment of the present invention, the suppression of CD8 + The expression of TGFB1 in T cells, or the inhibition of CD4 + BACH1 expression in T cells occurs via CRISPR-Cas9 knockout of CD8. + TGFB1 in T cells, or knockout of CD4 + BACH1 on T cells.

[0263] According to an embodiment of the present invention, the method includes inhibiting the expression of TGFB1 in the T cells; the T cells include CD8+. + T cells. As previously known, expression or high expression of TGFB1 leads to CD8... + T cells become regulatory KIR + CD8 + T cells. Therefore, according to embodiments of the present invention, this method helps to reduce regulatory KIR. + CD8 + The number of T cells to alleviate regulatory KIR + CD8 + T cells inhibit the anti-tumor ability of cytotoxic T cells.

[0264] According to an embodiment of the present invention, the method includes inhibiting the expression of BACH1 in the T cells; the T cells include CD4+. + T cells. As previously known, inhibiting BACH1 protein in T cells can downregulate the expression of FOXP3 in T cells, thereby inhibiting the transformation of T cells into regulatory T (Treg) cells. Therefore, according to embodiments of the present invention, this method helps to reduce the number of Treg cells, thereby alleviating the inhibition of the anti-tumor ability of cytotoxic T cells by Treg cells.

[0265] Use of inhibitors in the preparation of tumor therapeutic drugs In another aspect, the present invention provides the use of an inhibitor in the preparation of a tumor therapeutic drug. According to embodiments of the present invention, the inhibitor inhibits the expression of a target gene or a protein that inhibits the expression of a target gene, wherein the target gene is selected from at least one of BACH1, TGFB1, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KLRC2, KLRC3, IKZF2, and TYROBP.

[0266] According to embodiments of the present invention, the expression or high expression of BACH1 leads to the corresponding CD4... + T cells transform into immunosuppressive Treg cells. The expression or high expression of TGFB1, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KLRC2, KLRC3, IKZF2, or TYROBP leads to CD8+ transformation. + T cells become regulatory KIR + CD8 + Inhibiting the expression of these genes or the activity of the expressed proteins will help relieve the inhibitory effect of regulatory T cells on cytotoxic T cells, thereby achieving the effect of treating tumors.

[0267] Methods for enriching T cell subsets In another aspect of the invention, a method for enriching the T cell subsets described in the first aspect of the invention is provided. According to an embodiment of the invention, the method includes: contacting a population of immune cells infiltrating a tumor with a binding agent, said binding agent binding to at least one target gene or a protein or protein fragment expressed therein; and sorting the immune cells bound to said binding agent to enrich the T cell subsets; wherein the target gene is selected from the aforementioned characteristic genes.

[0268] According to an optional embodiment of the present invention, the method includes the following steps: contacting a population of immune cells infiltrating a tumor with a binding agent, the binding agent being bound to at least one of the aforementioned target genes or proteins or protein fragments expressed therein; and sorting the immune cells bound to the binding agent to provide an enriched subset of T cells.

[0269] According to an optional embodiment of the present invention, the binding agent includes nucleic acids, ligands, enzymes, substrates, antibodies, etc.

[0270] According to an optional embodiment of the present invention, CD8 is enriched. +The method for T cell subsets includes the following steps: contacting a population of immune cells infiltrating a tumor with a binding agent that binds to a target gene or its expressed protein or protein fragment; and sorting the immune cells bound to the binding agent to provide enriched CD8. + T cell subsets.

[0271] According to an optional embodiment of the present invention, the target gene (characteristic gene) includes GZMK and PDCD1. Preferably, the target gene further includes at least one of MT1X, MT1E, MT2A, CCL4, XCL1, and XCL2.

[0272] According to an optional embodiment of the present invention, the target gene (characteristic gene) includes GZMK, ZNF683, GPR183, and IL7R. Preferably, the target gene further includes at least one of CXCR3, CXCR4, CD44, CCR7, LMNA, XCL1, and XCL2.

[0273] According to an optional embodiment of the present invention, the target gene (characteristic gene) includes GZMK and CXCL13. Preferably, the target gene further includes at least one of CXCR4, DUSP4, DUSP1, IFNG, MT1X, MT1E, MT2A, XCL1, and XCL2.

[0274] According to an optional embodiment of the present invention, the target gene (characteristic gene) includes GZMK and ZNF683. Preferably, the target gene further includes at least one of CCL4L2, NR4A2, and CCL4.

[0275] According to an optional embodiment of the present invention, the target gene (characteristic gene) includes at least one of the KIR gene families KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL3. Preferably, the target gene further includes at least one of KLRC2, KLRC3, IKZF2, TYROBP, XCL1, and XCL2.

[0276] According to an optional embodiment of the present invention, CD4 is enriched. + The method for T cell subsets includes the following steps: contacting a population of immune cells infiltrating a tumor with a binding agent that binds to a target gene or its expressed protein or protein fragment; and sorting the immune cells bound to the binding agent to provide enriched CD4. + T cell subsets.

[0277] According to an optional embodiment of the present invention, the target gene (characteristic gene) includes FOXP3, TNFRSF9, and CTLA4. Preferably, the target gene further includes at least one of CTSC, IKZF2, STAM, and DUSP4.

[0278] According to an optional embodiment of the present invention, the target gene (characteristic gene) includes FOXP3 and BACH1. Preferably, the target gene further includes at least one of REL, ICOS, AHR, CREM, and NFE2L2.

[0279] In a preferred embodiment of the present invention, the sorting steps include fluorescence-activated cell sorting, magnetically assisted cell sorting, substrate-assisted cell sorting, laser-mediated cutting, fluorescence assay, flow cytometry, or microscopy.

[0280] Diagnostic or evaluation methods In another aspect of the invention, the invention provides a method for diagnosis or evaluation. According to an embodiment of the invention, the method includes (1) classifying a sample of immune cells infiltrating a tumor obtained from a subject as CD8. + T cells and CD4 + T cells; (2) the separated CD8 + T cells are contacted with at least one binding agent, which binds to a target gene or a protein or protein fragment expressed thereon, wherein the target gene is selected from the characteristic genes described in the first aspect of the present invention; (3) the separated CD4 cells are... + T cells are contacted with at least one binding agent, which binds to a target gene or a protein or protein fragment expressed thereon, wherein the target gene is selected from the characteristic genes described in the first aspect of the present invention.

[0281] According to an optional embodiment of the present invention, the method includes the following steps: 1) classifying immune cell samples obtained from the subject that have infiltrated the tumor as CD8. + T cells and CD4 + T cells; and, 2) the separated CD8 + T cells are contacted with at least one binding agent that binds to a target gene or its expressed protein or protein fragment, said target gene (characteristic gene) including GZMK and PDCD1; and / or, 3) the separated CD8 + T cells are contacted with at least one binding agent that binds to a target gene or its expressed protein or protein fragment, said target gene (characteristic gene) including GZMK, ZNF683, GPR183, and IL7R; and / or, 4) the separated CD8 +T cells are contacted with at least one binding agent that binds to a target gene or a protein or fragment thereof expressed therein, said target gene (characteristic gene) including GZMK and CXCL13; and / or, 5) the separated CD8 + T cells are contacted with at least one binding agent that binds to a target gene or its expressed protein or protein fragment, said target gene (characteristic gene) including GZMK and ZNF683; and / or, 6) the separated CD8 + T cells are contacted with at least one binding agent that binds to a target gene or a protein or fragment thereof expressed therein, said target gene (characteristic gene) including KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, or KIR3DL3; and / or, 7) the separated CD4 + T cells are contacted with at least one binding agent that binds to a target gene or a protein or fragment thereof expressed therein, said target gene (characteristic gene) including FOXP3, TNFRSF9, and CTLA4; and / or, 8) the separated CD4 + T cells contact at least one binding agent that binds to a target gene or its expressed protein or protein fragment, wherein the target gene (characteristic gene) includes FOXP3 and BACH1. The binding agent includes nucleic acids, ligands, enzymes, substrates, antibodies, etc. Preferably, in step 2), the target gene (characteristic gene) further includes at least one of MT1X, MT1E, MT2A, CCL4, XCL1, and XCL2. Preferably, in step 3), the target gene (characteristic gene) further includes at least one of CXCR3, CXCR4, CD44, CCR7, LMNA, XCL1, and XCL2. Preferably, in step 4), the target gene (characteristic gene) further includes at least one of CXCR4, DUSP4, DUSP1, IFNG, MT1X, MT1E, MT2A, XCL1, and XCL2. Preferably, in step 5), the target gene (characteristic gene) further includes at least one of CCL4L2, NR4A2, and CCL4. In preferred step 6), the target gene (characteristic gene) further includes at least one of KLRC2, KLRC3, IKZF2, TYROBP, XCL1, and XCL2. In preferred step 7), the target gene (characteristic gene) further includes at least one of CTSC, IKZF2, STAM, and DUSP4. In preferred step 8), the target gene (characteristic gene) further includes at least one of REL, ICOS, AHR, CREM, and NFE2L2.

[0282] In one embodiment of the invention, the method is performed before and after the patient receives anti-PD-1 combined with lenvatinib combination therapy. In other embodiments, the method may be performed before or after the subject receives other treatments, such as after chemotherapy, radiotherapy, surgery, and other immunotherapies.

[0283] In one embodiment of the present invention, the method is used to assess the therapeutic effect on a subject. For example, monitoring the expression of the target gene CD8 in the patient's peripheral blood or tumor microenvironment before, during, or after treatment. + or CD4 + Changes in T-cell subsets can serve as predictive indicators to differentiate between treatment-sensitive and insensitive patients, or to assess a patient's response to treatment. This allows for timely cessation and modification of treatment strategies, enabling patients to receive more appropriate treatment plans earlier.

[0284] According to embodiments of the present invention, the cancer includes at least one of the following: liver cancer, stomach cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphoma, esophageal cancer, intestinal cancer, bone marrow cancer, nasal cancer, bone cancer, head and neck cancer, oral cancer, kidney cancer, and Kaposi's sarcoma.

[0285] CD8 for diagnosis or monitoring + T cell biomarkers In another aspect, the present invention provides a CD8 for diagnosis or monitoring. + A biomarker set for T cells. According to embodiments of the present invention, the biomarker set comprises the characteristic gene described in the first aspect of the invention or the protein or protein fragment expressed by the characteristic gene.

[0286] According to embodiments of the present invention, the present invention provides a CD8 diagnostic or monitoring method. + T cell biomarkers, CD8 + T cells contain the characteristic genes GZMK and PDCD1, or the protein or protein fragment expressed by the characteristic genes.

[0287] According to an embodiment of the present invention, the biomarker set further comprises at least one of the characteristic genes MT1X, MT1E, MT2A, CCL4, XCL1 and XCL2, or a protein or protein fragment expressed by the at least one gene.

[0288] According to an embodiment of the present invention, the biomarker set comprises proteins or protein fragments expressed by the characteristic genes GZMK, PDCD1, MT1X, MT1E, MT2A, CCL4, XCL1, and XCL2.

[0289] According to an embodiment of the present invention, the diagnosis or monitoring may be a diagnosis or monitoring of CD8. + The effector or effector memory state of T cells. The diagnosis or monitoring could be used to diagnose or monitor the efficacy of tumor immunotherapy.

[0290] According to an embodiment of the present invention, CD8 in tumors of other patients + Compared to T cells, increased expression or expression of at least one of the above genes indicates that CD8 + The fact that T cells are in an effector or effector memory state further indicates that the patient's immunotherapy is effective.

[0291] According to embodiments of the present invention, the present invention provides a CD8 diagnostic or monitoring method. + T cell biomarkers, CD8 + T cells contain the characteristic genes GZMK, ZNF683, GPR183, and IL7R, or the proteins or protein fragments expressed by the characteristic genes.

[0292] According to an embodiment of the present invention, the biomarker set further comprises at least one of the characteristic genes CXCR3, CXCR4, CD44, CCR7, LMNA, XCL1 and XCL2, or a protein or protein fragment expressed by the at least one characteristic gene.

[0293] According to an embodiment of the present invention, the biomarker set comprises proteins or protein fragments expressed by the characteristic genes GZMK, ZNF683, GPR183, IL7R, CXCR3, CXCR4, CD44, CCR7, LMNA, XCL1, and XCL2.

[0294] According to an embodiment of the present invention, the diagnosis or monitoring may be a diagnosis or monitoring of CD8. + The circulating effector memory state of T cells. The diagnosis or monitoring could be used to diagnose or monitor the efficacy of tumor immunotherapy.

[0295] According to an embodiment of the present invention, CD8 in tumors of other patients + Compared to T cells, increased expression or expression of at least one of the above genes indicates that CD8 + The T cells are in a state of cyclic effector memory, which further indicates that the patient's immunotherapy is effective.

[0296] According to embodiments of the present invention, the present invention provides a CD8 diagnostic or monitoring method. + T cell biomarkers, CD8 + T cells contain the characteristic genes GZMK and CXCL13, or the protein or protein fragment expressed by the characteristic genes.

[0297] According to an embodiment of the present invention, the biomarker set further comprises at least one of the characteristic genes CXCR4, DUSP4, DUSP1, IFNG, MT1X, MT1E, MT2A, XCL1 and XCL2, or a protein or protein fragment expressed by the at least one characteristic gene.

[0298] According to an embodiment of the present invention, the biomarker set comprises proteins or protein fragments expressed by the characteristic genes GZMK, CXCL13, CXCR4, DUSP4, DUSP1, IFNG, MT1X, MT1E, MT2A, XCL1, and XCL2.

[0299] According to an embodiment of the present invention, the diagnosis or monitoring may be a diagnosis or monitoring of CD8. + The state of T cell progenitor cell depletion. The diagnosis or monitoring may be used to diagnose or monitor the efficacy of tumor immunotherapy.

[0300] According to an embodiment of the present invention, CD8 in tumors of other patients + Compared to T cells, the expression or increased expression of at least one of the above-mentioned characteristic genes indicates that CD8 + The fact that T cells are in a state of progenitor cell depletion further indicates that the patient's immunotherapy is effective.

[0301] According to embodiments of the present invention, the present invention provides a CD8 diagnostic or monitoring method. + T cell biomarkers, CD8 + T cells contain the characteristic genes GZMK and ZNF683, or the protein or protein fragment expressed by the characteristic genes.

[0302] According to an embodiment of the present invention, the biomarker set further comprises at least one of the characteristic genes CCL4L2, NR4A2 and CCL4, or a protein or protein fragment expressed by the at least one characteristic gene.

[0303] According to embodiments of the present invention, the biomarker set comprises proteins or protein fragments expressed by the characteristic genes GZMK, ZNF683, CCL4L2, NR4A2 and CCL4.

