Genetically engineered cells and their applications

Abstract

The present invention discloses a genetically engineered cell, which expresses an exogenous receptor and exogenous CCL21 that specifically bind to a target antigen, and can further express an IL-7R binding protein or exogenous IL-7 that promotes cell proliferation. Also disclosed is an expression construct including an exogenous CCL21 expression cassette, a vector containing the same, a virus, and a pharmaceutical composition including the above-mentioned cell. Also disclosed is the use of cells, expression constructs, vectors, and viruses in the preparation of drugs for inhibiting tumors or pathogens.

Description

[0001] This application claims the priority of Chinese patent application CN201810463564.7 filed on May 15, 2018, Chinese patent application CN201811088090.9 filed on September 18, 2018, Chinese patent application CN201811552806.6 filed on December 19, 2018, and Chinese patent application CN201910151930.X filed on February 28, 2019. This application cites the full text of the above Chinese patent application. Technical Field

[0002] The present invention belongs to the field of cell therapy, and relates to genetically engineered cells and applications. More specifically, the present invention relates to cells that specifically bind to exogenous receptors of target antigens and exogenous CCL21. Background Art

[0003] CAR-T cells can specifically kill tumors in an MHC-non-restricted manner and show good application prospects in tumor immunotherapy, but they still have many limitations, such as poor efficacy against solid tumors and candidate drugs that show excellent effects in vitro often fail to show corresponding effects in vivo.

[0004] Adachi et al. tried to use CAR-T cells expressing IL7 and CCL19 (IL-7and CCL19 expression inCAR-T cells improves immune cell infiltration and CAR-T cell survival in the tumor. Nature Biotechnology, 2018, 36(4), 346–351) to improve the anti-tumor ability of CAR-T cells. Summary of the invention

[0005] The object of the present invention is to provide a genetically engineered cell.

[0006] In a first aspect of the present invention, a genetically engineered cell is provided, wherein the cell comprises an exogenous receptor that specifically binds to a target antigen and exogenous CCL21.

[0007] In a specific embodiment, the cell expresses an exogenous receptor that specifically binds to a target antigen, exogenous CCL21, and a protein that promotes proliferation of the cell. Preferably, the protein that promotes proliferation of the cell is an IL-7R binding protein or exogenous IL-7.

[0008] In a specific embodiment, the IL-7R binding protein is an exogenous IL-7R binding protein, that is, the cell comprises an exogenous receptor that specifically binds to a target antigen, exogenous CCL21, and an exogenous IL-7R binding protein.

[0009] In a specific embodiment, the exogenous IL-7R binding protein can specifically bind to IL-7R and enhance IL-7R activity.

[0010] In a specific embodiment, the exogenous IL-7R binding protein is selected from an antibody of IL-7R. Preferably, the amino acid sequence of the exogenous IL-7R is as shown in SEQ ID NO:19.

[0011] In a specific embodiment, the exogenous CCL21 is natural CCL21, or a truncated fragment of natural CCL21 or a mutant of natural CCL21 having the same function as natural CCL21.

[0012] In a specific embodiment, the natural CCL21 has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 21, or is a truncated fragment of the amino acid sequence shown in SEQ ID NO: 21; or has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the amino acid sequence encoded by the nucleotides shown in SEQ ID NO: 14 or 15, or is a truncated fragment of the amino acid sequence encoded by the nucleotides shown in SEQ ID NO: 14 or 15. In a preferred example, the natural CCL21 is human CCL21, whose amino acid sequence is shown in SEQ ID NO: 21; or its amino acid sequence is encoded by the nucleotides shown in SEQ ID NO: 14 or 15.

[0013] In a specific embodiment, the exogenous CCL21 is constitutively expressed.

[0014] In a specific embodiment, the exogenous CCL21 is inducibly expressed. In a preferred embodiment, the inducible expression is initiated by an immune cell inducible promoter. In a preferred embodiment, the immune cell inducible promoter is a NFAT promoter.

[0015] In a specific embodiment, the exogenous IL-7 is natural IL-7, or a truncated fragment of natural IL-7 or a mutant of natural IL-7 having the same function as natural IL-7.

