WO2023123225A1 - Sirt1-7蛋白在免疫治疗中的应用 - Google Patents

Sirt1-7蛋白在免疫治疗中的应用 Download PDF

Info

Publication number
WO2023123225A1
WO2023123225A1 PCT/CN2021/143151 CN2021143151W WO2023123225A1 WO 2023123225 A1 WO2023123225 A1 WO 2023123225A1 CN 2021143151 W CN2021143151 W CN 2021143151W WO 2023123225 A1 WO2023123225 A1 WO 2023123225A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
sequence
functional mutant
gpc3
protein
Prior art date
Application number
PCT/CN2021/143151
Other languages
English (en)
French (fr)
Inventor
凌有国
吴昊天
Original Assignee
南京紫珑生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 南京紫珑生物科技有限公司 filed Critical 南京紫珑生物科技有限公司
Priority to PCT/CN2021/143151 priority Critical patent/WO2023123225A1/zh
Publication of WO2023123225A1 publication Critical patent/WO2023123225A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of immunology. Specifically, the present invention relates to the combination of one or more of SIRT1-7 protein or its functional mutant, CD258 protein or its functional mutant and SIRT1-7 protein or its functional mutant in immunotherapy in the application.
  • CAR Chimeric antigen receptor
  • the form of scFv is generally an antigen-binding region formed by connecting the heavy chain variable region (VH) and light chain variable region (VL) of a monoclonal antibody through a polypeptide linker, that is, VH-Linker-VL or VL-Linker -VH; the hinge region is usually composed of immunoglobulin superfamily, such as IgG4, CD8, IgG1, etc.; the transmembrane region is generally composed of CD8, CD28 or CD4, etc.; the intracellular signal transduction region is mainly composed of CD3 ⁇ chain and co-stimulatory signal molecules 41BB, CD28, ICOS, OX40, etc. Therefore, T cells expressing CAR do not need to pass through the antigen presentation mechanism, and can directly recognize TAA, which has the unique advantage of MHC (Major histocompatibility complex) independence.
  • VH-Linker-VL or VL-Linker -VH the hinge region is usually composed of immunoglobulin superfamily, such as IgG
  • CART Due to the excellent performance of CAR-T technology in the treatment of blood tumors, with CD19 as the target, CART has achieved amazing curative effect in the treatment of acute and chronic B lymphocytic leukemia, but the clinical efficacy in the treatment of solid tumors is very limited, and new therapies are urgently needed solution.
  • Sirtuins are a class of highly conserved protein deacetylases and ADP ribosyltransferases that depend on NAD+ and the core region, and play an important role in the regulation of histone acetylation/deacetylation gene expression.
  • SIRT1-7 means 7 kinds of SIRT proteins.
  • SIRT1 protein, SIRT6 protein and SIRT7 protein are located in the nucleus and mainly regulate biological processes such as transcription, terminal centromere chromatin structure, aging and metabolism.
  • SIRT1 protein is also present in the cytosol and mitochondria.
  • SIRT2 protein is mainly located in the cytoplasm, interacts with tubulin in the cytoplasm, and is one of the important factors regulating apoptosis;
  • SIRT3 protein, SIRT4 protein and SIRT5 protein are located in the mitochondria, mainly regulating the key points in the process of cell energy metabolism.
  • the acetylation modification of protein plays an important role in biological processes such as cellular oxidative phosphorylation, tricarboxylic acid cycle, fatty acid aerobic oxidation and amino acid degradation.
  • CD258 protein also known as tumor necrosis factor superfamily member 14 (Tumor necrosisfactor superfamily member 14, TNFSF14), is an inducible expression on T cells, and it competes with herpes simplex virus glycoproteins for binding to herpes simplex virus entry receptors. [Homologous to lymphotoxin, inducible expression, competes with herpes simplex virus (HSV) glycoprotein D for HSV entry mediator (HVEM), a receptor expressed on T lymphocytes, LIGHT] or HVEM ligand (Herpesvirus entry mediator-ligand, HVEM-L).
  • HSV herpes simplex virus
  • HVEM HSV entry mediator
  • LIGHT LIGHT
  • HVEM ligand Herpesvirus entry mediator-ligand
  • CD258 protein is mainly expressed on the surface of activated T cells, B cells, natural killer (NK) cells, immature dendritic cells (im DC) and monocytes, and has three forms:
  • the full-length type contains 240 amino acid residues and a type 2 transmembrane glycoprotein with a relative molecular weight of 29ku; it is composed of 204 amino acid residues, lacks a transmembrane region, and only activates T lymphocytes.
  • Non-glycosylated protein and cell surface metal Soluble CD258 protein under enzymatic cleavage.
  • the signaling pathway mediated by CD258 protein is closely related to the occurrence and development of inflammatory and autoimmune diseases, graft-versus-host disease, pathogen infection and tumor immune regulation.
  • the technical problem solved by the present invention is the application of one or more of SIRT1-7 protein or its functional mutant, CD258 protein or its functional mutant in immunotherapy, which is specifically manifested as promoting the formation of memory T cells and suppressing immune response.
  • the expression of negative regulatory proteins in cellular immunity can enhance the release of immune cell cytokines, enhance the ability of immune cells to kill tumor cells, and mobilize the body's own anti-tumor immune response to solve the problem of tumor heterogeneity and prevent tumor recurrence.
  • the present application provides a method for promoting immune cell proliferation, which comprises up-regulating one or more of SIRT1-7 protein or its functional mutant, CD258 protein or its functional mutant in the immune cell of expression.
  • the present application provides a method for promoting the production of memory immune cells, which comprises up-regulating one or more of SIRT1-7 protein or its functional mutants, CD258 protein or its functional mutants in the immune cells expression level, thereby promoting the differentiation of immune cells into memory immune cells.
  • the present application provides a method for inhibiting immune cell differentiation, which comprises up-regulating the expression of one or more of SIRT1-7 protein or its functional mutant, CD258 protein or its functional mutant in the immune cell , thereby inhibiting the differentiation of the immune cells into differentiated immune cells.
  • the present application provides a method for inhibiting the expression of immune negative regulatory proteins in immune cells, which comprises up-regulating one or more of SIRT1-7 protein or its functional mutants, CD258 protein or its functional mutants in the immune cells The expression level of the species, thereby inhibiting the expression of the immune negative regulatory protein of the immune cells.
  • the immune negative regulatory protein is selected from the group consisting of PD1, PDL1, TIM3 and LAG3.
  • the present application provides a method for enhancing immune cells to release cytokines, which comprises up-regulating one or more of SIRT1-7 protein or functional mutant thereof, CD258 protein or functional mutant thereof in the immune cells expression up.
  • the cytokine is selected from interleukins, interferons and/or tumor necrosis factors. In certain embodiments, the cytokine is selected from IL-2, IL4, IL6, IL7, IL10, IL12, TNF- ⁇ , and/or IFN ⁇ .
  • the present application provides a method for enhancing the ability of immune cells to kill tumors, which comprises up-regulating SIRT1-7 protein or its functional mutant, CD258 protein or its functional mutant, and SIRT1-7 protein in the immune cells
  • the combination of SIRT1-7 protein or its functional mutant and CD258 protein or its functional mutant is up-regulated.
  • the present application provides a method for solving tumor heterogeneity, the method comprising: administering immune cells to a subject, wherein the immune cells contain SIRT1-7 protein or its functional mutant, CD258 protein or its The expression level of one or more of the functional mutants is up-regulated.
  • the present application provides a method for preventing tumor recurrence in a subject, the method comprising: administering immune cells to the subject, wherein the immune cells contain SIRT1-7 protein or its functional mutant, CD258 protein The expression of one or more of its functional mutants is up-regulated.
  • the present application provides a method for treating a tumor in a subject in need, comprising the following steps: administering immune cells to the subject, wherein the immune cells contain SIRT1-7 protein or its function
  • the expression of one or more of the mutant, the CD258 protein or its functional mutant is up-regulated.
  • the tumor is selected from liver cancer, lung cancer, leukemia, and mesothelioma.
  • the methods include in vivo methods and in vitro methods.
  • the expression of SIRT1-7 protein or its functional mutant, CD258 protein or its functional mutant is up-regulated.
  • the immune cells are lymphocytes.
  • the immune cells are T cells, B cells, natural killer (Natural killer, NK) cells, immature dendritic cells (Immature dendritic cells, im DC), monocytes and macrophages cell.
  • the T cells are selected from memory stem cell-like T cells (TSCMs) and/or central memory T cells (TCMs).
  • TSCM memory stem cell-like T cells
  • TCMs central memory T cells
  • the TSCM is CCR7 + and/or CD62L + .
  • the TSCM also has one or more properties selected from the group consisting of CD45RA + or CD45RA ⁇ , CD45RO + or CD45RO ⁇ , CD27 + , CD28 + , CD127 + , CD122 + , CD95 + , CD3 + , CD4 + and CD8 + .
  • the term “memory immune cells” generally refers to immune cells with immune memory.
  • the immune memory can refer to the ability to generate a rapid and strong immune response when encountering the same antigen again after specific recognition and response to a certain antigen.
  • the memory immune cells may include memory T cells.
  • the memory T cells can be divided into memory stem cell-like T cells (TSCM) and central memory T cells (TCM).
  • the term "differentiated immune cells” generally refers to immune cells with a certain degree of differentiation.
  • the differentiated immune cells may be T cells with a certain degree of differentiation.
  • the differentiated immune cells can be obtained by culturing the immune cells to differentiate to a certain extent.
  • the differentiated immune cells may comprise regulatory T cells (Treg).
  • regulatory T cell generally refers to a group of lymphocytes that negatively regulate the body's immune response.
  • the molecular marker of the regulatory T cells may be a transcription factor Foxp3 + or CD127 ⁇ .
  • the regulatory T cells can be divided into two types: naturally occurring and induced. Among them, CD4 + CD25 + cells naturally exist, and TR1 cells and TH3 cells are induced.
  • T memory stem cells usually refers to memory T cells that are in the early differentiation stage, have stem cell characteristics, and have strong multidirectional differentiation potential. cell. After responding to antigenic stimulation, TSCM cells can differentiate into central memory T cells (TCM), effector memory T cells (TEM) and effector T cells (TEF) .
  • TCM central memory T cells
  • TEM effector memory T cells
  • TEF effector T cells
  • central memory T cells generally refers to T cells with long-term memory generated after antigen activation of naive T cells (Naive T cells).
  • the biomarkers of the TCM may comprise CD62L + and CD45RO + .
  • the central memory T cells can pass through the lymphatic shield and return to the lymph nodes while being activated by antigens.
  • the immune cell is selected from a genetically modified immune cell expressing a chimeric antigen receptor (CAR) or a T cell receptor (TCR).
  • the genetically modified immune cells are genetically modified T cells.
  • the TCR comprises a subunit selected from the group consisting of TCR ⁇ , TCR ⁇ , TCR ⁇ , and TCR ⁇ .
  • the subunit of the TCR includes an extracellular domain variable region that specifically binds and/or recognizes a tumor antigen.
  • the ectodomain variable region is selected from the group consisting of TCR ⁇ variable region fragment V ⁇ , TCR ⁇ variable region fragment J ⁇ , TCR ⁇ variable region fragment V ⁇ , TCR ⁇ variable region fragment D ⁇ , and TCR ⁇ variable region fragment J ⁇ .
  • the ectodomain variable region specifically binds and/or recognizes a target selected from the group consisting of MAGEA family members, CTA family members, HPV viruses, and tyrosinase.
  • the ectodomain variable region specifically binds and/or recognizes a target selected from the group consisting of MAGEA3, MAGEA4, NY-ESO-1, MART1, HPV16-E6, and the melanoma antigen tyrosine Acidase.
  • the CAR comprises an intracellular domain comprising a signaling domain and/or a co-stimulatory domain.
  • the signaling domain is selected from the group consisting of the following: a signaling domain of CD3 ⁇ (preferred nucleotide sequence as shown in SEQ ID NO: 11, amino acid sequence as shown in SEQ ID NO: 83), the signaling domain of CD3 ⁇ and the signaling domain of CD3 ⁇ .
  • the signaling domain comprises the sequence shown in SEQ ID NO: 83 or at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, A sequence of at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% homology, the homologous sequence still has the function of the sequence shown in SEQ ID NO: 83, or consists of it .
  • signaling domain generally refers to functional signaling of a protein selected from the group consisting of CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , FcR ⁇ (FCER1G), FcR ⁇ (Fc Epsilon R1b), CD79a, CD79b, Fc ⁇ RIIa, DAP10 and DAP12 domain.
  • the signaling domain may include: CD3 ⁇ , CD3 ⁇ and CD3 ⁇ .
  • the co-stimulatory domain is selected from the group consisting of: a co-stimulatory domain of CD27, a co-stimulatory domain of CD28 (the preferred nucleotide sequence is shown in SEQ ID NO: 9, The amino acid sequence is shown in SEQ ID NO:81) and the co-stimulatory domain of 4-1BB (the preferred nucleotide sequence is shown in SEQ ID NO:10, and the amino acid sequence is shown in SEQ ID NO:82).
  • the co-stimulatory domain comprises or is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, A sequence of at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology, or consisting of: the homologous sequences described in SEQ ID NO:81 and SEQ ID NO:82 Still have the sequence function shown in SEQ ID NO:81 or 82.
  • co-stimulatory domain generally refers to a functional signaling domain of a protein selected from one or more of the following: CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, Lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands specifically binding to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 ( KLRF1), CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a , LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB
  • the CAR comprises a hinge region.
  • the hinge region is selected from the group consisting of: the hinge region of IgG4, the hinge region of IgG1 and the hinge region of CD8 (preferred nucleotide sequence shown in SEQ ID NO: 7, amino acid The sequence is shown in SEQ ID NO:79).
  • the hinge region comprises or consists of the sequence shown in SEQ ID NO:79.
  • the term "hinge region” generally refers to the region between the CH1 and CH2 domains of an immunoglobulin heavy chain.
  • the hinge region is a region located between the scFv and the T cell membrane.
  • the hinge region is usually derived from the IgG family, for example, can be derived from IgG1 and IgG4, and can also be derived from IgD and CD8.
  • the hinge region may comprise a part selected from the group consisting of the hinge region of IgG4, the hinge region of IgG1 and the hinge region of CD8.
  • the CAR comprises a transmembrane region.
  • the transmembrane region is selected from the group consisting of: the transmembrane region of CD8 (preferred nucleotide sequence as shown in SEQ ID NO: 8, amino acid sequence as shown in SEQ ID NO: 80 shown), the transmembrane region of CD28 and the transmembrane region of CD24.
  • the transmembrane region comprises or consists of the sequence shown in SEQ ID NO:8.
  • transmembrane region usually refers to the transmembrane region connecting the extracellular antigen-binding domain and the intracellular signal domain, generally composed of dimeric membrane proteins, mainly including CD3 ⁇ , CD4, CD8, CD28, etc., which can anchor the CAR structure on the T cell membrane. Different designs of the transmembrane region can affect the expression of the introduced CAR gene.
  • the transmembrane region may comprise a part selected from the group consisting of the transmembrane region of CD8, the transmembrane region of CD28 and the transmembrane region of CD24.
  • the CAR comprises a targeting moiety.
  • the targeting moiety is a scFv.
  • the targeting moiety is selected from scFv against GPC3, scFv against CD19, scFv against BCMA, scFv against MSLN, and scFv against HER2.
  • the scFv against GPC3 comprises LCDR1 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:36) contained in the light chain variable region shown in SEQ ID NO:35, LCDR2 (preferably according to Kabat numbering system, sequence as shown in SEQ ID NO:37) and LCDR3 (preferably according to Kabat numbering system, sequence as shown in SEQ ID NO:38) and comprising the heavy chain variable region shown in SEQ ID NO:39 comprises HCDR1 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:40), HCDR2 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:41) and HCDR3 (preferably according to the Kabat numbering system, the sequence is as shown in SEQ ID NO:41) NO: 42), more preferably, the scFv against GPC3 comprises the nucleotide sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 34,
  • the scFv against CD19 comprises LCDR1 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:45) contained in the light chain variable region shown in SEQ ID NO:44, LCDR2 (preferably according to Kabat numbering system, the sequence is as shown in SEQ ID NO:46) and LCDR3 (preferably according to the Kabat numbering system, the sequence is as shown in SEQ ID NO:47) and comprising the heavy chain variable region shown in SEQ ID NO:48 HCDR1 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:49), HCDR2 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:50) and HCDR3 (preferably according to the Kabat numbering system, the sequence is as shown in SEQ ID NO:50) NO: 51), more preferably, the scFv against CD19 comprises the nucleotide sequence shown in SEQ ID NO: 3 or the amino acid sequence shown in SEQ ID NO:
  • the scFv against BCMA comprises LCDR1 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:54) contained in the light chain variable region shown in SEQ ID NO:53, LCDR2 (preferably according to Kabat numbering system, sequence as shown in SEQ ID NO:55) and LCDR3 (preferably according to Kabat numbering system, sequence as shown in SEQ ID NO:56) and comprising the heavy chain variable region shown in SEQ ID NO:57 comprises HCDR1 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:58), HCDR2 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:59) and HCDR3 (preferably according to the Kabat numbering system, the sequence is as shown in SEQ ID NO:59) NO: 60), more preferably, the scFv against BCMA comprises a nucleotide sequence as shown in SEQ ID NO: 4 or an amino acid sequence as shown in SEQ ID NO: 52
  • the scFv against MSLN comprises LCDR1 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:63) contained in the light chain variable region shown in SEQ ID NO:62, LCDR2 (preferably according to Kabat numbering system, the sequence is as shown in SEQ ID NO:64) and LCDR3 (preferably according to the Kabat numbering system, the sequence is as shown in SEQ ID NO:65) and comprising the heavy chain variable region shown in SEQ ID NO:66 HCDR1 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:67), HCDR2 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:68) and HCDR3 (preferably according to the Kabat numbering system, the sequence is as shown in SEQ ID NO:68) NO: 69), more preferably, the scFv against MSLN comprises the nucleotide sequence shown in SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 5
  • the scFv against HER2 comprises LCDR1 contained in the light chain variable region shown in SEQ ID NO:71 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:72), LCDR2 (preferably according to Kabat numbering system, sequence as shown in SEQ ID NO:73) and LCDR3 (preferably according to Kabat numbering system, sequence as shown in SEQ ID NO:74) and comprising the heavy chain variable region shown in SEQ ID NO:75 comprises HCDR1 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:76), HCDR2 (preferably according to the Kabat numbering system, the sequence is shown in SEQ ID NO:77) and HCDR3 (preferably according to the Kabat numbering system, the sequence is as shown in SEQ ID NO:77) NO: 78), more preferably, the scFv against HER2 comprises the nucleotide sequence shown in SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 70 or
  • the targeting moiety specifically binds and/or recognizes a tumor antigen. In certain embodiments, the targeting moiety specifically binds to and/or recognizes a target selected from the group consisting of B lymphocyte surface antigens, TNF family members, HER family members, and GPC family members. In certain embodiments, the targeting moiety specifically binds and/or recognizes a target selected from the group consisting of CD19, BCMA, HER2, Mesothelin, and GPC3.
  • the targeting moiety comprises the sequence shown in any one of the following: SEQ ID NO: 2, 3, 4, 5, 6, 34, 43, 52, 61 or 70 or at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous sequence, or consisting of it, the homologous sequence still has the sequence function shown in SEQ ID NO: 2, 3, 4, 5, 6, 34, 43, 52, 61 or 70.
  • tumor antigen generally refers to an antigenic substance within or produced by a tumor cell, which may have the ability to trigger an immune response in the host.
  • a tumor antigen may be a protein, polypeptide, peptide or fragment thereof that constitutes a part of a tumor cell and is capable of inducing tumor-specific cytotoxic T lymphocytes.
  • Tumor antigen peptides may be peptides produced due to the degradation of tumor antigens in tumor cells, and may induce or activate tumor-specific cytotoxic T lymphocytes after being expressed on the cell surface by binding to HLA molecules.
  • tumor antigen may also refer to biomolecules (e.g., proteins, carbohydrates, glycoproteins, etc.) that are exclusively or preferentially or differentially expressed on and/or associated with cancer cells thereby providing cancer preferential or specific targets.
