Application of SIRT1-7 protein in immunotherapy

Abstract

An application of a combination of SIRT1-7 protein or CD258 protein and SIRT1-7 protein for promoting immune cell proliferation is provided.

Claims

1. A n in vivo method or an in vitro method selected from the group consisting of: (1) a method to promote the proliferation of immune cell, (2) a method to promote the production of memory immune cells, (3) a method to inhibit the differentiation of immune cell, (4) a method to inhibit the expression of immune negative regulatory proteins in immune cells, (5) a method for enhancing immune cells to release cytokines, and (6) a method for enhancing the ability of immune cells to kill tumors, the method includes the following steps: up-regulating the expression level of one or the combination of more of SIRT1 or the functional mutant thereof, SIRT2 or the functional mutant thereof, SIRT3 or the functional mutant thereof, SIRT4 or the functional mutant thereof, SIRT5 or the functional mutant thereof, SIRT6 or the functional mutant thereof, SIRT7 protein or the functional mutant thereof in the immune cells, or up-regulation of the expression level of the CD258 protein or the functional mutant thereof and one or the combination of SIRT1 or the functional mutant thereof, SIRT2 or the functional mutant thereof, 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 in the immune cells; or a method selected from the group consisting of: (a) a method to solve tumor heterogeneity in subjects, (b) a method to prevent recurrence of tumors in subjects, and (c) a method to treat tumors in subjects in need, the method comprises administering immune cells to the subject, wherein up-regulating the expression level of one or the combination of more of SIRT1 or the functional mutant thereof, SIRT2 or the functional mutant thereof, SIRT3 or the functional mutant thereof, SIRT4 or the functional mutant thereof, SIRT5 or the functional mutant thereof, SIRT6 or the functional mutant thereof, SIRT7 protein or the functional mutant thereof in the immune cells, or up-regulation of the expression level of the CD258 protein or the functional mutant thereof and one or the combination of SIRT1 or the functional mutant thereof, SIRT2 or the functional mutant thereof, 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 in the immune cells.

2. The method of claim 1, wherein the tumor is selected from liver cancer, lung cancer, leukemia and mesothelioma, the negative immune regulatory protein is selected from PD1, PDL1, TIM3 and LAG3, and the cytokine is selected from interleukin, interferon and/or tumor necrosis factor, preferably, the cytokine is selected from IL-2, IL4, IL6, IL7, IL10, IL12, TNF- and/or IFN; the immune cell is a lymphocyte; preferably, the immune cells are T cells, B cells, natural killer cells, immature dendritic cells, monocytes and macrophages; further preferably, the T cells are selected from memory stem cell-like T cells and/or central memory T cells; preferably, the TSCM is CCR7.sup.+ and/or CD62L.sup.+, preferably, the TSCM also has one or more properties selected from: CD45RA.sup.+ or CD45RA.sup., CD45RO.sup.+ or CD45RO.sup., CD27.sup.+, CD28.sup.+, CD127.sup.+, CD122.sup.+, CD95.sup.+, CD3.sup.+, CD4.sup.+ and CD8.sup.+.

3. The method of claim 1, wherein the immune cells are selected from genetically modified immune cells, and the genetically modified immune cells express chimeric antigen receptors or T cell receptors, preferably the genetically modified immune cells are genetically modified T cells; preferably, the method includes the steps of isolating and activating the genetically modified immune cells, wherein the activation includes administering T cell culture medium to the isolated genetically modified immune cells, preferably, the T cell culture medium is selected from one or more of the following: DMEM culture medium, 1640 culture medium, MEM culture medium, X-VIVO culture medium and stem cells culture medium.

4. The method of claim 1, wherein the TCR comprises a subunit selected from: TCR, TCR, TCR and TCR, preferably, the subunits of the TCR include a variable region of extracellular domain that specifically binds and/or recognizes tumor antigens, preferably, the variable region of extracellular domain is selected from: variable region fragment V of TCR, variable region fragment J of TCR, variable region fragment V of TCR, variable region fragment D of TCR and variable region fragment J of TCR, preferably, the extracellular domain of the variable region specifically binds and/or recognizes a target selected from MAGEA family members, CTA family members, HPV viruses and tyrosinase, preferably, the extracellular domain of the variable region specifically binds and/or recognizes a target selected from: MAGEA3, MAGEA4, NY-ESO-1, MART1, HPV16-E6 and melanoma antigen tyrosinase.

5. The method of claim 1, the CAR comprising an intracellular domain which includes a signaling domain and/or a costimulatory domain, preferably, the signaling domain is selected from: the signaling domain of CD3 having the nucleotide sequence shown in SEQ ID NO: 11 and the amino acid sequence shown in SEQ ID NO: 83, the signaling domain of CD38 and the signaling domain of CD3; preferably, the signaling domain comprises or consists of the sequence shown as SEQ ID NO: 83 or is 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% homology thereto, and the homologous sequence still has the function of the sequence shown as SEQ ID NO: 83, preferably, the costimulatory domain 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, lymphoid Cell function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand that specifically binds 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, 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 and NKG2D; preferably, the costimulatory domain is selected from: CD27, CD28 and 4-1BB; more preferably, the costimulatory domain is selected from: the costimulatory domain of CD27, the costimulatory domain of CD28 having the nucleotide sequence is shown in as SEQ ID NO: 9 and the amino acid sequence shown in SEQ ID NO: 81 and the costimulatory domain of 4-1BB having the nucleotide sequence is shown as SEQ ID NO: 10 and the amino acid sequence shown in SEQ ID NO: 82; preferably, the costimulatory domain contains or consists of the sequence shown in any of: SEQ ID NO: 81 and SEQ ID NO: 82, or is 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% homology thereto, and the homologous sequence still has the function of the sequence shown as SEQ ID NO: 81 or SEQ ID NO: 82, preferably, the CAR comprises a hinge region, and more preferably the hinge region is selected from: the hinge region of IgG4, the hinge region of IgG1 and the hinge region of CD8 having the nucleotide sequence shown in SEQ ID NO:7 and the amino acid sequence shown in SEQ ID NO: 79; preferably, the hinge region includes or consists of the sequence shown in SEQ ID NO: 79, preferably, the CAR comprises a transmembrane region, preferably, the transmembrane region is selected from: the transmembrane region of CD8 having the nucleotide sequence is shown as SEQ ID NO: 8 and the amino acid sequence shown in SEQ ID NO: 80, the transmembrane region of CD28 and the transmembrane region of CD24, preferably, the transmembrane region includes or consists of the sequence shown in SEQ ID NO: 80, preferably, the CAR includes a targeting moiety, preferably the targeting moiety specifically binds and/or recognizes a tumor antigen, preferably, the tumor antigen 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, Fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLACI, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPVE6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, ie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutants, prostein, survivin and telomerase, PCTA-1/Galectin8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp702, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5 and IGLL1, more preferably, the targeting moiety specifically binds and/or recognizes a target selected from: B lymphocyte surface antigens, TNF family members, HER family members, and GPC family members, and further preferably, the targeting moiety specifically binds and/or recognizes a target selected from: CD19, BCMA, HER2, Mesothelin and GPC3.