[0304] According to an embodiment of the present invention, the diagnosis or monitoring may be a diagnosis or monitoring of HBV-specific CD8. + T cells. The diagnosis or monitoring may be used to diagnose or monitor the efficacy of tumor immunotherapy.

[0305] According to an embodiment of the present invention, CD8 in tumors of other patients+ Compared to T cells, the expression or increased expression of at least one of the above-mentioned characteristic genes indicates that CD8 + T cells are HBV-specific CD8 cells. + T cells further indicate that the patient's immunotherapy is effective.

[0306] According to embodiments of the present invention, the present invention provides a CD8 diagnostic or monitoring method. + T cell biomarkers, CD8 + T cells contain the characteristic genes KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL3, or the proteins or protein fragments expressed by the characteristic genes.

[0307] According to an embodiment of the present invention, the biomarker set further comprises at least one of the characteristic genes KLRC2, KLRC3, IKZF2, TYROBP, XCL1 and XCL2, or a protein or protein fragment expressed by the at least one characteristic gene.

[0308] According to embodiments of the present invention, the biomarker set comprises characteristic genes KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KLRC2, KLRC3, IKZF2, TYROBP, XCL1, and XCL2, or proteins or protein fragments expressed by the characteristic genes.

[0309] According to an embodiment of the present invention, the diagnosis or monitoring may be a diagnosis or monitoring of KIR. + CD8 + T cells. The diagnosis or monitoring may be used to diagnose or monitor the efficacy of tumor immunotherapy.

[0310] According to an embodiment of the present invention, CD8 in tumors of other patients + Compared to T cells, increased expression or expression of at least one of the above genes indicates that CD8 + T cells are KIR + CD8 + T cells, further, indicate poor efficacy of immunotherapy in patients.

[0311] CD4 for diagnosis or monitoring + T cell biomarkers In another aspect, the present invention provides a CD4 for diagnosis or monitoring. +A biomarker set for T cells. According to embodiments of the present invention, the biomarker set comprises the characteristic gene described in the first aspect of the invention or the protein or protein fragment expressed by the characteristic gene.

[0312] According to embodiments of the present invention, the present invention provides a CD4 for diagnosis or monitoring. + T cell biomarkers, CD4 + T cells contain the genes FOXP3, TNFRSF9, and CTLA4, or the proteins or protein fragments expressed by the characteristic genes.

[0313] According to an embodiment of the present invention, the biomarker set further comprises the characteristic genes CTSC, IKZF2, STAM and DUSP4, or the protein or protein fragment expressed by the characteristic genes.

[0314] According to embodiments of the present invention, the biomarker set includes the characteristic genes FOXP3, TNFRSF9, CTLA4, CTSC, IKZF2, STAM and DUSP4, or the proteins or protein fragments expressed by the characteristic genes.

[0315] According to an embodiment of the present invention, the diagnosis or monitoring may be a diagnosis or monitoring of CD4. + Whether the T cells are active regulatory T cells. The diagnosis or monitoring may be for diagnosing or monitoring the efficacy of tumor immunotherapy.

[0316] According to embodiments of the present invention, CD4 in normal tissue or peripheral blood of a patient + Compared to T cells, the expression or increased expression of the characteristic gene indicates that CD4+ + The T cells are active regulatory T cells, which further suggests that the patient's immunotherapy is not effective.

[0317] According to embodiments of the present invention, the present invention provides a CD4 for diagnosis or monitoring. + T cell biomarkers, CD4 + T cells contain the characteristic genes FOXP3 and BACH1, or the proteins or protein fragments expressed by the characteristic genes.

[0318] According to an embodiment of the present invention, the biomarker set further comprises proteins or protein fragments expressed by the characteristic genes REL, ICOS, AHR, CREM and NFE2L2, or the characteristic genes.

[0319] According to an embodiment of the present invention, the biomarker set comprises proteins or protein fragments expressed by the characteristic genes FOXP3, BACH1, REL, ICOS, AHR, CREM and NFE2L2.

[0320] According to an embodiment of the present invention, the diagnosis or monitoring may be a diagnosis or monitoring of CD4. + Whether the T cells are regulatory T cells with strong clonal expansion potential. The diagnosis or monitoring may be for diagnosing or monitoring the efficacy of tumor immunotherapy.

[0321] According to embodiments of the present invention, CD4 in normal tissue or peripheral blood of a patient + Compared to T cells, the expression or increased expression of the characteristic gene indicates that CD4+ + The T cells are regulatory T cells with strong clonal expansion potential, which further suggests that the patient's immunotherapy is ineffective.

[0322] According to embodiments of the present invention, the cancer includes at least one of the following: liver cancer, stomach cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphoma, esophageal cancer, intestinal cancer, bone marrow cancer, nasal cancer, bone cancer, head and neck cancer, oral cancer, kidney cancer, and Kaposi's sarcoma.

[0323] Test reagents for diagnosis or monitoring In another aspect of the invention, a diagnostic reagent is provided for diagnosis or monitoring. According to an embodiment of the invention, the diagnostic reagent comprises a binder that binds to the characteristic gene described in the first aspect of the invention or the protein or protein fragment expressed by the characteristic gene.

[0324] According to embodiments of the present invention, the present invention provides a diagnostic or monitoring reagent comprising a binder that binds to a protein or protein fragment containing the characteristic genes GZMK and PDCD1, or expressed by the characteristic genes.

[0325] According to an embodiment of the present invention, the detection reagent further comprises a binder that binds to at least one of the characteristic genes MT1X, MT1E, MT2A, CCL4, XCL1 and XCL2, or a protein or protein fragment expressed by the at least one characteristic gene.

[0326] According to an embodiment of the present invention, the detection reagent further comprises a binding agent that binds to proteins or protein fragments expressed by the characteristic genes GZMK, PDCD1, MT1X, MT1E, MT2A, CCL4, XCL1 and XCL2, or the characteristic genes.

[0327] According to embodiments of the present invention, the diagnosis or monitoring may be for diagnosing or monitoring the effector or effector memory state of CD8+ T cells. The diagnosis or monitoring may also be for diagnosing or monitoring the efficacy of tumor immunotherapy.

[0328] According to an embodiment of the present invention, CD8 in tumors of other patients + Compared to T cells, the expression or increased expression of at least one of the above-mentioned characteristic genes indicates that CD8 + The fact that T cells are in an effector or effector memory state further indicates that the patient's immunotherapy is effective.

[0329] According to embodiments of the present invention, the present invention provides a diagnostic or monitoring reagent comprising a binding agent that binds to characteristic genes GZMK, ZNF683, GPR183 and IL7R, or proteins or protein fragments expressed by the characteristic genes.

[0330] According to an embodiment of the present invention, the detection reagent further comprises a binder that binds to at least one of the characteristic genes CXCR3, CXCR4, CD44, CCR7, LMNA, XCL1 and XCL2, or a protein or protein fragment expressed by the at least one characteristic gene.

[0331] According to an embodiment of the present invention, the detection reagent comprises a binder that binds to the characteristic genes GZMK, ZNF683, GPR183, IL7R, CXCR3, CXCR4, CD44, CCR7, LMNA, XCL1, and XCL2, or to the proteins or protein fragments expressed by the characteristic genes.

[0332] According to an embodiment of the present invention, the diagnosis or monitoring may be a diagnosis or monitoring of CD8. + The circulating effector memory state of T cells. The diagnosis or monitoring could be used to diagnose or monitor the efficacy of tumor immunotherapy.

[0333] According to an embodiment of the present invention, CD8 in tumors of other patients + Compared to T cells, increased expression or expression of at least one of the above genes indicates that CD8 + The T cells are in a state of cyclic effector memory, which further indicates that the patient's immunotherapy is effective.

[0334] According to embodiments of the present invention, the present invention provides a CD8 diagnostic or monitoring method. + T-cell detection reagents, the detection reagents comprising binding agents that bind to the characteristic genes GZMK and CXCL13, or proteins or protein fragments expressed by the characteristic genes.

[0335] According to an embodiment of the present invention, the detection reagent further comprises a binder that binds to at least one of the characteristic genes CXCR4, DUSP4, DUSP1, IFNG, MT1X, MT1E, MT2A, XCL1, and XCL2, or a protein or protein fragment expressed by the at least one characteristic gene.

[0336] According to an embodiment of the present invention, the detection reagent comprises a binder that binds to proteins or protein fragments expressed by the characteristic genes GZMK, CXCL13, CXCR4, DUSP4, DUSP1, IFNG, MT1X, MT1E, MT2A, XCL1, and XCL2, or the characteristic genes themselves.

[0337] According to an embodiment of the present invention, the diagnosis or monitoring may be a diagnosis or monitoring of CD8. + The state of T cell progenitor cell depletion. The diagnosis or monitoring may be used to diagnose or monitor the efficacy of tumor immunotherapy.

[0338] According to an embodiment of the present invention, CD8 in tumors of other patients + Compared to T cells, the expression or increased expression of at least one of the above-mentioned characteristic genes indicates that CD8 + The fact that T cells are in a state of progenitor cell depletion further indicates that the patient's immunotherapy is effective.

[0339] According to embodiments of the present invention, the present invention provides a diagnostic reagent for diagnosis or monitoring. According to embodiments of the present invention, the diagnostic reagent comprises a binding agent that binds to the characteristic genes GZMK and ZNF683, or the protein or protein fragment expressed by the characteristic genes.

[0340] According to an embodiment of the present invention, the detection reagent further comprises a binder that binds to at least one of the characteristic genes CCL4L2, NR4A2 and CCL4, or a protein or protein fragment expressed by the at least one characteristic gene.

[0341] According to embodiments of the present invention, the detection reagent comprises a binding agent that binds to proteins or protein fragments expressed by the characteristic genes GZMK, ZNF683, CCL4L2, NR4A2 and CCL4.

[0342] According to an embodiment of the present invention, the diagnosis or monitoring may be a diagnosis or monitoring of HBV-specific CD8. + T cells. The diagnosis or monitoring may be used to diagnose or monitor the efficacy of tumor immunotherapy.

[0343] According to an embodiment of the present invention, CD8 in tumors of other patients +Compared to T cells, the expression or increased expression of at least one of the above-mentioned characteristic genes indicates that CD8 + T cells are HBV-specific CD8 cells. + T cells further indicate that the patient's immunotherapy is effective.

[0344] According to embodiments of the present invention, the present invention provides a diagnostic or monitoring reagent comprising a binding agent that binds to characteristic genes KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL3, or proteins or protein fragments expressed by the characteristic genes.

[0345] According to an embodiment of the present invention, the detection reagent further comprises a binder that binds to at least one of the characteristic genes KLRC2, KLRC3, IKZF2, TYROBP, XCL1 and XCL2, or a protein or protein fragment expressed by said at least one gene.

[0346] According to embodiments of the present invention, the detection reagent comprises a binder that binds to the characteristic genes KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KLRC2, KLRC3, IKZF2, TYROBP, XCL1, and XCL2, or the protein or protein fragment expressed by the characteristic genes.

[0347] According to an embodiment of the present invention, the diagnosis or monitoring may be a diagnosis or monitoring of KIR. + CD8 + T cells. The diagnosis or monitoring may be used to diagnose or monitor the efficacy of tumor immunotherapy.

[0348] According to an embodiment of the present invention, CD8 in tumors of other patients + Compared to T cells, the expression or increased expression of at least one of the above-mentioned characteristic genes indicates that CD8 + T cells are KIR + CD8 + T cells, further, indicate poor efficacy of immunotherapy in patients.

[0349] Test reagents for diagnosis or monitoring In another aspect, the present invention provides a diagnostic reagent for diagnosis or monitoring.

[0350] According to embodiments of the present invention, the detection reagent comprises a binder that binds to the characteristic genes FOXP3, TNFRSF9, and CTLA4, or the protein or protein fragment expressed by the characteristic genes.

[0351] According to embodiments of the present invention, the detection reagent further comprises a binding agent that binds to the characteristic genes CTSC, IKZF2, STAM and DUSP4, or the protein or protein fragment expressed by the characteristic genes.

[0352] According to embodiments of the present invention, the detection reagent comprises a binder that binds to the characteristic genes FOXP3, TNFRSF9, CTLA4, CTSC, IKZF2, STAM, and DUSP4, or the protein or protein fragment expressed by the characteristic genes.

[0353] According to an embodiment of the present invention, the diagnosis or monitoring may be a diagnosis or monitoring of CD4. + Whether the T cells are active regulatory T cells. The diagnosis or monitoring may be for diagnosing or monitoring the efficacy of tumor immunotherapy.

[0354] According to embodiments of the present invention, CD4 in normal tissue or peripheral blood of a patient + Compared to T cells, the expression or increased expression of the characteristic gene indicates that CD4+ + The T cells are active regulatory T cells, which further suggests that the patient's immunotherapy is not effective.

[0355] According to embodiments of the present invention, the present invention provides a diagnostic reagent for diagnosis or monitoring. According to embodiments of the present invention, the diagnostic reagent comprises a binding agent that binds to the characteristic genes FOXP3 and BACH1, or the protein or protein fragment expressed by the characteristic genes.

[0356] According to an embodiment of the present invention, the detection reagent further comprises a binding agent that binds to proteins or protein fragments expressed by the characteristic genes REL, ICOS, AHR, CREM and NFE2L2, or the characteristic genes.

[0357] According to an embodiment of the present invention, the detection reagent comprises a binding agent that binds to proteins or protein fragments expressed by the characteristic genes FOXP3, BACH1, REL, ICOS, AHR, CREM and NFE2L2.

[0358] According to an embodiment of the present invention, the diagnosis or monitoring may be a diagnosis or monitoring of CD4. + Whether the T cells are regulatory T cells with strong clonal expansion potential. The diagnosis or monitoring may be for diagnosing or monitoring the efficacy of tumor immunotherapy.

[0359] According to embodiments of the present invention, CD4 in normal tissue or peripheral blood of a patient + Compared to T cells, the expression or increased expression of the characteristic gene indicates that CD4+ +The T cells are regulatory T cells with strong clonal expansion potential, which further suggests that the patient's immunotherapy is ineffective.

[0360] According to embodiments of the present invention, the cancer includes at least one of the following: liver cancer, stomach cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphoma, esophageal cancer, intestinal cancer, bone marrow cancer, nasal cancer, bone cancer, head and neck cancer, oral cancer, kidney cancer, and Kaposi's sarcoma.

[0361] According to an embodiment of the present invention, the detection reagent comprises a set of primers capable of specifically amplifying each gene (characteristic gene) in the biomarker set.

[0362] According to an embodiment of the present invention, the detection reagent comprises a set of probes capable of hybridizing with each gene in the biomarker group.

[0363] According to embodiments of the present invention, the binding agent is a nucleic acid, ligand, enzyme, substrate, and / or antibody.