  • the preferential expression can be a general preferential expression, or a preferential expression within a particular region of the organism (eg, within a particular organ or tissue) compared to any other cell in the organism.
  • the tumor antigens may include TSHR, CD19, CD123, CD138, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, TnAg, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, Mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR- ⁇ , SSEA-4, CD20, Folate receptor ⁇ , ERBB2(Her2/neu), MUC1, EGFR, NCAM, Prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, rock Cocosyl GM1, sLe, GM3, TGS5, HMWMAA,
  • the method further includes the step of: isolating peripheral blood mononuclear cells PBMC, CD3 + T lymphocytes, CD8 + T lymphocytes, CD4 + T lymphocytes or regulatory T cells.
  • the method further comprises: adding one or more T cell stimulators to the isolated PBMCs.
  • the T cell stimulating factor is selected from the group consisting of antibodies to B lymphocyte surface antigens, anti-TNF antibodies, intracellular polyesters, and antibiotics.
  • the T cell stimulating factor is selected from the group consisting of anti-CD3 antibody, anti-CD28 antibody, anti-4-1BB antibody, anti-CD80 antibody, anti-CD86 antibody, PHA, PMA, and Ionomycin white.
  • the T cell stimulating factor is an anti-CD3 antibody, and the concentration of the anti-CD3 antibody is 1-10000 ng/mL. In some embodiments, the T cell stimulating factor is an anti-CD28 antibody, and the concentration of the anti-CD28 antibody is 1-10000 ng/mL.
  • the method further comprises: adding one or more cytokines to the isolated PBMCs.
  • the cytokine is an interleukin.
  • interleukin generally refers to a secreted protein or signaling molecule capable of promoting the development and differentiation of T and/or B lymphocytes and/or hematopoietic cells. Interleukins can be synthesized by helper CD4 T lymphocytes, as well as by monocytes, macrophages and endothelial cells.
  • interleukins may include IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL- 23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35 and /or IL-36.
  • interleukin may include full-length interleukins or fragments (e.g., truncated forms) or variants thereof that substantially retain the biological activity of the corresponding wild-type interleukin (e.g., have a biological activity of at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% % of the biological activity of the corresponding wild-type interleukin).
  • the interleukins used herein may be from any mammalian species.
  • the interleukin is from a species selected from the group consisting of human, equine, bovine, murine, porcine, rabbit, cat, dog, rat, goat, sheep, and non-human primate.
  • the interleukins may be in mutated forms.
  • the interleukin can be super IL-2 (also known as sIL2, see Nature 484, 529-533, 2612), which can be obtained by modifying IL-2 to increase its binding affinity for IL-2R ⁇ .
  • sIL-2 Mutations in sIL-2 are predominantly cytokine core, and molecular dynamics simulations suggest that evolutionary mutations stabilize IL-2, reducing the flexibility of the helix in the IL-2R ⁇ -binding site to resemble an optimized receptor-binding conformation When bound to CD25.
  • sIL-2 induces superior expansion of cytotoxic T cells, leading to improved antitumor responses in vivo, causing less expansion of T regulatory cells, and less pulmonary edema.
  • the cytokines may comprise IL-2, IL4, IL6, IL7, IL10, IL21, TNF- ⁇ and/or IFN ⁇ .
  • the interleukin is selected from one or more of the group consisting of IL2, IL21, IL7, and IL15.
  • the interleukin is IL2, and the concentration of the IL2 is 0.1-10000 U/mL.
  • the interleukin is IL21, and the concentration of the IL21 is 0.01-1000 ng/mL.
  • the interleukin is IL7, and the concentration of the IL7 is 0.01-1000 ng/mL.
  • the interleukin is IL15, and the concentration of the IL15 is 0.01-1000 ng/mL.
  • the SIRT1-7 or functional mutants thereof, CD258 or functional mutants thereof are derived from humans.
  • SIRT1 protein has 2 nuclear localization signals (PLRKRPRR shown in SEQ ID NO:105 and PPKRKKRK shown in SEQ ID NO:106), the preferred mutation is any amino acid substitution to A or deletion, the more preferred mutant is SEQ PLRKRPAA shown in ID NO:107 and PPKRAAAA shown in SEQ ID NO:108); preferred deletion mutations are PLRKRPRR shown in SEQ ID NO:105 and complete deletion of PPKRKKRK shown in SEQ ID NO:106; SIRT1 protein There are 2 nuclear nuclear signals (LLLTDGLL shown in SEQ ID NO:109 and VDLLIVI shown in SEQ ID NO:110), the preferred mutant is any amino acid substitution to A or deletion, the more preferred mutant is SEQ AAATDGAA shown in ID NO:111 and ADAAAAA shown in SEQ ID NO:112 (see THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.282, NO.9, pp.6823-6832), the preferred deletion mutation is SEQ ID NO:109 Complete deletion of LLLT
  • the SIRT1 sequence is a truncation of the original sequence (UniProtKB number: Q96EB6-1), from the small molecule sirtuin-activating compounds binding domain (Small molecule sirtuin-activating compounds binding domain, SBD, original sequence 183-229) of the original sequence, to The acetylase domain (Deacetylase domain, No. 229-516 of the original sequence) and the C-terminal regulatory segment (C-terminal regulatory segment, CTR, No. 641-665 of the original sequence) are sequentially spliced; SIRT2-7 is the original sequence, see https://www.uniprot.org/uniprot/Q96EB6 .
  • the SIRT1 protein, SIRT2 protein, SIRT3 protein, SIRT4 protein, SIRT5 protein, SIRT6 protein, SIRT7 protein respectively comprise the sequence shown in any of the following: SEQ ID NO:15-21 or SEQ ID NO:15-21 ID NO:86-92, or at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, A sequence with at least 98%, at least 99% homology, or consisting of it, said homologous sequence still has the sequence function shown in any one of SEQ ID NO: 15-21 or SEQ ID NO: 86-92.
  • the functional mutant of the SIRT1-7 protein is mutated in the domain of the SIRT1-7 protein selected from the following group: deacetylase domain (Deacetylase domain), small molecule Sirtuin activation Agent binding domain (Small molecule sirtuin-activating compounds binding domain, SBD), and C-terminal regulatory segment (CTR).
  • deacetylase domain Deacetylase domain
  • small molecule Sirtuin activation Agent binding domain Small molecule Sirtuin activation Agent binding domain
  • SBD small molecule sirtuin-activating compounds binding domain
  • CTR C-terminal regulatory segment
  • the SIRT1 functional mutant comprises the sequence shown in any of the following: SEQ ID NO:22-30 or SEQ ID NO:93-101 or at least 80%, at least 85%, or consisting of sequences of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homology,
  • the homologous sequence still has the sequence function shown in any one of SEQ ID NO: 22-30 or SEQ ID NO: 93-101.
  • the CD258 protein comprises or has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, or at least 80% of the sequence shown in SEQ ID NO:31 or SEQ ID NO:102. At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous sequence, or consisting of it, said homologous sequence still possesses SEQ ID NO: 31 Or the sequence function shown in SEQ ID NO:102.
  • the CD258 protein functional mutants include membrane-bound CD258 (preferred sequence as shown in SEQ ID NO: 32 or SEQ ID NO: 103), secreted CD258 (preferred sequence as shown in SEQ ID NO: 33 or SEQ ID NO: 104) shown) and the intracellular region of CD258.
  • said membrane-bound CD258 comprises a deletion or site-directed mutation of said CD258 proteolytic site.
  • the proteolysis site is the QL site at positions 82-83 of the sequence shown in SEQ ID NO:102, preferably, the deletion mutation is the complete deletion of the CD258 proteolysis site or the sequence shown in SEQ ID NO:102
  • the EQLI at positions 81-84 in EQLI is deleted; the substitution mutation is that Q and/or L amino acids are replaced by A, more preferably, QL amino acids are replaced by AA.
  • the SIRT1-7 protein or its functional mutant, CD258 protein or its functional mutant comprises the sequence shown in any one of the following: SEQ ID NO: 15-33 or at least 80% thereof , at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous to the sequence,
  • the homologous sequence still has the sequence function shown in any one of SEQ ID NO:15-33, preferably comprises the sequence shown in any of the following: SEQ ID NO:86-104 or at least 80%, at least 85% thereof %, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous sequences, said homologous
  • the source sequence still has the sequence function shown in any one of SEQ ID NO:86-104.
  • the present application provides a genetically modified immune cell, wherein the genetic modification makes one or more of SIRT1-7 protein or its functional mutant, CD258 protein or its functional mutant in the immune cell , and the expression levels of their combinations were up-regulated.
  • the up-regulation of the expression level is achieved by:
  • the activator is selected from the group consisting of: SRT2104 (CAS No. 1093403-33- 8, chemical formula C 26 H 24 N 6 O 2 S 2 ); CAY10602 (CAS No. 374922-43-7, chemical formula C 22 H 15 FN 4 O 2 S); OSS-128167 (CAS No. 887686-02-4, chemical formula C 19 H 14 N 2 O 6 );
  • SIRT1-7 protein or a functional mutant thereof by transfecting into the immune cells an expression vector comprising nucleic acid encoding one or more of the SIRT1-7 protein or a functional mutant thereof, CD258 protein or a functional mutant thereof to increase the immune cells
  • the amount of one or more of the SIRT1-7 protein or its functional mutants, CD258 protein or its functional mutants, preferably, the SIRT1 or its functional mutants, SIRT2 or its functional mutants, SIRT3 or Functional mutants thereof, SIRT4 or functional mutants thereof, SIRT5 or functional mutants thereof, SIRT6 or functional mutants thereof, SIRT7 protein or functional mutants thereof, and CD258 protein or functional mutants thereof exist in the form of monomers or by linking It exists in the form of conjugates where the elements are linked.
  • the vector is selected from the group consisting of retroviral vectors, lentiviral vectors, and transposon plasmids, preferably, the vector further comprises a CAR, more preferably, the CAR is linked by a linking element A regulatory unit, the regulatory unit is selected from one or more of the SIRT1-7 protein or its functional mutant, CD258 protein or its functional mutant, or a combination thereof.
  • the term "2A sequence” generally refers to a protease-independent self-cleaving amino acid sequence.
  • the 2A sequence may facilitate transcription to produce both proteins.
  • the connecting element is a 2A sequence, selected from the group consisting of: T2A (preferred nucleotide sequence as shown in SEQ ID NO: 12, amino acid sequence as shown in SEQ ID NO: 84 ), P2A (the preferred nucleotide sequence is as shown in SEQ ID NO:13, and the amino acid sequence is as shown in SEQ ID NO:85), F2A, E2A and IRES (the preferred nucleotide sequence is as shown in SEQ ID NO:14) .
  • the linking element comprises or consists of the sequences shown in SEQ ID NO: 84, 85 and 14.
  • the present application provides a composition comprising the genetically modified immune cells.
  • the composition also includes, optionally, a pharmaceutically acceptable carrier.
  • the application provides one or more of the SIRT1-7 protein or its functional mutants, CD258 protein or its functional mutants, or a combination thereof in the preparation of drugs for improving the efficacy of preventing and/or treating tumors.
  • the drug is genetically modified immune cells (preferably CAR-T cells).
  • the present application also provides a use of the genetically modified immune cells and/or the composition in the preparation of medicines, wherein the medicines are used for treating and/or preventing tumors.
  • the tumor is selected from liver cancer, lung cancer, leukemia, and mesothelioma.
  • the genetically modified immune cells are selected from lymphocytes. In certain embodiments, the genetically modified immune cell expresses a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the method comprises the step of isolating and activating said genetically modified immune cells, wherein said activating comprises administering a T cell culture medium to said isolated genetically modified immune cells.
  • the T cell culture medium is selected from one or more of the following groups: DMEM medium, 1640 medium, MEM medium, X-VIVO medium and stem cell medium.
  • Figure 1 is a schematic diagram of different CAR structures
  • Figure 2 shows that SIRT1 protein promotes the expansion of specific CAR-T cells induced by tumor antigens
  • FIG. 3 shows that SIRT1 protein inhibits the expression of immune negative regulatory proteins
  • Fig. 4 is that SIRT1 protein promotes the proliferation of memory T cells in CD3 cells
  • FIG. 5 shows that SIRT1 protein promotes the proliferation of memory T cells in CD4 and CD8 cells
  • Figure 6-8 shows the expression of SIRT1 protein functional mutants and their combinations to inhibit the expression of immune negative regulatory proteins
  • FIGS 9-10 show that SIRT1 protein functional mutants and their combinations promote the proliferation of memory T cells in CD3 cells
  • FIG 11 shows that SIRT1 protein functional mutants and their combinations promote the proliferation of memory T cells in CD4 cells
  • Figure 12 shows that SIRT1 protein functional mutants and their combinations promote the proliferation of memory T cells in CD8 cells
  • FIG. 13 shows that SIRT1 protein functional mutants and their combinations promote the proliferation of T cells
  • FIG. 14 shows that SIRT1 protein functional mutants and their combinations promote the resistance of T cells to the immunosuppressive microenvironment
  • FIG. 15 shows that CD258 protein and its functional mutants enhance the killing ability of CAR-T cells against tumor cells
  • Figure 16 shows that the combination of CD258 protein and SIRT1 protein promotes the expansion of specific CAR-T cells induced by tumor antigens
  • Figure 17 is CD258 protein combined with SIRT1 protein to promote the proliferation of memory T cells in CD3 cells (GPC3);
  • Figure 18 is CD258 protein combined with SIRT1 protein to promote the proliferation of memory T cells in CD4 and CD8 cells (GPC3);
  • FIG 19 shows that the combination of CD258 protein and SIRT1 protein inhibits the expression of immune negative regulatory protein (CD19);
  • Figure 20 is CD258 protein combined with SIRT1 protein to promote the proliferation of memory T cells in CD8 cells (CD19);
  • Figure 21 is CD258 protein combined with SIRT1 protein to promote the proliferation of memory T cells in CD3 and CD4 cells (CD19);
  • FIG 22 shows that the combination of CD258 protein and SIRT1 protein inhibits the expression of immune negative regulatory protein (MSLN);
  • Fig. 23 is that the combination of CD258 protein and SIRT1 protein promotes the proliferation of memory T cells in CD3 cells (MSLN);
  • Figure 24 shows that the combination of CD258 protein and SIRT1 protein enhances the killing ability of CAR-T cells on tumor cells
  • Figure 25 shows that the combination of CD258 protein and SIRT1 protein enhances the anti-tumor effect of CAR-T cells
  • Figure 26 is a combination of CD258 protein and SIRT1 protein to promote the proliferation of granulocytes and monocytes in vivo;
  • Figure 27 shows that the combination of CD258 protein and SIRT1 protein promotes the proliferation of CAR-T cells in vivo and the release of Th1 cytokines.
  • the CAR-T targets GPC3, CD19, and Mesothelin (MSLN) as an example, artificially synthesizes a fragment containing the CAR structure, and constructs it into a lentiviral vector (LV100A, System Biosciences Company), and then transfects it according to the method described in its instructions
  • the lentivirus was obtained, and GPC3-CAR, GPC3-S1, GPC3-S2, GPC3-S3, GPC3-S6, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S1A-S3, GPC3-C8, GPC3-C8A, GPC3-C8B, GPC3-S1A-C8A, CD19-CAR, CD
  • GPC3-CAR is sequentially spliced and synthesized by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11.
  • GPC3-S1 consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 15 are sequentially spliced and synthesized.
  • GPC3-S2 consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 16 are sequentially spliced and synthesized.
  • GPC3-S3 consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 17 are sequentially spliced and synthesized.
  • GPC3-S6 consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 20 are sequentially spliced and synthesized.
  • GPC3-S1A consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 22 are sequentially spliced and synthesized.
  • GPC3-S1B consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 23 are sequentially spliced and synthesized.
  • GPC3-S1B1 consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 24 are sequentially spliced and synthesized.
  • GPC3-S1C consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 25 spliced and synthesized sequentially.
  • GPC3-S1C1 consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 26 are sequentially spliced and synthesized.
  • GPC3-S1D consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 27 are sequentially spliced and synthesized.
  • GPC3-S1D1 consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 28 are sequentially spliced and synthesized.
  • GPC3-S1E consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 29 were spliced and synthesized sequentially.
  • GPC3-S1E1 consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 30 are sequentially spliced and synthesized.
  • GPC3-S1-S3 consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:15, SEQ ID NO:13, and SEQ ID NO:17 were sequentially spliced and synthesized.
  • GPC3-S1A-S3 consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:13, and SEQ ID NO:17 were sequentially spliced and synthesized.
  • GPC3-C8 consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 31 are sequentially spliced and synthesized.
  • GPC3-C8A consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 32 are sequentially spliced and synthesized.
  • GPC3-C8B consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 33 are sequentially spliced and synthesized.
  • GPC3-S1A-C8A consists of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:13, and SEQ ID NO:32 were sequentially spliced and synthesized.
  • CD19-CAR is sequentially spliced and synthesized by SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11.
  • CD19-S1A consists of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 22 are sequentially spliced and synthesized.
  • CD19-C8A consists of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 32 are sequentially spliced and synthesized.
  • CD19-S1A-C8A consists of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:13, and SEQ ID NO:32 were sequentially spliced and synthesized.
  • MSLN-CAR is sequentially spliced and synthesized by SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, and SEQ ID NO:11.
  • MSLN-S1A consists of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 22 are sequentially spliced and synthesized.
  • MSLN-C8 consists of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 31 are sequentially spliced and synthesized.
  • MSLN-C8A consists of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 32 are sequentially spliced and synthesized.
  • MSLN-C8B consists of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO: 33 are sequentially spliced and synthesized.
  • MSLN-S1A-C8A consists of SEQ ID NO:1, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:13, and SEQ ID NO:32 were sequentially spliced and synthesized.
  • Example 2 Lentivirus infection of T cells.
  • PBMC Peripheral blood mononuclear lymphocytes
  • Dilute anti-human CD3 and anti-human CD28 antibodies (purchased from Shanghai Nearshore Technology Co., Ltd.) with PBS, the final concentration is 1ug/ml, add the diluted antibody mixture to the cell culture dish, make it cover the culture dish, and incubate at room temperature 2 hours. After 2 hours, wash once with PBS and set aside.
  • T lymphocyte culture medium (Xvivo15 medium + 5% FBS + 100 U/ml IL2 + 20ng/ml IL21 + 20ng/ml IL7) to a final concentration of 1 ⁇ 106 cells/ml, and place Put them into the culture dish treated in step 2 for culture, the culture condition is 37° C.+5% CO 2 , and the culture time is 24 hours.
  • T cell culture solution Take a certain amount of T cell culture solution, add synperonic F108 (Sigma company) with a final concentration of 1mg/ml, mix well, and heat the water bath to 37°C for use.
  • synperonic F108 Sigma company
  • Dilute activated T cells with the infection reagent prepared in 1), and add lentivirus according to MOI 3, and mix well. Spread evenly in the culture dish treated in 2).
  • Example 3 SIRT1 protein promotes tumor antigen-induced expansion of specific CAR-T cells.
  • T cells expressing GPC3-CAR, GPC3-S2, GPC3-S3 or GPC3-S6 were mixed with HepG2 cells (purchased from the Cell Bank of Chinese Academy of Sciences) irradiated (X-RAD cell irradiator, irradiation dose: 30Gy) by 1
  • the cell number ratio of :1 was co-cultured with Xvivo15 medium, and irradiated HepG2 was re-supplemented every 4 days for stimulation, and the stimulation was 3 times.
  • Count each time by trypan blue staining, and the cell proliferation is shown in Figure 2A.
  • the results showed that the expansion factor of T cells expressing GPC3-S2, GPC3-S3 or GPC3-S6 was much higher than that of T cells expressing GPC3-CAR in the control group.
  • T cells expressing GPC3-CAR, GPC3-S2, GPC3-S3 or GPC3-S6 were combined with irradiated (X-RAD cell irradiator, irradiation dose was 30Gy) Huh7 cells (purchased from the Cell Bank of Chinese Academy of Sciences) ) were co-cultured with Xvivo15 medium at a cell number ratio of 1:1, and irradiated Huh7 was re-stimulated every 4 days for 3 stimulations. Each count was performed by staining with trypan blue, and the cell proliferation was shown in Figure 2B. The results also showed that the expansion factor of T cells expressing GPC3-S2, GPC3-S3 or GPC3-S6 was much higher than that of T cells expressing GPC3-CAR in the control group.
  • Example 4 SIRT1 protein inhibits the expression of immune negative regulatory proteins.
  • T cells expressing GPC3-CAR, GPC3-S2, GPC3-S3 or GPC3-S6 obtained in Example 2 were cultured in vitro at 37°C in a 5% CO2 cell incubator for 9 days or 12 days.
  • BD flow cytometry was used to detect the expression of CD3, CD4, CD8, PD1, PDL1, TIM3, and LAG3 proteins in T cells.