6. The method of claim 5, wherein the targeting moiety is scFv, preferably the targeting moiety is selected from scFv against GPC3, scFv against CD19, scFv against BCMA, scFv against MSLN, scFv against HER2 scFv, preferably, the scFv against GPC3 includes LCDR1 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 36), LCDR2 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 37) and LCDR3 (preferably according to the Kabat numbering system, the sequence as shown in SEQ ID NO: 38) contained in the light chain variable region shown in SEQ ID NO: 35 and HCDR1 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 40), HCDR2 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 41) and HCDR3 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 42) contained in the heavy chain variable region shown in SEQ ID NO: 39, more preferably, the scFv against GPC3 comprises or consists of the nucleotide sequence shown in SEQ ID NO: 2 or the amino acid sequence shown in SEQ ID NO: 34, or has 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% homology thereto, and the homologous sequence still has the function of the sequence shown as SEQ ID NO: 2 or SEQ ID NO: 34; the scFv against CD19 includes LCDR1 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 45), LCDR2 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 46) and LCDR3 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 47) contained in the light chain variable region shown in SEQ ID NO: 44 and HCDR1 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 49), HCDR2 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 50) and HCDR3 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 51) contained in the heavy chain variable region shown in SEQ ID NO: 48, more preferably, the scFv against CD19 comprises or consists of the nucleotide sequence as shown in SEQ ID NO: 3 or the amino acid sequence as shown in SEQ ID NO: 43 or has 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% homology thereto, the sequence still has the function of the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 43; the scFv against BCMA includes LCDR1 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 54), LCDR2 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO:55) and LCDR3 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO:56) contained in the light chain variable region shown in SEQ ID NO: 53 and HCDR1 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 58), HCDR2 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 59) and HCDR3 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 60) contained in the heavy chain variable region shown in SEQ ID NO: 57, more preferably, the scFv against BCMA contains or consists of the nucleotide sequence as shown in SEQ ID NO:4 or the amino acid sequence shown in SEQ ID NO:52 or has 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% homology thereto, the sequence still has the function of the sequence shown in SEQ ID NO: 4 or SEQ ID NO: 52; the scFv against MSLN includes LCDR1 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 63), LCDR2 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 64) and LCDR3 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO:65) contained in the light chain variable region shown in SEQ ID NO: 62 and HCDR1 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 67), HCDR2 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 68) and HCDR3 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 69) comprising the heavy chain variable region shown in SEQ ID NO: 66, more preferably, the scFv against MSLN comprises or consists of the nucleotide sequence as shown in SEQ ID NO: 5 or the amino acid sequence shown in SEQ ID NO: 61 or has 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% homology thereto, the sequence still has the function of the sequence shown as SEQ ID NO: 5 or SEQ ID NO: 61; the scFv against HER2 includes LCDR1 (preferably according to the Kabat numbering system, the sequence is shown as SEQ ID NO: 72), LCDR2 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 73) and LCDR3 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 74) contained in the light chain variable region shown in SEQ ID NO: 71 and HCDR1 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 76), HCDR2 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 77) and HCDR3 (preferably according to the Kabat numbering system, the sequence shown in SEQ ID NO: 78) contained in the heavy chain variable region shown in SEQ ID NO: 75, more preferably, the scFv against HER2 comprises or consists of the nucleotide sequence as shown in SEQ ID NO: 6 or the amino acid sequence shown in SEQ ID NO: 70 or has 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% homology thereto, the sequence still has the function of the sequence shown as SEQ ID NO: 6 or SEQ ID NO: 70.

7. The method of claim 1, wherein the method further Includes-includes the following steps: isolate and obtain peripheral blood mononuclear cells PBMC, CD3.sup.+ T lymphocytes, CD8.sup.+ T lymphocytes, CD4.sup.+ T lymphocytes or regulatory T cells, preferably, the method further includes: adding one or more T cell stimulating factors to the isolated PBMCs, preferably, the T cell stimulating factors are selected from: antibodies against the surface antigens of B lymphocyte, anti-TNF antibodies, intracellular polyesters and antibiotics, preferably, the T cell stimulating factor is selected from: anti-CD3 antibodies, anti-CD28 antibodies, anti-4-1BB antibody, anti-CD80 antibody, anti-CD86 antibody, PHA, PMA and ionomycin, more preferably, the T cell stimulating factor is an anti-CD3 antibody, and the concentration of the anti-CD3 antibody is 1-10000 ng/mL, or the T cell stimulating factor is an anti-CD28 antibody, and the concentration of the anti-CD28 antibody is 1-10000 ng/mL.

8. The method of claim 1, wherein the method further includes: adding one or more cytokines to the isolated PBMC, preferably, the cytokine is interleukin, more preferably, the interleukin is 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; more preferably, the interleukin is IL2, and the concentration of IL2 is 0.1-10000 U/mL, or the interleukin is IL21, and the concentration of IL21 is 0.01-1000 ng/mL, or the interleukin is IL7, and the concentration of IL7 is 0.01-1000 ng/mL, or the interleukin is IL15, and the concentration of IL15 is 0.01-1000 ng/mL.

9. The method of claim 1, wherein the SIRT1 or the functional mutant thereof, SIRT2 or the functional mutant thereof, SIRT3 or the functional mutant thereof, SIRT4 or the functional mutant thereof, SIRT5 or the functional mutant thereof, SIRT6 or the functional mutant thereof, SIRT7 protein or the functional mutant thereof, CD258 or the functional mutant thereof are derived from a human.

10. The method of claim 1, wherein the SIRT1 protein, SIRT2 protein, SIRT3 protein, SIRT4 protein, SIRT5 protein, SIRT6 protein, and SIRT7 protein respectively comprise or consisted of the sequence shown in any one of the following: SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 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%, at least 98%, or at least 99% homology thereto, the sequence still has the function of the sequence shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, the functional mutant of the SIRT1 protein is selected from: (1) the functional mutants of the SIRT1 protein, SIRT2 protein, SIRT3 protein, SIRT4 protein, SIRT5 protein, SIRT6 protein, and SIRT7 protein have mutations in the domains of the SIRT1 protein, SIRT2 protein, SIRT3 protein, SIRT4 protein, SIRT5 protein, SIRT6 protein, and SIRT7 protein selected from: sirtuin domain, the small molecule Sirtuin activator binding domain, and/or the C-terminal regulatory domain, (2) the SIRT1 protein, wherein any one or more amino acids in the sequence shown in SEQ ID NO: 105 and the sequence shown in SEQ ID NO: 106 are substituted by A to obtain the functional mutant, and the substitution results in the loss of function of the protein having the sequence shown in SEQ ID NO: 105 and the loss of function of the protein having the sequence shown in SEQ ID NO: 106, and the more preferred functional mutants are PLRKRPAA as shown in SEQ ID NO: 107 and PPKRAAAA as shown in SEQ ID NO: 108, (3) the SIRT1 protein, wherein functional mutants are obtained by completely deleting the sequence PLRKRPRR as shown in SEQ ID NO: 105 and the sequence PPKRKKRK as shown in SEQ ID NO: 106, (4) the SIRT1 protein, wherein any one or more amino acids in the sequence shown in SEQ ID NO: 109 and the sequence shown in SEQ ID NO: 110 are replaced with A to obtain the functional mutant, and the substitution results in the loss of function of the protein having that the sequence shown in SEQ ID NO: 109 and the loss of function of the protein having the sequence shown in SEQ ID NO: 110, and the more preferred mutants are AAATDGAA as shown in SEQ ID NO: 111 and ADAAAA as shown in SEQ ID NO: 112, (5) the SIRT1 protein, wherein a functional mutant is obtained by completely deleting the sequence shown in SEQ ID NO: 109 and the sequence shown in SEQ ID NO: 110, more preferably, the SIRT1 functional mutant comprises or consists of the sequence shown in any one of the following: SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, or is 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 thereto, the homologous sequence still has the function of the sequence shown in any one of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101.