[0364] According to an embodiment of the present invention, the binding agent is a nucleic acid probe capable of binding to the target gene (characteristic gene). According to an embodiment of the present invention, the binding agent is a primer capable of specifically amplifying a target gene (characteristic gene). According to an embodiment of the present invention, the binding agent is an antibody capable of binding to a protein or protein fragment encoded by the target gene (characteristic gene). According to embodiments of the present invention, the binder further binds an indicator molecule, such as a fluorescent substance, a radioactive substance, and / or an enzyme. Kits for diagnosing or monitoring tumor prognosis In another aspect, the present invention provides a kit for diagnosing or monitoring tumor prognosis. According to embodiments of the present invention, the kit comprises the aforementioned detection reagents.

[0365] According to embodiments of the present invention, a kit for diagnosing or monitoring tumor prognosis is provided. According to embodiments of the present invention, the kit contains components capable of interacting with CD8... + A binding agent that binds to the T cell characteristic gene GZMK or its expressed protein or protein fragments, and a binding agent that can bind to CD8. + A binding agent that binds to the T cell's characteristic gene PDCD1 or its expressed protein or protein fragments.

[0366] According to an embodiment of the present invention, the kit further comprises components capable of interacting with CD8. + A binding agent that binds to the T cell's characteristic gene MT1X or its expressed protein or protein fragments, and / or can bind to CD8.+ A binding agent that binds to the T cell's characteristic gene MT1E or its expressed protein or protein fragment, and / or can bind to CD8. + A binding agent that binds to the T cell characteristic gene MT2A or its expressed protein or protein fragments, and / or a binding agent that can bind to CD8. + A binding agent that binds to the T cell's characteristic gene CCL4 or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the characteristic gene XCL1 of T cells or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the characteristic gene XCL2 of T cells or its expressed protein or protein fragments.

[0367] According to an embodiment of the present invention, the kit contains components capable of interacting with CD8. + A binding agent that binds to the T cell's characteristic gene GZMK or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene PDCD1 or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene MT1X or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene MT1E or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene MT1E or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene CCL4 or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell characteristic gene XCL1 or its expressed protein or protein fragments, and a binding agent that can bind to CD8. + A binding agent that binds to the characteristic gene XCL2 of T cells or its expressed protein or protein fragments.

[0368] According to embodiments of the present invention, a kit for diagnosing or monitoring tumor prognosis is provided. According to embodiments of the present invention, the kit contains components capable of interacting with CD8... + A binding agent that binds to the T cell's characteristic gene GZMK or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene ZNF683 or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell characteristic gene GPR183 or its expressed protein or protein fragments, and a binding agent that can bind to CD8. + A binding agent that binds to the T cell's characteristic gene IL7R or its expressed protein or protein fragments.

[0369] According to an embodiment of the present invention, the kit further comprises components capable of interacting with CD8. + A binding agent that binds to the T cell's characteristic gene CXCR3 or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the T cell's characteristic gene CXCR4 or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the T cell's characteristic gene CD44 or its expressed protein or protein fragments, and / or a binding agent that can bind to CD8. + A binding agent that binds to the T cell's characteristic gene CCR7 or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the T cell's characteristic gene LMNA or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the characteristic gene XCL1 of T cells or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the characteristic gene XCL2 of T cells or its expressed protein or protein fragments.

[0370] According to an embodiment of the present invention, the kit contains components capable of interacting with CD8. + A binding agent that binds to the T cell's characteristic gene GZMK or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene ZNF683 or its expressed protein or protein fragments, and can bind to CD8 + A binding agent that binds to the T cell's characteristic gene GPR183 or its expressed protein or protein fragments, and can bind to CD8 + A binding agent that binds to the T cell's characteristic gene IL7R or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene CXCR3 or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene CXCR4 or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene CD44 or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene CCR7 or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene LMNA or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene XCL1 or its expressed protein or protein fragments, and can bind to CD8. +A binding agent that binds to the characteristic gene XCL2 of T cells or its expressed protein or protein fragments.

[0371] According to embodiments of the present invention, a kit for diagnosing or monitoring tumor prognosis is provided. According to embodiments of the present invention, the kit contains components capable of interacting with CD8... + A binding agent that binds to the T cell characteristic gene GZMK or its expressed protein or protein fragments, and a binding agent that can bind to CD8. + A binding agent that binds to the T cell's characteristic gene CXCL13 or its expressed protein or protein fragments.

[0372] According to an embodiment of the present invention, the kit further comprises components capable of interacting with CD8. + A binding agent that binds to the T cell's characteristic gene CXCR4 or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the T cell's characteristic gene DUSP4 or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the T cell characteristic gene DUSP1 or its expressed protein or protein fragments, and / or a binding agent that can bind to CD8. + A binding agent that binds to the T cell's characteristic gene IFNG or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the T cell's characteristic gene MT1X or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the T cell's characteristic gene MT1E or its expressed protein or protein fragment, and / or can bind to CD8. + A binding agent that binds to the T cell's characteristic gene MT2A or its expressed protein or protein fragments, and / or a binding agent that can bind to CD8. + A binding agent that binds to the characteristic gene XCL1 of T cells or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the characteristic gene XCL2 of T cells or its expressed protein or protein fragments.

[0373] According to an embodiment of the present invention, the kit contains components capable of interacting with CD8. + A binding agent that binds to the T cell's characteristic gene GZMK or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene CXCL13 or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene CXCR4 or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene DUSP4 or its expressed protein or protein fragments, and can bind to CD8.+ A binding agent that binds to the T cell's characteristic gene DUSP1 or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene IFNG or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene MT1X or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene MT1E or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene MT2A or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene XCL1 or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the characteristic gene XCL2 of T cells or its expressed protein or protein fragments.

[0374] According to embodiments of the present invention, a kit for diagnosing or monitoring tumor prognosis is provided. According to embodiments of the present invention, the kit contains components capable of interacting with CD8... + A binding agent that binds to the T cell characteristic gene GZMK or its expressed protein or protein fragments, and a binding agent that can bind to CD8. + A binding agent that binds to the characteristic gene ZNF683 of T cells or its expressed protein or protein fragments.

[0375] According to an embodiment of the present invention, the kit further comprises components capable of interacting with CD8. + A binding agent that binds to the characteristic gene CCL4L2 of T cells or its expressed protein or protein fragments, and / or a binding agent that can bind to CD8. + A binding agent that binds to the T cell's characteristic gene NR4A2 or its expressed protein or protein fragments, and / or a binding agent that can bind to CD8. + A binding agent that binds to the characteristic gene CCL4 of T cells or its expressed protein or protein fragments.

[0376] According to an embodiment of the present invention, the kit contains components capable of interacting with CD8. + A binding agent that binds to the T cell's characteristic gene GZMK or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell's characteristic gene ZNF683 or its expressed protein or protein fragments, and can bind to CD8 + A binding agent that binds to the T cell's characteristic gene CCL4L2 or its expressed protein or protein fragments, and can bind to CD8. + A binding agent that binds to the T cell characteristic gene NR4A2 or its expressed protein or protein fragments, and a binding agent that can bind to CD8. +A binding agent that binds to the characteristic gene CCL4 of T cells or its expressed protein or protein fragments.

[0377] According to embodiments of the present invention, a kit for diagnosing or monitoring tumor prognosis is provided. According to embodiments of the present invention, the kit contains components capable of interacting with CD8... + A binding agent that binds to the T cell characteristic gene KIR2DL1 or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the T cell characteristic gene KIR2DL3 or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the T cell characteristic gene KIR2DL4 or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the T cell characteristic gene KIR3DL1 or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the T cell characteristic gene KIR3DL2 or its expressed protein or protein fragments, and / or a binding agent that can bind to CD8. + A binding agent that binds to the characteristic gene KIR3DL3 of T cells or its expressed protein or protein fragments.

[0378] According to an embodiment of the present invention, the kit further comprises components capable of interacting with CD8. + A binding agent that binds to the characteristic gene KLRC2 of T cells or its expressed protein or protein fragments, and / or a binding agent that can bind to CD8. + A binding agent that binds to the T cell's characteristic gene KLRC3 or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the T cell characteristic gene IKZF2 or its expressed protein or protein fragments, and / or a binding agent that can bind to CD8. + A binding agent that binds to the T cell's characteristic gene TYROBP or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the characteristic gene XCL1 of T cells or its expressed protein or protein fragments, and / or can bind to CD8. + A binding agent that binds to the characteristic gene XCL2 of T cells or its expressed protein or protein fragments.

[0379] Kits for diagnosing or monitoring tumor prognosis In another aspect, the present invention provides a kit for diagnosing or monitoring tumor prognosis. According to an embodiment of the invention, the kit comprises components capable of interacting with CD4... + A binding agent that binds to the T cell's characteristic gene FOXP3 or its expressed protein or protein fragments, and can bind to CD4. +A binding agent that binds to the T cell characteristic gene TNFRSF9 or its expressed protein or protein fragments, and a binding agent that can bind to CD4. + A binding agent that binds to the T cell's characteristic gene CTLA4 or its expressed protein or protein fragments.

[0380] According to an embodiment of the present invention, the kit further comprises components capable of interacting with CD4. + A binding agent that binds to the T cell's characteristic gene CTSC or its expressed protein or protein fragments, and / or can bind to CD4. + A binding agent that binds to the T cell's characteristic gene IKZF2 or its expressed protein or protein fragments, and / or a binding agent that can bind to CD4+. + A binding agent that binds to the STAM gene, a characteristic gene of T cells, or to the protein or protein fragments it expresses, and / or, to CD4+. + A binding agent that binds to the characteristic T cell gene DUSP4 or its expressed protein or protein fragments.

[0381] According to embodiments of the present invention, a kit for diagnosing or monitoring tumor prognosis is provided. According to embodiments of the present invention, the kit contains components capable of interacting with CD4. + A binding agent that binds to the T cell characteristic gene FOXP3 or its expressed protein or protein fragments, and a binding agent that can bind to CD4. + A binding agent that binds to the characteristic T cell gene BACH1 or its expressed protein or protein fragments.

[0382] According to an embodiment of the present invention, the kit further comprises components capable of interacting with CD4. + A binding agent that binds to the T cell's characteristic gene ICOS or its expressed protein or protein fragments, and / or can bind to CD4. + A binding agent that binds to the T cell's characteristic gene AHR or its expressed protein or protein fragment, and / or can bind to CD4. + A binding agent that binds to the T cell's characteristic gene CREM or its expressed protein or protein fragments, and / or can bind to CD4. + A binding agent that binds to the T cell's characteristic gene NFE2L2 or its expressed protein or protein fragments.

[0383] According to embodiments of the present invention, the binding agent includes nucleic acids, ligands, enzymes, substrates, antibodies, etc.

[0384] In one embodiment of the invention, the kit includes one or more containers containing one or more of the binders. More preferably, it also includes instructional materials for using the kit, such as an instruction manual.

[0385] According to embodiments of the present invention, the cancer includes at least one of the following: liver cancer, stomach cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphoma, esophageal cancer, intestinal cancer, bone marrow cancer, nasal cancer, bone cancer, head and neck cancer, oral cancer, kidney cancer, and Kaposi's sarcoma.

[0386] According to embodiments of the present invention, the binding agent is a nucleic acid, ligand, enzyme, substrate, and / or antibody.

[0387] According to an embodiment of the present invention, the binding agent is a nucleic acid probe capable of binding to the target gene (characteristic gene). According to an embodiment of the present invention, the binding agent is a primer capable of specifically amplifying a target gene (characteristic gene). According to an embodiment of the present invention, the binding agent is an antibody capable of binding to a protein or protein fragment encoded by the target gene (characteristic gene). According to embodiments of the present invention, the binder further binds an indicator molecule, such as a fluorescent substance, a radioactive substance, and / or an enzyme. Methods for identifying novel antitumor effector T cell subsets In another aspect, the present invention provides a method for identifying novel antitumor effector T cell subsets. According to an embodiment of the present invention, the method includes: (1) For HBV patients receiving neoadjuvant therapy with anti-PD-1 combined with lenvatinib + T cells from HCC patients were analyzed using single-cell RNA sequencing (scRNA-seq), single-cell TCR sequencing (scTCR-seq), and flow cytometry. (2) Analyze the single-cell gene expression profile of T cells in cancer tissues and paired peripheral blood, and isolate and characterize T cell subsets that reflect the body's tumor immune status; (3) Determine the characteristic genes expressed by the T cell subsets and the relationship between these cell subsets and drug response.

[0388] According to an embodiment of the present invention, the T cell subset includes: GZMK + CD8 + Teff / Tem cells, the GZMK + CD8 + Teff / Tem cells are adapted for large-scale clonal expansion in tumors of responders after treatment; Tpex cells, adapted to exhibit higher CXCL13 expression levels compared to terminally exhausted T cells (tTex); cTem cells, adapted to exhibit higher levels of expression of effector memory marker genes; HBV-specific CD8+. +T cells, the HBV-specific CD8 + T cells are adapted to preferentially maintain an effector or memory state and are enriched in cTem cell populations; KIR + CD8 + T cell subsets, the KIR + CD8 + T cell subsets are associated with resistance to combination therapy; activated CD4 + Treg cell subsets, the activated CD4 + Treg cell subsets are more readily observed in tumors after combination therapy in non-responders; CD4 cells expressing BACH1 + Treg cells, CD4 cells expressing BACH1 + Treg cells are adapted to be highly expressed in tumors in non-responders before treatment.

[0389] Methods for screening drugs In another aspect, the present invention provides a method for screening drugs. According to an embodiment of the present invention, the method includes the following steps: 1) mixing a test chemical substance with the characteristic gene or a protein expressed therein, or mixing the test chemical substance with CD8 expressing the characteristic gene. + T cells or CD4 + 2) Detect changes in the activity of the expressed protein, or whether the test chemical substance binds to the characteristic gene or the protein it expresses, or changes in the activity of the cell, or changes in the expression level of the characteristic gene in the cell; the characteristic gene is selected from at least one of the following genes: BACH1, TGFB1, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KLRC2, KLRC3, IKZF2, TYROBP, GZMK, MT1X, MT1E, MT2A, CCL4, XCL1, XCL2, GPR183, IL7R, ZNF683, CXCL13, CCL4L2 and NR4A2.

[0390] In a preferred embodiment of the present invention, the drug is used to treat tumors. According to embodiments of the present invention, the cancer includes at least one of the following: liver cancer, stomach cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphoma, esophageal cancer, intestinal cancer, bone marrow cancer, nasal cancer, bone cancer, head and neck cancer, oral cancer, kidney cancer, and Kaposi's sarcoma.