  • the results are shown in Figure 3.
  • T cells expressing GPC3-S2, GPC3-S3, or GPC3-S6 Among them, the proportion of cells expressing two negative regulatory proteins at the same time was significantly lower than that of the GPC3-CAR control group.
  • SIRT1 protein promotes the proliferation of memory T cells in CD3 cells.
  • T cells expressing GPC3-CAR, GPC3-S2, GPC3-S3 or GPC3-S6 obtained in Example 2 were cultured in vitro at 37°C in a 5% CO2 cell incubator for 9 days or 12 days.
  • BD flow cytometry was used to detect the expression of CD3, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells, as shown in Figure 4, the expression of GPC3-S2, GPC3-S3 or GPC3-
  • the proportion of memory stem cell-like T cells (TSCM) in the CD3 cells of S6 was significantly higher than that of the GPC3-CAR control group.
  • SIRT1 protein promotes the proliferation of memory T cells in CD4 and CD8 cells.
  • T cells expressing GPC3-CAR, GPC3-S2, GPC3-S3 or GPC3-S6 obtained in Example 2 were cultured in vitro at 37°C in a 5% CO2 cell incubator for 9 days or 12 days.
  • the expression of CD4, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells was detected by BD flow cytometry, as shown in Figure 5A, the expression of GPC3-S2, GPC3-S3 or GPC3-
  • the proportion of memory stem cell-like T cells (TSCM) in the CD4 cells of S6 was significantly higher than that of the GPC3-CAR control group.
  • T cells expressing GPC3-CAR, GPC3-S2, GPC3-S3 or GPC3-S6 obtained in Example 2 were cultured in vitro at 37°C in a 5% CO2 cell incubator, and co-cultured for 9 days or 12 days. sky.
  • the expression of CD8, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells was detected by BD flow cytometry, as shown in Figure 5B, the expression of GPC3-S2, GPC3-S3 or GPC3-
  • the proportion of memory stem cell-like T cells (TSCM) in the CD8 cells of S6 was also significantly higher than that of the GPC3-CAR control group.
  • Example 7 SIRT1 protein functional mutants and their combinations inhibit the expression of immune negative regulatory proteins.
  • Example 2 At 37°C, in a 5% CO2 cell culture incubator, the cells obtained in Example 2 were cultured in vitro to express GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, T cells of GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S1A-S3 were co-cultured for 9 days or 12 days.
  • BD flow cytometry was used to detect the expression of CD3, CD4, CD8, PD1, PDL1, TIM3, and LAG3 proteins in T cells.
  • Example 8 SIRT1 protein functional mutants and combinations thereof promote the proliferation of memory T cells in CD3 cells.
  • Example 2 At 37°C, in a 5% CO2 cell culture incubator, the cells obtained in Example 2 were cultured in vitro to express GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, T cells of GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S1A-S3 were co-cultured for 9 days or 12 days.
  • GPC3-CAR GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, T cells of GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S
  • T cells The expression of CD3, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells was detected by BD flow cytometry, as shown in Figure 9-10, the expression of GPC3-S1, GPC3-S1A, Memory stem cells in CD3 cells of GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S1A-S3
  • the proportion of T-like T cells (TSCM) in the total cells was significantly higher than that of the GPC3-CAR control group.
  • Example 9 SIRT1 protein functional mutants and their combinations promote the proliferation of memory T cells in CD4 cells.
  • Example 2 At 37°C, in a 5% CO2 cell culture incubator, the cells obtained in Example 2 were cultured in vitro to express GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, T cells of GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S1A-S3 were co-cultured for 9 days or 12 days.
  • GPC3-CAR GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, T cells of GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S
  • BD flow cytometry was used to detect the expression of CD4, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells, as shown in Figure 11, the expression of GPC3-S1, GPC3-S1A, GPC3- Memory stem cell-like T in CD4 cells of S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S1A-S3
  • the proportion of cells (TSCM) to the total cells was significantly higher than that of the GPC3-CAR control group.
  • Example 10 SIRT1 protein functional mutants and their combinations promote the proliferation of memory T cells in CD8 cells.
  • Example 2 At 37°C, in a 5% CO2 cell culture incubator, the cells obtained in Example 2 were cultured in vitro to express GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, T cells of GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S1A-S3 were co-cultured for 9 days or 12 days.
  • GPC3-CAR GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, T cells of GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S
  • BD flow cytometry was used to detect the expression of CD8, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells, as shown in Figure 12, the expression of GPC3-S1, GPC3-S1A, GPC3- Memory stem cell-like T in CD8 cells of S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S1A-S3
  • the proportion of cells (TSCM) to the total cells was significantly higher than that of the GPC3-CAR control group.
  • Example 11 SIRT1 protein functional mutants and their combinations promote the proliferation of T cells.
  • Example 2 At 37°C, in a 5% CO2 cell culture incubator, the cells obtained in Example 2 were cultured in vitro to express GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, T cells of GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S1A-S3 were co-cultured for 11 days, counted and passaged every 2-3 days with trypan blue staining, Cell proliferation is shown in Figure 13A.
  • Example 2 Similarly, at 37°C, 5% CO 2 cell culture incubator in vitro culture obtained in Example 2 respectively express GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3 -S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S1A-S3 T cells were co-cultured for 7 days, and the cells and Xvivo15 medium were collected on the 7th day After co-incubating for 24 h, CCK8 (MCE company) was used to detect the proliferation effect of T cells.
  • MCE company MCE company
  • Example 12 SIRT1 protein functional mutants and their combinations promote the resistance of T cells to the immunosuppressive microenvironment.
  • Example 2 At 37°C, in a 5% CO2 cell culture incubator, the cells obtained in Example 2 were cultured in vitro to express GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S1A-S3 T cells were co-cultured for 8 days, and the cells were collected on the 8th day and 1640 glucose-free and serum-free After co-incubating with culture medium (Gibco Company) for 24 h, CCK8 (MCE Company) was used to detect the proliferation effect of T cells.
  • culture medium Gibco Company
  • CCK8 MCE Company
  • Example 2 Similarly, at 37°C, 5% CO 2 cell culture incubator in vitro culture obtained in Example 2 respectively express GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3 -S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, GPC3-S1A-S3 T cells were co-cultured for 8 days, and the cells were collected on the 8th day and 1640 without glucose , serum-free medium (Gibco Company) and 20mM lactic acid (pH value dropped from normal 7.4 to 6.5, Sigma Company) were co-incubated for 24h, and CCK8 (MCE Company) was used to detect the proliferation effect of T cells.
  • GPC3-CAR GPC3-S1, GPC3-S1A, GPC3-S1B,
  • Example 13 CD258 protein and its functional mutants enhance the killing ability of CAR-T cells on tumor cells.
  • T cells expressing GPC3-CAR, GPC3-C8, GPC3-C8A or GPC3-C8B and Huh7 cells purchased from the Chinese Academy of Sciences Cell Bank
  • Xvivo15 medium obtained from the Chinese Academy of Sciences Cell Bank
  • Violet staining was used to detect the killing effect of CAR-T cells on tumor cells.
  • the killing effect of T cells expressing GPC3-C8 or GPC3-C8A on tumor cells was much higher than that of the control group expressing GPC3- CAR T cells, in which T cells expressing GPC3-C8A have a superior killing effect compared with T cells expressing GPC3-C8.
  • T cells expressing GPC3-CAR, GPC3-C8, GPC3-C8A or GPC3-C8B and Huh7 cells purchased from the Chinese Academy of Sciences Cell Bank
  • 1640 glucose-free, serum-free ( Gibco Company) and 20mM lactic acid (pH value dropped from normal 7.4 to 6.5, Sigma Company) medium were co-cultured for 7 days, and the killing effect of CAR-T cells on tumor cells was detected by crystal violet staining (MCE Company), and the results are shown in Figure 15B
  • MCE Company crystal violet staining
  • the killing effect of T cells expressing GPC3-C8 or GPC3-C8A on tumor cells was also significantly higher than that of T cells expressing GPC3-CAR in the control group.
  • T cells expressing GPC3-C8 Cells also have a superior killing effect.
  • Example 14 The combination of CD258 protein and SIRT1 protein promotes the expansion of specific CAR-T cells induced by tumor antigens.
  • T cells expressing GPC3-CAR, GPC3-S1A, GPC3-C8, GPC3-C8A, GPC3-C8B or GPC3-S1A-C8A were combined with Huh7 irradiated (X-RAD cell irradiator, irradiation dose was 30Gy)
  • Huh7 irradiated X-RAD cell irradiator, irradiation dose was 30Gy
  • the cells purchased from the Cell Bank of the Chinese Academy of Sciences
  • irradiated Huh7 was added for stimulation every 3-4 days, and stimulated 4 times.
  • Counting was performed each time by staining with trypan blue, and the cell proliferation is shown in Figure 16 .
  • GPC3-S1A-C8A is the coupling of GPC3, S1A and C8A sequentially through the 2A sequence.
  • Example 15 The combination of CD258 protein and SIRT1 protein promotes the proliferation of memory T cells in CD3 cells (GPC3).
  • T cells expressing GPC3-CAR, GPC3-S1A, GPC3-C8, GPC3-C8A, GPC3-C8B or GPC3-S1A-C8A obtained in Example 2 in vitro at 37°C in a 5% CO2 cell incubator , co-cultured for 9 days or 12 days.
  • BD flow cytometry was used to detect the expression of CD3, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells, as shown in Figure 17, the expression of GPC3-S1A, GPC3-C8, GPC3-
  • the proportion of memory stem cell-like T cells (TSCM) in the CD3 cells of C8A, GPC3-C8B or GPC3-S1A-C8A was significantly higher than that of the GPC3-CAR control group.
  • Example 16 The combination of CD258 protein and SIRT1 protein promotes the proliferation of memory T cells in CD4 and CD8 cells (GPC3).
  • T cells expressing GPC3-CAR, GPC3-S1A, GPC3-C8, GPC3-C8A, GPC3-C8B or GPC3-S1A-C8A obtained in Example 2 in vitro at 37°C in a 5% CO2 cell incubator , co-cultured for 9 days or 12 days.
  • BD flow cytometry was used to detect the expression of CD4, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells, as shown in Figure 18A, expressing GPC3-S1A, GPC3-C8, GPC3-
  • the proportion of memory stem cell-like T cells (TSCM) in the CD4 cells of C8A, GPC3-C8B or GPC3-S1A-C8A was significantly higher than that of the GPC3-CAR control group.
  • Example 2 Similarly, at 37°C, 5% CO 2 cell culture incubator in vitro culture obtained in Example 2 respectively express GPC3-CAR, GPC3-S1A, GPC3-C8, GPC3-C8A, GPC3-C8B or GPC3-S1A-C8A T cells were co-cultured for 9 days or 12 days.
  • BD flow cytometry was used to detect the expression of CD8, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells, as shown in Figure 18B, expressing GPC3-S1A, GPC3-C8, GPC3-
  • the proportion of memory stem cell-like T cells (TSCM) in the CD8 cells of C8A, GPC3-C8B or GPC3-S1A-C8A was also significantly higher than that of the GPC3-CAR control group.
  • Example 17 The combination of CD258 protein and SIRT1 protein inhibits the expression of immune negative regulatory protein (CD19).
  • BD flow cytometry was used to detect the expression of CD3, CD4, CD8, PD1, PDL1, TIM3, and LAG3 proteins in T cells. The results are shown in Figure 19. In T cells, the proportion of cells expressing two negative regulatory proteins at the same time was significantly lower than that of the CD19-CAR control group.
  • Example 18 The combination of CD258 protein and SIRT1 protein promotes the proliferation of memory T cells in CD8 cells (CD19).
  • the expression of CD8, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells was detected by BD flow cytometry, as shown in Figure 20, the expression of CD19-S1A, CD19-C8A or CD19-
  • the proportion of memory stem cell-like T cells (TSCM) in the CD8 cells of S1A-C8A was significantly higher than that of the CD19-CAR control group.
  • Example 19 The combination of CD258 protein and SIRT1 protein promotes the proliferation of memory T cells in CD3 and CD4 cells (CD19).
  • BD flow cytometry was used to detect the expression of CD3, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells.
  • TSCM memory stem cell-like T cells
  • T cells expressing CD19-CAR, CD19-S1A, CD19-C8A or CD19-S1A-C8A obtained in Example 2 were cultured in vitro at 37°C in a 5% CO2 cell incubator, and co-cultured for 9 days or 12 days.
  • BD flow cytometry was used to detect the expression of CD4, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells, as shown in Figure 21B, the expression of CD19-S1A, CD19-C8A or CD19-
  • the proportion of memory stem cell-like T cells (TSCM) in the CD4 cells of S1A-C8A was also significantly higher than that of the CD19-CAR control group.
  • Example 20 The combination of CD258 protein and SIRT1 protein inhibits the expression of immune negative regulatory protein (MSLN).
  • MSLN immune negative regulatory protein
  • BD flow cytometry was used to detect the expression of CD3, CD4, CD8, PD1, PDL1, TIM3, and LAG3 proteins in T cells. The results are shown in Figure 22.
  • MSLN-C8A MSLN-C8A
  • MSLN-C8B In S1A or MSLN-S1A-C8A T cells, the proportion of cells expressing two negative regulatory proteins was significantly lower than that of the MSLN-CAR control group.
  • Example 21 The combination of CD258 protein and SIRT1 protein promotes the proliferation of memory T cells in CD3 cells (MSLN).
  • BD flow cytometry was used to detect the expression of CD3, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells, as shown in Figure 23, the expression of MSLN-C8, MSLN-C8A, MSLN-
  • the proportion of memory stem cell-like T cells (TSCM) in the CD3 cells of C8B, MSLN-S1A or MSLN-S1A-C8A was significantly higher than that of the MSLN-CAR control group.
  • Example 22 The combination of CD258 protein and SIRT1 protein enhances the killing ability of CAR-T cells on tumor cells.
  • T cells expressing GPC3-S1A or GPC3-S1A-C8A and Huh7 cells purchased from the Chinese Academy of Sciences Cell Bank
  • Xvivo15 medium obtained from the Chinese Academy of Sciences Cell Bank
  • MCE company crystal violet
  • T cells expressing GPC3-CAR or GPC3-S1A-C8A and Huh7 cells purchased from the Cell Bank of the Chinese Academy of Sciences
  • Xvivo15 medium obtained from the Cell Bank of the Chinese Academy of Sciences
  • crystal violet MCE Company
  • Example 23 The combination of CD258 protein and SIRT1 protein enhances the anti-tumor effect of CAR-T cells.
  • mice purchased from Biocytogen
  • Huh7 cells (1 ⁇ 10 7 cells/mouse).
  • the tumor volume of the mice was measured to be about 200 mm 3 .
  • the mice were divided into 5 groups, namely T, GPC3-CAR and GPC3-S1A-C8A groups, with 6-8 mice in each group.
  • T cells (9 ⁇ 10 5 cells/body) were injected into the T cell group through the tail vein
  • CAR-T cells (3 ⁇ 10 5 cells/body or 9 ⁇ 10 5 cells) expressing GPC3-CAR were injected into the GPC3-CAR group through the tail vein.
  • CAR-T cells expressing GPC3-S1A-C8A (3 ⁇ 10 5 or 9 ⁇ 10 5 /only) were injected into the GPC3-S1A-C8A group.
  • the tumor volume was measured on Monday and Thursday every week, and the death of mice was recorded.
  • the results are shown in Figure 25, the tumor-inhibiting effect of the GPC3-S1A-C8A group was significantly higher than that of the other control groups.
  • Example 24 The combination of CD258 protein and SIRT1 protein promotes the proliferation of granulocytes and monocytes in vivo.
  • mice purchased from Biocytogen
  • Huh7 cells (1 ⁇ 10 7 cells/mouse).
  • the tumor volume of the mice was measured to be about 200 mm 3 .
  • the mice were divided into 5 groups, namely T, GPC3-CAR and GPC3-S1A-C8A groups, with 6 mice in each group.
  • T cells (9 ⁇ 10 5 cells/body) were injected into the T cell group through the tail vein
  • CAR-T cells (3 ⁇ 10 5 cells/body or 9 ⁇ 10 5 cells) expressing GPC3-CAR were injected into the GPC3-CAR group through the tail vein.
  • GPC3-S1A-C8A group was injected with CAR-T cells expressing GPC3-S1A-C8A (3 ⁇ 10 5 or 9 ⁇ 10 5 per mouse), and the mice were collected from the tail on the 7th day. 50 ⁇ l of blood was used to detect the number and size of monocytes and neutrophils in each group by BD flow cytometry. The results are shown in Figure 26. The number and size of monocytes and neutrophils in the GPC3-S1A-C8A group were much higher than those in the GPC3-CAR control group.
  • Example 25 The combination of CD258 protein and SIRT1 protein promotes the proliferation of CAR-T cells in vivo and the release of Th1 cytokines.
  • mice purchased from Biocytogen
  • Huh7 cells (1 ⁇ 10 7 cells/mouse).
  • the tumor volume of the mice was measured to be about 200 mm 3 .
  • the mice were divided into 5 groups, namely T, GPC3-CAR and GPC3-S1A-C8A groups, with 6 mice in each group.
  • T cells (9 ⁇ 10 5 cells/body) were injected into the T cell group through the tail vein
  • CAR-T cells (3 ⁇ 10 5 cells/body or 9 ⁇ 10 5 cells) expressing GPC3-CAR were injected into the GPC3-CAR group through the tail vein.
  • GPC3-S1A-C8A group was injected with CAR-T cells expressing GPC3-S1A-C8A (3 ⁇ 10 5 or 9 ⁇ 10 5 per mouse), and the mice were collected from the tail on the 14th day 50 ⁇ l of blood was used to detect the expression of human CD3, CD4 and CD8 protein in each group by BD flow cytometry.
  • the results are shown in Figure 27A, Figure 27B and Figure 27C, in the group expressing CD3, CD4 and CD8, the proportion of cells expressing CD3, CD4 or CD8 protein in the GPC3-S1A-C8A group was much higher than that of the total number of cells GPC3-CAR control group.
  • mice purchased from Biocytogen
  • Huh7 cells (1 ⁇ 10 7 cells/mouse).
  • the tumor volume of the mice was measured to be about 200 mm 3 .
  • the mice were divided into 5 groups, namely T, GPC3-CAR and GPC3-S1A-C8A groups, with 6 mice in each group.
  • T cells (9 ⁇ 10 5 cells/body) were injected into the T cell group through the tail vein
  • CAR-T cells (3 ⁇ 10 5 cells/body or 9 ⁇ 10 5 cells) expressing GPC3-CAR were injected into the GPC3-CAR group through the tail vein.
  • GPC3-S1A-C8A group was injected with CAR-T cells expressing GPC3-S1A-C8A (3 ⁇ 10 5 or 9 ⁇ 10 5 per mouse), and the mice were collected from the tail on the 7th day. 50 ⁇ l of blood was used to detect the expression of human IL-2, IL4, IL6, IL10, TNF- ⁇ and IFN- ⁇ cytokines in each group by BD flow cytometry. Results As shown in Figure 27D, the expression of IFN- ⁇ cytokine in the GPC3-S1A-C8A group was significantly higher than that in the GPC3-CAR control group.
  • SEQ ID NO.1 Leader sequence
  • SEQ ID NO.2 Nucleotide sequence of GPC3 scFv
  • the underline marks are CDR1 ( RSSQSLVHSNGNTYLH , SEQ ID NO:36), CDR2 ( KVSNRFS , SEQ ID NO:37) and CDR3 (SQNTHVPPT, SEQ ID NO:38);
  • the underline marks are CDR1(DYEMH, SEQ ID NO:40), CDR2(ALDPKTGDTAYSQKFKG, SEQ ID NO:41) and CDR3(FYSYTYW, SEQ ID NO:42))
  • SEQ ID NO.3 Nucleotide sequence of CD19scFv
  • the underline marks are CDR1 (RASQDISKYLN, SEQ ID NO:45), CDR2 (HTSRLHS, SEQ ID NO:46) and CDR3 (QQGNTLPYT, SEQ ID NO:47);
  • the underline marks are CDR1 (LPDYGVS, SEQ ID NO:49), CDR2 (VIWGSETTYYNSALKS, SEQ ID NO:50) and CDR3 (HYYYGGSYAMDYW, SEQ ID NO:51))
  • SEQ ID NO.4 Nucleotide sequence of BCMA scFv
  • BCMA scFv amino acid sequence SEQ ID NO:52
  • the underline marks are CDR1 (RASESVSVIGAHLIH, SEQ ID NO:54), CDR2 (LASNLET, SEQ ID NO:55) and CDR3 (LQSRIFPRT, SEQ ID NO:56);
  • the underline marks are CDR1(DYSIN, SEQ ID NO:58), CDR2(WINTETREPAYAYDFRG SEQ ID NO:59) and CDR3(DYSYAMDYW, SEQ ID NO:60))
  • SEQ ID NO.5 Nucleotide sequence of MSLN scFv
  • the underline marks are CDR1 (TLRSGINVGPYRIY, SEQ ID NO:63), CDR2 (YKSDSDKQQGS, SEQ ID NO:64) and CDR3 (MIWHSSAAV, SEQ ID NO:65);
  • VH SEQ ID NO:66
  • the underline marks are CDR1 (SNSATWN, SEQ ID NO:67), CDR2 (RTYYRSKWYNDYAVSVKS, SEQ ID NO:68) and CDR3 (GMMTYYYGMDVW, SEQ ID NO:69))
  • SEQ ID NO.6 Nucleotide sequence of HER2 scFv
  • HER2 scFv amino acid sequence SEQ ID NO: 70
  • the underline marks are CDR1 (KASQDVYNAVA, SEQ ID NO:72), CDR2 (SASSRYT, SEQ ID NO:73) and CDR3 (QQHFRTPFT, SEQ ID NO:74);
  • the underline marks are CDR1 (NYGMN, SEQ ID NO:76), CDR2 (WINTSTGESTFADDFKG, SEQ ID NO:77) and CDR3 (WEVYHGYVPYW, SEQ ID NO:78))
  • SEQ ID NO.7 Nucleotide sequence of CD8 hinge region
  • SEQ ID NO.8 Nucleotide sequence of CD8 transmembrane region
  • SEQ ID NO.9 Nucleotide sequence of CD28 co-stimulatory domain
  • SEQ ID NO.10 Nucleotide sequence of the co-stimulatory domain of 4-1BB
  • Amino acid sequence of the co-stimulatory domain of 4-1BB SEQ ID NO: 82
  • SEQ ID NO.11 Nucleotide sequence of the signaling domain of CD3 ⁇
  • Amino acid sequence of the signaling domain of CD3 ⁇ SEQ ID NO: 83
  • SEQ ID NO.12 Nucleotide sequence of T2A
  • SEQ ID NO.13 Nucleotide sequence of P2A
  • SEQ ID NO.14 Nucleotide sequence of IRES
  • SEQ ID NO.15 Nucleotide sequence of S1
  • SEQ ID NO.16 Nucleotide sequence of S2
  • SEQ ID NO.17 Nucleotide sequence of S3
  • SEQ ID NO.18 Nucleotide sequence of S4
  • SEQ ID NO.19 Nucleotide sequence of S5
  • SEQ ID NO.20 Nucleotide sequence of S6
  • SEQ ID NO.21 Nucleotide sequence of S7
  • SEQ ID NO.22 Nucleotide sequence of S1A
  • SEQ ID NO.23 Nucleotide sequence of S1B
  • SEQ ID NO.24 Nucleotide sequence of S1B1
  • SEQ ID NO.25 Nucleotide sequence of S1C
  • SEQ ID NO.26 Nucleotide sequence of S1C1
  • SEQ ID NO.27 Nucleotide sequence of S1D
  • SEQ ID NO.28 Nucleotide sequence of S1D1
  • SEQ ID NO.29 Nucleotide sequence of S1E
  • SEQ ID NO.30 Nucleotide sequence of S1E1
  • SEQ ID NO.31 Nucleotide sequence of C8
  • SEQ ID NO.32 Nucleotide sequence of C8A
  • SEQ ID NO.33 Nucleotide sequence of C8B
  • SIRT1 protein nuclear localization signal SEQ ID NO: 105
  • SIRT1 protein nuclear localization signal SEQ ID NO: 106
  • SIRT1 protein nuclear localization signal mutant SEQ ID NO:107
  • SIRT1 protein nuclear localization signal mutant SEQ ID NO: 108
  • SIRT1 protein nuclear export nuclear signal mutant SEQ ID NO:111
  • SIRT1 protein nuclear export nuclear signal mutant SEQ ID NO:112