11. The method of claim 1, wherein the CD258 protein comprises or consists of the sequence shown as SEQ ID NO: 31 or SEQ ID NO: 102 or is 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 thereto, and the homologous sequences still has the function of the sequence shown in SEQ ID NO: 31 or SEQ ID NO: 102, the CD258 protein functional mutant is selected from: (1) a functional mutant obtained by replacing one or both of Q and L at positions 82-83 with A (preferably the QL amino acids are replaced with AA) in the sequence shown as SEQ ID NO: 102, and the functional mutation makes the protein always expressed on the cell membrane in a membrane-bound form and reduces the expression of the secreted form of the protein; (2) a functional mutant obtained by deleting Q and L at positions 82-83 in the sequence shown in SEQ ID NO: 102, or deleting EQLI at positions 81-84 in the sequence shown in SEQ ID NO: 102, and the functional mutant enables the protein always expressed on the cell membrane in a membrane-bound form and reduces the expression of the secreted form; (3) the sequence shown in SEQ ID NO: 32, SEQ ID NO: 33 or the sequence shown in SEQ ID NO: 103, SEQ ID NO: 104; (4) intracellular region of CD258.

12. Genetically modified immune cells, wherein the genetic modification is such that the expression level of one or more of SIRT1 or the functional mutants thereof, SIRT2 or the functional mutants thereof, SIRT3 or the functional mutants thereof, SIRT4 or the functional mutants thereof, SIRT5 or the functional mutant thereof, SIRT6 or the functional mutant thereof, SIRT7 protein or the functional mutant thereof is increased in the immune cells, or the genetic modification is such that the expression level of the CD258 protein or one or more of SIRT1 or the functional mutants thereof, SIRT2 or the functional mutants thereof, SIRT3 or the functional mutants thereof, SIRT4 or the functional mutants thereof, SIRT5 or the functional mutant thereof, SIRT6 or the functional mutant thereof, SIRT7 protein or the functional mutant thereof is increased in the immune cells.

13. The genetically modified immune cell of claim 12, wherein the up-regulation of expression is achieved by: (1) stimulating the immune cells themselves to increase the expression of one or more of SIRT1 or the functional mutant thereof, SIRT2 or the functional mutant thereof, SIRT3 or the functional mutant thereof, SIRT4 or the functional mutant thereof, SIRT5 or the functional mutant thereof, SIRT6 or the functional mutant thereof, SIRT7 protein or the functional mutant thereof by adding an activator to the immune cells, the activator increases the expression of SIRT1 or the functional mutant thereof, SIRT2 or the functional mutant thereof, SIRT3 or the functional mutant thereof, SIRT4 or the functional mutant thereof, SIRT5 or the functional mutant thereof, SIRT6 protein or the functional mutant thereof, SIRT7 protein or the functional mutant thereof, preferably the activator is selected from: SRT2104 (chemical formula C.sub.26H.sub.24N.sub.6O.sub.2S.sub.2); CAY10602 (chemical formula C.sub.22H.sub.15FN.sub.4O.sub.2S); OSS-128167 (chemical formula C19H.sub.14N.sub.2O.sub.6); and/or (2) transfecting the immune cell with the expression vector which encodes the nucleic acid of SIRT1 or the functional mutant thereof, SIRT2 or the functional mutant thereof, SIRT3 or the functional mutant thereof, SIRT4 or the functional mutant thereof, SIRT5 or the functional mutant thereof, SIRT6 or the functional mutants thereof, SIRT7 protein or the functional mutants thereof, CD258 protein or the functional mutants thereof, to increase the amount of the SIRT1 or functional mutants thereof, SIRT2 or the functional mutant thereof, SIRT3 or the functional mutant thereof, SIRT4 or the functional mutant thereof, SIRT5 or the functional mutant thereof, SIRT6 or the functional mutant thereof, SIRT7 protein or the functional mutant thereof, CD258 protein or the functional mutant thereof in the immune cells, preferably, the SIRT1 or its functional mutant, SIRT2 or its functional mutant, SIRT3 or its functional mutant, SIRT4 or its functional mutant, SIRT5 or its functional mutant, SIRT6 or its functional mutant, SIRT7 protein or the functional mutants thereof, and CD258 protein or the functional mutants thereof are in the form of monomers or in the form of conjugates connected through connecting elements.

14. The genetically modified immune cell of claim 13, wherein the vector is selected from: a retroviral vector, a lentiviral vector or a transposon plasmid, preferably the vector further comprises a CAR, more preferably, the CAR is connected to a regulatory unit through a connecting element, and the regulatory unit is selected from one or more of the SIRT1 or the functional mutant thereof, SIRT2 or the functional mutant thereof, SIRT3 or the functional mutant thereof, SIRT4 or the functional mutant thereof, SIRT5 or the functional mutant thereof, SIRT6 or the functional mutant thereof, SIRT7 protein or the functional mutant thereof, CD258 protein or the functional mutant thereof.

15. The genetically modified immune cell of claim 12, further comprising a connecting element, wherein the connecting element is selected from: T2A (preferably the nucleotide sequence as shown in SEQ ID NO: 12 and the amino acid sequence shown in SEQ ID NO:84), P2A (the preferred nucleotide sequence shown in SEQ ID NO: 13 and the amino acid sequence shown in SEQ ID NO:85), F2A, E2A and IRES (the preferred nucleotide sequence shown in SEQ ID NO: 14), preferably, the connecting element includes or consists of the sequences shown in SEQ ID NO: 84, SEQ ID NO: 85 and SEQ ID NO: 14.