[0391] The sequence list of the present invention is shown below. The sequence details involved in this invention are shown in the table below: sequence list

[0392] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0393] The chemical reagents used in the following examples are all conventional reagents and are commercially available. The analysis software used and its sources are as follows: Cell Ranger (http: / / 10xgenomics.com); Scrublet (https: / / github.com / swolock / scrublet); Scanpy (https: / / github.com / scverse / scanpy); STARTRAC (https: / / github.com / Japrin / STARTRAC); BBKNN (https: / / github.com / Teichlab / bbknn); Celltypist (https: / / github.com / Teichlab / celltypist); COSG (https: / / github.com / genecell / COSG); Seurat (https: / / satijalab.org / seurat); Coin (http: / / coin.r-forge.r-project.org); Fitdistrplus (https: / / github.com / aursiber / fitdistrplus); Mixtools (https: / / github.com / dsy109 / mixtools); FastQC (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc); Trim Galore (https: / / www.bioinformatics.babraham.ac.uk / projects / trim_galore); Salmon (https: / / combine-lab.github.io / salmon); Tximport (https: / / bioconductor.org / packages / release / bioc / html / tximport.html); DESeq2 (https: / / bioconductor.org / packages / release / bioc / html / DESeq2.html); OptiType (https: / / github.com / FRED-2 / OptiType); EnhancedVolcano (https: / / bioconductor.org / packages / release / bioc / html / EnhancedVolcano.html); clusterProfiler (https: / / bioconductor.org / packages / release / bioc / html / clusterProfiler.html); AUCell (https: / / bioconductor.org / packages / release / bioc / html / AUCell.html); ComplexHeatmap (https: / / bioconductor.org / packages / release / bioc / html / ComplexHeatmap.html); SCENIC (https: / / scenic.aertslab.org); Igraph (https: / / r.igraph.org); Scvelo (https: / / github.com / theislab / scvelo); Python (https: / / www.python.org); DexTRAC (https: / / github.com / lijxug / DexTRAC). Example 1: Identification of T cell functional subsets 1. Construction and sequencing of single-cell libraries. First, single cells were isolated from peripheral blood and tissues using density gradient centrifugation and tissue digestion, respectively. Then, CD45 cells were sorted using a flow cytometry (FACS) system. + Immune cells were collected and then subjected to single-cell sequencing and data analysis. The specific procedures were as follows: Single cells were collected by cutting fresh tumor biopsy samples and surgically removed tumor tissue into fragments of approximately 1-2 mm³ in an environment containing RPMI-1640 medium (Gibco). The tissue fragments were enzymatically digested at 37°C using a gentleMACS instrument (Miltenyi) on a vortex mixer for 60 minutes. The digested cells were filtered through a 100-μm SmartStrainer (Miltenyi) and centrifuged at 400g for 5 minutes. The precipitated cells were resuspended in erythrocyte lysis buffer (Miltenyi) and incubated on ice for 1-2 minutes to remove erythrocytes. After washing twice with PBS, the cell pellet was resuspended in FBS (Gibco) containing 10% DMSO (Sigma) and stored in liquid nitrogen for preservation. Fresh peripheral blood PBMCs were separated using HISTOPAQUE-1077 (Sigma) solution and washed twice with PBS. Erythrocytes were removed using the same procedure described above. CD45 was sorted using a BD FACSAria III flow cytometer. + Live cells were used, and libraries were constructed according to the standard operating procedures of the 10x Chromium single-cell 5' sequencing platform (10x Genomics) and the human variable region, differentiation region and junction region (VDJ) kit. The libraries were then analyzed using an Illumina NovaSeq 6000 sequencer (using 150-bp paired-end sequencing mode).

[0394] A total of 9 patients were matched with CD45 samples from tumors and peripheral blood. + Single-cell transcriptome libraries of immune cells and their accompanying T-cell receptor (TCR) libraries.

[0395] 2. CD4 + T cells and CD8 +Identification of T cell populations. In this embodiment, the generated scRNA-seq data was processed using the Cell Ranger single-cell toolkit (version 6.1.2) and aligned to the GRCh38 human reference genome (refdata-gex-GRCh38-2020-A). Preliminary filtered raw count data generated by Cell Ranger was used for subsequent analysis. In this embodiment, the count data was normalized using scanpy (v1.8.2), batch-corrected using BBKNN (v1.5.1), and further identified as major cell populations using the Leiden clustering method in scanpy. In the first round of clustering, unsupervised clustering was used to distinguish T / natural killer cells (T / NK cells), which are characterized by their highly expressed marker genes CD3D / CD3G. Further differentiation of CD4... + T cells, CD8 + T cells, whose cell types are characterized by the marker genes CD4 and CD8A / CD8B, respectively. In the second round of clustering, T cell functional subsets are determined by combining cell types predicted by celltypist (v0.2.0), expression levels of classic marker genes, and marker genes identified by COSG (v1.0.1). In this embodiment of the invention, the following two methods are used to identify cell marker genes: the FindMarkers function of the Seurat (v4.3.0) package and COSG (v1.0.1).

[0396] The results showed that the embodiments of the present invention obtained 11 CD8 genes with different characteristic genes and tissue distribution patterns. + T cell subsets, results see [link to results] Figure 1 A, 1B. Two of these cell subpopulations originated from the blood and were defined as naive / central memory T cells (Tn / Tcm) due to their high expression of CCR7, LEF1, SELL, TCF7, and GPR183 genes. Another cell subpopulation, primarily distributed in the blood, possessed high levels of CX3CR1 and other genes related to T cell effector function (such as GZMB and GZMH), and was therefore defined as recently activated effector memory or effector T cells (Temra). See the results below. Figure 1 B. Two cell subpopulations, primarily derived from blood and tumor, respectively, were identified as KIR cells due to their high expression of the inhibitory killer cell immunoglobulin-like receptor (KIR) gene. + CD8 + T cells, results see Figure 1B. A cell subset was distributed in both the tumor and the bloodstream, and highly expressed chemokines (XCL1 and XCL2) and metallothionein genes (MT1X, MT1E, and MT2A). Compared with Tn / Tcm cells, this cell subset showed higher levels of marker genes of effector or effector memory T cells (Teff / Tem) (such as GZMK, PRDM1, IFNG, and TNF). Compared with terminally exhausted T cells (tTex) mainly located in the tumor, the Teff / Tem cells in this subset showed lower expression levels of exhaustion-related genes (such as PDCD1, LAYN, TOX, and ENTPD1). See [see results]. Figure 1 B.

[0397] Furthermore, in this embodiment of the invention, nine CD4 clusters were obtained through unsupervised clustering analysis. + T cell subsets were identified, and two types of Treg cells were recognized within them. See the results below. Figure 1 C. A group of resting Treg cells (CD4_C08_FOXP3) mainly originated from peripheral blood; a group of activated Treg cells (CD4_C07_CTLA4) were mainly distributed in tumor tissue. Furthermore, activated Treg cells exhibited higher levels of repressive molecule expression, including CTLA4 and TNFRSF9; see [see results]. Figure 1 D.

[0398] Example 2: Effector or effector memory T cells (Teff / Tem) expressing the characteristic gene GZMK contain a group of progenitor exhausted T cells (Tpex) expressing the characteristic gene CXCL13 and a group of effector memory T cells (Tem) with peripheral circulation characteristics.

[0399] 1. Identification of T cell receptor (TCR) sequences. In this embodiment of the invention, the generated TCR sequence data were processed using Cell Ranger software (v.6.1.2) and its provided human VDJ reference genome (refdata-cellranger-vdj-GRCh38-alts-ensembl-5.0.0). The amino acid sequences of the complementarity-determining region 3 (CDR3) of the TCR α and β chains contained in each sample were used for downstream analysis. For each patient, T cells with the same VJ and VDJCDR3 nucleotide sequences and using the same TRAV, TRBV, TRAJ, and TRBJ gene fragments were defined as originating from the same clone.

[0400] The results show that this embodiment uses 118,130 CD3s + 58,872 T cells with paired TCR α and β chains were identified in the T cell group.

[0401] 2. Identification of Tpex and cTem based on whether they share a TCR sequence with terminally exhausted T cells (tTex). In this embodiment of the invention, tumor-infiltrating Teff / Tem cells from Example 1 are further divided into progenitor exhausted T cells (Tpex) and circulating effector memory T cells (cTem). Tpex cells are identified by their identical TCR to tTex cells, while cTem cells primarily share a TCR with blood-derived Temra or Teff / Tem cells, but do not share a TCR with tTex cells. See [link to results]. Figure 2 A. Furthermore, the inventors compared the expression levels of specific genes in Tpex cells and cTem cells, and used the average expression levels of four exhausted genes—HAVCR2, ENTPD1, ​​LAYN, and LAG3—as the exhaustion score.

[0402] The results showed that, unlike cTem cells, both Tpex cells and tTex cells exhibited high levels of CXCL13 expression. (See attached results). Figure 2 B. Tpex cells showed higher exhaustion scores and higher TOX expression levels; see results below. Figure 2 C, 2D. TOX is a hallmark gene associated with exhaustion, and these results support the developmental trajectory of Tpex cells to tTex cells.

[0403] 3. Furthermore, the inventors analyzed the differentially expressed characteristic genes of cTem and Tpex cells in tumors. The specific steps were as follows: first, the "FindMarkers" function in the Seurat software package was used to compare the differentially expressed characteristic genes between the two groups of cells; then, the significance of each gene was assessed using the Wilcoxon rank-sum test; and multiple hypothesis testing was performed using the Benjamini-Hochberg method for correction. Finally, differentially expressed characteristic genes with adjusted p-values ​​less than 0.01 or 0.05 were identified.

[0404] The results showed that, compared with Tpex cells and tTex cells, cTem cells exhibited higher levels of expression of effector memory marker genes, including GNLY and IL7R. (See attached results). Figure 2 D indicates its memory potential during combined treatment.

[0405] 4. The inventors compared the effects of several genes related to T cell function on CD8 cells in different tumor infiltration sites. + The expression levels of T cells can be used to further distinguish these cell groups.

[0406] The results showed that a series of specific genes can be used to differentiate four CD8+ genes within tumors. + T cell population, including Temra cells (CX3CR1) + GZMK- ZNF683 + CXCL13 - ENTPD1 - GPR183 - ), cTem cells (CX3CR1) - GZMK + ZNF683 + CXCL13 - ENTPD1 - GPR183 hi Tpex cells (CX3CR1) - GZMK + ZNF683 - CXCL13 + ENTPD1 lo GPR183 lo ) and tTex cells (CX3CR1) - GZMK + ZNF683 - CXCL13 + ENTPD1 hi GPR183 - (See results) Figure 2 D.

[0407] 5. Tpex cells and cTem cells have different transcriptomic characteristics. To evaluate the enrichment of characteristic genes of previously reported tTex (terminally exhausted T cells), Mem (memory T cells), Eff-like (effector T cell-like), and Eff (effector T cell) subsets (Giles et al., Nat Immunol, 2022) in the Tpex, cTem, and tTex subsets of the dataset, this embodiment of the invention performed gene set enrichment analysis (GSEA). The specific steps are as follows: First, the "FindAllMarkers" function of the Seurat (v4.3.0) package was used to extract differentially expressed characteristic genes from the tTex, Mem, Eff-like, and Eff subsets. The screening criteria were an adjusted P-value < 0.01 and log2(FC) > 0.5. These genes were considered significant characteristic genes to characterize their respective cell subsets. Second, single cells with both transcriptomic and TCR data were screened from the scRNA-seq dataset. For the Tpex analysis, cells were divided into two groups: Tpex and other cells. Then, genes with an average expression level exceeding 0.1 in either group were selected, and the fold change (FC) of expression levels between the Tpex group and other cell groups was calculated, with genes sorted in descending order. Finally, the "GSEA" function from the clusterProfiler (v.3.18.1) package was used to perform gene set enrichment analysis on the Tpex, cTem, and tTex subpopulations, based on the sorted gene list and the characteristic gene sets of the tTex, Mem, Eff-like, and Eff subpopulations, respectively.

[0408] The results showed that the transcriptome of Tpex cells was more enriched with exhausted T cells in mice infected with chronic lymphocytic choriomeningitis virus (LCMV) strain C113. Compared with Tpex cells and tTex cells, the transcriptome of cTem cells was more enriched with antigen-specific effector memory T cells in mice infected with acute LCMV strain Armstrong, but less correlated with exhausted T cells in mice infected with chronic LCMV. This finding suggests that Tpex cells can differentiate into tTex cells, while cTem cells are not associated with exhaustion. (See attached results). Figure 2 E.

[0409] 6. Through the TCR clonality analysis in step 1 of this embodiment, the inventors compared different tumor-infiltrating CD8... + T cell populations and peripheral blood CD8 + The inventors further quantified the transformation relationships between T cell subsets using the STARTRAC algorithm (Zhang et al., Nature, 2018).

[0410] The results showed that, compared with Tpex and tTex cells, cTem cells had a higher concentration of CD8+ in the blood. + T cells share more TCR clones; results can be found in [link to results]. Figure 2 F indicates that cTem cells have circulating potential. Further analysis using the STARTRAC algorithm revealed that Temra cells, primarily distributed in the blood, and cTem cells exhibited the highest transformation levels; see [see results]. Figure 2 G further indicates that cTem originates from peripheral blood, while Tpex cells tend to originate from other sites and subsequently differentiate into tTex cells within the tumor.

[0411] Example 3: The Teff / Tem cell subset expressing the characteristic gene GZMK participates in the antitumor response of both combination therapy and anti-PD-1 monotherapy. In this embodiment, "combination therapy" refers to treatment with a combination of lenvatinib and anti-PD-1 monotherapy.

[0412] By tracking changes in cell proportions before and after treatment, cell populations associated with treatment response and drug resistance can be identified. Previous studies have generally suggested that cell populations with increased proportions in responders after treatment are associated with antitumor response, while those with decreased proportions are associated with treatment resistance.

[0413] 1. In the embodiments of this invention, the inventors calculated the CD8 infiltrating CD8 in all tumors. + The CD8 cells described in Example 1 in T cells + The proportion of T cell subsets. Specifically, this proportion is the percentage of CD8+ cells in each tumor or peripheral blood sample. + Using the number of T cell subsets as the molecule, and considering all CD8 cells in this sample... + The number of T cells is the denominator.

[0414] The results showed that, after combination therapy, Teff / Tem cells in the responders of Example 1 significantly expanded in the tumors, while no similar phenomenon was observed in the non-responders. (See attached results). Figure 3 A, 3B. Simultaneously, the proportion of tTex cells in responders decreased. This suggests that the expansion of Teff / Tem cells may be an important characteristic of treatment response, while the reduction of tTex cells may be associated with an improved tumor immune microenvironment.

[0415] 2. To further elucidate the association between T-cell response and the clinical efficacy of combination therapy, and to compare it with anti-PD-1 monotherapy, this embodiment of the invention systematically compared patients' CD8 levels by integrating combination therapy data with reported anti-PD-1 monotherapy data (Magenet al., Nat Med, 2023). +T-cell responses. The inventors used Harmony to remove batch effects from the two datasets, then defined cell subpopulations using the clustering method in Example 1, and finally calculated the CD8+ responses in the anti-PD-1 monotherapy data using the method in step 1 of this example. + T cell subsets in all tumor-infiltrating CD8 + The proportion of T cells.