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Epidemiology (AREA)

Abstract

一种SIRT1-7蛋白或CD258蛋白与SIRT1-7蛋白的组合用于促进免疫细胞增殖的应用。

Description

SIRT1-7蛋白在免疫治疗中的应用 技术领域
本发明涉及免疫学领域。具体地，本发明涉及SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体与SIRT1-7蛋白或其功能突变体的一种或多种的组合在免疫治疗中的应用。
背景技术
嵌合抗原受体(Chimeric antigen receptor,CAR)T细胞疗法是近年来发展十分迅速的新一代肿瘤免疫治疗技术。CAR的结构主要由特异性识别肿瘤相关抗原((Tumor associated antigen,TAA)的单链可变区结构域(Single chain variable fragment,scFv)、铰链区、跨膜区和胞内信号传导区四个部分构成。scFv的形式一般为单克隆抗体的重链可变区(VH)与轻链可变区(VL)通过多肽接头连接起来构成的抗原结合区，即VH-Linker-VL或VL-Linker-VH；铰链区通常由免疫球蛋白超家族组成，如IgG4、CD8、IgG1等；跨膜区一般由CD8、CD28或CD4等组成；胞内信号传导区主要由CD3ζ链和协同共刺激信号分子41BB、CD28、ICOS、OX40等组成。因此表达CAR的T细胞不需要通过抗原递呈机制，可直接识别TAA，具有MHC(Major histocompatibility complex)非依赖性的独特优势。
由于CAR-T技术在血液肿瘤治疗上表现出色，以CD19为靶点，CART治疗急性和慢性B淋巴细胞白血病更是取得惊人疗效，但对实体瘤的治疗临床疗效十分有限，亟需需新的解决方案。
Sirtuins是一类依赖于NAD+和核心区域高度保守的蛋白去乙酰化酶和ADP核糖基转移酶，在组蛋白的乙酰化/去乙酰化基因表达调控中起重要作用。哺乳动物中主要有七种Sirtuins蛋白亚型，即SIRT1蛋白、SIRT2蛋白、SIRT3蛋白、SIRT4蛋白、SIRT5蛋白、SIRT6蛋白、SIRT7蛋白，有时以“SIRT1-7”表示7种SIRT蛋白。SIRT1蛋白、SIRT6蛋白和SIRT7蛋白定位于细胞核，主要调节转录、末端着丝粒染色质结构、衰老以及代谢等生物学过程。此外，SIRT1蛋白在胞浆和线粒体中也存在。SIRT2蛋白主要定位于胞浆，与胞浆的微管蛋白相互作用，是调控细胞凋亡的重要因子之一；SIRT3蛋白、SIRT4蛋 白和SIRT5蛋白定位于线粒体内，主要调节细胞能量代谢过程中关键蛋白的乙酰化修饰，并在细胞氧化磷酸化、三羧酸循环、脂肪酸有氧氧化及氨基酸降解等生物学过程中发挥着重要作用。
CD258蛋白又名肿瘤坏死因子超家族成员14(Tumor necrosisfactor superfamily member 14，TNFSF14)、T细胞上可诱导表达的、与单纯疱疹病毒糖蛋白竞争结合单纯疱疹病毒侵入受体的淋巴毒素同源类似物[Homologous to lymphotoxin，inducible expression，competes with herpes simplex virus(HSV)glycoprotein D for HSV entry mediator(HVEM)，a receptor expressed on T lymphocytes，LIGHT]或HVEM配体(Herpesvirusentry mediator-ligand，HVEM-L)。CD258蛋白主要表达在活化的T细胞、B细胞、自然杀伤(Natural killer，NK)细胞、未成熟的树突状细胞(Immature dendritic cells，im DC)和单核细胞表面，有3种存在形式:全长型包含240个氨基酸残基、相对分子质量29ku的2型跨膜糖蛋白；由204个氨基酸残基组成、缺少跨膜区并仅激活T淋巴细胞的非糖基化蛋白及细胞表面金属酶剪切作用下可溶性的CD258蛋白。CD258蛋白介导的信号通路与炎症性及自身免疫性疾病的发生、发展、移植物抗宿主病、病原体感染以及肿瘤的免疫调节有密切关系。
发明内容
本发明解决的技术问题为SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种在免疫治疗中的应用，具体表现为促进记忆性T细胞的形成，抑制免疫细胞免疫负调控蛋白的表达，增强免疫细胞细胞因子的释放，增强免疫细胞对肿瘤细胞的杀伤能力，以及调动机体自身抗肿瘤免疫反应，解决肿瘤异质性问题，预防肿瘤的复发。
具体地，一方面，本申请提供了一种促进免疫细胞增殖的方法，其包含上调所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量。
一方面，本申请提供了一种促进记忆性免疫细胞产生的方法，其包含上调所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量，从而促进所述免疫细胞分化为记忆性免疫细胞。
一方面，本申请提供了一种抑制免疫细胞分化的方法，其包含上调所述 免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量，从而抑制所述免疫细胞分化为分化型免疫细胞。
一方面，本申请提供了一种抑制免疫细胞免疫负调控蛋白表达的方法，其包含上调所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量，从而抑制所述免疫细胞免疫负调控蛋白表达。在某些实施方式中，所述免疫负调控蛋白选自PD1、PDL1、TIM3和LAG3组成的组。
一方面，本申请提供了一种增强免疫细胞释放细胞因子的方法，其包含上调所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量上调。
在某些实施方式中，所述细胞因子选自白介素、干扰素和/或肿瘤坏死因子。在某些实施方式中，所述细胞因子选自IL-2、IL4、IL6、IL7、IL10、IL12、TNF-α和/或IFNγ。
一方面，本申请提供了一种增强免疫细胞对肿瘤杀伤能力的方法，其包含上调所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白蛋白或其功能突变体、SIRT1-7蛋白或其功能突变体的联合、以及SIRT1-7蛋白或其功能突变体与CD258蛋白或其功能突变体的联合的表达量上调。
一方面，本申请提供了一种解决肿瘤异质性的方法，所述方法包含：向受试者施用免疫细胞，其中所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量上调。
一方面，本申请提供了一种预防受试者中肿瘤复发的方法，所述方法包含：向受试者施用免疫细胞，其中所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量上调。
一方面，本申请提供了一种治疗有需要的受试者中的肿瘤的方法，其包含以下步骤：向所述受试者施用免疫细胞，其中所述免疫细胞中SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的表达量上调。
在某些实施方式中，所述肿瘤选自肝癌、肺癌、白血病和间皮瘤。
在某些实施方式中，所述方法包括体内方法和体外方法。
在本发明的一个或多个实施方式中，所述SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体表达量的上调。
在发明的一个或多个实施方式中，所述免疫细胞为淋巴细胞。在某些实施方式中，所述免疫细胞为T细胞、B细胞、自然杀伤(Natural killer，NK)细胞、未成熟的树突状细胞(Immature dendritic cells，im DC)、单核细胞和巨噬细胞。在某些实施方式中，所述T细胞选自记忆性干细胞样T细胞(TSCM)和/或中央记忆性T细胞(TCM)。在某些实施方式中，所述TSCM为CCR7 +和/或CD62L +。在某些实施方式中，所述TSCM还具有选自下组的一种或多种性质：CD45RA +或CD45RA -、CD45RO +或CD45RO -、CD27 +、CD28 +、CD127 +、CD122 +、CD95 +、CD3 +、CD4 +和CD8 +。
在本申请中，术语“记忆性免疫细胞”通常是指具备免疫记忆的免疫细胞。所述免疫记忆可以指对某一抗原产生特异性识别及应答后，再次遇到同一抗原能够产生快速和强烈的免疫应答。在本申请中，所述记忆性免疫细胞可以包括记忆型T细胞。所述记忆型T细胞可以分为记忆性干细胞样T细胞(TSCM)和中央记忆性T细胞(TCM)。
在本申请中，术语“分化型免疫细胞”通常是指具有一定分化程度的免疫细胞。例如，所述分化型免疫细胞可以为具备一定分化程度的T细胞。在本申请中，所述分化型免疫细胞可以通过培养所述免疫细胞使其分化至一定程度而获得。例如，所述分化型免疫细胞可以包含调节性T细胞(Treg)。
在本申请中，术语“调节性T细胞”(Regulatory T cell，Treg)通常是指一群具有负调节机体免疫反应的淋巴细胞。所述调节性T细胞的分子标记可以为种转录因子Foxp3 +或CD127 -。在本申请中，所述调节性T细胞可被分为自然存在和诱导产生两类。其中自然存在为CD4 +CD25 +细胞，诱导产生为TR1细胞和TH3细胞。
在本申请中，术语“记忆性干细胞样T细胞”(T memory stem cells,TSCM)通常是指通常是指处于记忆性T细胞早期分化阶段，具有干细胞特征，具有较强的多向分化潜能的细胞。TSCM细胞在应答抗原刺激后，能够分化为中央记忆性T细胞(Central memory T cells,TCM)、效应记忆性T细胞(Effector memory T cells,TEM)和效应性T细胞(Effector T cells,TEF)。
在本申请中，术语“中央记忆性T细胞”(Central memory T cells,TCM)通常是指是幼稚T细胞(Naive T Cell)经过抗原激活后，产生的具有长期记忆性的T细胞。所述TCM的生物标记可以包含CD62L +和CD45RO +。所述中央记忆性T细胞能够通过淋巴屏蔽，回归淋巴结，同时处于被抗原激活的状 态。
在某些实施方式中，所述免疫细胞选自经遗传修饰的免疫细胞，且所述经遗传修饰的免疫细胞表达嵌合抗原受体(CAR)或T细胞受体(TCR)。在某些实施方式中，所述经遗传修饰的免疫细胞为经遗传修饰的T细胞。
在某些实施方式中，所述TCR包含选自下组的亚基：TCRα、TCRβ、TCRγ和TCRδ。
在某些实施方式中，所述TCR的亚基包括特异性结合和/或识别肿瘤抗原的胞外域可变区。在某些实施方式中，胞外域可变区选自以下组：TCRα可变区片段Vα、TCRα可变区片段Jα、TCRβ可变区片段Vβ、TCRβ可变区片段Dβ和TCRβ可变区片段Jβ。
在某些实施方式中，所述胞外域可变区特异性结合和/或识别选自下组的靶标：MAGEA家族成员、CTA家族成员、HPV病毒和酪氨酸酶。
在某些实施方式中，所述胞外域可变区特异性结合和/或识别选自下组的靶标：MAGEA3、MAGEA4、NY-ESO-1、MART1、HPV16-E6和黑素瘤抗原酪氨酸酶。
在某些实施方式中，所述CAR包含细胞内结构域，所述细胞内结构域包括信号传导结构域和/或共刺激结构域。
在某些实施方式中，所述信号传导结构域选自以下各项组成的组：CD3ζ的信号传导结构域(优选核苷酸序列如SEQ ID NO:11所示，氨基酸序列如SEQ ID NO:83所示)、CD3δ的信号传导结构域和CD3ε的信号传导结构域。在某些实施方式中，所述信号传导结构域包含SEQ ID NO:83所示的序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，所述同源序列仍具备SEQ ID NO:83所示序列功能，或由其组成。
术语“信号传导结构域”通常是指是指选自CD3ζ、CD3γ、CD3δ、CD3ε、FcRγ(FCER1G)、FcRβ(Fc Epsilon R1b)、CD79a、CD79b、FcγRIIa、DAP10和DAP12的蛋白质的功能性信号传导结构域。在本申请中，所述信号传导结构域可以包括：CD3ζ、CD3δ和CD3ε。
在某些实施方式中，所述共刺激结构域选自以下各项组成的组：CD27的共刺激结构域、CD28的共刺激结构域(优选核苷酸序列如SEQ ID NO:9所示，氨基酸序列如SEQ ID NO:81所示)和4-1BB的共刺激结构域(优选核苷酸序 列如SEQ ID NO:10所示，氨基酸序列如SEQ ID NO:82所示)。在某些实施方式中，所述共刺激结构域包含下述任一项所示的序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，或由其组成：SEQ ID NO:81和SEQ ID NO:82所述同源序列仍具备SEQ ID NO:81或82所示序列功能。
术语“共刺激结构域”通常是指选自以下一个或多个的蛋白质的功能性信号传导结构域:CD27、CD28、4-1BB(CD137)、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3、特异结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46和NKG2D。在本申请中，所述共刺激结构域可以包括：CD27、CD28和4-1BB。
在某些实施方式中，所述CAR包含铰链区。在某些实施方式中，所述铰链区选自以下各项组成的组：IgG4的铰链区、IgG1的铰链区和CD8的铰链区(优选核苷酸序列如SEQ ID NO:7所示，氨基酸序列如SEQ ID NO:79所示)。在某些实施方式中，所述铰链区包含SEQ ID NO:79所示的序列或由其组成。
术语“铰链区”通常是指免疫球蛋白重链CHl和CH2功能区之间的区域。铰链区为位于scFv和T细胞膜之间的一段区域。铰链区通常来源于IgG家族，例如，可来源于IgG1和IgG4，还可以来源于IgD和CD8。在本申请中，铰链区可以包含选自下组的部分：IgG4的铰链区、IgG1的铰链区和CD8的铰链区。
在某些实施方式中，所述CAR包含跨膜区。在某些实施方式中，所述跨 膜区选自以下各种组成的组：CD8的跨膜区(优选核苷酸序列如SEQ ID NO:8所示，氨基酸序列如SEQ ID NO:80所示)、CD28的跨膜区和CD24的跨膜区。在某些实施方式中，所述跨膜区包含SEQ ID NO:8所示的序列或由其组成。
术语“跨膜区”通常是指跨膜区连接胞外抗原结合域和胞内信号域，一般由二聚体膜蛋白组成，主要包括CD3ζ、CD4、CD8、CD28等，能将CAR结构锚定于T细胞膜上。跨膜区不同的设计能影响导入的CAR基因的表达。在本申请中，所述跨膜区可以包含选自下组的部分：CD8的跨膜区、CD28的跨膜区和CD24的跨膜区。
在某些实施方式中，所述CAR包含靶向部分。在某些实施方式中，所述靶向部分为scFv。优选地，所述靶向部分选自针对GPC3的scFv，针对CD19的scFv，针对BCMA的scFv，针对MSLN的scFv，针对HER2的scFv。
在一些实施方案中，所述针对GPC3的scFv包含SEQ ID NO:35所示轻链可变区包含的LCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:36所示)，LCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:37所示)和LCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:38所示)和包含SEQ ID NO:39所示的重链可变区包含的HCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:40所示)，HCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:41所示)和HCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:42所示)，更优选地，针对GPC3的scFv包含如SEQ ID NO:2所示的核苷酸序列或如SEQ ID NO:34所示的氨基酸序列，或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:2或34所示序列功能。
在一些实施方案中，所述针对CD19的scFv包含SEQ ID NO:44所示轻链可变区包含的LCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:45所示)，LCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:46所示)和LCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:47所示)和包含SEQ ID NO:48所示的重链可变区包含的HCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:49所示)，HCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:50所示)和HCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:51所示)，更 优选地，针对CD19的scFv包含如SEQ ID NO:3所示的核苷酸序列或如SEQ ID NO:43所示的氨基酸序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:3或43所示序列功能。
在一些实施方案中，所述针对BCMA的scFv包含SEQ ID NO:53所示轻链可变区包含的LCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:54所示)，LCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:55所示)和LCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:56所示)和包含SEQ ID NO:57所示的重链可变区包含的HCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:58所示)，HCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:59所示)和HCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:60所示)，更优选地，针对BCMA的scFv包含的如SEQ ID NO:4所示核苷酸序列或如SEQ ID NO:52所示的氨基酸序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:4或52所示序列功能。
在一些实施方案中，所述针对MSLN的scFv包含SEQ ID NO:62所示轻链可变区包含的LCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:63所示)，LCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:64所示)和LCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:65所示)和包含SEQ ID NO:66所示的重链可变区包含的HCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:67所示)，HCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:68所示)和HCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:69所示)，更优选地，针对MSLN的scFv包含如SEQ ID NO:5所示的核苷酸序列或如SEQ ID NO:61所示的氨基酸序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:5或61所示序列功能。
在一些实施方案中，所述针对HER2的scFv包含SEQ ID NO:71所示轻链可变区包含的LCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:72所示)， LCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:73所示)和LCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:74所示)和包含SEQ ID NO:75所示的重链可变区包含的HCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:76所示)，HCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:77所示)和HCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:78所示)，更优选地，针对HER2的scFv包含如SEQ ID NO:6所示的核苷酸序列或如SEQ ID NO:70所示的氨基酸序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:6或70所示序列功能。
在某些实施方式中，所述靶向部分特异性结合和/或识别肿瘤抗原。在某些实施方式中，所述靶向部分特异性结合和/或识别选自下组的靶标：B淋巴细胞表面抗原、TNF家族成员、HER家族成员和GPC家族成员。在某些实施方式中，所述靶向部分特异性结合和/或识别选自下组的靶标：CD19、BCMA、HER2、Mesothelin和GPC3。在某些实施方式中，所述靶向部分包含下述任一项所示的序列：SEQ ID NO:2、3、4、5、6、34、43、52、61或70或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:2、3、4、5、6、34、43、52、61或70所示序列功能。
术语“肿瘤抗原”通常是指是指在肿瘤细胞内或由肿瘤细胞产生的抗原物质，其可能具有在宿主中触发免疫应答的能力。例如，肿瘤抗原可以是构成肿瘤细胞的一部分并且能够诱导肿瘤特异性细胞毒性T淋巴细胞的蛋白质，多肽，肽或其片段。肿瘤抗原肽可以是由于肿瘤细胞中的肿瘤抗原降解而产生的肽，并且可以通过结合HLA分子在细胞表面上表达后诱导或激活肿瘤特异性细胞毒性T淋巴细胞。在一些实施方案中，术语“肿瘤抗原”还可以指在癌细胞上专门或优先或差异表达和/或与癌细胞相关的生物分子(例如，蛋白质，碳水化合物，糖蛋白等)从而提供癌症优先或特异性的靶标。例如，与生物体中的任何其他细胞相比，优先表达可以为常规的优先表达，或者在生物体的特定区域内的优先表达(例如在特定器官或组织内)。例如，所述肿瘤抗原可以包括TSHR、CD19、CD123、CD138、CD22、CD30、CD171、CS-1、CLL-1、 CD33、EGFRvIII、GD2、GD3、BCMA、TnAg、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-11Ra、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、叶酸受体α、ERBB2(Her2/neu)、MUC1、EGFR、NCAM、Prostase、PAP、ELF2M、肝配蛋白B2、I GF-I受体、CAIX、LMP2、gp100、bcr-abl、酪氨酸酶、EphA2、岩藻糖基GM1、sLe、GM3、TGS5、HMWMAA、邻乙酰基-GD2、叶酸受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD179a、ALK、聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-1a、MAGE-A1、legumain、HPVE6、E7、MAGE A1、ETV6-AML、精子蛋白17、XAGE1、ie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、prostein、存活蛋白和端粒酶、PCTA-1/Galectin 8、MelanA/MART1、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、雄激素受体、细胞周期蛋白B1、MYCN、RhoC、TRP-2、CYP1B1、BORIS、SART3、PAX5、OY-TES1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠羧基酯酶、mut hsp702、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5和IGLL1。
在某些实施方式中，所述方法还包括以下步骤：分离获得外周血单核细胞PBMC、CD3 +T淋巴细胞、CD8 +T淋巴细胞、CD4 +T淋巴细胞或调节T细胞。
在某些实施方式中，所述方法还包括：向经分离的所述PBMC中加入一种或多种T细胞刺激因子。在某些实施方式中，所述T细胞刺激因子选自以下各项组成的组：针对B淋巴细胞表面抗原的抗体、抗TNF抗体、细胞内聚酯和抗生素。在某些实施方式中，所述T细胞刺激因子选自以下各项组成的组：抗CD3抗体、抗CD28抗体、抗4-1BB抗体、抗CD80抗体、抗CD86抗体、PHA、PMA和离子霉素。
在某些实施方式中，所述T细胞刺激因子为抗CD3抗体，且所述抗CD3抗体的浓度为1-10000ng/mL。在某些实施方式中，所述T细胞刺激因子为抗CD28抗体，且所述抗CD28抗体的浓度为1-10000ng/mL。
在某些实施方式中，所述方法还包括：向经分离的所述PBMC中加入一 种或多种细胞因子。
在某些实施方式中，所述细胞因子为白细胞介素。
术语“白细胞介素”通常是指能够促进T和/或B淋巴细胞和/或造血细胞的发育和分化的分泌蛋白或信号分子。白介素可以由辅助CD4T淋巴细胞，以及通过单核细胞，巨噬细胞和内皮细胞合成。如本文所用，白介素(IL)可以包括IL-1，IL-2，IL-3，IL-4，IL-5，IL-6，IL-7，IL-8，IL-9，IL-10，IL-11，IL-12，IL-13，IL-14，IL-15，IL-16，IL-17，IL-18，IL-19，IL-20，IL-21，IL-22，IL-23，IL-24，IL-25，IL-26，IL-27，IL-28，IL-29，IL-30，IL-31，IL-32，IL-33，IL-34，IL-35和/或IL-36。如本文所用，术语“白细胞介素”可以包括全长白介素或片段(例如截短形式)或其变体，其基本上保持相应野生型白细胞介素的生物学活性(例如，具有生物活性至少80％，至少85％、至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％，或甚至100％的相应野生型白细胞介素的生物活性)。本文所用的白细胞介素可以来自任何哺乳动物物种。在某些实施方式中，白细胞介素来自选自人、马、牛、鼠、猪、兔、猫、狗、大鼠、山羊、绵羊和非人灵长类动物的物种。在某些实施方式中，白细胞介素可以是突变形式。例如，白细胞介素可以是超IL-2(也称为sIL2，参见Nature 484,529-533,2612)，其可以通过修饰IL-2以增加其对IL-2Rβ的结合亲和力而获得。sIL-2中的突变主要是细胞因子的核心，分子动力学模拟表明，进化型突变使IL-2稳定，将IL-2Rβ结合位点中螺旋的灵活性降低到类似于优化的受体结合构象当绑定到CD25。与IL-2相比，sIL-2诱导细胞毒性T细胞的优异扩增，导致体内抗肿瘤反应改善，引起T调节细胞扩张少，肺水肿减少。例如，在本申请中，所述细胞因子可以包含IL-2、IL4、IL6、IL7、IL10、IL21、TNF-α和/或IFNγ。