[0016] In a specific embodiment, the amino acid sequence of the natural IL-7 has at least 90% identity with the sequence shown in SEQ ID NO: 18, or is a truncated fragment of the amino acid sequence shown in SEQ ID NO: 18; or has at least 90% identity with the amino acid sequence encoded by the nucleotides shown in SEQ ID NO: 13, or is a truncated fragment of the amino acid sequence encoded by the nucleotides shown in SEQ ID NO: 13.

[0017] In a specific embodiment, the exogenous IL-7R binding protein or exogenous IL-7 is constitutively expressed.

[0018] In a preferred embodiment, the exogenous IL-7R binding protein or exogenous IL-7 is expressed inducibly.

[0019] In a preferred embodiment, the inducible expression is initiated by an immune cell inducible promoter. In a preferred embodiment, the immune cell inducible promoter is a NFAT promoter.

[0020] In a specific embodiment, the cell is an immune effector cell. In a specific embodiment, the immune effector cell is selected from T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells or bone marrow-derived phagocytes or a combination thereof; preferably, the immune effector cell is selected from T cells and NK cells; more preferably, the immune effector cell is a T cell.

[0021] In a specific embodiment, the cells are derived from autologous cells; preferably, they are autologous T cells or autologous NK cells; more preferably, they are autologous T cells.

[0022] In a specific embodiment, the cells are derived from allogeneic cells; preferably, they are allogeneic T cells or allogeneic NK cells (also including NK cell lines, such as NK92 cells).

[0023] In a specific embodiment, the target antigen is a tumor antigen or a pathogen antigen.

[0024] In a specific embodiment, the target antigen is a tumor antigen. In a preferred embodiment, the tumor antigen is selected from: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit α (IL-13Rα); interleukin 11 receptor α (IL-11Rα); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; proteinase serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor β (PD GFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor; tumor endothelial marker 25 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); Claudin 6, Claudin18.2 (CLD18A2), Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; kappa light chain (kappa light chain); CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tenascin; oncofetal variant of tumor necrosis; G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1);Hexose moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor 5 beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2) T-2); Fos-related antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocytic virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); I Fc fragment of gA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1). ;

[0025] In a preferred example, the tumor antigen is GPC3, EGFR, EGFRvIII or Claudin18.2.

[0026] In a specific embodiment, the target antigen is a pathogen antigen. In a preferred embodiment, the pathogen antigen is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites. In a specific embodiment, the viral antigen is selected from: cytomegalovirus antigens, Epstein-Barr virus antigens, human immunodeficiency virus antigens, or influenza virus antigens.

[0027] In a specific embodiment, the exogenous receptor is a chimeric receptor, which comprises an antigen binding domain, a transmembrane domain and an intracellular domain.

[0028] In a specific embodiment, the exogenous receptor is a chimeric receptor, which is selected from: a chimeric antigen receptor (CAR), a modified T cell (antigen) receptor (TCR), a T cell fusion protein (TFP), a T cell antigen coupler (TAC) or a combination thereof.

[0029] In a preferred embodiment, the exogenous receptor is a chimeric antigen receptor, the antigen binding domain of which comprises: an antibody, an antibody fragment, scFv, Fv, Fab, (Fab')2, a single domain antibody (SDAB), a VH or VL domain, or a camelid VHH domain, or a natural ligand of the corresponding antigen, or a combination thereof.

[0030] In a preferred embodiment, the exogenous receptor is a chimeric antigen receptor, the transmembrane domain of which comprises a transmembrane domain of a protein selected from the group consisting of: α, β or ζ chain of a T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2 , CD27, LFA-1(CD11a, CD18), ICOS(CD278), 4-1BB(CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7 , NKp80(KLRF1), CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA -6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD2 9. ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEAC AM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C.

[0031] In a preferred embodiment, the exogenous receptor is a chimeric antigen receptor, the intracellular domain of which includes: a primary signaling domain and / or a co-stimulatory signaling domain, wherein: (1) the primary signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεR1b), CD79a, CD79b, FcγRIIa, DAP10, and DAP12, or a combination thereof; and / or (2) the co-stimulatory signaling domain comprises a protein selected from the group consisting of: Functional signaling domains of proteins: CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, CD83-specific ligand, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2 Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITG AM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), C D84, 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, or a combination thereof.