[0416] The results showed that GZMK was found in the tumors of responders after anti-PD-1 monotherapy. + Tem / Teff cells showed a significantly higher proportion, compared to a lower proportion in non-responder tumors. (See results below.) Figure 3 C. This indicates that GZMK + Tem / Teff cells demonstrated antitumor responses in both combination therapy and anti-PD-1 monotherapy.

[0417] Example 4: Tpex cell subsets expressing the characteristic gene CXCL13 participate in the antitumor response of both combination therapy and anti-PD-1 monotherapy. 1. In the embodiments of the present invention, CD8 infiltrating cells in all tumors were calculated. + The proportion of Tpex cell subsets in T cells. Specifically, this proportion is calculated by using the number of Tpex cell subsets in each tumor sample as the numerator, and the proportion of all CD8+ cells in that sample as the numerator. + The number of T cells is the denominator.

[0418] The results showed that, after combination therapy, the proportion of Tpex cells in Example 2 significantly increased from an average of 4.1% in the tumor before treatment to 11.6% in the tumor of responders after treatment, and was higher than the proportion of Tpex cells in non-responders after treatment. (See attached results). Figure 4 A.

[0419] 2. Based on the integrated analysis results in Example 3, the tumor-infiltrating Tpex cells in all tumor-infiltrating CD8 groups were calculated from the anti-PD-1 monotherapy data. + The proportion of T cells.

[0420] The results showed that Tpex cells were enriched in tumors of responders after anti-PD-1 monotherapy. (See attached results). Figure 4 B. This indicates that Tpex cells are involved in both combination therapy and anti-PD-1 monotherapy in the anti-tumor response.

[0421] Example 5: cTem cell subset-specific involvement in the antitumor response of combination therapy 1. In the embodiments of the present invention, CD8 infiltrating cells in all tumors were calculated. +The proportion of cTem cells among T cells. Specifically, this proportion is calculated using the number of cTem cells in each tumor sample as the numerator and the total number of CD8 cells in that sample as the denominator. + The number of T cells is the denominator.

[0422] The results showed that the proportion of cTem cells in the responder's tumors significantly increased after treatment, from 32.3% before treatment to 49.9% after treatment. (See attached results). Figure 5 A.

[0423] 2. Based on the integrated analysis results in Example 3, the tumor-infiltrating cTem cells in all tumor-infiltrating CD8 groups were calculated from the anti-PD-1 monotherapy data. + The proportion of T cells.

[0424] The results showed no significant difference in the proportion of cTem cells in tumors of responders and non-responders after anti-PD-1 monotherapy. (See attached results.) Figure 5 B. Previous reports have shown that combination therapy is more effective than anti-PD-1 monotherapy, therefore this result indicates that cTem cells specifically participate in the anti-tumor response of combination therapy and play a key role in improving the clinical efficacy of combination therapy.

[0425] Example 6: Combined with lenvatinib, it enhanced tumor angiogenesis normalization after anti-PD-1 therapy, thereby promoting CD8. + Teff cell infiltration To further explore the mechanism by which cTem cells enhanced their response to combination therapy in Example 5, this embodiment of the invention integrates bulk RNA-seq data from 15 tumor samples after combination therapy in this study, as well as tumor data from HCC patients who received anti-PD-1 monotherapy (Li et al., Hepatology, 2024). Based on previous reports that dual PD-1 and VEGFR2 blockade can promote vascular normalization and increase immune infiltration in mouse models, the inventors hypothesized that combining lenvatinib with anti-PD-1 therapy may enhance T-cell infiltration in HCC patients by promoting vascular normalization. To verify this hypothesis, this embodiment uses a vascular normalization index based on previous studies on liver cancer tumor angiogenesis (Tian et al., Nature, 2017).

[0426] The results showed that, compared with anti-PD-1 monotherapy, combination therapy significantly enhanced vascular normalization in patients' tumors. (See attached results). Figure 6 A. Furthermore, this embodiment, through analysis of differentially expressed genes using DESeq2 software, revealed that in post-treatment tumors of combination therapy responders, differentially expressed genes were associated with T cells and CD8. +The expression levels of genes associated with Teff cell characteristics were significantly higher than those treated with anti-PD-1 monotherapy. (See results below.) Figure 6 B indicates CD8 + Enhanced Teff cell infiltration. These results collectively suggest that the combination of lenvatinib and anti-PD-1 therapy can improve the efficacy of anti-PD-1 monotherapy by promoting circulating Teff cell infiltration through vascular normalization.

[0427] Example 7: CD8 that specifically recognizes HBV antigen + Identification and characteristics of T cells 1. Currently, there are no reports on the role of HBV-specific T cells in ICB therapy. To study HBV-specific T cells, the inventors first aligned Fastq files generated from whole-transcriptome RNA sequencing (bulk RNA-seq) data with the default HLA reference genome using Razer S3, and used the alignment results as input to the bioinformatics tool OptiType to determine a 4-digit HLA typing for each patient. Subsequently, the inventors designed a set of decamers targeting the human leukocyte antigen (HLA) HLA-A*02:01 type, containing HBV peptides and DNA barcodes adapted for single-cell RNA sequencing analysis. Furthermore, the decamers are linked to a PE fluorescent group. Specifically, this set of decamers contains 31 antigenic epitope peptides with a length of 9 to 10 amino acids. These antigenic epitope peptides are derived from the core protein, envelope protein, polymerase, or X protein sequence of HBV and have been reported to induce CD8+. + T cell response. HBV peptide barcode decamer compatible with the 10x Chromium Single Cell 5' Transcriptome and Human Variable Region, Diversity Region and Connecting Region (VDJ) Kit (10x Genomics) is manufactured by Immudex.

[0428] Subsequently, the inventors performed decamer staining and FASC sorting on the cell samples according to the manufacturer's instructions. Specifically, 0.5 to 2 million cryopreserved and revived cells were added to 100 μl of staining buffer (PBS containing 2% FBS and 0.1 g / L herring sperm DNA, pH 7.4) and all dextramer, and incubated at room temperature for 10 minutes in the dark. Then, anti-human CD45 (BV421), CD3 (PerCP-Cy5.5), and CD8 (AF700) antibodies and a fixative active dye (eFluor 660, diluted 1:1000) were added to the cell suspension, and the cells were incubated for an additional 20 minutes. Afterward, the cells were washed twice with washing buffer (PBS containing 2% FBS, pH 7.4), and the cell pellet was resuspended in sorting buffer (PBS containing 2% FBS, pH 7.4). The CD45 staining of the patients was then performed using a BD FACSAria III flow cytometer. + CD3 + CD8 + Cells, CD45 + CD8-cells, CD45 + Cells or CD45 + CD3 + Cells were sorted, and finally, single-cell libraries were constructed and sequenced according to the method described in step 1 of Example 1. PE-fluorescent CD8+ cells were used. + T cell characterization of HBV-specific CD8 + T cells, results see Figure 7 A. Finally, the inventors used FACS to identify and sort CD8 cells containing decamer-positive cells from the tumor, adjacent tissue, and peripheral blood of HLA-A*02:01 HBV-positive HCC patients. + T cells, results see Figure 7 B.

[0429] The results showed that, compared to CD4 + T cells, these decamers affect CD8 + T cells showed stronger binding ability; see results below. Figure 7 C. It is worth noting that although HBV-specific T cells could be identified from the flow cytometry results, the inventors observed CD8 cells belonging to the same clone in subsequent data analysis. + T cells exhibit different binding patterns to the aforementioned HBV-derived epitope peptides, and different T cells generate binding signals for multiple epitope peptides, making it difficult to determine the binding mode of each CD8 antigen. + The main antigenic epitope peptides recognized by T cell clones.

[0430] 2. Identification of HBV-specific CD8 using bioinformatics analysis +T cell clones. To further address the signal clutter issue observed in step 1 of this embodiment regarding T cell binding to multiple peptides, the inventors used the bioinformatics analysis tool DexTRAC (https: / / github.com / lijxug / DexTRAC) to identify HBV-specific CD8. + T cell clones and the specific antigenic epitope peptide sequences they recognize.

[0431] The results showed that among the 31 HBV antigenic epitope peptides mentioned above, 10 antigenic epitope peptides (HBVgp2_gcgg_pol, HBVgp4_ag_core, HBVgp1_ccag_pol, HBVgp1_aa_pol, HBVgp2_ac_env, HBVgp1_ccat_pol, HBVgp2_cg_env, HBVgp2_gcac_env, HBVgp3_ag_x, HBVgp1_tg_pol) were mediated by CD8. + T cell-specific recognition; the amino acid sequences of the 10 antigenic epitope peptides are shown in SEQ ID NO: 249~258. In 3,903 amplified CD8... + Within T-cell clonal types (with at least 2 cells detected in each clonal type), DexTRAC identified 24 HBV-specific CD8 clonal types. + T cells (i.e., CD8) + T cells express HBV-specific clonal CD8+ cells containing TCR-1, TCR-2, TCR-3, TCR-4, TCR-5, TCR-6, TCR-7, TCR-8, TCR-9, TCR-10, TCR-11, TCR-12, TCR-13, TCR-14, TCR-15, TCR-16, TCR-17, TCR-18, TCR-19, TCR-20, TCR-21, TCR-22, TCR-23, and TCR-24, respectively. + T cells). These 24 HBV-specific clonal CD8 cells. + T cells (i.e., CD8) + T cells express HBV-specific clonal CD8+ cells containing TCR-1, TCR-2, TCR-3, TCR-4, TCR-5, TCR-6, TCR-7, TCR-8, TCR-9, TCR-10, TCR-11, TCR-12, TCR-13, TCR-14, TCR-15, TCR-16, TCR-17, TCR-18, TCR-19, TCR-20, TCR-21, TCR-22, TCR-23, and TCR-24, respectively. +T cells can specifically recognize the aforementioned 10 antigenic epitope peptides (HBVgp2_gcgg_pol, HBVgp4_ag_core, HBVgp1_ccag_pol, HBVgp1_aa_pol, HBVgp2_ac_env, HBVgp1_ccat_pol, HBVgp2_cg_env, HBVgp2_gcac_env, HBVgp3_ag_x, HBVgp1_tg_pol) on the CD8+ of these HBV-specific clones. + In T cells, one HBV-specific clone of CD8 + T cells showed cross-reactivity to two antigenic epitope peptides, while the remaining HBV-specific clonal CD8 cells exhibited cross-reactivity. + T cells showed significant recognition ability for only one antigenic epitope peptide (HBVgp1_tg_pol, SEQ ID NO:258), results are shown in [link to results]. Figure 7 D.

[0432] 3. Furthermore, the inventors have developed HBV-specific CD8... + The functional status of T cells was analyzed. Specifically, the functional status of HBV-specific CD8 cells was calculated. + T cells belong to each CD8 in Example 1 + The proportion of T cell subsets, i.e., per CD8 + The number of cells in the T cell subset is used as the molecule, based on all HBV-specific CD8 cells. + The number of T cells was used as the denominator. Subsequently, the inventors also calculated the HBV-specific CD8+ content in tumor-infiltrating cTem cells or Tpex cells. + The proportion of T cells.

[0433] The results showed that in 31,023 CD8 + A total of 3,180 HBV-specific T cells were identified among the T cells. These HBV-specific cells were all derived from responders, primarily from the Temra and Teff / Tem subsets in blood, tumors, and adjacent normal tissues. (See attached results). Figure 7 E. In tumors, 79.4% of HBV-specific T cells belonged to the Teff / Tem subset, of which 61.9% were cTem cells and 17.5% were Tpex cells. (See attached results). Figure 7 F. Compared with Tpex, the proportion of HBV-specific T cells in the cTem population was significantly higher; see results below. Figure 7 G. Notably, only one HBV-specific T cell belonged to the tTex subset; see results below. Figure 7 F. These results indicate that HBV-specific CD8 +T cells are preferentially maintained in effector or memory states and are enriched in cTem cell populations.

[0434] 4. Since all HBV-specific clones originate from responders, the inventors hypothesize that HBV-specific CD8+... + T cells participated in the anti-tumor response of the aforementioned combination therapy. To verify this hypothesis, the inventors first used the bioinformatics tool STARTRAC to quantify HBV-specific CD8. + T cells and other CD8+ cells + The clonal expansion level and migration potential of T cells are used to reflect the degree of cell activation. Considering HBV-specific CD8... + T cells are primarily enriched in the cTem cell population. The inventors, using the method described in step 3 of Example 2, identified genes highly expressed in HBV-specific cTem cells compared to other cTem cells. The inventors further analyzed genes highly expressed in HBV-specific cTem cells and previously reported HBV-specific CD8+ genes. + A series of genes were extracted from T cell-highly expressed genes and used as HBV-specific biomarker scores. An HBV-specific biomarker score was established to estimate the presence of HBV-specific cells before and after treatment in non-HLA-A*02:01 HCC patients, and these inferred HBV-specific CD8 cells were tracked. + T cells are present in all tumor-infiltrating CD8 cells. + The proportion of T cells (based on HBV-specific CD8 inferred from each tumor sample) + The number of T cells was used as the numerator, and the total number of CD8 cells in the sample was used as the molecule. + The number of T cells is the denominator.

[0435] The results showed that HBV-specific CD8 in the tumor + Teff / Tem cells exhibited a higher level of clonal expansion compared to other Teff / Tem cells. (See results below.) Figure 7 H. Further focusing on cTem cells, HBV-specific cTem cells showed higher clonal expansion levels and migration potential than other cTem cells in the Teff / Tem subset. (See results below.) Figure 7 I. Pathway enrichment analysis showed that HBV-specific cTem cells expressed higher levels of CD8+, which is associated with cytotoxicity. + T cell activation, migration, and antiviral activity-related genes, such as CCL4, CCL4L2, KDM6B, and VIM, are discussed in the results. Figure 7 These results indicate that HBV-specific cTem cells are capable of active clonal expansion and migration during treatment.

[0436] In addition, the inventors selected 17 genes and used the average expression level of these genes as the HBV-specific marker score. These 17 genes include CCL4L2, NR4A2, CCL4, CCL3L1, TNFAIP3, ZNF331, CD69, CREM, SAT1, RASGEF1B, CRIP1, KDM68, MYADM, VIM, GLUD1, LMNA, and PCBP2. By comparing the inferred proportion of HBV-specific cells in tumors before and after treatment in non-HLA-A*02:01 type HCC patients, the inventors found that the proportion of HBV-specific T cells in tumors after treatment in responders was significantly increased compared to before treatment, and this increase was significantly higher than that in non-responders. (See attached results). Figure 7 K. These results indicate that in HBV-infected HCC patients, HBV-specific T cells enriched in the cTem population and maintaining an effector or effector memory state produced an antitumor response to combination therapy.