在某些实施方式中，所述白细胞介素选自下组的一种或多种：IL2、IL21、IL7和IL15。在某些实施方式中，所述白细胞介素为IL2，且所述IL2的浓度为0.1-10000U/mL。在某些实施方式中，所述白细胞介素为IL21，且所述IL21的浓度为0.01-1000ng/mL。在某些实施方式中，所述白细胞介素为IL7，且所述IL7的浓度为0.01-1000ng/mL。在某些实施方式中，所述白细胞介素为IL15，且所述IL15的浓度为0.01-1000ng/mL。
在某些实施方式中，所述SIRT1-7或其功能突变体、CD258或其功能突 变体来源于人。
SIRT1蛋白有2条核定位信号(SEQ ID NO:105所示的PLRKRPRR和SEQ ID NO:106所示的PPKRKKRK)，优选的突变为任一氨基酸置换为A或缺失，更优选的突变体为SEQ ID NO:107所示的PLRKRPAA和SEQ ID NO:108所示的PPKRAAAA)；优选的缺失突变为SEQ ID NO:105所示的PLRKRPRR和SEQ ID NO:106所示的PPKRKKRK的完全缺失；SIRT1蛋白有2条核出核信号(SEQ ID NO:109所示的LLLTDGLL和SEQ ID NO:110所示的VDLLIVI)，优选的突变体为任一氨基酸置换为A或缺失，更优选的突变体为SEQ ID NO:111所示的AAATDGAA和SEQ ID NO:112所示的ADAAAAA(参见THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL.282,NO.9,pp.6823–6832)，优选的缺失突变为SEQ ID NO:109所示的LLLTDGLL和SEQ ID NO:110所示的VDLLIVI的完全缺失。通过对核定位信号和核出核信号的改变，调节蛋白在细胞质以及细胞核的表达量，进而调节蛋白的功能活性。
SIRT1序列为原始序列(UniProtKB编号：Q96EB6-1)截短体，由原始序列的小分子Sirtuin激活剂结合区(Small molecule sirtuin-activating compounds binding domain，SBD，原始序列第183-229位)，去乙酰化酶区(Deacetylase domain，原始序列第229-516位)和C端调控区(C-terminal regulatory segment，CTR，原始序列第641-665位)依次拼接构成；SIRT2-7为原始序列，参见 https://www.uniprot.org/uniprot/Q96EB6。
在某些实施方式中，所述SIRT1蛋白、SIRT2蛋白、SIRT3蛋白、SIRT4蛋白、SIRT5蛋白、SIRT6蛋白、SIRT7蛋白分别包含下述任一项所示的序列：SEQ ID NO:15-21或SEQ ID NO:86-92，或与其具备至少80％、至少85％、至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:15-21或SEQ ID NO:86-92任一项所示序列功能。
在某些实施方式中，所述SIRT1-7蛋白的功能性突变体在所述SIRT1-7蛋白的选自下组的结构域进行突变：去乙酰化酶区(Deacetylase domain)，小分子Sirtuin激活剂结合区(Small molecule sirtuin-activating compounds binding domain，SBD)，和C端调控区(C-terminal regulatory segment，CTR)。
在某些实施方式中，所述SIRT1功能突变体包含下述任一项所示的序列：SEQ ID NO:22-30或SEQ ID NO:93-101或与其具备至少80％、至少85％、至 少90％、至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:22-30或SEQ ID NO:93-101任一项所示序列功能。
在某些实施方式中，所述CD258蛋白包含SEQ ID NO:31或SEQ ID NO:102所示的序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:31或SEQ ID NO:102所示序列功能。所述CD258蛋白功能突变体包括膜结合型CD258(优选序列如SEQ ID NO:32或SEQ ID NO:103所示)、分泌型CD258(优选序列如SEQ ID NO:33或SEQ ID NO:104所示)和CD258的胞内区。在某些实施方式中所述膜结合型CD258包括所述CD258蛋白水解位点(Proteolytic site)的缺失或定点突变。蛋白水解位点为SEQ ID NO:102所示序列的第82-83位的QL位点，优选地，所述缺失突变为CD258蛋白水解位点的完全缺失或在SEQ ID NO:102所示序列中第81-84位的EQLI缺失；所述置换突变为Q和/或L氨基酸置换为A，更优选地，QL氨基酸被置换为AA。通过对该蛋白水解位点进行置换突变或缺失，使该蛋白始终以膜结合形式表达在细胞膜上，减少分泌形式的表达(参见 https://www.uniprot.org/uniprot/O43557)。
在某些实施方式中，所述SIRT1-7蛋白或其功能突变体、CD258蛋白或其功能突变体包含下述任一项所示的序列：SEQ ID NO:15-33或与其具备至少80％、至少85％、至少90％、至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％同源性的序列，所述同源序列仍具备SEQ ID NO:15-33任一项所示序列功能，优选包含下述任一项所示的序列：SEQ ID NO:86-104或与其具备至少80％、至少85％、至少90％、至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％同源性的序列，所述同源序列仍具备SEQ ID NO:86-104任一项所示序列功能。
另一方面，本申请提供了一种经遗传修饰的免疫细胞，其中所述遗传修饰使得该免疫细胞中SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体、以及其组合的表达量上调。
在某些实施方式中，所述表达量上调通过以下方式实现：
(1)通过向所述免疫细胞中添加增加所述SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体的表达的激活剂以刺激所述免疫细胞自身增加所述SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体的表达，优选所述激活剂选自以下各项组成的组：SRT2104(CAS号1093403-33-8，化学式C 26H 24N 6O 2S 2)；CAY10602(CAS号374922-43-7，化学式C 22H 15FN 4O 2S)；OSS-128167(CAS号887686-02-4，化学式C 19H 14N 2O 6)；
和/或
(2)通过向所述免疫细胞中转染包含编码所述SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体的核酸的表达载体以增加所述免疫细胞中所述SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体的量，优选地，所述SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体、和CD258蛋白或其功能突变体以单体形式存在或通过连接元件连接的缀合物的形式存在。在某些实施方式中，所述载体选自以下组：逆转录病毒载体、慢病毒载体和转座子质粒，优选地，所述载体进一步包含CAR，更优选地，所述CAR通过连接元件连接调节单元，所述调节单元选自所述SIRT1-7蛋白或其功能突变体的一种或多种、CD258蛋白或其功能突变体、或其组合。
术语“2A序列”通常是指一段不依赖于蛋白酶的自剪切氨基酸序列。所述2A序列可以有助于转录产生两种蛋白。
在某些实施方式中，所述连接元件为2A序列，选自下列各项组成的组：T2A(优选核苷酸序列如SEQ ID NO:12所示，氨基酸序列如SEQ ID NO:84所示)、P2A(优选核苷酸序列如SEQ ID NO:13所示，氨基酸序列如SEQ ID NO:85所示)、F2A、E2A和IRES(优选核苷酸序列如SEQ ID NO:14所示)。在某些实施方式中，所述连接元件包含SEQ ID NO:84、85和14所示的序列或由其组成。
另一方面，本申请提供了一种组合物，其包含所述的经遗传修饰的免疫细胞。
在某些实施方式中，所述组合物还包含任选地药学上可接受的载体。
另一方面，本申请提供了所述SIRT1-7蛋白或其功能突变体的一种或多 种、CD258蛋白或其功能突变体、或其组合在制备提高药物预防和/或治疗肿瘤的功效的试剂中的应用，优选地，所述药物为经遗传修饰的免疫细胞(优选CAR-T细胞)。
另一方面，本申请还提供了一种所述经遗传修饰的免疫细胞和/或所述的组合物在制备药物中的用途，其中所述药物用于治疗和/或预防肿瘤。
在某些实施方式中，所述肿瘤选自肝癌、肺癌、白血病和间皮瘤。
在某些实施方式中，所述经遗传修饰的免疫细胞选自淋巴细胞。在某些实施方式中，所述经遗传修饰的免疫细胞表达嵌合抗原受体(CAR)。
在某些实施方式中，所述方法包括分离并激活所述经遗传修饰的免疫细胞的步骤，其中所述激活包括向经分离的所述经遗传修饰的免疫细胞施用T细胞培养基。
在某些实施方式中，所述T细胞培养基选自以下组中的一种或多种：DMEM培养基、1640培养基、MEM培养基、X-VIVO培养基和干细胞培养基。
附图说明
图1为不同CAR结构示意图；
图2为SIRT1蛋白促进肿瘤抗原诱导的特异性CAR-T细胞扩增；
图3为SIRT1蛋白抑制免疫负调控蛋白的表达；
图4为SIRT1蛋白促进CD3细胞中记忆性T细胞的增殖；
图5为SIRT1蛋白促进CD4、CD8细胞中记忆性T细胞的增殖；
图6-8为SIRT1蛋白功能突变体及其组合抑制免疫负调控蛋白的表达；
图9-10为SIRT1蛋白功能突变体及其组合促进CD3细胞中记忆性T细胞的增殖；
图11为SIRT1蛋白功能突变体及其组合促进CD4细胞中记忆性T细胞的增殖；
图12为SIRT1蛋白功能突变体及其组合促进CD8细胞中记忆性T细胞的增殖；
图13为SIRT1蛋白功能突变体及其组合促进T细胞的增殖；
图14为SIRT1蛋白功能突变体及其组合促进T细胞对免疫抑制微环境的抵抗能力；
图15为CD258蛋白及其功能突变体增强CAR-T细胞对肿瘤细胞的杀伤能力；
图16为CD258蛋白与SIRT1蛋白组合促进肿瘤抗原诱导的特异性CAR-T细胞扩增；
图17为CD258蛋白与SIRT1蛋白组合促进CD3细胞中记忆性T细胞的增殖(GPC3)；
图18为CD258蛋白与SIRT1蛋白组合促进CD4、CD8细胞中记忆性T细胞的增殖(GPC3)；
图19为CD258蛋白与SIRT1蛋白组合抑制免疫负调控蛋白的表达(CD19)；
图20为CD258蛋白与SIRT1蛋白组合促进CD8细胞中记忆性T细胞的增殖(CD19)；
图21为CD258蛋白与SIRT1蛋白组合促进CD3、CD4细胞中记忆性T细胞的增殖(CD19)；
图22为CD258蛋白与SIRT1蛋白组合抑制免疫负调控蛋白的表达(MSLN)；
图23为CD258蛋白与SIRT1蛋白组合促进CD3细胞中记忆性T细胞的增殖(MSLN)；
图24为CD258蛋白与SIRT1蛋白组合增强CAR-T细胞对肿瘤细胞的杀伤能力；
图25为CD258蛋白与SIRT1蛋白组合增强CAR-T细胞抗肿瘤效果；
图26为CD258蛋白与SIRT1蛋白组合促进粒细胞和单核细胞体内增殖；
图27为CD258蛋白与SIRT1蛋白组合促进CAR-T细胞体内增殖以及Th1细胞因子的释放。
具体实施方式
以下通过实施例来进一步阐述本发明。但是应该理解，所述实施例只是举例说明的目的，并不意欲限制本发明的范围和精神。
本领域技术人员能够从下文的详细描述中容易地洞察到本发明的其它方面和优势。下文的详细描述中仅显示和描述了本发明的示例性实施方式。如本领域技术人员将认识到的，本发明的内容使得本领域技术人员能够对所公开的 具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
实施例1 慢病毒载体的构建
所述CAR-T以靶向GPC3、CD19、Mesothelin(MSLN)为例，人工合成包含CAR结构的片段，并构建到慢病毒载体(LV100A，System Biosciences公司)，随后依照其说明书记载的方式转染获得慢病毒，分别得到GPC3-CAR、GPC3-S1、GPC3-S2、GPC3-S3、GPC3-S6、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3、GPC3-C8、GPC3-C8A、GPC3-C8B、GPC3-S1A-C8A、CD19-CAR、CD19-S1A、CD19-C8A、CD19-S1A-C8A、MSLN-CAR、MSLN-C8、MSLN-C8A、MSLN-C8B、MSLN-S1A、MSLN-S1A-C8A慢病毒。各CAR结构示意图如图1所示。
GPC3-CAR由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11依次拼接合成。
GPC3-S1由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:15依次拼接合成。
GPC3-S2由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:16依次拼接合成。
GPC3-S3由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:17依次拼接合成。
GPC3-S6由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:20依次拼接合成。
GPC3-S1A由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22依次拼接合成。
GPC3-S1B由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:23依次拼接合成。
GPC3-S1B1由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:24依次拼接合成。
GPC3-S1C由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:25依次拼接合成。
GPC3-S1C1由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:26依次拼接合成。
GPC3-S1D由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:27依次拼接合成。
GPC3-S1D1由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:28依次拼接合成。
GPC3-S1E由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:29依次拼接合成。
GPC3-S1E1由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:30依次拼接合成。
GPC3-S1-S3由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:15、SEQ ID NO:13、SEQ ID NO:17依次拼接合成。
GPC3-S1A-S3由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:13、SEQ ID NO:17依次拼接合成。
GPC3-C8由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID  NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:31依次拼接合成。
GPC3-C8A由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:32依次拼接合成。
GPC3-C8B由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:33依次拼接合成。
GPC3-S1A-C8A由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:13、SEQ ID NO:32依次拼接合成。
CD19-CAR由SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11依次拼接合成。
CD19-S1A由SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22依次拼接合成。
CD19-C8A由SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:32依次拼接合成。
CD19-S1A-C8A由SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:13、SEQ ID NO:32依次拼接合成。
MSLN-CAR由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11依次拼接合成。
MSLN-S1A由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22依次拼接合成。
MSLN-C8由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:31依次拼接合成。
MSLN-C8A由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID  NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:32依次拼接合成。
MSLN-C8B由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:33依次拼接合成。
MSLN-S1A-C8A由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:13、SEQ ID NO:32依次拼接合成。
实施例2：慢病毒感染T细胞。
感染实验按照本领域技术人员已知的常规方法进行。简述感染步骤如下：
1.外周血单核淋巴细胞(PBMC)的获得，通过血液单采系统获得大于1×10 7的细胞。
2.抗人CD3/CD28抗体处理细胞培养皿。
用PBS稀释抗人CD3及抗人CD28抗体(购自上海近岸科技公司)，终浓度为1ug/ml，向细胞培养皿中加入稀释后的抗体混合液，使其铺满培养皿，室温孵育2小时。2小时后用PBS洗一次，备用。
3.对T细胞进行激活处理
将分离的PBMC用T淋巴细胞培养液(Xvivo15培养基+5％FBS+100U/mlIL2+20ng/mlIL21+20ng/mlIL7)进行重悬，使终浓度为1×10 6个细胞/ml，并放入步骤2中处理过的培养皿中培养，培养条件为37℃+5％CO 2，培养时间为24小时。
4.对激活的T细胞进行感染
1)感染试剂配制。
取一定量的T细胞培养液，加入终浓度为1mg/ml的synperonic F108(Sigma公司)，混匀，水浴锅加热至37℃待用。
2)培养板处理。
取1mg/ml抗人CD3抗体及0.5mg/ml抗人CD28抗体按1:1000体积比稀释至适量的PBS缓冲液中，并取retronectin(1mg/ml，Takara公司)试剂，按1:40体积比稀释至该PBS缓冲液中，混匀后均匀铺至细胞皿，室温孵育2小时。2小时后用PBS进行洗涤并待用。
3)慢病毒感染T细胞
用1)中所配感染试剂稀释已激活的T细胞，并按MOI＝3加入慢病毒，并混匀。均匀铺在2)中所处理的培养皿中。
感染后监测细胞密度，使细胞维持在1×10 6个细胞/ml，一般14天，可扩增30-1000倍。
实施例3：SIRT1蛋白促进肿瘤抗原诱导的特异性CAR-T细胞扩增。
将表达GPC3-CAR、GPC3-S2、GPC3-S3或GPC3-S6的T细胞与辐照(X-RAD细胞辐照仪，辐照剂量为30Gy)的HepG2细胞(中科院细胞库购得)按1:1的细胞个数比用Xvivo15培养基进共培养，每4天重新补加辐照的HepG2进行刺激，刺激3次。每次用台盼蓝染色进行计数，细胞增殖情况如图2A所示。结果显示，表达GPC3-S2、GPC3-S3或GPC3-S6的T细胞的扩增倍数远高于对照组表达GPC3-CAR的T细胞。
同样的，将表达GPC3-CAR、GPC3-S2、GPC3-S3或GPC3-S6的T细胞与辐照(X-RAD细胞辐照仪，辐照剂量为30Gy)的Huh7细胞(中科院细胞库购得)按1:1的细胞个数比用Xvivo15培养基进共培养，每4天重新补加辐照的Huh7进行刺激，刺激3次。每次用台盼蓝染色进行计数，细胞增殖情况如图2B所示。结果亦表明，表达GPC3-S2、GPC3-S3或GPC3-S6的T细胞的扩增倍数远高于对照组表达GPC3-CAR的T细胞。
实施例4：SIRT1蛋白抑制免疫负调控蛋白的表达。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S2、GPC3-S3或GPC3-S6的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD4、CD8、PD1、PDL1、TIM3、LAG3蛋白的表达情况，结果如图3所示，表达GPC3-S2、GPC3-S3或GPC3-S6的T细胞中，同时表达两个负调控蛋白的细胞占比均显著低于GPC3-CAR对照组。
实施例5：SIRT1蛋白促进CD3细胞中记忆性T细胞的增殖。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S2、GPC3-S3或GPC3-S6的T细胞，共培养9天或12天。 用BD流式细胞仪检测T细胞中CD3、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图4所示，表达GPC3-S2、GPC3-S3或GPC3-S6的CD3细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
实施例6：SIRT1蛋白促进CD4、CD8细胞中记忆性T细胞的增殖。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S2、GPC3-S3或GPC3-S6的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD4、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图5A所示，表达GPC3-S2、GPC3-S3或GPC3-S6的CD4细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
同样的，于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S2、GPC3-S3或GPC3-S6的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD8、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图5B所示，表达GPC3-S2、GPC3-S3或GPC3-S6的CD8细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例亦均显著高于GPC3-CAR对照组。
实施例7：SIRT1蛋白功能突变体及其组合抑制免疫负调控蛋白的表达。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD4、CD8、PD1、PDL1、TIM3、LAG3蛋白的表达情况，结果如图6-8所示，表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3或GPC3-S1A-S3的T细胞中，同时表达两个负调控蛋白的细胞占比均显著低于GPC3-CAR对照组。
实施例8：SIRT1蛋白功能突变体及其组合促进CD3细胞中记忆性T细胞的 增殖。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图9-10所示，表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的CD3细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
实施例9：SIRT1蛋白功能突变体及其组合促进CD4细胞中记忆性T细胞的增殖。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD4、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图11所示，表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的CD4细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
实施例10：SIRT1蛋白功能突变体及其组合促进CD8细胞中记忆性T细胞的增殖。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD8、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图12所示，表达GPC3-S1、GPC3-S1A、GPC3-S1B、 GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的CD8细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
实施例11：SIRT1蛋白功能突变体及其组合促进T细胞的增殖。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞，共培养11天，每2-3天用台盼蓝染色进行计数传代，细胞增殖情况如图13A所示。结果显示，表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞的扩增倍数远高于对照组表达GPC3-CAR的T细胞。
同样的，于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞，共培养7天，在第7天分别取细胞与Xvivo15培养基共孵育24h后，采用CCK8(MCE公司)检测T细胞增殖效果。结果如图13B显示，表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞的增殖效果亦远高于对照组表达GPC3-CAR的T细胞。
实施例12：SIRT1蛋白功能突变体及其组合促进T细胞对免疫抑制微环境的抵抗能力。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞，共培养8天，在第8天分别取细胞与1640无葡萄糖、无血清培养基(Gibco公司)共孵育24h后，采用CCK8(MCE公司)检测T细胞增殖效果。结果如图14A显示，表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、 GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞的增殖效果远高于对照组表达GPC3-CAR的T细胞。