16. The genetically modified immune cell of claim 12, wherein the immune cell comprises a fragment selected from: GPC3-S1, consisting 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, and SEQ ID NO: 15 which are spliced and synthesized in sequence, GPC3-S2, consisting 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, and SEQ ID NO: 16 which are spliced and synthesized in sequence, GPC3-S3, consisting 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, and SEQ ID NO: 17 which are spliced and synthesized in sequence, GPC3-S6, consisting 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, and SEQ ID NO: 20 which are spliced and synthesized in sequence, GPC3-S1A, consisting 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, and SEQ ID NO: 22 which are spliced and synthesized in sequence, GPC3-S1B, consisting 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, and SEQ ID NO: 23 which are spliced and synthesized in sequence, GPC3-S1B1, consisting 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, and SEQ ID NO: 24 which are spliced and synthesized in sequence, GPC3-S1C, consisting 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, and SEQ ID NO: 25 which are spliced and synthesized in sequence, GPC3-S1C1, consisting 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, and SEQ ID NO: 26 which are spliced and synthesized in sequence, GPC3-S1D, consisting 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, and SEQ ID NO: 27 which are spliced and synthesized in sequence, GPC3-S1D1, consisting 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, and SEQ ID NO: 28 which are spliced and synthesized in sequence, GPC3-S1E, consisting 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, and SEQ ID NO: 29 which are spliced and synthesized in sequence, GPC3-S1E1, consisting 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, and SEQ ID NO: 30 which are spliced and synthesized in sequence, GPC3-S1-S3, consisting 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 which are spliced and synthesized in sequence. GPC3-S1A-S3, consisting 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 which are spliced and synthesized in sequence. GPC3-C8, consisting 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, and SEQ ID NO: 31 which are spliced and synthesized in sequence, 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, and SEQ ID NO: 32 which are spliced and synthesized in sequence, GPC3-C8B, consisting 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, and SEQ ID NO: 33 which are spliced and synthesized in sequence, GPC3-S1A-C8A, consisting 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 which are spliced and synthesized in sequence. CD19-S1A, consisting 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, and SEQ ID NO: 22 which are spliced and synthesized in sequence, CD19-C8A, consisting 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, and SEQ ID NO: 32 which are spliced and synthesized in sequence, CD19-S1A-C8A, consisting 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 which are spliced and synthesized in sequence. MSLN-S1A, consisting 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, and SEQ ID NO: 22 which are spliced and synthesized in sequence, MSLN-C8, consisting 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, and SEQ ID NO: 31 which are spliced and synthesized in sequence. MSLN-C8A, consisting 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, and SEQ ID NO: 32 which are spliced and synthesized in sequence, MSLN-C8B, consisting 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, and SEQ ID NO: 33 which are spliced and synthesized in sequence, and MSLN-S1A-C8A, consisting 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 which were spliced and synthesized in sequence.

17. A composition comprising the genetically modified immune cells of claim 12, optionally, the composition further comprises a pharmaceutically acceptable carrier.

18. A method of improving an efficacy of drugs in preventing and/or treating tumors comprising the method according to claim 10 11 mutant, or its functional mutants in the preparation of reagents that improve the efficacy of drugs in preventing and/or treating tumors, preferably, the reagents are genetically modified immune cells (preferably CAR-T cells).

19. A method of treating and/or preventing tumors comprising the step of using the genetically modified immune cells according to claim 12, wherein the tumor is selected from the group consisting of liver cancer, lung cancer, leukemia and mesothelioma, preferably, the genetically modified immune cells are selected from lymphocytes, preferably, the genetically modified immune cells express chimeric antigen receptors (CAR).

20. A method of improving an efficacy of drugs in preventing and/or treating tumors comprising the method according to claim 11 in the preparation of reagents that improve the efficacy of drugs in preventing and/or treating tumors, preferably, the reagents are genetically modified immune cells (preferably CAR-T cells).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0080] FIG. 1 is a schematic diagram of different CAR structures;

[0081] FIG. 2 is a diagram showing that SIRT1 protein promotes the expansion of specific CAR-T cells induced by tumor antigens;

[0082] FIG. 3 is a diagram showing that SIRT1 protein inhibits the expression of immune negative regulatory proteins;

[0083] FIG. 4 is a diagram showing that SIRT1 protein promotes the proliferation of memory T cells in CD3 cells;

[0084] FIG. 5 is a diagram showing that SIRT1 protein promotes the proliferation of memory T cells in CD4 and CD8 cells;

[0085] FIGS. 6-8 are diagrams showing that functional mutants of SIRT1 protein and their combinations inhibit the expression of negative immune regulatory proteins;

[0086] FIGS. 9-10 are diagrams showing that functional mutants of SIRT1 protein and their combinations promote the proliferation of memory T cells in CD3 cells;

[0087] FIG. 11 shows that the functional mutants of SIRT1 protein and their combinations promote the proliferation of memory T cells in CD4 cells;

[0088] FIG. 12 shows that the functional mutants of SIRT1 protein and their combinations promote the proliferation of memory T cells in CD8 cells;

[0089] FIG. 13 shows that the functional mutants of SIRT1 protein and their combinations promote the proliferation of T cells;

[0090] FIG. 14 shows that the functional mutants of SIRT1 protein and their combinations promote the resistance of T cells to immune suppressive micro-environment;

[0091] FIG. 15 shows that CD258 protein and the functional mutant thereof enhance the killing ability of CAR-T cells against tumor cells;

[0092] FIG. 16 shows that the combination of CD258 protein and SIRT1 protein promotes the expansion of specific CAR-T cells induced by tumor antigens;

[0093] FIG. 17 shows that the combination of CD258 protein and SIRT1 protein promotes the proliferation of memory T cells in CD3 cells (GPC3);

[0094] FIG. 18 shows that the combination of CD258 protein and SIRT1 protein promotes the proliferation of memory T cells in CD4 and CD8 cells (GPC3);

[0095] FIG. 19 shows that the combination of CD258 protein and SIRT1 protein inhibits the expression of immune negative regulatory proteins (CD19);

[0096] FIG. 20 shows that the combination of CD258 protein and SIRT1 protein promotes the proliferation of memory T cells in CD8 cells (CD19);

[0097] FIG. 21 shows that the combination of CD258 protein and SIRT1 protein promotes the proliferation of memory T cells in CD3 and CD4 cells Proliferation (CD19);

[0098] FIG. 22 shows that the combination of CD258 protein and SIRT1 protein inhibits the expression of negative immune regulatory proteins (MSLN);

[0099] FIG. 23 shows that the combination of CD258 protein and SIRT1 protein promotes the proliferation of memory T cells in CD3 cells (MSLN);

[0100] FIG. 24 shows that the combination of CD258 protein and SIRT1 protein enhances the killing ability of CAR-T cells against tumor cells;

[0101] FIG. 25 shows that the combination of CD258 protein and SIRT1 protein enhances the anti-tumor effects of CAR-T cells;

[0102] FIG. 26 shows that the combination of CD258 protein and SIRT1 protein promotes the proliferation of granulocytes and monocytes in vivo;

[0103] FIG. 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.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0104] The present invention is further described below by examples. However, it should be understood that the examples are for illustrative purposes only and are not intended to limit the scope and spirit of the present invention.