[0437] Example 8: KIR in tumors + CD8 + Characteristic analysis of T cell subsets Two types of KIR were discovered in Embodiment 1 of this invention. + CD8 + T cell subsets: This subset exhibits high levels of expression of the KIR gene and other marker genes such as IKZF2, KLRC2, KLRC3, and TYROBP, but almost no expression of CCR7 and CD27. (See attached results). Figure 8 A, 8B.

[0438] 1. In this embodiment of the invention, the top 2,000 highly variable genes for each sample are screened and integrated using the SelectIntegrationFeatures function in the Seurat R package. Subsequently, the average gene expression level of these highly variable genes in each group of cells is calculated using the AverageExpression function in Seurat. To assess the similarity between the four groups of cells, this embodiment of the invention calculates the Pearson correlation coefficient based on the average gene expression level of each group of cells using the cor function in R.

[0439] The results showed that KIR was mainly distributed in the blood. + CD8 + T cell subsets share similar characteristics with cells previously reported in patients with infections and autoimmune diseases (Li et al., Science, 2022), while KIR cells in tumors... + CD8 + T cell subsets, on the other hand, exhibited similarity to KIR in the blood. + CD8 +The distinct transcriptional signatures of T cells are shown in the results below. Figure 8 B.

[0440] 2. The embodiments of the present invention further compare the two KIRs. + CD8 + Characterization of T cell subsets. To identify differentially expressed characteristic genes between two cell groups, this embodiment of the invention uses the 'FindMarkers' function in the Seurat package to assess the significance of each gene based on the Wilcoxon rank-sum test, and performs multiple hypothesis correction using the Benjamini–Hochberg method. Genes with adjusted p-values ​​less than 0.01 or 0.05 are considered differentially expressed characteristic genes.

[0441] The results showed that KIR in the blood + CD8 + T cell subsets exhibited more Temra-like features, including higher expression levels of CX3CR1, GZMB, GZMH, and GNLY genes; in contrast, KIR cells in tumors... + CD8 + T cell subsets showed higher levels of GZMK, XCL1, XCL2, and IL7R gene expression, indicating a Teff / Tem-like state. (See attached results.) Figure 8 C. Specifically, although KIR levels in the blood of patients with liver cancer and autoimmune diseases... + CD8 + T cells all showed high levels of KIR2DL3 expression, but KIR in tumors + CD8 + T cells specifically express KIR2DL4, a gene that is an atypical KIR family member with both activating and repressive functions. (See results below.) Figure 8 D.

[0442] 3. Based on the TCR clonality analysis in step 1 of Example 2, the inventors compared the tumor-infiltrating KIR. + CD8 + T cell populations and other CD8 + Transformation relationships between T cell subsets. Furthermore, the inventors used the same STARTRAC transformation analysis as in step 6 of Example 2 to identify KIRs associated with tumor invasion. + CD8 + T cells are a subset of T cells with a strong transformation relationship.

[0443] The results showed that KIR in the tumor + CD8 + T cell subsets and CD8 in the blood +T cells showed significant TCR sharing and were associated with KIR in the blood. + CD8 + T cell subsets exhibited high transformation levels; see results below. Figure 8 E, 8F, indicating KIR in the blood + CD8 + T cells may be KIR in tumors. + CD8 + The origin of T cells.

[0444] Example 9: KIR in tumors + CD8 + T cell subsets are specifically associated with resistance to combination therapy. 1. In the embodiments of the present invention, calculations were performed on all CD8... + Two KIRs in T cells + CD8 + The proportion of T cell subsets. Specifically, this proportion is based on the percentage of each KIR in each tumor or peripheral blood sample. + CD8 + Using the number of T cell subsets as the molecule, and considering all CD8 cells in this sample... + The number of T cells is the denominator.

[0445] The results showed that, compared with responders who received combination therapy, non-responders had lower levels of KIR in their tumors after treatment. + CD8 + T cell subsets in all CD8 + The proportion of T cells was significantly higher, as shown in the results. Figure 9 A. However, this difference was not observed in tumors after treatment in patients who received anti-PD-1 monotherapy; see results below. Figure 9 B indicates KIR in the tumor. + CD8 + T-cell subset specificity is associated with resistance to combination therapies.

[0446] 2. Furthermore, embodiments of the present invention systematically compared KIR in tumors of responders and non-responders. + CD8 + Transcriptomic characteristics of T cell subsets. The specific steps are the same as those in step 1 of Example 8, involving correlation analysis.

[0447] The results showed that KIR was higher in tumors of non-responders before combination therapy and responders before and after treatment. + CD8 + Compared to T-cell subsets, KIR in tumors of non-responders after combination therapy + CD8 + T cell subsets have unique transcriptomic characteristics; see results below. Figure 9C.

[0448] 3. The inventors further analyzed the KIR in tumors of non-responders after combined therapy using the method in step 2 of Example 8. + CD8 + The gene expression of T cell subsets differed from that of the subsets in tumors of responders after treatment. Subsequently, the expression of these two KIR subsets in tumors of non-responders and responders after treatment was compared. + CD8 + T cell gene expression was analyzed to identify upregulated genes based on a fold change > 1.25 and an adjusted p-value < 0.01. Subsequently, the enrichGO function in the R package clusterProfiler (v.4.6.1) was used to perform functional annotation and overexpression analysis on these upregulated genes.

[0449] The results showed that, compared with responders, non-responders had lower levels of KIR in their tumors after treatment. + CD8 + T cells showed higher levels of JUND, CD81, TGFB1, REL, PRF1, CXCR6, and ZEB2. (See attached results.) Figure 9 D, These genes are enriched in pathways related to T cell differentiation and activation.

[0450] 4. Based on the list of differentially characterized genes in step 3 of this embodiment, and in conjunction with a literature review, the inventors identified genes that may be involved in regulating KIR in tumors and peripheral blood. + CD8 + Genes related to T cell function.

[0451] The results showed that KIR levels in tumors and blood were elevated in non-responders after combination therapy. + CD8 + T cells showed significantly higher expression of TGFB1, as shown in the results below. Figure 9 E. There are previous reports of TGFB1's effect on CD8 in mice. + The differentiation of regulatory T cells is crucial and can promote the expression of IKZF2 (also known as Helios), which is a regulator of CD8. + and CD4 + T cell repression transcription factors (Mishra et al., J Exp Med, 2021; Kim et al., Science, 2015). These results suggest that high expression of TGFB1 and its related genes may contribute to KIR. + CD8 + T-cell-mediated resistance to combination therapy.

[0452] Example 10: Activated CD4 in tumors+ Treg cell subsets are specifically associated with resistance to combination therapy. There have been reports on the role of Treg cells in suppressing CD8. + The role of CD4 in T cell effector function was investigated in this embodiment of the invention. + Dynamic changes in T cell subsets.

[0453] 1. Identification of Treg cell subsets. Nine CD4 subpopulations were identified in Example 1. + In the T cell subsets, two types of Treg cells were identified in this embodiment of the invention; the results are detailed below. Figure 1 C, where resting Treg cells mainly originate from peripheral blood, while activated Treg cells are mainly distributed in tumors. Activated Treg cells exhibit higher levels of inhibitory molecules, including CTLA4 and TNFRSF9; see [see results]. Figure 1 D.

[0454] 2. The inventors calculated the CD4 infiltration rates in all tumor-infiltrating cells in the embodiments of this invention. + The CD4 cells described in Example 1 in T cells + The proportion of T cell subsets. Specifically, this proportion is based on the percentage of CD4+ cells in each tumor or peripheral blood sample. + Using the number of T cell subsets as the molecule, and considering all CD4 cells in the sample... + The number of T cells is the denominator.

[0455] The results showed that although the proportion of these Treg cells was comparable in both groups of patients before treatment, the proportion of activated Treg cells in the tumor was higher in non-responders after combination therapy compared to responders, and the proportion of resting Treg cells in the blood also increased significantly after treatment. (See attached results). Figure 10 .

[0456] 3. Through the TCR clonal analysis identification in step 1 of Example 2, the inventors compared all Treg cell clonal types in the tumors of responders and non-responders before and after combination therapy. The size of the clonal type was reflected by comparing the number of cells in each clonal type. If the clonal type was larger after treatment compared to before treatment, it indicated that the clonal type had undergone clonal expansion after treatment.

[0457] The results showed that, compared with responders, non-responders exhibited clonal expansion of activated Treg cells in their tumors after treatment. Since activated Treg cells have an inhibitory function against tumor-killing effector cells, this result suggests that the enrichment of Treg cells in non-responders after treatment is associated with drug resistance.

[0458] Example 11: BACH1 regulates activated CD4 in tumors +Treg cell differentiation and inhibitory function 1. Identifying regulatory genes associated with the enrichment of activated Treg cells in tumors. First, the gene expression differences of tumor-infiltrating Treg cells before and after treatment in responders and non-responders were compared using the method described in step 3 of Example 9. Second, the transcriptomic similarity of tumor-infiltrating Treg cells before and after treatment in responders and non-responders was calculated and compared using the Pearson correlation coefficient described in step 1 of Example 8. Then, in this embodiment of the invention, the SCENIC algorithm was used to identify gene regulatory networks or regulators and to evaluate CD4. + The activity (AUC score) of activation-related transcription factors in Treg cells was evaluated and ranked from highest to lowest. Then, transcription factors with the highest AUC scores were identified from 158 genes upregulated in non-responders compared to responders in pre-treatment tumor-infiltrating Treg cells (selection criteria: corrected P-value < 0.01 and fold change > 1.5). Finally, a gene regulatory network was constructed using the R package igraph, utilizing the selected transcription factors and their target genes among the 158 differentially expressed genes.

[0459] The results showed that Treg cells in non-responder tumors after combination therapy exhibited increased expression levels of genes related to negative regulation of immune responses and cell proliferation, such as ICOS, REL, CTLA4, IKZF2, and TNFRSF9. (See attached results). Figure 11 A. The inventors unexpectedly discovered that Treg cells in tumors of non-responders before treatment already exhibited unique transcriptomic characteristics, manifested in low Pearson correlation coefficients (<0.9) between their transcriptomes and those of Treg cells in tumors of non-responders after treatment or in tumors of responders before and after treatment, while the transcriptomic Pearson correlation coefficients among the latter three groups were all high (>0.9). Furthermore, the inventors found that Treg cells in tumors of non-responders before treatment had higher levels of gene expression related to activation, recruitment, and inhibition functions, including REL and BATF, compared to responders. (See attached results). Figure 11 B. Notably, among the top 10 most active regulators of Treg cells in tumors, three transcription factors (BACH1, BATF, and AHR) were upregulated in pre-treatment tumors of non-responders compared to responders. (See attached results). Figure 11 C. Regulatory network analysis showed that BACH1, BATF, and AHR actively interact through TNFRSF9, JAK1, and FOXP1. (See attached results.) Figure 11D. These results indicate that the three transcription factors mentioned above may regulate the enrichment of tumor-infiltrating activated Treg cells during combination therapy. Inhibition of REL and BATF enhances the efficacy of anti-PD-1 therapy in the treatment of melanoma in mice. Therefore, BACH1 likely also plays a key regulatory role in tumor-activated Treg cells.

[0460] 2. Further exploration of the clinical relevance of BACH1. First, the inventors downloaded single-cell transcriptome data from triple-negative breast cancer patients receiving anti-PD-L1 therapy (Zhang et al., Cancer Cell, 2021) and compared the BACH1 expression levels in tumor-infiltrating Treg cells of responding and non-responding breast cancer patients before and after treatment using gene expression data. Second, the inventors downloaded clinical information from 182 hepatocellular carcinoma patients in the TCGA database, including overall survival, gender, and age. Then, based on the median ratio of BACH1 expression to FOXP3 expression, the TCGA hepatocellular carcinoma patients were grouped, and their overall survival curves were plotted.

[0461] The results showed that, compared to responders, the BACH1 gene was highly expressed in tumor-infiltrating Treg cells of non-responder breast cancer patients before treatment. (See attached results). Figure 11 E. Further survival analysis of the TCGA hepatocellular carcinoma cohort showed that, after adjusting for Treg proportions based on FOXP3 gene expression levels, patients with high BACH1 expression had significantly lower overall survival compared to those with low expression. (See attached results). Figure 11 F.

[0462] 3. To verify the regulatory function of BACH1 on Treg cells, CRISPR / Cas9 technology was used to induce Treg cells in mice with naïve CD4+ cells. +BACH1 knockout experiments were conducted in T cells (two sgRNA sequences: TACTTCCACTCGAGAATCGT (SEQ ID NO:243) and TCTGGCCTACGATTCTCGAG (SEQ ID NO:244)), and the cells were cultured under Treg differentiation conditions to study its function in Treg cells. Based on previous reports, the inventors used knockout groups of two genes that regulate Treg function, AHR and TGFBR2, as positive controls (sgRNA sequences: GAACACAGAGTTAGACCGCC (SEQ ID NO:245) and GGCCGCTGCATATCGTCCTG (SEQ ID NO:246)), a knockout group of the Treg cell marker gene FOXP3 as a positive control (sgRNA sequence: TCTACCCACAGGGATCAATG (SEQ ID NO:247)), and a knockout group of the random sequence H11 (sgRNA sequence: AACACTAGTGCACTTATCC (SEQ ID NO:248)) that does not target any genomic location as a negative control. The specific steps were as follows: Each sgRNA was cloned into the pWKO mTagBFP2 retroviral expression vector and co-transfected with the pCL-ECO packaging plasmid into the 293T cell line. Retroviruses were collected from the supernatant 48 hours after transfection. Subsequently, naïve CD4 was isolated and purified from the spleen and lymph nodes of Rosa26-Cas9 knock-in mice (from the Jackson laboratory). + T cells (CD4) + CD25- CD62L + CD44- cells were then activated for 20 hours by stimulation with coated anti-CD3 (clone 145-2C11, 5 μg / ml; BioXCell) and anti-CD28 (clone 37.51, 1 μg / ml; BioXCell). The activated cells were then co-incubated with retroviral infection medium containing polybrominated polybromide (1 μg / ml) and centrifuged at 32°C, 1800 rpm for 90 minutes. The medium was replaced with fresh complete medium one hour after infection. Forty-eight hours after infection, retroviral-infected positive cells (BFP) were sorted by flow cytometry. + ), and in a 96-well plate coated with anti-CD3 / anti-CD28, at 5 × 10⁻⁶ ppm per well. 4 Cells were induced to develop CD4 using Treg differentiation medium (200-300 μl). +Cells differentiated into Treg cells, and the culture medium was supplemented with IL-2 (1 ng / ml), TGF-β (2 ng / ml), anti-IFN-γ (10 µg / ml), and anti-IL-4 (10 µg / ml). After 3 days, half of the culture medium was replaced daily with complete medium. After 4 days of culture, cells were collected for staining analysis. For staining, cells were resuspended in 100 μl PBS containing 2% FBS (Gibco). Fixed Viability Dye (eFluor 660, diluted 1:1000) and anti-mouse CD25 antibody (PE-Cy7) were added to the cell suspension. Anti-mouse FOXP3 (FITC) staining was then performed using a Foxp3 / transcription factor staining buffer kit (eBioscience). After staining, cells were washed twice with PBS and resuspended in PBS, followed by analysis on a BD LSRFortessa X-20 flow cytometer.