[0032] In a specific embodiment, the chimeric antigen receptor comprises: (i) an antibody or a fragment thereof that specifically binds to the target antigen, a transmembrane domain of CD28 or CD8, a costimulatory signaling domain of CD28, and CD3ζ; or (ii) an antibody or a fragment thereof that specifically binds to the target antigen, a transmembrane domain of CD28 or CD8, a costimulatory signaling domain of 4-1BB, and CD3ζ; or (iii) an antibody or a fragment thereof that specifically binds to the target antigen, a transmembrane domain of CD28 or CD8, a costimulatory signaling domain of CD28, a costimulatory signaling domain of 4-1BB, and CD3ζ.

[0033] In a specific embodiment, the amino acid sequence of the antigen binding domain of the exogenous receptor is at least 90% identical to the sequence shown in SEQ ID NO:2.

[0034] In a specific embodiment, the amino acid sequence of the exogenous receptor is at least 90% identical to the sequence shown in SEQ ID NO:26, 27 or 35.

[0035] In a specific embodiment, the exogenous receptor, and / or exogenous IL-7R binding protein, and / or exogenous CCL21 are expressed using a viral vector. Preferably, the viral vector includes: a lentiviral vector, a retroviral vector or an adenoviral vector.

[0036] In the second aspect of the present invention, an expression construct is provided, which comprises sequentially connected: an expression cassette 1 of an exogenous receptor that specifically binds to a target antigen, an expression cassette 2 of an exogenous IL-7R binding protein or an exogenous IL-7, and an expression cassette 3 of an exogenous CCL21; preferably, the expression cassettes are connected by a tandem fragment selected from F2A, PA2, T2A, and / or E2A. The nucleic acid sequences of F2A and P2A are shown in SEQ ID NO: 11 and SEQ ID NO: 16, respectively.

[0037] In the third aspect of the present invention, an expression vector is provided, which comprises the expression construct described in the second aspect of the present invention.

[0038] In the fourth aspect of the present invention, a virus is provided, wherein the virus comprises the expression vector described in the third aspect of the present invention.

[0039] In the fifth aspect of the present invention, a method for improving the viability of immune response cells is provided, the method comprising co-expressing in immune response cells: a chimeric antigen receptor that specifically binds to a target antigen as described in the first aspect of the present invention, an exogenous IL-7R binding protein or exogenous IL-7, and exogenous CCL21.

[0040] In the sixth aspect of the present invention, there is provided a use of the cell described in the first aspect of the present invention, or the expression construct described in the second aspect of the present invention, or the expression vector described in the third aspect of the present invention, or the virus described in the fourth aspect of the present invention, for preparing a drug for inhibiting tumors, inhibiting pathogens, or enhancing the immune tolerance of a subject. In a specific embodiment, it is used to prepare a drug for inhibiting tumors. In a preferred embodiment, the drug for inhibiting tumors is used in combination with a chemotherapeutic drug.

[0041] In a specific embodiment, the tumor is a blood tumor.

[0042] In a specific embodiment, the tumor is a solid tumor.

[0043] In a specific embodiment, the tumor is selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, malignant melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, Thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of said cancers and metastatic lesions of said cancers.

[0044] In a preferred embodiment, the solid tumor is selected from colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulva cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma.

[0045] More preferably, the solid tumor is selected from colon cancer, rectal cancer, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, pancreatic cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer. More preferably, the solid tumor is gastric cancer, pancreatic cancer, or esophageal cancer.

[0046] In the seventh aspect of the present invention, a pharmaceutical composition is provided, comprising the cell according to the first aspect of the present invention and a pharmaceutically acceptable carrier or excipient.

[0047] In the eighth aspect of the present invention, a kit is provided, comprising kit A and kit B, wherein kit A comprises genetically engineered cells, wherein the cells comprise the exogenous receptor expressing specific binding to the target antigen as described in the first aspect of the present invention; and kit B comprises CCL21, and / or, a protein that promotes the proliferation of the cells. Preferably, the protein that promotes cell proliferation comprises the IL-7R binding protein or IL-7 as described in the first aspect of the present invention. More preferably, the administration of kit A and kit B is not in order.

[0048] In a preferred embodiment, the drug kit A comprises immune effector cells modified with chimeric receptors. Preferably, the chimeric receptor is a chimeric antigen receptor.

[0049] In a preferred example, the immune effector cells are T cells, NK cells or NKT cells.