[0105] Those skilled in the art can easily gain insight into other aspects and advantages of the present invention from the detailed description below. Only exemplary embodiments of the present invention are shown and described in the detailed description below. As those skilled in the art will recognize, the content of the present invention enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention involved in this application.

[0106] Accordingly, the descriptions in the drawings and specifications of this application are merely exemplary and not restrictive.

Example 1 Construction of Lentivirus Vector

[0107] The CAR-T targeting GPC3, CD19, and Mesothelin (MSLN) is used as an example, artificially synthesize a fragment containing the CAR structure and construct it into a lentivirus vector (LV100A, System Biosciences), then transfect in accordance with the method described in its specification to obtain the lentivirus comprising 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. The schematic diagram of each CAR structure is shown in FIG. 1.

[0108] GPC3-CAR is synthesized by splicing 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 in sequence.

[0109] GPC3-S1 was synthesized by splicing 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, and SEQ ID NO: 15 in sequence. [0110] GPC3-S2 was synthesized by splicing 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, and SEQ ID NO:16 in sequence. [0111] GPC3-S3 was synthesized by splicing 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, and SEQ ID NO:17 in sequence. [0112] GPC3-S6 was synthesized by splicing 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, and SEQ ID NO:20 in sequence. [0113] GPC3-S1A was synthesized by splicing 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, and SEQ ID NO:22 in sequence. [0114] 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 spliced and synthesized in sequence. [0115] 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 was spliced and synthesized in sequence. [0116] GPC3-SIC 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 was spliced and synthesized in sequence. [0117] 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 was spliced and synthesized in sequence. [0118] 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 was spliced and synthesized in sequence. [0119] 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 was spliced and synthesized in sequence. [0120] 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 was spliced and synthesized in sequence. [0121] 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 was spliced and synthesized in sequence. [0122] 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 spliced and synthesized in sequence. [0123] 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 spliced and synthesized in sequence. [0124] 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 was spliced and synthesized in sequence. [0125] 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 was spliced and synthesized in sequence. [0126] 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 was spliced and synthesized in sequence. [0127] 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 spliced and synthesized in sequence. [0128] CD19-CAR is synthesized by splicing 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 in sequence. [0129] CD19-S1A is composed 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 spliced and synthesized in sequence. [0130] 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 was spliced and synthesized in sequence. [0131] 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 spliced and synthesized in sequence. [0132] MSLN-CAR is synthesized by splicing 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 in sequence. [0133] 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 was spliced and synthesized in sequence. [0134] 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 was spliced and synthesized in sequence. [0135] 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 was spliced and synthesized in sequence. [0136] 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 was spliced and synthesized in sequence. [0137] 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: Infecting T Cells with Lentivirus

[0138] Infection experiment was performed according to the conventional approach known by those skilled in the art. The infection steps are briefly described as follows: [0139] 1. Obtain peripheral blood mononuclear lymphocytes (PBMCs), obtain more than 110.sup.7 cells through a blood apheresis system. [0140] 2. Treat the cell culture dish with anti-human CD3/CD28 antibodies. [0141] Dilute the anti-human CD3 and anti-human CD28 antibodies (purchased from Shanghai Jinan Technology Co., Ltd.) with PBS to a final concentration of 1 ug/ml, add the diluted antibody mixture to the cell culture dish to cover the entire dish, and incubate at room temperature for 2 hours. 2 hours later, wash once with PBS and set aside. [0142] 3. Activate T cells [0143] Resuspend the separated PBMC with T lymphocyte culture medium (Xvivo15 medium+5% FBS+100 U/mlIL2+20 ng/mlIL21+20 ng/mlIL7) to a final concentration of 110.sup.6 cells/ml, and cultured in the culture dish which are treated in step 2 at 37 C.+5% CO.sub.2, and culture for 24 hours. [0144] 4. Infect the activated T cells [0145] 1) Prepare the infection reagent. [0146] Take a certain amount of T cell culture medium and add synperonic F108 (Sigma) at a final concentration of 1 mg/ml, mix well, and heat to 37 C. in a water bath for later use. [0147] 2) treating the culture plates. [0148] Take 1 mg/ml anti-human CD3 antibody and 0.5 mg/ml anti-human CD28 antibody and dilute them in an appropriate amount of PBS buffer at a volume ratio of 1:1000, and take retronectin (1 mg/ml, Takara) reagent, dilute it into the PBS buffer at a volume ratio of 1:40, mix well and evenly spread it on the cell dish, incubate at room temperature for 2 hours. After 2 hours, wash it with PBS and set aside. [0149] 3) Infecting T cells with Lentivirus. [0150] Dilute the activated T cells with the infection reagent prepared in 1), add lentivirus at an MOI of 3, and mix well. Evenly spread in the culture dish treated in 2). [0151] After infecting, monitor the cell density and maintain the cell density at 110.sup.6 cells/ml. Generally, the cell density can be expanded 30-1000 times in 14 days.

Example 3: SIRT1 Protein Promotes Tumor Antigen-Induced Expansion of Specific CAR-T Cells

[0152] T cells expressing GPC3-CAR, GPC3-S2, GPC3-S3 or GPC3-S6 were co-cultured with irradiated (X-RAD cell irradiator, irradiation dose of 30Gy) HepG2 cells (purchased from the Cell Bank of Chinese Academy of Sciences) at a cell number ratio of 1:1 using Xvivo15 medium, and irradiated HepG2 was re-added every 4 days for stimulation, and the stimulation was repeated 3 times. Each time, the cells were counted by trypan blue staining, and the cell proliferation is shown in FIG. 2A. The results showed that the expansion multiples of T cells expressing GPC3-S2, GPC3-S3 or GPC3-S6 were much higher than those of T cells expressing GPC3-CAR in the control group.

[0153] Similarly, T cells expressing GPC3-CAR, GPC3-S2, GPC3-S3 or GPC3-S6 were co-cultured with irradiated (X-RAD cell irradiator, irradiation dose of 30Gy) Huh7 cells (purchased from the cell bank of the Chinese Academy of Sciences) at a cell number ratio of 1:1 using Xvivo15 medium, and irradiated Huh7 was re-added every 4 days for stimulation, and the stimulation was repeated 3 times. Each time, the cells were counted by trypan blue staining, and the cell proliferation is shown in FIG. 2B. The results also showed that the expansion multiples of T cells expressing GPC3-S2, GPC3-S3 or GPC3-S6 were much higher than those of T cells expressing GPC3-CAR in the control group.