[0463] The results showed that knocking out the random sequence H11 did not affect CD4. + The expression level of the FOXP3 gene in T cells, and the CD4 count in the FOXP3 knockout group. + T cells rarely express the characteristic gene FOXP3, indicating that gene knockout in this embodiment is highly efficient. Knockout of AHR and TGFBR2 significantly reduced CD4+. + The expression level of FOXP3 in T cells. Notably, FOXP3 expression was significantly higher in the BACH1 knockout group. + CD25 high The proportion of T cells was also significantly lower than that in the H11 control group. (See results below.) Figure 11 G, which suggests that BACH1 may play an important role in regulating Treg cell differentiation.

[0464] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0465] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A CD8 + T cell subsets, characterized by include: The genes GZMK and PDCD1 are expressed. Optionally, the CD8 + T cell subsets further express at least one of the characteristic genes MT1X, MT1E, MT2A, CCL4, XCL1, and XCL2.

2. A CD8 + T cell subsets, characterized by include: The expression of characteristic genes GZMK, GPR183, IL7R and ZNF683; Optionally, the CD8 + T cell subsets further express at least one of the characteristic genes CXCR3, CXCR4, CD44, CCR7, LMNA, XCL1, and XCL2.

3. A CD8 + T cell subsets, characterized by include: The characteristic genes GZMK and CXCL13 are expressed; Optionally, the CD8 + T cell subsets further express at least one of the characteristic genes CXCR4, DUSP4, DUSP1, IFNG, MT1X, MT1E, MT2A, XCL1, and XCL2.

4. A CD8 + T cell subsets, characterized by include: The characteristic genes GZMK and ZNF683 are expressed; Optionally, the CD8 + T cell subsets further express at least one of the characteristic genes CCL4L2, NR4A2, and CCL4.

5. A CD8 + T cell subsets, characterized by include: Express at least one of KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, and KIR3DL3; Optionally, the CD8 + T cell subsets further express at least one of the characteristic genes KLRC2, KLRC3, IKZF2, TYROBP, XCL1, and XCL2.

6. A CD4 + T cell subsets, characterized by include: The genes expressing the characteristic genes FOXP3, TNFRSF9, and CTLA4 are: Optionally, the CD4 + T cell subsets further express at least one of the characteristic genes CTSC, IKZF2, STAM, and DUSP4.

7. A CD4 + T cell subsets, characterized by include: The genes FOXP3 and BACH1 are expressed. Optionally, the CD4 + T cell subsets further express at least one of the characteristic genes REL, ICOS, AHR, CREM, and NFE2L2.

8. An antigenic epitope peptide, characterized in that, Includes at least one of the amino acid sequences shown in SEQ ID NO:249~258; or at least one of the amino acid sequences that have 90% sequence identity with it.

9. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antigenic epitope peptide of claim 8.

10. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 9.

11. A recombinant cell, characterized in that, include: Carrying the nucleic acid molecule of claim 9 or the expression vector of claim 10; or, Express the antigenic epitope peptide as described in claim 8.

12. An antigen, characterized in that, The antigen includes the antigenic epitope polypeptide of claim 8.

13. The use of the antigenic epitope peptide of claim 8, the nucleic acid molecule of claim 9, the expression vector of claim 10, or the recombinant cell of claim 11 in the preparation of a medicament for the prevention of hepatitis B virus infection or the treatment of hepatitis B virus-related diseases.

14. A pharmaceutical composition, characterized in that, Includes the antigenic epitope polypeptide of claim 8.

15. Use of the antigenic epitope peptide of claim 8, the nucleic acid molecule of claim 9, the expression vector of claim 10, the recombinant cell of claim 11, or the pharmaceutical composition of claim 14 in screening TCRs that specifically recognize hepatitis B virus.

16. A separated TCR, characterized in that, include: TCRα chain variable region and / or TCRβ chain variable region, The TCRα chain variable region includes a CDR3 sequence selected from at least one of the following: The amino acid sequences of SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:39, SEQ ID NO:42, SEQ ID NO:45, SEQ ID NO:48, SEQ ID NO:51, SEQ ID NO:54, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:63, SEQ ID NO:66, SEQ ID NO:69 and SEQ ID NO:72 or their conserved modified forms; The variable region of the TCRβ chain includes a CDR3 sequence selected from at least one of the following: The amino acid sequences of SEQ ID NO:75, SEQ ID NO:78, SEQ ID NO:81, SEQ ID NO:84, SEQ ID NO:87, SEQ ID NO:90, SEQ ID NO:93, SEQ ID NO:96, SEQ ID NO:99, SEQ ID NO:102, SEQ ID NO:105, SEQ ID NO:108, SEQ ID NO:111, SEQ ID NO:114, SEQ ID NO:117, SEQ ID NO:120, SEQ ID NO:123, SEQ ID NO:126, SEQ ID NO:129, SEQ ID NO:132, SEQ ID NO:135, SEQ ID NO:138, SEQ ID NO:141 and SEQ ID NO:144 or their conserved modified forms; Optionally, the TCRα chain variable region further comprises a CDR1 sequence selected from at least one of the following: The amino acid sequences shown or their conserved modified forms as shown in SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:19, SEQ ID NO:22, SEQ ID NO:25, SEQ ID NO:28, SEQ ID NO:31, SEQ ID NO:34, SEQ ID NO:37, SEQ ID NO:40, SEQ ID NO:43, SEQ ID NO:46, SEQ ID NO:49, SEQ ID NO:52, SEQ ID NO:55, SEQ ID NO:58, SEQ ID NO:61, SEQ ID NO:64, SEQ ID NO:67 and SEQ ID NO:70; And / or, the TCRα chain variable region further comprises a CDR2 sequence selected from at least one of the following: The amino acid sequences shown in or conservatively modified forms of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:11, SEQ ID NO:14, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, SEQ ID NO:35, SEQ ID NO:38, SEQ ID NO:41, SEQ ID NO:44, SEQ ID NO:47, SEQ ID NO:50, SEQ ID NO:53, SEQ ID NO:56, SEQ ID NO:59, SEQ ID NO:62, SEQ ID NO:65, SEQ ID NO:68 and SEQ ID NO:71; And / or, the TCRβ chain variable region further comprises a CDR1 sequence selected from at least one of the following: The amino acid sequences shown in or conservatively modified forms of SEQ ID NO:73, SEQ ID NO:76, SEQ ID NO:79, SEQ ID NO:82, SEQ ID NO:85, SEQ ID NO:88, SEQ ID NO:91, SEQ ID NO:94, SEQ ID NO:97, SEQ ID NO:100, SEQ ID NO:103, SEQ ID NO:106, SEQ ID NO:109, SEQ ID NO:112, SEQ ID NO:115, SEQ ID NO:118, SEQ ID NO:121, SEQ ID NO:124, SEQ ID NO:127, SEQ ID NO:130, SEQ ID NO:133, SEQ ID NO:136, SEQ ID NO:139 and SEQ ID NO:142; And / or, the TCRβ chain variable region further comprises a CDR2 sequence selected from at least one of the following: The amino acid sequences shown in or conservatively modified forms of SEQ ID NO:74, SEQ ID NO:77, SEQ ID NO:80, SEQ ID NO:83, SEQ ID NO:86, SEQ ID NO:89, SEQ ID NO:92, SEQ ID NO:95, SEQ ID NO:98, SEQ ID NO:101, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:110, SEQ ID NO:113, SEQ ID NO:116, SEQ ID NO:119, SEQ ID NO:122, SEQ ID NO:125, SEQ ID NO:128, SEQ ID NO:131, SEQ ID NO:134, SEQ ID NO:137, SEQ ID NO:140 and SEQ ID NO:143; Optionally, the variable region of the TCRα chain includes: The amino acid sequences shown in SEQ ID NO: 1-3 or the CDR1, CDR2, and CDR3 sequences having at least 90% homology with them, respectively; The CDR1, CDR2, and CDR3 sequences are shown as amino acid sequences of SEQ ID NO:4-6 or amino acid sequences having at least 90% homology with them, respectively. The CDR1, CDR2, and CDR3 sequences are shown as in SEQ ID NO: 7-9 or as amino acid sequences having at least 90% homology with them, respectively. The amino acid sequences shown in SEQ ID NO: 10-12 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 13-15 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 16-18 or the amino acid sequences having at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 19-21 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 22-24 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 25-27 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 28-30 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 31-33 or the amino acid sequences having at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 34-36 or the amino acid sequences having at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 37-39 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 40-42 or the amino acid sequences having at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 43-45 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 46-48 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 49-51 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 52-54 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 55-57 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 58-60 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 61-63 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 64-66 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 67-69 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. or The amino acid sequences shown in SEQ ID NO: 70-72 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. And / or, the variable region of the TCRβ chain includes: The amino acid sequences shown in SEQ ID NO: 73-75 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The CDR1, CDR2, and CDR3 sequences are shown as the amino acid sequences of SEQ ID NO:76-78 or amino acid sequences having at least 90% homology with them, respectively. The amino acid sequences shown in SEQ ID NO: 79-81 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 82-84 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 85-87 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The CDR1, CDR2, and CDR3 sequences are shown in SEQ ID NO: 88-90 or amino acid sequences that have at least 90% homology with them, respectively. The amino acid sequences shown in SEQ ID NO: 91-93 or the amino acid sequences having at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 94-96 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The CDR1, CDR2, and CDR3 sequences are shown as in SEQ ID NO: 97-99 or as amino acid sequences having at least 90% homology with them, respectively. The amino acid sequences shown in SEQ ID NO: 100-102 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 103-105 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 106-108 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 109-111 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 112-114 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 115-117 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 118-120 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 121-123 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 124-126 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 127-129 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 130-132 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 133-135 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 136-138 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. The amino acid sequences shown in SEQ ID NO: 139-141 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. or The amino acid sequences shown in SEQ ID NO: 142-144 or the amino acid sequences that have at least 90% homology with them are CDR1, CDR2, and CDR3 sequences, respectively. Optionally, the CDR1, CDR2, and CDR3 sequences of the TCRα chain variable region shown in the amino acid sequences of SEQ ID NO: 1-3 or amino acid sequences having at least 90% homology therewith, and the CDR1, CDR2, and CDR3 sequences of the TCRβ chain variable region shown in the amino acid sequences of SEQ ID NO: 73-75 or amino acid sequences having at least 90% homology therewith; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 4-6 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 76-78 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 7-9 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 79-81 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 10-12 or amino acid sequences having at least 90% homology with them, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 82-84 or amino acid sequences having at least 90% homology with them; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 13-15 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 85-87 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 16-18 or amino acid sequences having at least 90% homology with them, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 88-90 or amino acid sequences having at least 90% homology with them; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 19-21 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 91-93 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 22-24 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 94-96 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 25-27 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 97-99 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 28-30 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 100-102 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 31-33 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 103-105 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 34-36 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 106-108 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 37-39 or amino acid sequences having at least 90% homology with them, respectively; and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 109-111 or amino acid sequences having at least 90% homology with them, respectively. The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 40-42 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 112-114 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 43-45 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 115-117 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 46-48 or amino acid sequences having at least 90% homology with them, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 118-120 or amino acid sequences having at least 90% homology with them; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 49-51 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 121-123 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 52-54 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 124-126 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 55-57 or amino acid sequences having at least 90% homology with them, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 127-129 or amino acid sequences having at least 90% homology with them; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 58-60 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 130-132 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 61-63 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 133-135 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 64-66 or amino acid sequences having at least 90% homology with them, respectively, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 136-138 or amino acid sequences having at least 90% homology with them, respectively; The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 67-69 or amino acid sequences having at least 90% homology with them, respectively; and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 139-141 or amino acid sequences having at least 90% homology with them, respectively; or The amino acid sequences of the TCRα chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 70-72 or amino acid sequences having at least 90% homology with them, and the amino acid sequences of the TCRβ chain variable regions CDR1, CDR2, and CDR3 shown in SEQ ID NO: 142-144 or amino acid sequences having at least 90% homology with them; Optionally, the separated TCR further includes: TCRα chain constant region and / or TCRβ chain constant region; Optionally, the isolated TCR is isolated, purified, or recombinant; And / or, the isolated TCR is human; And / or, the isolated TCR is monoclonal; And / or, the separated TCR is single-stranded; And / or, the separated TCR contains two chains; And / or, the isolated TCR is in a cell-bound form or in a soluble form, preferably in a soluble form; And / or, the isolated TCR binds to the antigenic epitope peptide-HLA complex, preferably, the amino acid sequence of the antigenic epitope peptide is shown in at least one of SEQ ID NO:249~258.

17. A nucleic acid molecule, characterized in that, Encodes the separate TCR as described in claim 16.

18. An expression carrier, characterized in that, Carrying the nucleic acid molecule as described in claim 17.

19. A recombinant cell, characterized in that, include: Carrying the nucleic acid molecule of claim 17 or the expression vector of claim 18; or, The separated TCR as described in claim 16 is expressed.

20. A pharmaceutical composition, characterized in that, It includes the isolated TCR as described in claim 16, the nucleic acid molecule as described in claim 17, the expression vector as described in claim 18, or the recombinant cell as described in claim 19.

21. Use of the isolated TCR of claim 16, the nucleic acid molecule of claim 17, the expression vector of claim 18, the recombinant cell of claim 19, or the pharmaceutical composition of claim 20 in the preparation of a medicament for the prevention and / or treatment of hepatitis B virus infection and / or related diseases caused by hepatitis B virus.

22. A method for preventing and / or treating hepatitis B virus infection and / or related diseases caused by hepatitis B virus, characterized in that, The pharmaceutical composition of claim 20 is described, which is administered to a subject in a pharmaceutically acceptable amount.

23. A method for screening the separated TCRs as described in claim 16, characterized in that, include: (i) HLA and antigenic epitope peptides are combined to form an HLA-antigenic epitope peptide complex, and the HLA-antigenic epitope peptide complex is contacted with T cells to screen for target T cells that specifically respond to the HLA-antigenic epitope peptide complex. (ii) Analyze the TCR sequence and characteristic genes of the target T cells with specific responses in order to screen isolated TCRs.