同样的，于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞，共培养8天，在第8天分别取细胞与1640无葡萄糖、无血清培养基(Gibco公司)以及20mM乳酸(PH值从正常7.4降至6.5，Sigma公司)共孵育24h后，采用CCK8(MCE公司)检测T细胞增殖效果。结果如图14B显示，表达GPC3-S1、GPC3-S1A、GPC3-S1B、GPC3-S1B1、GPC3-S1C、GPC3-S1C1、GPC3-S1D、GPC3-S1D1、GPC3-S1E、GPC3-S1E1、GPC3-S1-S3、GPC3-S1A-S3的T细胞的增殖效果亦远高于对照组表达GPC3-CAR的T细胞。
实施例13：CD258蛋白及其功能突变体增强CAR-T细胞对肿瘤细胞的杀伤能力。
将表达GPC3-CAR、GPC3-C8、GPC3-C8A或GPC3-C8B的T细胞与Huh7细胞(中科院细胞库购得)按1:15的细胞个数比用Xvivo15培养基共培养7天，采用结晶紫染色(MCE公司)检测CAR-T细胞对肿瘤细胞的杀伤效果，结果如图15A所示，表达GPC3-C8或GPC3-C8A的T细胞对肿瘤细胞的杀伤效果远高于对照组表达GPC3-CAR的T细胞，其中表达GPC3-C8A的T细胞相较于表达GPC3-C8的T细胞具有更为优越的杀伤效果。
同样的，将表达GPC3-CAR、GPC3-C8、GPC3-C8A或GPC3-C8B的T细胞与Huh7细胞(中科院细胞库购得)按1:15的细胞个数比用1640无葡萄糖、无血清(Gibco公司)以及20mM乳酸(PH值从正常7.4降至6.5，Sigma公司)培养基共培养7天，采用结晶紫染色(MCE公司)检测CAR-T细胞对肿瘤细胞的杀伤效果，结果如图15B所示，表达GPC3-C8或GPC3-C8A的T细胞对肿瘤细胞的杀伤效果亦显著高于对照组表达GPC3-CAR的T细胞，表达GPC3-C8A的T细胞相较于表达GPC3-C8的T细胞亦具有更为优越的杀伤效果。
实施例14：CD258蛋白与SIRT1蛋白组合促进肿瘤抗原诱导的特异性CAR-T细胞扩增。
将表达GPC3-CAR、GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的T细胞与辐照(X-RAD细胞辐照仪，辐照剂量为30Gy)的Huh7细胞(中科院细胞库购得)按1:1的细胞个数比用Xvivo15培养基共培养，每3-4天重新补加辐照的Huh7进行刺激，刺激4次。每次用台盼蓝染色进行计数，细胞增殖情况如图16所示。结果表明，表达GPC3-S1A、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的T细胞的扩增倍数远高于对照组表达GPC3-CAR的T细胞。
GPC3-S1A-C8A为GPC3、S1A和C8A依次通过2A序列偶联。
实施例15：CD258蛋白与SIRT1蛋白组合促进CD3细胞中记忆性T细胞的增殖(GPC3)。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图17所示，表达GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的CD3细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
实施例16：CD258蛋白与SIRT1蛋白组合促进CD4、CD8细胞中记忆性T细胞的增殖(GPC3)。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD4、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图18A所示，表达GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的CD4细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于GPC3-CAR对照组。
同样的，于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达GPC3-CAR、GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的T细胞，共培养9天或12天。用BD流式细胞仪检测T细 胞中CD8、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图18B所示，表达GPC3-S1A、GPC3-C8、GPC3-C8A、GPC3-C8B或GPC3-S1A-C8A的CD8细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例亦均显著高于GPC3-CAR对照组。
实施例17：CD258蛋白与SIRT1蛋白组合抑制免疫负调控蛋白的表达(CD19)。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达CD19-CAR、CD19-S1A、CD19-C8A或CD19-S1A-C8A的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD4、CD8、PD1、PDL1、TIM3、LAG3蛋白的表达情况，结果如图19所示，表达CD19-S1A、CD19-C8A或CD19-S1A-C8A的T细胞中，同时表达两个负调控蛋白的细胞占比均显著低于CD19-CAR对照组。
实施例18：CD258蛋白与SIRT1蛋白组合促进CD8细胞中记忆性T细胞的增殖(CD19)。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达CD19-CAR、CD19-S1A、CD19-C8A或CD19-S1A-C8A的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD8、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图20所示，表达CD19-S1A、CD19-C8A或CD19-S1A-C8A的CD8细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于CD19-CAR对照组。
实施例19：CD258蛋白与SIRT1蛋白组合促进CD3、CD4细胞中记忆性T细胞的增殖(CD19)。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达CD19-CAR、CD19-S1A、CD19-C8A或CD19-S1A-C8A的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图21A所示，表达CD19-S1A、CD19-C8A或CD19-S1A-C8A的CD3细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于CD19-CAR对照组。
同样的，于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达CD19-CAR、CD19-S1A、CD19-C8A或CD19-S1A-C8A的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD4、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图21B所示，表达CD19-S1A、CD19-C8A或CD19-S1A-C8A的CD4细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例亦均显著高于CD19-CAR对照组。
实施例20：CD258蛋白与SIRT1蛋白组合抑制免疫负调控蛋白的表达(MSLN)。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达MSLN-CAR、MSLN-C8、MSLN-C8A、MSLN-C8B、MSLN-S1A或MSLN-S1A-C8A的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD4、CD8、PD1、PDL1、TIM3、LAG3蛋白的表达情况，结果如图22所示，表达MSLN-C8、MSLN-C8A、MSLN-C8B、MSLN-S1A或MSLN-S1A-C8A的T细胞中，同时表达两个负调控蛋白的细胞占比均显著低于MSLN-CAR对照组。
实施例21：CD258蛋白与SIRT1蛋白组合促进CD3细胞中记忆性T细胞的增殖(MSLN)。
于37℃，5％CO 2细胞培养箱中体外培养实施例2中所得的分别表达MSLN-CAR、MSLN-C8、MSLN-C8A、MSLN-C8B、MSLN-S1A或MSLN-S1A-C8A的T细胞，共培养9天或12天。用BD流式细胞仪检测T细胞中CD3、CD45RO、CD45RA、CD62L、CCR7、CD95、CD122、CD127、CD27、CD28蛋白的表达，如图23所示，表达MSLN-C8、MSLN-C8A、MSLN-C8B、MSLN-S1A或MSLN-S1A-C8A的CD3细胞中记忆性干细胞样T细胞(TSCM)占总细胞比例均显著高于MSLN-CAR对照组。
实施例22：CD258蛋白与SIRT1蛋白组合增强CAR-T细胞对肿瘤细胞的杀伤能力。
将表达GPC3-S1A或GPC3-S1A-C8A的T细胞与Huh7细胞(中科院细胞库购得)按1:15的细胞个数比用Xvivo15培养基共培养7天，采用结晶紫 染色(MCE公司)检测CAR-T细胞对肿瘤细胞的杀伤效果，结果如图24A所示，表达GPC3-S1A-C8A的T细胞对肿瘤细胞的杀伤效果远高于对照组表达GPC3-S1A的T细胞。
同样的，将表达GPC3-CAR或GPC3-S1A-C8A的T细胞与Huh7细胞(中科院细胞库购得)按1:15的细胞个数比用Xvivo15培养基共培养7天，采用结晶紫染色(MCE公司)检测CAR-T细胞对肿瘤细胞的杀伤效果，结果如图24B所示，表达GPC3-S1A-C8A的T细胞对肿瘤细胞的杀伤效果亦远高于对照组表达GPC3-CAR的T细胞。
实施例23：CD258蛋白与SIRT1蛋白组合增强CAR-T细胞抗肿瘤效果。
向NSG小鼠(百奥赛图公司购得)皮下接种Huh7细胞(1×10 7个/只)，14天后，测得小鼠瘤体积约200mm 3。此时将小鼠分为5组，分别为T、GPC3-CAR和GPC3-S1A-C8A组，每组6-8只。然后通过尾静脉分别向T细胞组注射T细胞(9×10 5个/只)，向GPC3-CAR组注射表达GPC3-CAR的CAR-T细胞(3×10 5个/只或9×10 5个/只)，向GPC3-S1A-C8A组注射表达GPC3-S1A-C8A的CAR-T细胞(3×10 5个/只或9×10 5个/只)。于每周的周一和周四分别测量瘤体积大小，并记录小鼠死亡情况。结果如图25所示，GPC3-S1A-C8A组抑瘤效果显著高于其他对照组。
实施例24：CD258蛋白与SIRT1蛋白组合促进粒细胞和单核细胞体内增殖。
向NSG小鼠(百奥赛图公司购得)皮下接种Huh7细胞(1×10 7个/只)，14天后，测得小鼠瘤体积约200mm 3。此时将小鼠分为5组，分别为T、GPC3-CAR和GPC3-S1A-C8A组，每组6只。然后通过尾静脉分别向T细胞组注射T细胞(9×10 5个/只)，向GPC3-CAR组注射表达GPC3-CAR的CAR-T细胞(3×10 5个/只或9×10 5个/只)，向GPC3-S1A-C8A组注射表达GPC3-S1A-C8A的CAR-T细胞(3×10 5个/只或9×10 5个/只)，于第7天从小鼠尾部取血50μl，用BD流式检测每组中单核细胞和中性粒细胞的细胞数以及细胞大小。结果如图26所示，GPC3-S1A-C8A组中单核细胞和中性粒细胞的细胞数以及细胞大小均远高于GPC3-CAR对照组。
实施例25：CD258蛋白与SIRT1蛋白组合促进CAR-T细胞体内增殖以及Th1 细胞因子的释放。
向NSG小鼠(百奥赛图公司购得)皮下接种Huh7细胞(1×10 7个/只)，14天后，测得小鼠瘤体积约200mm 3。此时将小鼠分为5组，分别为T、GPC3-CAR和GPC3-S1A-C8A组，每组6只。然后通过尾静脉分别向T细胞组注射T细胞(9×10 5个/只)，向GPC3-CAR组注射表达GPC3-CAR的CAR-T细胞(3×10 5个/只或9×10 5个/只)，向GPC3-S1A-C8A组注射表达GPC3-S1A-C8A的CAR-T细胞(3×10 5个/只或9×10 5个/只)，于第14天从小鼠尾部取血50μl，用BD流式检测每组中人CD3、CD4和CD8蛋白的表达。结果如图27A、图27B和图27C所示，在表达了CD3、CD4和CD8的组中，GPC3-S1A-C8A组中表达CD3、CD4或CD8蛋白的细胞占总细胞数的比例远高于GPC3-CAR对照组。
同样的，向NSG小鼠(百奥赛图公司购得)皮下接种Huh7细胞(1×10 7个/只)，14天后，测得小鼠瘤体积约200mm 3。此时将小鼠分为5组，分别为T、GPC3-CAR和GPC3-S1A-C8A组，每组6只。然后通过尾静脉分别向T细胞组注射T细胞(9×10 5个/只)，向GPC3-CAR组注射表达GPC3-CAR的CAR-T细胞(3×10 5个/只或9×10 5个/只)，向GPC3-S1A-C8A组注射表达GPC3-S1A-C8A的CAR-T细胞(3×10 5个/只或9×10 5个/只)，于第7天从小鼠尾部取血50μl，用BD流式检测每组中人IL-2、IL4、IL6、IL10、TNF-α和IFN-γ细胞因子的表达。结果如图27D所示，GPC3-S1A-C8A组中IFN-γ细胞因子的表达显著高于GPC3-CAR对照组。
序列表：
SEQ ID NO.1：前导序列
Figure PCTCN2021143151-appb-000001
前导序列的氨基酸序列:SEQ ID NO:105
Figure PCTCN2021143151-appb-000002
SEQ ID NO.2：GPC3 scFv的核苷酸序列
Figure PCTCN2021143151-appb-000003
GPC3 scFv的氨基酸序列：SEQ ID NO:34
Figure PCTCN2021143151-appb-000004
(VL，SEQ ID NO:35：
Figure PCTCN2021143151-appb-000005
下划线标注依次是CDR1( RSSQSLVHSNGNTYLH,SEQ ID NO:36)、 CDR2( KVSNRFS,SEQ ID NO:37)和CDR3(SQNTHVPPT,SEQ ID NO:38)；
VH，SEQ ID NO:39：
Figure PCTCN2021143151-appb-000006
下划线标注依次是CDR1(DYEMH,SEQ ID NO:40)、CDR2(ALDPKTGDTAYSQKFKG，SEQ ID NO:41)和CDR3(FYSYTYW，SEQ ID NO:42))
SEQ ID NO.3：CD19scFv的核苷酸序列
Figure PCTCN2021143151-appb-000007
CD19 scFv的氨基酸序列：SEQ ID NO:43
Figure PCTCN2021143151-appb-000008
(VL，SEQ ID NO:44：
Figure PCTCN2021143151-appb-000009
Figure PCTCN2021143151-appb-000010
下划线标注依次是CDR1(RASQDISKYLN，SEQ ID NO:45)、CDR2(HTSRLHS，SEQ ID NO:46)和CDR3(QQGNTLPYT，SEQ ID NO:47)；
VH，SEQ ID NO:48：
Figure PCTCN2021143151-appb-000011
下划线标注依次是CDR1(LPDYGVS，SEQ ID NO:49)、CDR2(VIWGSETTYYNSALKS，SEQ ID NO:50)和CDR3(HYYYGGSYAMDYW，SEQ ID NO:51))
SEQ ID NO.4：BCMA scFv的核苷酸序列
Figure PCTCN2021143151-appb-000012
BCMA scFv氨基酸序列：SEQ ID NO:52
Figure PCTCN2021143151-appb-000013
(VL，SEQ ID NO:53：
Figure PCTCN2021143151-appb-000014
下划线标注依次是CDR1(RASESVSVIGAHLIH，SEQ ID NO:54)、CDR2(LASNLET，SEQ ID NO:55)和CDR3(LQSRIFPRT，SEQ ID NO:56)；
VH，SEQ ID NO:57：
Figure PCTCN2021143151-appb-000015
下划线标注依次是CDR1(DYSIN，SEQ ID NO:58)、CDR2(WINTETREPAYAYDFRG SEQ ID NO:59)和CDR3(DYSYAMDYW，SEQ ID NO:60))
SEQ ID NO.5：MSLN scFv的核苷酸序列
Figure PCTCN2021143151-appb-000016
MSLN scFv氨基酸序列：SEQ ID NO:61
Figure PCTCN2021143151-appb-000017
(VL，SEQ I NO:62：
Figure PCTCN2021143151-appb-000018
下划线标注依次是CDR1(TLRSGINVGPYRIY，SEQ ID NO:63)、CDR2(YKSDSDKQQGS，SEQ ID NO:64)和CDR3(MIWHSSAAV，SEQ ID NO:65)；
VH：SEQ ID NO:66
Figure PCTCN2021143151-appb-000019
下划线标注依次是CDR1(SNSATWN，SEQ ID NO:67)、CDR2(RTYYRSKWYNDYAVSVKS，SEQ ID NO:68)和CDR3(GMMTYYYGMDVW，SEQ ID NO:69))
SEQ ID NO.6：HER2 scFv的核苷酸序列
Figure PCTCN2021143151-appb-000020
Figure PCTCN2021143151-appb-000021
HER2 scFv氨基酸序列：SEQ ID NO:70
Figure PCTCN2021143151-appb-000022
(VL，SEQ ID NO:71：
Figure PCTCN2021143151-appb-000023
下划线标注依次是CDR1(KASQDVYNAVA，SEQ ID NO:72)、CDR2(SASSRYT，SEQ ID NO:73)和CDR3(QQHFRTPFT，SEQ ID NO:74)；
VH，SEQ ID NO:75：
Figure PCTCN2021143151-appb-000024
下划线标注依次是CDR1(NYGMN，SEQ ID NO:76)、CDR2(WINTSTGESTFADDFKG，SEQ ID NO:77)和CDR3(WEVYHGYVPYW，SEQ ID NO:78))
SEQ ID NO.7：CD8铰链区的核苷酸序列
Figure PCTCN2021143151-appb-000025
CD8铰链区氨基酸序列：SEQ ID NO:79
Figure PCTCN2021143151-appb-000026
SEQ ID NO.8：CD8跨膜区的核苷酸序列
Figure PCTCN2021143151-appb-000027
CD8跨膜区的氨基酸序列：SEQ ID NO:80
Figure PCTCN2021143151-appb-000028
SEQ ID NO.9：CD28共刺激结构域的核苷酸序列
Figure PCTCN2021143151-appb-000029
CD28共刺激结构域的氨基酸序列：SEQ ID NO:81
Figure PCTCN2021143151-appb-000030
SEQ ID NO.10：4-1BB的共刺激结构域的核苷酸序列
Figure PCTCN2021143151-appb-000031
4-1BB的共刺激结构域的氨基酸序列：SEQ ID NO:82
Figure PCTCN2021143151-appb-000032
SEQ ID NO.11：CD3ζ的信号传导结构域的核苷酸序列
Figure PCTCN2021143151-appb-000033
CD3ζ的信号传导结构域的氨基酸序列：SEQ ID NO:83
Figure PCTCN2021143151-appb-000034
SEQ ID NO.12：T2A的核苷酸序列
Figure PCTCN2021143151-appb-000035
T2A的氨基酸序列：SEQ ID NO:84
Figure PCTCN2021143151-appb-000036
SEQ ID NO.13：P2A的核苷酸序列
Figure PCTCN2021143151-appb-000037
P2A的氨基酸序列：SEQ ID NO:85
Figure PCTCN2021143151-appb-000038
SEQ ID NO.14：IRES的核苷酸序列
Figure PCTCN2021143151-appb-000039
核糖体跳跃位点，不翻译成氨基酸
SEQ ID NO.15：S1的核苷酸序列
Figure PCTCN2021143151-appb-000040
Figure PCTCN2021143151-appb-000041
S1的氨基酸序列：SEQ ID NO:86
Figure PCTCN2021143151-appb-000042
SEQ ID NO.16：S2的核苷酸序列
Figure PCTCN2021143151-appb-000043
Figure PCTCN2021143151-appb-000044
S2的氨基酸序列：SEQ ID NO:87
Figure PCTCN2021143151-appb-000045
SEQ ID NO.17：S3的核苷酸序列
Figure PCTCN2021143151-appb-000046
Figure PCTCN2021143151-appb-000047
S3的氨基酸序列：SEQ ID NO:88
Figure PCTCN2021143151-appb-000048
SEQ ID NO.18：S4的核苷酸序列
Figure PCTCN2021143151-appb-000049
S4的氨基酸序列：SEQ ID NO:89
Figure PCTCN2021143151-appb-000050
Figure PCTCN2021143151-appb-000051
SEQ ID NO.19：S5的核苷酸序列
Figure PCTCN2021143151-appb-000052
S5的氨基酸序列：SEQ ID NO:90
Figure PCTCN2021143151-appb-000053
SEQ ID NO.20：S6的核苷酸序列
Figure PCTCN2021143151-appb-000054
Figure PCTCN2021143151-appb-000055
S6的氨基酸序列：SEQ ID NO:91
Figure PCTCN2021143151-appb-000056
SEQ ID NO.21：S7的核苷酸序列
Figure PCTCN2021143151-appb-000057
Figure PCTCN2021143151-appb-000058
S7的氨基酸序列：SEQ ID NO:92
Figure PCTCN2021143151-appb-000059
SEQ ID NO.22：S1A的核苷酸序列
Figure PCTCN2021143151-appb-000060
S1A的氨基酸序列：SEQ ID NO:93
Figure PCTCN2021143151-appb-000061
SEQ ID NO.23：S1B的核苷酸序列
Figure PCTCN2021143151-appb-000062
S1B的氨基酸序列：SEQ ID NO:94
Figure PCTCN2021143151-appb-000063
Figure PCTCN2021143151-appb-000064
SEQ ID NO.24：S1B1的核苷酸序列
Figure PCTCN2021143151-appb-000065
S1B1的氨基酸序列：SEQ ID NO:95
Figure PCTCN2021143151-appb-000066
SEQ ID NO.25：S1C的核苷酸序列
Figure PCTCN2021143151-appb-000067
S1C的氨基酸序列：SEQ ID NO:96
Figure PCTCN2021143151-appb-000068
SEQ ID NO.26：S1C1的核苷酸序列
Figure PCTCN2021143151-appb-000069
Figure PCTCN2021143151-appb-000070
S1C1的氨基酸序列：SEQ ID NO:97
Figure PCTCN2021143151-appb-000071
SEQ ID NO.27：S1D的核苷酸序列
Figure PCTCN2021143151-appb-000072
Figure PCTCN2021143151-appb-000073
S1D的氨基酸序列：SEQ ID NO:98
Figure PCTCN2021143151-appb-000074
SEQ ID NO.28：S1D1的核苷酸序列
Figure PCTCN2021143151-appb-000075
Figure PCTCN2021143151-appb-000076
S1D1的氨基酸序列：SEQ ID NO:99
Figure PCTCN2021143151-appb-000077
SEQ ID NO.29：S1E的核苷酸序列
Figure PCTCN2021143151-appb-000078
Figure PCTCN2021143151-appb-000079
S1E的氨基酸序列：SEQ ID NO:100
Figure PCTCN2021143151-appb-000080
SEQ ID NO.30：S1E1的核苷酸序列
Figure PCTCN2021143151-appb-000081
S1E1的氨基酸序列：SEQ ID NO:101
Figure PCTCN2021143151-appb-000082
SEQ ID NO.31：C8的核苷酸序列
Figure PCTCN2021143151-appb-000083
C8的氨基酸序列：SEQ ID NO:102
Figure PCTCN2021143151-appb-000084
SEQ ID NO.32：C8A的核苷酸序列
Figure PCTCN2021143151-appb-000085
Figure PCTCN2021143151-appb-000086
C8A的氨基酸序列：SEQ ID NO:103
Figure PCTCN2021143151-appb-000087
SEQ ID NO.33：C8B的核苷酸序列
Figure PCTCN2021143151-appb-000088
C8B的氨基酸序列：SEQ ID NO:104
Figure PCTCN2021143151-appb-000089
SIRT1蛋白核定位信号：SEQ ID NO:105
Figure PCTCN2021143151-appb-000090
SIRT1蛋白核定位信号：SEQ ID NO:106
Figure PCTCN2021143151-appb-000091
SIRT1蛋白核定位信号突变体：SEQ ID NO:107
Figure PCTCN2021143151-appb-000092
SIRT1蛋白核定位信号突变体：SEQ ID NO:108
Figure PCTCN2021143151-appb-000093
SIRT1蛋白核出核信号：SEQ ID NO:109
Figure PCTCN2021143151-appb-000094
SIRT1蛋白核出核信号：SEQ ID NO:110
Figure PCTCN2021143151-appb-000095
SIRT1蛋白核出核信号突变体：SEQ ID NO:111
Figure PCTCN2021143151-appb-000096
SIRT1蛋白核出核信号突变体：SEQ ID NO:112
Figure PCTCN2021143151-appb-000097