Example 4: SIRT1 Protein Inhibits the Expression of Immune Negative Regulatory Proteins

[0154] The T cells expressing GPC3-CAR, GPC3-S2, GPC3-S3 or GPC3-S6 obtained in Example 2 were cultured in vitro at 37 C. and 5% CO.sub.2 in a cell culture incubator for 9 or 12 days. The expression of CD3, CD4, CD8, PD1, PDL1, TIM3 and LAG3 proteins in T cells was detected by BD flow cytometer. The results are shown in FIG. 3. In T cells expressing GPC3-S2, GPC3-S3 or GPC3-S6, the proportion of cells expressing two negative regulatory proteins at the same time was significantly lower than that of the GPC3-CAR control group.

Example 5: SIRT1 Protein Promotes the Proliferation of Memory T Cells in CD3 Cells

[0155] The T cells expressing GPC3-CAR, GPC3-S2, GPC3-S3 or GPC3-S6 obtained in Example 2 were cultured in vitro at 37 C. and 5% CO.sub.2 in a cell culture incubator for 9 or 12 days. The expression of CD3, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27 and CD28 proteins in T cells was detected by BD flow cytometer. As shown in FIG. 4, the proportion of memory stem cell-like T cells (TSCM) in CD3 cells expressing GPC3-S2, GPC3-S3 or GPC3-S6 in the total cells was significantly higher than that in the GPC3-CAR control group.

Example 6: SIRT1 Protein Promotes the Proliferation of Memory T Cells in CD4 and CD8 Cells

[0156] The T cells expressing GPC3-CAR, GPC3-S2, GPC3-S3 or GPC3-S6 obtained in Example 2 were cultured in vitro in a cell culture incubator at 37 C. and 5% CO.sub.2 for 9 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 cytometer. As shown in FIG. 5A, the proportion of memory stem cell-like T cells (TSCM) in CD4 cells expressing GPC3-S2, GPC3-S3 or GPC3-S6 in the total cells was significantly higher than that in the GPC3-CAR control group.

[0157] Similarly, the T cells expressing GPC3-CAR, GPC3-S2, GPC3-S3 or GPC3-S6 obtained in Example 2 were cultured in vitro at 37 C. and 5% CO.sub.2 in a cell culture incubator for 9 or 12 days. The expression of CD8, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells was detected by BD flow cytometer. As shown in FIG. 5B, the proportion of memory stem cell-like T cells (TSCM) in CD8 cells expressing GPC3-S2, GPC3-S3 or GPC3-S6 in the total cells was significantly higher than that in the GPC3-CAR control group.

Example 7: The Functional Mutants of SIRT1 Protein and their Combinations Inhibit the Expression of Immune Negative Regulatory Proteins

[0158] The T cells expressing GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-SIB, GPC3-S1B1, GPC3-SIC, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 and GPC3-S1A-S3 respectively obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator for 9 days or 12 days. The expression of CD3, CD4, CD8, PD1, PDL1, TIM3, and LAG3 proteins in T cells was detected by BD flow cytometer. The results are shown in FIGS. 6-8. In T cells expressing GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 or GPC3-S1A-S3, the proportion of cells expressing two negative regulatory proteins at the same time was significantly lower than that of the GPC3-CAR control group.

Example 8: The Functional Mutants of SIRT1 Protein and their Combinations Promote the Proliferation of Memory T Cells in CD3 Cells

[0159] The T cells expressing GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-SIB, GPC3-S1B1, GPC3-SIC, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 and GPC3-S1A-S3 obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator for 9 days or 12 days. The expression of CD3, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27 and CD28 proteins in T cells was detected by BD flow cytometer. As shown in FIGS. 9-10, the proportion of memory stem cell-like T cells (TSCM) in CD3 cells expressing GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 and GPC3-S1A-S3 was significantly higher than that in the GPC3-CAR control group.

Example 9: The Functional Mutants of SIRT1 Protein and Combinations Thereof Promote the Proliferation of Memory T Cells in CD4 Cells

[0160] The T cells expressing GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-SIB, GPC3-S1B1, GPC3-SIC, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 and GPC3-S1A-S3 respectively obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture 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 cytometer. As shown in FIG. 11, the proportion of memory stem cell-like T cells (TSCM) in CD4 cells expressing GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 and GPC3-S1A-S3 was significantly higher than that in the GPC3-CAR control group.

Example 10: The Functional Mutants of SIRT1 Protein and Combinations Thereof Promote the Proliferation of Memory T Cells in CD8 Cells

[0161] The T cells expressing GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-SIB, GPC3-S1B1, GPC3-SIC, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 and GPC3-S1A-S3 respectively obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator for 9 days or 12 days. The expression of CD8, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27 and CD28 proteins in T cells was detected by BD flow cytometer. As shown in FIG. 12, the proportion of memory stem cell-like T cells (TSCM) in the CD8 cells expressing GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 and GPC3-S1A-S3 was significantly higher than that in the GPC3-CAR control group.

Example 11: The Functional Mutants of SIRT1 Protein and Combinations Thereof Promote the Proliferation of T Cells

[0162] The T cells expressing GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-SIB, GPC3-S1B1, GPC3-SIC, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, and GPC3-S1A-S3 respectively obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator for 11 days. The cells were counted using trypan blue staining and passaged every 2-3 days. The cell proliferation is shown in FIG. 13A. The results showed that the expansion multiples of T cells expressing GPC3-S1, GPC3-S1A, GPC3-SIB, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 and GPC3-S1A-S3 were much higher than those of T cells expressing GPC3-CAR in the control group.

[0163] Similarly, the T cells expressing GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-SIB, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 and GPC3-S1A-S3 obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator and cultured for 7 days. On the 7th day, the cells were taken and incubated with Xvivo15 culture medium for 24 hours, and CCK8 (MCE Company) was used to detect the proliferation effect of the T cell. As shown in FIG. 13B, the proliferation effect of T cells expressing GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 and GPC3-S1A-S3 was also much higher than that of T cells expressing GPC3-CAR in the control group.

Example 12: The Functional Mutants of SIRT1 Protein and Combinations Thereof Promote the Resistance of T Cells to Immunosuppressive Microenvironment

[0164] The T cells expressing GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-SIB, GPC3-S1B1, GPC3-SIC, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 and GPC3-S1A-S3 respectively obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator for 8 days. On the 8th day, the cells were taken and incubated with 1640 glucose-free and serum-free culture medium (Gibco) for 24 hours, and CCK8 (MCE) was used to detect the proliferation effect of the T cell. The results are shown in FIG. 14A. The proliferation effect of T cells expressing GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-S1C, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 and GPC3-S1A-S3 was much higher than that of T cells expressing GPC3-CAR in the control group.

[0165] Similarly, the T cells expressing GPC3-CAR, GPC3-S1, GPC3-S1A, GPC3-S1B, GPC3-S1B1, GPC3-SIC, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3 and GPC3-S1A-S3 obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator and cultured for 8 days. On the 8th day, the cells were taken and incubated with 1640 glucose-free and serum-free culture medium (Gibco) and 20 mM lactic acid (PH value dropped from normal 7.4 to 6.5, Sigma) for 24 hours, and CCK8 (MCE) was used to detect the T cell proliferation effect. As shown in FIG. 14B, the proliferation effect of T cells expressing GPC3-S1, GPC3-S1A, GPC3-SIB, GPC3-S1B1, GPC3-SIC, GPC3-S1C1, GPC3-S1D, GPC3-S1D1, GPC3-S1E, GPC3-S1E1, GPC3-S1-S3, and GPC3-S1A-S3 was also much higher than that of T cells expressing GPC3-CAR in the control group.