24. A method for screening the antigenic epitope peptide of claim 8, characterized in that, include: HLA and the antigen epitope peptide to be screened are formed into an HLA-antigen epitope peptide complex. The HLA-antigen epitope peptide is then treated by contacting T cells to screen for antigen epitope peptides.

25. A T cell expressing the isolated TCR of claim 16 or carrying the nucleic acid molecule of claim 17.

26. A method for screening T cells according to claim 25, characterized in that, include: HLA and antigenic epitope peptides are combined to form an HLA-antigenic epitope peptide complex. The HLA-antigenic epitope peptide complex is then contacted with T cells to screen for T cells that specifically respond to the HLA-antigenic epitope peptide complex.

27. A method for identifying T cells with HBV antigen specificity, characterized in that, include: (1) Obtain gene expression information in the T cells to be identified by staining and / or sequencing; (2) Determine whether the T cells to be identified have HBV antigen specificity based on the characteristic genes described in claim 4; (3) Analyze and obtain T cells with HBV antigen specificity among the T cells to be identified.

28. A method for identifying TCRs with HBV antigen specificity, characterized in that, include: (1) Obtain gene expression information in the T cells to be identified by staining and / or sequencing; (2) Determine whether the T cells to be identified have HBV antigen specificity based on the characteristic genes described in claim 4; (3) Analyze and obtain the TCR sequence of the T cells to be identified that have HBV antigen specificity.

29. A method for activating a T cell subset according to any one of claims 1-7, characterized in that, include: The T cell subsets according to any one of claims 1-7 are co-cultured with artificial antigen-presenting cells that have been genetically engineered to express co-stimulatory molecules, or exogenous cytokines are administered to activate the T cell subsets.

30. A method for suppressing T cell subsets according to any one of claims 1-7, characterized in that, include: The T cell subsets are identified by using the proteins that are specifically highly expressed by the T cell subsets according to any one of claims 1-7, and their invasion is blocked by targeting specific chemokines or receptors on their surface, or by using small molecule inhibitors to interfere with the regulation of downstream signal transduction by key intracellular signaling molecules, so as to inhibit the T cell subsets.

31. A method for inducing T cell subsets according to any one of claims 1-7, characterized in that, include: Antigen epitope peptides or exogenous cytokines are added to an in vitro co-culture system with antigen-presenting cells to induce the generation of the T cell subsets.

32. A method for amplifying the T cell subset according to any one of claims 1-7, characterized in that, include: The T cell subsets are identified and enriched using the characteristic genes described in any one of claims 1-7, and antibodies or exogenous cytokines are added to promote the proliferation of T cell subsets.

33. A method for in vitro expansion of antigen-specific T cells, characterized in that, include: (1) T cells are isolated from peripheral blood of a patient or donor or from tumor tissue of a patient using the characteristic gene described in claim 4; (2) Culture the T cells in vitro and add at least one of the following: antibody, cytokine, antigen epitope peptide-MHC complex and antigen presenting cells to promote the expansion of the T cells; (3) Detect the tumor killing function of the expanded T cells; wherein the antigen-specific T cells are the T cells identified by the method of claim 27.

34. Use of the T cells of claim 33 in the preparation of medicaments for treating and / or preventing cancer.

35. A method for preparing T cells containing TCR, characterized in that, include: (1) Introduce the TCR α or TCR β gene into a viral or non-viral vector for gene expression; (2) Create a virus that introduces the genes from a retroviral vector expressing the TCR α and TCR β genes; (3) Infect lymphocytes collected from a patient independently and sequentially with the virus carrying the TCR α and TCR β genes for transfection, or create a gene expression retroviral vector including the TCR α and TCR β genes to transform the two genes at once; introduce the DNA or mRNA of the TCR into T cells using electroporation or liposomes; (4) Demonstrate that the TCR α / TCR β heterodimer is expressed on the surface of T cells; wherein the TCR α or TCR β gene is derived from the TCR of the T cells described in any one of claims 1-7, the isolated TCR of claim 16, the TCR screened by the method of claim 23, or the TCR identified by the method of claim 28.

36. A method for adoptive cell therapy, characterized in that, include: Cells are obtained from a patient or donor, and specific T cell subsets are screened according to the characteristic genes described in claims 1-7. The T cells are genetically modified or not modified, and the T cell subsets are induced or expanded in vitro. The expanded T cell subsets are then reinfused into the patient for treatment. The T cell subsets used for adoptive cell therapy are selected from the T cells described in claim 25, the T cells identified by the method described in claim 27, or the T cell subsets activated, expanded, or induced by the method described in any one of claims 29-32.

37. A method for a novel antitumor therapy by inhibiting or eliminating regulatory T cells, characterized in that, include: Regulatory T cells are identified by utilizing the characteristic genes of T cells according to any one of claims 1-7, and the regulatory T cells identified by the target gene are screened and knocked out to inhibit or eliminate regulatory T cells; wherein, the regulatory T cells include the CD8+ as described in claim 5. + T cell subsets, CD4 as described in claim 6 + T cells and CD4 as described in claim 7 + At least one of the T cell subsets.

38. A method for preparing a cancer vaccine, characterized in that, include: (1) Synthesize the antigenic epitope peptide according to claim 8; (2) Mix the antigenic epitope peptide with an adjuvant or a carrier, or extract the patient's mononuclear cells, culture them in vitro, and load them with antigen; (3) Present the antigenic epitope peptide to antigen-specific T cells in vitro, and test the killing activity of antigen-specific T cells or the level of secreted IFN-γ and TNF-α cytokines to verify the immunogenicity of the antigenic epitope peptide; (4) Verify the immunogenicity in a tumor model or an immune humanization model; (5) Deliver it to the patient's tumor or lymph node.

39. A method for treating cancer using virus-specific T cells, characterized in that, include: (1) Prepare T cells using peripheral blood mononuclear cells or donor-derived blood; (2) Stimulate T cells with viral peptides, virus-infected target cells, or vectors encoding viral antigens; (3) Expand the corresponding virus-specific T cells under culture conditions containing supplemental cytokines; (4) Ensure that the expanded T cells have high specificity and low toxicity and can effectively kill target cells by in vitro killing experiments; (5) Infuse the expanded T cells directly into the patient via intravenous injection, or infuse them into the patient after combining them with specific targeting technologies to enhance their specificity and function; wherein the T cells are derived from the T cells identified by the method described in claim 27.

40. A method for using bispecific or trispecific antibodies targeting T cells and other cells, characterized in that, include: Antigen targets are selected on T cells and other cells to design and construct antibodies, and in vitro affinity tests and functional assays are performed to evaluate their efficacy and toxicity. Clinical trials are also conducted to verify the efficacy. The T cells are derived from the T cells described in claim 25, the T cells identified by the method described in claim 27, or the T cells activated, expanded, or induced by any one of the methods described in claims 29-33. The other cells include immune cells or non-immune cells, including immune cells such as T cells, B cells, dendritic cells, macrophages, monocytes, NK cells, and ILC cells, or at least one of tumor cells, tumor-associated fibroblasts, endothelial cells, and epithelial cells.

41. A method of combined treatment, characterized in that, include: The method of any one of claims 17-21 and 35-40 is combined with an immune checkpoint inhibitor or a small molecule drug; wherein the immune checkpoint inhibitor includes at least one of CTLA-4 inhibitors, PD-1 inhibitors and PD-L1 inhibitors; and the small molecule drug includes at least one of methotrexate, fluorouracil and imatinib.

42. The use of any one of claims 1-7 in the treatment of autoimmune diseases, characterized in that, include: By inhibiting or eliminating effector CD8 + T cell subsets can reduce inflammation, increase the number or function of regulatory T cells to suppress excessive immune responses, or simultaneously target CD8. + Combined therapy of T cells and regulatory T cells.

43. A method for inhibiting the transformation of T cells into regulatory T cells, characterized in that, include: Inhibit CD8 + The expression of TGFB1 in T cells, or the inhibition of CD4 + Expression of BACH1 in T cells.

44. Use of an inhibitor in the preparation of a tumor therapeutic agent, wherein the inhibitor inhibits the expression of a target gene or a protein that inhibits the expression of a target gene, said target gene being selected from at least one of BACH1, TGFB1, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KLRC2, KLRC3, IKZF2, and TYROBP.

45. A method for enriching T cell subsets according to any one of claims 1-7, characterized in that, include: The immune cell population infiltrating the tumor is contacted with a binding agent, the binding agent being bound to at least one target gene or its expressed protein or protein fragment; and the immune cells bound to the binding agent are sorted to enrich a subset of T cells; wherein the target gene is selected from the characteristic genes described in any one of claims 1-7.

46. ​​A method for diagnosis or evaluation, characterized in that, include: (1) Immune cell samples infiltrating tumors obtained from subjects were classified as CD8 + T cells and CD4 + T cells; (2) the separated CD8 + T cells are contacted with at least one binding agent, which binds to a target gene or a protein or protein fragment expressed therein, wherein the target gene is selected from the characteristic genes described in claims 1-5; (3) the separated CD4 cells are... + T cells are contacted with at least one binding agent, which binds to a target gene or a protein or protein fragment expressed thereon, wherein the target gene is selected from the characteristic genes described in claims 6-7.

47. A CD8 assay for diagnosis or monitoring + The biomarker set of T cells, characterized by... It includes the characteristic gene as described in claims 1-5 or the protein or protein fragment expressed by the characteristic gene.

48. A CD4 assay for diagnosis or monitoring + The biomarker set of T cells, characterized by... It includes the characteristic gene as described in claims 6-7 or the protein or protein fragment expressed by the characteristic gene.

49. A diagnostic reagent for diagnosis or monitoring, characterized in that, The detection reagent includes a binder that binds to the characteristic gene or the protein or protein fragment expressed therein as described in claims 1-5.

50. A diagnostic reagent for diagnosis or monitoring, characterized in that, The detection reagent includes a binder that binds to the characteristic gene or the protein or protein fragment expressed therein as described in claims 6-7.

51. A kit for diagnosing or monitoring tumor prognosis, characterized in that, The kit comprises the detection reagent as described in claim 49.

52. A kit for diagnosing or monitoring tumor prognosis, characterized in that, The kit comprises the detection reagent as described in claim 50.

53. The biomarker set according to claim 47, the detection reagent according to claim 49, or the kit according to claim 51, characterized in that, The biomarker set, the detection reagent, or the kit is used for the diagnosis or monitoring of CD8. + T cell effector or effector memory state; Or used for the diagnosis or monitoring of CD8 + T cell cyclic effector memory state; Or used for the diagnosis or monitoring of CD8 + T cell progenitor cell exhaustion state; Or it can be used for the diagnosis or monitoring of HBV-specific CD8. + T cells.

54. The biomarker group according to claim 48, the detection reagent according to claim 50, or the kit according to claim 52, characterized in that, The biomarker set or the kit is used for the diagnosis or monitoring of CD4. + Are the T cells active regulatory T cells? Or it can be used for the diagnosis or monitoring of CD4. + Are the T cells regulatory T cells with strong clonal expansion potential? 55. The biomarker set according to claim 47 or 48, the detection reagent according to claim 49 or 50, or the kit according to claim 51 or 52, characterized in that, The tumor in question is liver cancer, particularly hepatocellular carcinoma.

56. The detection reagent according to claim 49 or 50, characterized in that, The detection reagent contains a set of primers that can specifically amplify each gene in the biomarker set; And / or, the detection reagent comprises a set of probes capable of hybridizing with each gene in the biomarker group.

57. The detection reagent according to claim 49 or 50, or the kit according to claim 51 or 52, wherein the binding agent is a nucleic acid, ligand, enzyme, substrate, and / or antibody; And / or, the binding agent is a nucleic acid probe capable of binding to the target gene; And / or, the binding agent is a primer capable of specifically amplifying the target gene; And / or, the binding agent is an antibody capable of binding to a protein or protein fragment encoded by the target gene; And / or, the binder further binds an indicator molecule, such as a fluorescent substance, a radioactive substance, and / or an enzyme.

58. A method for identifying novel antitumor effector T cell subsets, characterized in that, include: (1) For HBV patients receiving neoadjuvant therapy with anti-PD-1 combined with lenvatinib + Single-cell RNA sequencing, single-cell TCR sequencing, and flow cytometry analysis were performed on T cells from HCC patients. (2) Analyze the single-cell gene expression profile of T cells in cancer tissues and paired peripheral blood, and isolate and characterize T cell subsets that reflect the body's tumor immune status; (3) Determine the characteristic genes expressed by the T cell subsets and the relationship between these cell subsets and drug response.

59. The method according to claim 58, characterized in that, The T cell subsets include: GZMK + CD8 + Teff / Tem cells, the GZMK + CD8 + Teff / Tem cells are well-suited for large-scale clonal expansion in tumors of responders after treatment; Tpex cells, which are adapted to exhibit higher CXCL13 expression levels than terminally exhausted T cells; cTem cells, which are adapted to express higher levels of effector memory marker genes; HBV-specific CD8 + T cells, the HBV-specific CD8 + T cells are adapted to preferentially maintain an effector or memory state and are enriched in cTem cell populations; KIR + CD8 + T cell subsets, the KIR + CD8 + T cell subsets are well-suited for association with resistance to combination therapy; Activated CD4 + Treg cell subsets, the activated CD4 + Treg cell subsets are well-suited to be present in higher proportions in tumors after combination therapy in non-responders; CD4 expressing BACH1 + Treg cells, CD4 cells expressing BACH1 + Treg cells are adapted to be highly expressed in tumors in non-responders before treatment.

60. A method for screening drugs, characterized in that, include: a) Mixing the test chemical substance with the characteristic gene or the protein it expresses, or mixing the test chemical substance with CD8 expressing the characteristic gene. + T cells or CD4 + T-cell mixing; b) Detect changes in the activity of the expressed protein, or whether the test chemical substance binds to the characteristic gene or the protein it expresses, or changes in the activity of the cell, or changes in the expression level of the characteristic gene in the cell; The characteristic gene is selected from at least one of the following genes: BACH1, TGFB1, KIR2DL1, KIR2DL3, KIR2DL4, KIR3DL1, KIR3DL2, KIR3DL3, KLRC2, KLRC3, IKZF2, TYROBP, GZMK, MT1X, MT1E, MT2A, CCL4, XCL1, XCL2, GPR183, IL7R, ZNF683, CXCL13, CCL4L2, and NR4A2; Optionally, the drug is used to treat tumors; Optionally, the tumor is selected from at least one of the following: liver cancer, stomach cancer, lung cancer, pancreatic cancer, breast cancer, cervical cancer, skin cancer, prostate cancer, melanoma, thyroid cancer, uterine fibroids, lymphoma, esophageal cancer, intestinal cancer, bone marrow cancer, nasal cancer, bone cancer, head and neck cancer, oral cancer, kidney cancer, and Kaposi's sarcoma.

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