Claims (19)

  1. 一种方法，优选所述方法为体内方法或体外方法，所述方法选自以下各项组成的组：
    (1)促进免疫细胞增殖的方法，(2)促进记忆性免疫细胞产生的方法，(3)抑制免疫细胞分化的方法，(4)抑制免疫细胞免疫负调控蛋白表达的方法，(5)增强免疫细胞释放细胞因子的方法，和(6)增强免疫细胞对肿瘤杀伤能力的方法，
    所述方法包含以下步骤：上调所述免疫细胞中选自SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体的一种或多种的表达量、或上调所述免疫细胞中CD258蛋白或其功能突变体与选自SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体的一种或多种的组合的表达量；
    或者一种方法，所述方法选自以下各项组成的组：
    (a)解决受试者肿瘤异质性的方法，(b)预防受试者中肿瘤复发的方法，和(c)治疗有需要的受试者中的肿瘤的方法，
    所述方法包含向受试者施用免疫细胞，其中所述免疫细胞中选自SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体的一种或多种的表达量上调，或所述免疫细胞中CD258蛋白或其功能突变体与选自SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体的一种或多种的表达量上调。
  2. 权利要求1所述的方法，其中所述肿瘤选自肝癌、肺癌、白血病和间皮瘤，所述免疫负调控蛋白选自PD1、PDL1、TIM3和LAG3组成的组，所述细胞因子选自白介素、干扰素和/或肿瘤坏死因子，优选地，所述细胞因子选自IL-2、IL4、IL6、IL7、IL10、IL12、TNF-α和/或IFNγ；所述免疫细胞为淋巴细胞；优选地，所述免疫细胞为T细胞、B细胞、自然杀伤细胞、未成熟 的树突状细胞、单核细胞和巨噬细胞。进一步优选地，所述T细胞选自记忆性干细胞样T细胞和/或中央记忆性T细胞，优选地，所述TSCM为CCR7 +和/或CD62L +，优选地，所述TSCM还具有选自下组的一种或多种性质：CD45RA +或CD45RA -、CD45RO +或CD45RO -、CD27 +、CD28 +、CD127 +、CD122 +、CD95 +、CD3 +、CD4 +和CD8 +。
  3. 权利要求1-2任一项所述的方法，所述免疫细胞选自经遗传修饰的免疫细胞，且所述经遗传修饰的免疫细胞表达嵌合抗原受体或T细胞受体，优选地，所述经遗传修饰的免疫细胞为经遗传修饰的T细胞，优选地，所述方法包括分离并激活所述经遗传修饰的免疫细胞的步骤，其中所述激活包括向经分离的所述经遗传修饰的免疫细胞施用T细胞培养基，优选地，所述T细胞培养基选自以下组中的一种或多种：DMEM培养基、1640培养基、MEM培养基、X-VIVO培养基和干细胞培养基。
  4. 权利要求1-3任一项所述的方法，所述TCR包含选自下组的亚基：TCRα、TCRβ、TCRγ和TCRδ，
    优选地，所述TCR的亚基包括特异性结合和/或识别肿瘤抗原的胞外域可变区优选地，胞外域可变区选自以下组：TCRα可变区片段Vα、TCRα可变区片段Jα、TCRβ可变区片段Vβ、TCRβ可变区片段Dβ和TCRβ可变区片段Jβ，
    优选地，所述胞外域可变区特异性结合和/或识别选自下组的靶标：MAGEA家族成员、CTA家族成员、HPV病毒和酪氨酸酶，
    优选地，所述胞外域可变区特异性结合和/或识别选自下组的靶标：MAGEA3、MAGEA4、NY-ESO-1、MART1、HPV16-E6和黑素瘤抗原酪氨酸酶。
  5. 权利要求1-3任一项所述的方法，所述CAR包含细胞内结构域，所述细胞内结构域包括信号传导结构域和/或共刺激结构域，
    优选地，所述信号传导结构域选自以下各项组成的组：CD3ζ的信号传导结构域(优选核苷酸序列如SEQ ID NO:11所示，氨基酸序列如SEQ ID NO:83所示)、CD3δ的信号传导结构域和CD3ε的信号传导结构域。在某些实施方式中，所述信号传导结构域包含SEQ ID NO:83所示的序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，所述同源序列仍具备SEQ ID NO:83所示序列功能，或由其组成，
    优选地，共刺激结构域是指选自以下一个或多个的蛋白质的功能性信号传导结构域:CD27、CD28、4-1BB(CD137)、OX40、CD30、CD40、PD-1、ICOS、淋巴细胞功能相关抗原1(LFA-1)、CD2、CD7、LIGHT、NKG2C、B7-H3、特异结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46和NKG2D优选地，所述共刺激结构域选自：CD27、CD28和4-1BB，
    更优选地，所述共刺激结构域选自以下各项组成的组：CD27的共刺激结构域、CD28的共刺激结构域(优选核苷酸序列如SEQ ID NO:9所示，氨基酸序列如SEQ ID NO:81所示)和4-1BB的共刺激结构域(优选核苷酸序列如SEQ ID NO:10所示，氨基酸序列如SEQ ID NO:82所示)优选地，所述共刺激结构域包含下述任一项所示的序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，或由其组成：SEQ ID NO:81和SEQ ID NO:82，所述同源序列仍具备SEQ ID NO:81或82所示序列功能，
    优选地，所述CAR包含铰链区，更优选地，所述铰链区选自以下各项组成的组：IgG4的铰链区、IgG1的铰链区和CD8的铰链区(优选核苷酸序列如SEQ ID NO:7所示，氨基酸序列如SEQ ID NO:79所示)优选地，所述铰链区包含SEQ ID NO:79所示的序列或由其组成，
    优选地，所述CAR包含跨膜区优选地，所述跨膜区选自以下各种组成的组：CD8的跨膜区(优选核苷酸序列如SEQ ID NO:8所示，氨基酸序列如SEQ ID NO:80所示)、CD28的跨膜区和CD24的跨膜区优选地，所述跨膜区包含SEQ ID NO:80所示的序列或由其组成，
    优选地，所述CAR包含靶向部分，优选所述靶向部分特异性结合和/或识 别肿瘤抗原，
    优选地，所述肿瘤抗原可以包括TSHR、CD19、CD123、CD138、CD22、CD30、CD171、CS-1、CLL-1、CD33、EGFRvIII、GD2、GD3、BCMA、TnAg、PSMA、ROR1、FLT3、FAP、TAG72、CD38、CD44v6、CEA、EPCAM、B7H3、KIT、IL-13Ra2、间皮素、IL-11Ra、PSCA、PRSS21、VEGFR2、LewisY、CD24、PDGFR-β、SSEA-4、CD20、叶酸受体α、ERBB2(Her2/neu)、MUC1、EGFR、NCAM、Prostase、PAP、ELF2M、肝配蛋白B2、I GF-I受体、CAIX、LMP2、gp100、bcr-abl、酪氨酸酶、EphA2、岩藻糖基GM1、sLe、GM3、TGS5、HMWMAA、邻乙酰基-GD2、叶酸受体β、TEM1/CD248、TEM7R、CLDN6、GPRC5D、CXORF61、CD97、CD179a、ALK、聚唾液酸、PLAC1、GloboH、NY-BR-1、UPK2、HAVCR1、ADRB3、PANX3、GPR20、LY6K、OR51E2、TARP、WT1、NY-ESO-1、LAGE-1a、MAGE-A1、legumain、HPVE6、E7、MAGE A1、ETV6-AML、精子蛋白17、XAGE1、ie 2、MAD-CT-1、MAD-CT-2、Fos相关抗原1、p53、p53突变体、prostein、存活蛋白和端粒酶、PCTA-1/Galectin8、MelanA/MART1、Ras突变体、hTERT、肉瘤易位断点、ML-IAP、ERG(TMPRSS2ETS融合基因)、NA17、PAX3、雄激素受体、细胞周期蛋白B1、MYCN、RhoC、TRP-2、CYP1B1、BORIS、SART3、PAX5、OY-TES1、LCK、AKAP-4、SSX2、RAGE-1、人端粒酶逆转录酶、RU1、RU2、肠羧基酯酶、mut hsp702、CD79a、CD79b、CD72、LAIR1、FCAR、LILRA2、CD300LF、CLEC12A、BST2、EMR2、LY75、GPC3、FCRL5和IGLL1，
    更优选所述靶向部分特异性结合和/或识别选自下组的靶标：B淋巴细胞表面抗原、TNF家族成员、HER家族成员和GPC家族成员，进一步优选地，所述靶向部分特异性结合和/或识别选自下组的靶标：CD19、BCMA、HER2、Mesothelin和GPC3。
  6. 权利要求5所述的方法，其中所述靶向部分为scFv，优选地，所述靶向部分选自针对GPC3的scFv，针对CD19的scFv，针对BCMA的scFv，针对MSLN的scFv，针对HER2的scFv，
    优选所述针对GPC3的scFv包含SEQ ID NO:35所示轻链可变区包含的LCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:36所示)，LCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:37所示)和LCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:38所示)和包含SEQ ID NO:39所示的重 链可变区包含的HCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:40所示)，HCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:41所示)和HCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:42所示)，更优选地，针对GPC3的scFv包含如SEQ ID NO:2所示的核苷酸序列或如SEQ ID NO:34所示的氨基酸序列，或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:2或34所示序列功能；
    所述针对CD19的scFv包含SEQ ID NO:44所示轻链可变区包含的LCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:45所示)，LCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:46所示)和LCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:47所示)和包含SEQ ID NO:48所示的重链可变区包含的HCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:49所示)，HCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:50所示)和HCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:51所示)，更优选地，针对CD19的scFv包含如SEQ ID NO:3所示的核苷酸序列或如SEQ ID NO:43所示的氨基酸序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:3或43所示序列功能；
    所述针对BCMA的scFv包含SEQ ID NO:53所示轻链可变区包含的LCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:54所示)，LCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:55所示)和LCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:56所示)和包含SEQ ID NO:57所示的重链可变区包含的HCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:58所示)，HCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:59所示)和HCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:60所示)，更优选地，针对BCMA的scFv包含的如SEQ ID NO:4所示核苷酸序列或如SEQ ID NO:52所示的氨基酸序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:4 或52所示序列功能；
    所述针对MSLN的scFv包含SEQ ID NO:62所示轻链可变区包含的LCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:63所示)，LCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:64所示)和LCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:65所示)和包含SEQ ID NO:66所示的重链可变区包含的HCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:67所示)，HCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:68所示)和HCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:69所示)，更优选地，针对MSLN的scFv包含如SEQ ID NO:5所示的核苷酸序列或如SEQ ID NO:61所示的氨基酸序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:5或61所示序列功能；
    所述针对HER2的scFv包含SEQ ID NO:71所示轻链可变区包含的LCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:72所示)，LCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:73所示)和LCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:74所示)和包含SEQ ID NO:75所示的重链可变区包含的HCDR1(优选按照Kabat编号系统，序列如SEQ ID NO:76所示)，HCDR2(优选按照Kabat编号系统，序列如SEQ ID NO:77所示)和HCDR3(优选按照Kabat编号系统，序列如SEQ ID NO:78所示)，更优选地，针对HER2的scFv包含如SEQ ID NO:6所示的核苷酸序列或如SEQ ID NO:70所示的氨基酸序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％或至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:6或70所示序列功能。
  7. 权利要求1-6任一项所述的方法，其中所述方法还包括以下步骤：分离获得外周血单核细胞PBMC、CD3 +T淋巴细胞、CD8 +T淋巴细胞、CD4 +T淋巴细胞或调节T细胞，优选地，所述方法还包括：向经分离的所述PBMC中加入一种或多种T细胞刺激因子，优选地，所述T细胞刺激因子选自以下各项组成的组：针对B淋巴细胞表面抗原的抗体、抗TNF抗体、细胞内聚酯和抗生素优选地，所述T细胞刺激因子选自以下各项组成的组：抗CD3抗体、 抗CD28抗体、抗4-1BB抗体、抗CD80抗体、抗CD86抗体、PHA、PMA和离子霉素，更优选地，所述T细胞刺激因子为抗CD3抗体，且所述抗CD3抗体的浓度为1-10000ng/mL，或所述T细胞刺激因子为抗CD28抗体，且所述抗CD28抗体的浓度为1-10000ng/mL。
  8. 权利要求1-7任一项所述的方法，其中所述方法还包括：向经分离的所述PBMC中加入一种或多种细胞因子，优选地，所述细胞因子为白细胞介素，更优选地，所述白细胞介素为IL-1，IL-2，IL-3，IL-4，IL-5，IL-6，IL-7，IL-8，IL-9，IL-10，IL-11，IL-12，IL-13，IL-14，IL-15，IL-16，IL-17，IL-18，IL-19，IL-20，IL-21，IL-22，IL-23，IL-24，IL-25，IL-26，IL-27，IL-28，IL-29，IL-30，IL-31，IL-32，IL-33，IL-34，IL-35和/或IL-36；更优选地，所述白细胞介素为IL2，且所述IL2的浓度为0.1-10000U/mL，或所述白细胞介素为IL21，且所述IL21的浓度为0.01-1000ng/mL，或所述白细胞介素为IL7，且所述IL7的浓度为0.01-1000ng/mL，或所述白细胞介素为IL15，且所述IL15的浓度为0.01-1000ng/mL。
  9. 权利要求1-8任一项所述的方法，其中所述SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体、CD258或其功能突变体来源于人。
  10. 权利要求1-9任一项所述的方法，其中所述SIRT1蛋白、SIRT2蛋白、SIRT3蛋白、SIRT4蛋白、SIRT5蛋白、SIRT6蛋白、SIRT7蛋白分别包含下述任一项所示的序列：SEQ ID NO:15-21或SEQ ID NO:86-92，或与其具备至少80％、至少85％、至少90％，至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:15-21或SEQ ID NO:86-92任一项所示序列功能，
    所述SIRT1蛋白的功能突变体选自以下各项组成的组：
    (1)所述SIRT1蛋白、SIRT2蛋白、SIRT3蛋白、SIRT4蛋白、SIRT5蛋白、SIRT6蛋白、SIRT7蛋白的功能性突变体在所述SIRT1蛋白、SIRT2蛋白、SIRT3蛋白、SIRT4蛋白、SIRT5蛋白、SIRT6蛋白、SIRT7蛋白的选自下组的结构域进行突变：去乙酰化酶区，小分子Sirtuin激活剂结合区，和/或C端调控区，
    (2)SIRT1蛋白中，在SEQ ID NO:105所示的序列和SEQ ID NO:106所示的序列中任意一个或多个氨基酸置换为A获得的功能突变体，所述置换导致SEQ ID NO:105所示的序列和SEQ ID NO:106所示的序列丧失功能，更优选的功能突变体为SEQ ID NO:107所示的PLRKRPAA和SEQ ID NO:108所示的PPKRAAAA，
    (3)SIRT1蛋白中，SEQ ID NO:105所示的PLRKRPRR和SEQ ID NO:106所示的PPKRKKRK的完全缺失获得的功能突变体，
    (4)SIRT1蛋白中，在SEQ ID NO:109所示的序列和SEQ ID NO:110所示的序列中任意一个或多个氨基酸置换为A获得的功能突变体，所述置换导致SEQ ID NO:109所示的序列和SEQ ID NO:110所示的序列丧失功能，更优选的突变体为SEQ ID NO:111所示的AAATDGAA和SEQ ID NO:112所示的ADAAAAA，
    (5)SIRT1蛋白中，在SEQ ID NO:109所示的序列和SEQ ID NO:110所示的序列的完全缺失获得的功能突变体，
    更优选地，所述SIRT1功能突变体包含下述任一项所示的序列：SEQ ID NO:22-30或SEQ ID NO:93-101，或与其具备至少80％、至少85％、至少90％、至少91％，至少92％，至少93％，至少94％，至少95％，至少96％，至少97％，至少98％，至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:22-30或SEQ ID NO:93-101任一项所示序列功能。
  11. 权利要求1-9任一项所述的方法，其中所述CD258蛋白包含SEQ ID NO:31或SEQ ID NO:102所示的序列或与其具备至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％同源性的序列，或由其组成，所述同源序列仍具备SEQ ID NO:31或SEQ ID NO:102所示序列功能，
    所述CD258蛋白功能突变体选自以下各项组成的组：
    (1)在SEQ ID NO:102所示序列中第82-83位的Q和L的一种或两种置换为A(优选QL氨基酸被置换为AA)获得的功能突变体，所述功能突变体使该蛋白始终以膜结合形式表达在细胞膜上，减少分泌形式的表达，
    (2)在SEQ ID NO:102所示序列中第82-83位的Q和L缺失，或在SEQ ID NO:102所示序列中第81-84位的EQLI缺失获得的功能突变体，所述功能突变体使该蛋白始终以膜结合形式表达在细胞膜上，减少分泌形式的表达，
    (3)如SEQ ID NO:32-33所示的序列或如SEQ ID NO:103-104所示的序列，
    (4)CD258的胞内区。
  12. 经遗传修饰的免疫细胞，其中所述遗传修饰使得所述免疫细胞中选自SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体的一种或多种的表达量上调，或所述遗传修饰使得所述免疫细胞中CD258蛋白或其功能突变体与选自SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体的一种或多种的表达量上调。
  13. 权利要求1所述的方法或权利要求12所述的经遗传修饰的免疫细胞，其中所述表达量上调通过以下方式实现：
    (1)通过向所述免疫细胞中添加增加所述SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体的一种或多种的表达的激活剂以刺激所述免疫细胞自身增加所述SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体的一种或多种的表达，优选所述激活剂选自以下各项组成的组：SRT2104(化学式C 26H 24N 6O 2S 2)；CAY10602(化学式C 22H 15FN 4O 2S)；OSS-128167(化学式C 19H 14N 2O 6)；
    和/或
    (2)通过向所述免疫细胞中转染包含编码所述SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种的核酸的表达载体以增加所述免疫细胞中所述SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体、CD258蛋白或其功能突变体的量，优选地，所述SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白 或其功能突变体、和CD258蛋白或其功能突变体以单体形式存在或通过连接元件连接的缀合物的形式存在。
  14. 权利要求12或13所述的经遗传修饰的免疫细胞，其中所述载体选自以下组：逆转录病毒载体、慢病毒载体和转座子质粒，优选地，所述载体进一步包含CAR，更优选地，所述CAR通过连接元件连接调节单元，所述调节单元选自所述SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体、CD258蛋白或其功能突变体的一种或多种。
  15. 权利要求12-14任一项所述的经遗传修饰的免疫细胞，其中所述连接元件选自下列各项组成的组：T2A(优选核苷酸序列如SEQ ID NO:12所示，氨基酸序列如SEQ ID NO:84所示)、P2A(优选核苷酸序列如SEQ ID NO:13所示，氨基酸序列如SEQ ID NO:85所示)、F2A、E2A和IRES(优选核苷酸序列如SEQ ID NO:14所示)，优选地，所述连接元件包含SEQ ID NO:84、85和14所示的序列或由其组成。
  16. 权利要求12-15任一项所述的经遗传修饰的免疫细胞，其中所述免疫细胞包含选自以下各项组成的组的片段：
    GPC3-S1，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:15依次拼接合成，
    GPC3-S2，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:16依次拼接合成，
    GPC3-S3，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:17依次拼接合成，
    GPC3-S6，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:20依次拼接合成，
    GPC3-S1A，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22依次 拼接合成，
    GPC3-S1B，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:23依次拼接合成，
    GPC3-S1B1，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:24依次拼接合成，
    GPC3-S1C，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:25依次拼接合成，
    GPC3-S1C1，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:26依次拼接合成，
    GPC3-S1D，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:27依次拼接合成，
    GPC3-S1D1，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:28依次拼接合成，
    GPC3-S1E，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:29依次拼接合成，
    GPC3-S1E1，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:30依次拼接合成，
    GPC3-S1-S3，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:15、SEQ ID NO:13、SEQ ID NO:17依次拼接合成，
    GPC3-S1A-S3，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:13、SEQ ID NO:17依次拼接合成，
    GPC3-C8，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:31依次拼接合成，
    GPC3-C8A由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:32依次拼接合成，
    GPC3-C8B，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:33依次拼接合成，
    GPC3-S1A-C8A，由SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:13、SEQ ID NO:32依次拼接合成，
    CD19-S1A，由SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22依次拼接合成，
    CD19-C8A，由SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:32依次拼接合成，
    CD19-S1A-C8A，由SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:13、SEQ ID NO:32依次拼接合成，
    MSLN-S1A，由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22依次拼接合成，
    MSLN-C8，由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:31依次拼接合成。
    MSLN-C8A，由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:32依次拼接合成，
    MSLN-C8B，由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID  NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:33依次拼接合成，和
    MSLN-S1A-C8A，由SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:22、SEQ ID NO:13、SEQ ID NO:32依次拼接合成。
  17. 组合物，其包含权利要求12-16任一项所述的经遗传修饰的免疫细胞，任选地，所述组合物还包含药学上可接受的载体。
  18. 权利要求10所述方法中涉及的所述SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体的一种或多种，或权利要求10所述方法中涉及的所述SIRT1或其功能突变体、SIRT2或其功能突变体、SIRT3或其功能突变体、SIRT4或其功能突变体、SIRT5或其功能突变体、SIRT6或其功能突变体、SIRT7蛋白或其功能突变体的一种或多种与权利要求11所述方法中涉及的所述CD258蛋白或其功能突变体的组合在制备提高药物预防和/或治疗肿瘤的功效的试剂中的应用，优选地，所述药物为经遗传修饰的免疫细胞(优选CAR-T细胞)。
  19. 权利要求12-16任一项所述的经遗传修饰的免疫细胞和/或权利要求17所述的组合物在制备药物中的用途，其中所述药物用于治疗和/或预防肿瘤，优选地，所述肿瘤选自肝癌、肺癌、白血病和间皮瘤，优选地，所述经遗传修饰的免疫细胞选自淋巴细胞，优选地，所述经遗传修饰的免疫细胞表达嵌合抗原受体(CAR)。
PCT/CN2021/143151 2021-12-30 2021-12-30 Sirt1-7蛋白在免疫治疗中的应用 WO2023123225A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/143151 WO2023123225A1 (zh) 2021-12-30 2021-12-30 Sirt1-7蛋白在免疫治疗中的应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/143151 WO2023123225A1 (zh) 2021-12-30 2021-12-30 Sirt1-7蛋白在免疫治疗中的应用