Example 13: CD258 Protein and the Functional Mutant Thereof Enhance the Killing Ability of CAR-T Cells Against Tumor Cells

[0166] T cells expressing GPC3-CAR, GPC3-C8, GPC3-C8A or GPC3-C8B were co-cultured with Huh7 cells (purchased from the cell bank of the Chinese Academy of Sciences) at a cell number ratio of 1:15 using Xvivo15 medium for 7 days, and crystal violet staining (MCE) was used to detect the killing effect of CAR-T cells on tumor cells. The results are shown in FIG. 15A. The killing effect of T cells expressing GPC3-C8 or GPC3-C8A on tumor cells was much higher than that of T cells expressing GPC3-CAR in the control group, among which T cells expressing GPC3-C8A had a more superior killing effect than T cells expressing GPC3-C8.

[0167] Similarly, T cells expressing GPC3-CAR, GPC3-C8, GPC3-C8A or GPC3-C8B were co-cultured with Huh7 cells (purchased from the Cell Bank of Chinese Academy of Sciences) at a cell number ratio of 1:15 using 1640 glucose-free serum-free (Gibco) and 20 mM lactate (PH value dropped from normal 7.4 to 6.5, Sigma) medium for 7 days, and crystal violet staining (MCE) was used to detect the killing effect of CAR-T cells on tumor cells. The results are shown in FIG. 15B. The killing effect of T cells expressing GPC3-C8 or GPC3-C8A on tumor cells is also significantly higher than that of T cells expressing GPC3-CAR in the control group. T cells expressing GPC3-C8A also have a superior killing effect compared to T cells expressing GPC3-C8.

Example 14: The Combination of CD258 Protein and SIRT1 Protein Promotes Tumor Antigen-Induced Specific CAR-T Cell Expansion

[0168] T cells expressing GPC3-CAR, GPC3-S1A, GPC3-C8, GPC3-C8A, GPC3-C8B or GPC3-S1A-C8A were co-cultured with irradiated (X-RAD cell irradiator, irradiation dose of 30Gy) Huh7 cells (purchased from the Cell Bank of Chinese Academy of Sciences) at a 1:1 cell number ratio using Xvivo15 medium, and irradiated Huh7 was re-added every 3-4 days for stimulation, and the stimulation was repeated 4 times. Each time, the cells were counted by trypan blue staining, and the cell proliferation is shown in FIG. 16. The results showed that the expansion multiples of T cells expressing GPC3-S1A, GPC3-C8A, GPC3-C8B or GPC3-S1A-C8A were much higher than those of T cells expressing GPC3-CAR in the control group.

[0169] GPC3-S1A-C8A is consisted of GPC3, S1A and C8A which are coupled 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)

[0170] The T cells expressing GPC3-CAR, GPC3-S1A, GPC3-C8, GPC3-C8A, GPC3-C8B or GPC3-S1A-C8A obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator for 9 or 12 days. The expression of CD3, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27 and CD28 proteins in T cells was detected by BD flow cytometer. As shown in FIG. 17, the proportion of memory stem cell-like T cells (TSCM) in the CD3 cells expressing GPC3-S1A, GPC3-C8, GPC3-C8A, GPC3-C8B or GPC3-S1A-C8A was significantly higher than that in 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)

[0171] T cells expressing GPC3-CAR, GPC3-S1A, GPC3-C8, GPC3-C8A, GPC3-C8B or GPC3-S1A-C8A obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator for 9 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 cytometer. As shown in FIG. 18A, the proportion of memory stem cell-like T cells (TSCM) in the CD4 cells expressing GPC3-S1A, GPC3-C8, GPC3-C8A, GPC3-C8B, or GPC3-S1A-C8A was significantly higher than that in the GPC3-CAR control group. Similarly, the T cells expressing GPC3-CAR, GPC3-S1A, GPC3-C8, GPC3-C8A, GPC3-C8B or GPC3-S1A-C8A obtained in Example 2 were cultured in vitro at 37 C. and 5% CO.sub.2 in a cell culture incubator for 9 or 12 days. The expression of CD8, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27 and CD28 proteins in T cells was detected by BD flow cytometer. As shown in FIG. 18B, the proportion of memory stem cell-like T cells (TSCM) in the CD8 cells expressing GPC3-S1A, GPC3-C8, GPC3-C8A, GPC3-C8B or GPC3-S1A-C8A was significantly higher than that in the GPC3-CAR control group.

Example 17: The Combination of CD258 Protein and SIRT1 Protein Inhibits the Expression of Negative Immune Regulatory Proteins (CD19)

[0172] The T cells expressing CD19-CAR, CD19-S1A, CD19-C8A or CD19-S1A-C8A obtained in Example 2 were cultured in vitro in a cell culture incubator at 37 C. and 5% CO.sub.2 for 9 or 12 days. The expression of CD3, CD4, CD8, PD1, PDL1, TIM3 and LAG3 proteins in T cells was detected by BD flow cytometer. The results are shown in FIG. 19. In T cells expressing CD19-S1A, CD19-C8A or CD19-S1A-C8A, the proportion of cells expressing two negative regulatory proteins at the same time was significantly lower than that in 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)

[0173] The T cells expressing CD19-CAR, CD19-S1A, CD19-C8A, or CD19-S1A-C8A obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator for 9 or 12 days. The expression of CD8, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27 and CD28 proteins in T cells was detected by BD flow cytometer. As shown in FIG. 20, the proportion of memory stem cell-like T cells (TSCM) in the total cells in CD8 cells expressing CD19-S1A, CD19-C8A or CD19-S1A-C8A was significantly higher than that in 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)

[0174] The T cells expressing CD19-CAR, CD19-S1A, CD19-C8A or CD19-S1A-C8A respectively obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator for 9 or 12 days. The expression of CD3, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27, and CD28 proteins in T cells was detected by BD flow cytometer. As shown in FIG. 21A, the proportion of memory stem cell-like T cells (TSCM) in the total cells in CD3 cells expressing CD19-S1A, CD19-C8A or CD19-S1A-C8A was significantly higher than that in the CD19-CAR control group. Similarly, the T cells expressing CD19-CAR, CD19-S1A, CD19-C8A or CD19-S1A-C8A obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator for 9 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 cytometer. As shown in FIG. 21B, the proportion of memory stem cell-like T cells (TSCM) in the total cells in CD4 cells expressing CD19-S1A, CD19-C8A or CD19-S1A-C8A was also significantly higher than that in the CD19-CAR control group.