Publications (1)

Publication Number Publication Date
WO2023123225A1 true WO2023123225A1 (zh) 2023-07-06

Family

ID=86997026

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/143151 WO2023123225A1 (zh) 2021-12-30 2021-12-30 Sirt1-7蛋白在免疫治疗中的应用

Country Status (1)

Country Link
WO (1) WO2023123225A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008100376A2 (en) * 2007-02-15 2008-08-21 Sirtris Pharmaceuticals, Inc. Truncation variants of sirt1 and methods of use thereof
CN103391777A (zh) * 2011-02-02 2013-11-13 普林斯顿大学理事会 作为病毒产生调节剂的去乙酰化酶调节剂
US20140371307A1 (en) * 2011-10-18 2014-12-18 Amorepacific Corporation Sirt 1 activator including syringaresinol
WO2020214936A1 (en) * 2019-04-17 2020-10-22 University Of Houston System Methods of improving the longevity of immune cells
CN112522394A (zh) * 2019-09-17 2021-03-19 中国科学院上海营养与健康研究所 新型外泌体释放相关靶点及其在监测和抑制肿瘤中的应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008100376A2 (en) * 2007-02-15 2008-08-21 Sirtris Pharmaceuticals, Inc. Truncation variants of sirt1 and methods of use thereof
CN103391777A (zh) * 2011-02-02 2013-11-13 普林斯顿大学理事会 作为病毒产生调节剂的去乙酰化酶调节剂
US20140371307A1 (en) * 2011-10-18 2014-12-18 Amorepacific Corporation Sirt 1 activator including syringaresinol
WO2020214936A1 (en) * 2019-04-17 2020-10-22 University Of Houston System Methods of improving the longevity of immune cells
CN112522394A (zh) * 2019-09-17 2021-03-19 中国科学院上海营养与健康研究所 新型外泌体释放相关靶点及其在监测和抑制肿瘤中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HAI-YAN CHEN, ZHU XIAO-XIA, XU XUE, XUE YU, YANG XUE, ZOU HE-JIAN: "Research Progress of Sirt1 in Immune Regulation", PROGRESS IN MODERN BIOMEDICINE, vol. 13, 10 May 2013 (2013-05-10), pages 2570 - 2574, XP093074087, DOI: 10.13241/j.cnki.pmb.2013.13.005 *
HANYU LI: "Research Progress on the Relationship between Sirtuins and Tumor", CHINESE JOURNAL OF CLINICIANS, CN, vol. 7, no. 6, 15 March 2013 (2013-03-15), CN , pages 173 - 174, XP093074540, ISSN: 1674-0785 *
HAODI YUE, ZHU XIAOFEI: "Research Progress on the Regulatory Effect and Mechanism of Sirtuin1 on Immune Cells", JOURNAL OF XINXIANG MEDICAL UNIVERSITY, vol. 34, no. 6, 5 June 2017 (2017-06-05), pages 455 - 459, XP093074092 *

Similar Documents

Publication Publication Date Title
WO2017027392A1 (en) Treatment of cancer using chimeric cd3 receptor proteins
CN109280086B (zh) 一种肿瘤微环境特异性活化的缺氧诱导型嵌合抗原受体
US11299552B2 (en) Eliminating MHC restriction from the T cell receptor as a strategy for immunotherapy
WO2020057641A1 (zh) 表达有趋化因子的细胞及用途
EP4066842A1 (en) Application of car t-cells in preparing drug for treating cancer
JP2020527036A (ja) 癌免疫療法用ｍｒ１制限ｔ細胞受容体
TW202039832A (zh) γδＴ細胞及其用途
JP2024518011A (ja) 多様な免疫細胞のための単鎖および多鎖合成抗原受容体
US20230143947A1 (en) Immune effector cell in which expression is regulated by cytokines
TW201930342A (zh) 包含nkg2d結構域的多特異性嵌合受體及其使用方法
KR20230084470A (ko) 면역 세포 기능의 향상
CN115485369A (zh) γδT细胞及其用途
CN116411025A (zh) Sirt1-7蛋白在免疫治疗中的应用
WO2022037562A1 (en) Engineered immunoresponsive cells and uses thereof
WO2023123225A1 (zh) Sirt1-7蛋白在免疫治疗中的应用
WO2023125945A1 (zh) Cd258蛋白在免疫治疗中的应用
US20240009310A1 (en) A CHIMERIC ANTIGEN RECEPTOR CONSTRUCT ENCODING A CHECKPOINT INHIBITORY MOLECULE AND AN IMMUNE STIMULATORY CYTOKINE AND CAR-EXPRESSING CELLS RECOGNIZING CD44v6
WO2022229884A2 (en) Recombinant antigen presenting cells
KR20230155521A (ko) 면역 세포 기능의 향상
EP4199958A1 (en) Chimeric antigen receptor (car)-expressing cells recognizing cea
US20240216426A1 (en) Recombinant antigen presenting cells
WO2024051831A1 (zh) 组成型嵌合细胞因子受体及表达其的免疫细胞及应用
WO2024011335A1 (zh) 经修饰的免疫细胞
WO2020085480A1 (ja) 高効率な遺伝子改変細胞の作製方法
CN117210410A (zh) 一种t细胞、其用途及药物组合物

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21969586

Country of ref document: EP

Kind code of ref document: A1