Example 20: The Combination of CD258 Protein and SIRT1 Protein Inhibits the Expression of Immune Negative Regulatory Proteins (MSLN)

[0175] The T cells expressing MSLN-CAR, MSLN-C8, MSLN-C8A, MSLN-C8B, MSLN-S1A or MSLN-S1A-C8A respectively obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator for 9 or 12 days. The expression of CD3, CD4, CD8, PD1, PDL1, TIM3, and LAG3 proteins in T cells was detected by BD flow cytometer. The results are shown in FIG. 22. In T cells expressing MSLN-C8, MSLN-C8A, MSLN-C8B, MSLN-S1A or MSLN-S1A-C8A, the proportion of cells expressing two negative regulatory proteins at the same time 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)

[0176] The T cells expressing MSLN-CAR, MSLN-C8, MSLN-C8A, MSLN-C8B, MSLN-S1A or MSLN-S1A-C8A respectively obtained in Example 2 were cultured in vitro in a 37 C., 5% CO.sub.2 cell culture incubator for 9 or 12 days. The expression of CD3, CD45RO, CD45RA, CD62L, CCR7, CD95, CD122, CD127, CD27 and CD28 proteins in T cells was detected by BD flow cytometer. As shown in FIG. 23, the proportion of memory stem cell-like T cells (TSCM) in the CD3 cells expressing MSLN-C8, MSLN-C8A, MSLN-C8B, MSLN-S1A or MSLN-S1A-C8A was significantly higher than that in the MSLN-CAR control group.

Example 22: The Combination of CD258 Protein and SIRT1 Protein Enhances the Killing Ability of CAR-T Cells Against Tumor Cells

[0177] T cells expressing GPC3-S1A or GPC3-S1A-C8A were co-cultured with Huh7 cells (purchased from the cell bank of the Chinese Academy of Sciences) at a cell number ratio of 1:15 using Xvivo15 medium for 7 days, and crystal violet staining (MCE) was used to detect the killing effect of CAR-T cells on tumor cells. The results are shown in FIG. 24A. The killing effect of T cells expressing GPC3-S1A-C8A on tumor cells was much higher than that of T cells expressing GPC3-S1A in the control group.

[0178] Similarly, T cells expressing GPC3-CAR or GPC3-S1A-C8A were co-cultured with Huh7 cells (purchased from the cell bank of the Chinese Academy of Sciences) at a cell number ratio of 1:15 for 7 days using Xvivo15 medium, and crystal violet staining (MCE) was used to detect the killing effect of CAR-T cells on tumor cells. The results are shown in FIG. 24B. The killing effect of T cells expressing GPC3-S1A-C8A on tumor cells is also much higher than that of T cells expressing GPC3-CAR in the control group.

Example 23: The Combination of CD258 Protein and SIRT1 Protein Enhances the Anti-Tumor Effect of CAR-T Cells

[0179] Huh7 cells (110.sup.7/mouse) were subcutaneously inoculated into NSG mice (purchased from Biocytogen). After 14 days, the tumor volume of the mice was measured to be about 200 mm.sup.3. At this time, the mice were divided into 5 groups, namely T, GPC3-CAR and GPC3-S1A-C8A groups, with 6-8 mice in each group. Then, T cells (910.sup.5/mouse) were injected into the T cell group through the tail vein, CAR-T cells expressing GPC3-CAR were injected into the GPC3-CAR group (310.sup.5/mouse or 910.sup.5/mouse), and CAR-T cells expressing GPC3-S1A-C8A were injected into the GPC3-S1A-C8A group (310.sup.5/mouse or 910.sup.5/mouse). The tumor volume was measured on Mondays and Thursdays of each week, and the death of mice was recorded. As shown in FIG. 25, the tumor inhibition 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

[0180] Huh7 cells (110.sup.7/mouse) were subcutaneously inoculated into NSG mice (purchased from Biocytogen). After 14 days, the tumor volume of the mice was measured to be about 200 mm.sup.3. At this time, the mice were divided into 5 groups, namely T, GPC3-CAR and GPC3-S1A-C8A groups, with 6 mice in each group. Then, T cells (910.sup.5/mouse) were injected into the T cell group through the tail vein, CAR-T cells expressing GPC3-CAR (310.sup.5/mouse or 910.sup.5/mouse) were injected into the GPC3-CAR group, and CAR-T cells expressing GPC3-S1A-C8A (310.sup.5/mouse or 910.sup.5/mouse) were injected into the GPC3-S1A-C8A group. On the 7th day, 50 l of blood was collected from the tail of the mouse, and the number and size of monocytes and neutrophils in each group were detected by BD flow cytometry. As shown in FIG. 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

[0181] Huh7 cells (110.sup.7/mouse) were subcutaneously inoculated into NSG mice (purchased from Biocytogen). After 14 days, the tumor volume of the mice was measured to be about 200 mm.sup.3. At this time, the mice were divided into 5 groups, namely T, GPC3-CAR and GPC3-S1A-C8A groups, with 6 mice in each group. Then, T cells (910.sup.5/mouse) were injected into the T cell group through the tail vein, CAR-T cells expressing GPC3-CAR were injected into the GPC3-CAR group (310.sup.5/mouse or 910.sup.5/mouse), and CAR-T cells expressing GPC3-S1A-C8A were injected into the GPC3-S1A-C8A group (310.sup.5/mouse or 910.sup.5/mouse). On the 14th day, 50 l of blood was collected from the tail of the mice, and the expression of human CD3, CD4 and CD8 proteins in each group was detected by BD flow cytometry. The results are shown in FIGS. 27A, 27B and 27C. In the groups expressing CD3, CD4 and CD8, the proportion of cells expressing CD3, CD4 or CD8 proteins in the GPC3-S1A-C8A group to the total number of cells was much higher than that in the GPC3-CAR control group.

[0182] Similarly, Huh7 cells (110.sup.7/mouse) were subcutaneously inoculated into NSG mice (purchased from Biocytogen). After 14 days, the tumor volume of the mice was measured to be about 200 mm.sup.3. At this time, the mice were divided into 5 groups, namely T, GPC3-CAR and GPC3-S1A-C8A groups, with 6 mice in each group. Then, T cells (910.sup.5/mouse) were injected into the T cell group through the tail vein, CAR-T cells expressing GPC3-CAR (310.sup.5/mouse or 910.sup.5/mouse) were injected into the GPC3-CAR group, and CAR-T cells expressing GPC3-S1A-C8A (310.sup.5/mouse or 910.sup.5/mouse) were injected into the GPC3-S1A-C8A group. On the 7th day, 50 l of blood was collected from the tail of the mice, and the expression of human IL-2, IL4, IL6, IL10, TNF- and IFN- cytokines in each group was detected by BD flow cytometry. The results are shown in FIG. 27D. The expression of IFN- cytokine in the GPC3-S1A-C8A group was significantly higher than that in the GPC3-CAR control group.