[0050] In the ninth aspect of the present invention, a method for inhibiting tumors or pathogens or strengthening the immune tolerance of a subject is provided, which comprises administering the cell described in the first aspect of the present invention, or the pharmaceutical composition described in the seventh aspect of the present invention, or the drug kit described in the eighth aspect of the present invention. Preferably, it also comprises administering a chemotherapeutic drug.

[0051] Beneficial effects of the present invention:

[0052] 1. The cells provided by the present invention can improve the survival and accumulation capabilities of the cells by co-expressing an exogenous receptor that specifically binds to a target antigen, an exogenous IL-7R binding protein or exogenous IL-7, and exogenous CCL21.

[0053] 2. The immune effector cells prepared by the technical solution of the present invention have excellent tumor cell killing ability.

[0054] 3. The cells prepared by the technical solution of the present invention can fight against the immunosuppression in the cancer microenvironment in the treatment of cancer, thereby significantly enhancing the effect on solid tumors. It also has a good effect on refractory and progressive cancers. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1A is the plasmid map of MSCV-hu8E5(2I)-m28Z;

[0056] Figure 1B This is the plasmid map of MSCV-hu8E5(2I)-m28Z-F2A-mIL-7-P2A-mCCL21a;

[0057] Figure 1C This is the plasmid map of MSCV-hu8E5(2I)-m28Z-F2A-mIL7-P2A-mCCL21b;

[0058] Figure 1D This is the plasmid map of MSCV-hu8E5(2I)-mBBZ;

[0059] Figure 1E This is the plasmid map of MSCV-hu8E5(2I)-mBBZ-F2A-mIL-7-P2A-mCCL21a;

[0060] Figure 1F This is the plasmid map of MSCV-hu8E5(2I)-m28Z-F2A-mIL7-P2A-mCCL21b;

[0061] Figure 2 The results of in vitro cytokine IL7 and CCL21 assays are shown;

[0062] Figure 3A and 3B The secretion of PD-1 by cells in different groups is shown; Figure 3C and 3D The secretion of LAG-3 by cells from different groups is shown; Figure 3E and 3F The secretion of TIM-3 by cells in different groups is shown;

[0063] Figure 4A 4B shows the in vitro killing results of BBZ;

[0064] Figure 5 Results of in vitro proliferation assays are shown;

[0065] Figure 6 The results of in vivo tumor treatment experiments in mice are shown;

[0066] Figure 7 This is the plasmid map of mBBZ-7*19;

[0067] Fig. 8A The in vivo killing comparison results of CAR-T cells expressing IL7 and CCL21 and CAR-T cells expressing IL7 and CCL19 are shown; Figure 8B Changes in mouse body weight are shown; Figure 8C Comparative results of tumor weights are shown; Fig.8D The CAR-T cell copy number after pancreatic cancer PANC02-A2 treatment in mice is shown; Fig. 8E The results of immunohistochemical detection of CD8+ cells in mouse pancreatic cancer are shown;

[0068] Fig.9A Shows the changes in tumor volume of mouse breast cancer E0771-A2 orthotopic transplanted tumors after CAR-T cell treatment;

[0069] Fig. 9B The tumor weight of mouse breast cancer E0771-A2 orthotopic xenografts after treatment is shown;

[0070] Fig. 9C The CAR-T cell copy number after breast cancer treatment in mice is shown;

[0071] Fig.9D The results of immunohistochemical detection of CD8+ cells in mouse breast cancer are shown;

[0072] Fig. 10A Shows the changes in tumor volume after treatment of mouse liver cancer Hepal-6-A2 transplanted tumors;

[0073] Fig. 10B The tumor weights of mouse hepatocellular carcinoma Hepa1-6-A2 xenografts after treatment are shown;

[0074] Fig. 10C The CAR-T cell copy number after liver cancer treatment in mice is shown;

[0075] Fig. 10D The results of immunohistochemical detection of mouse liver cancer CD8+ cells are shown;

[0076] Fig.11 The results of in vitro IFN-γ assays are shown;

[0077] Fig. 12A The changes in tumor volume after CAR-T cell treatment in the subcutaneous tumor-clearing lymphoma model of pancreatic cancer in mice are shown; Fig. 12B The tumor weight after treatment of the mouse pancreatic cancer subcutaneous tumor ablation model is shown; Fig. 12C The CAR-T cell copy number after subcutaneous tumor ablation in pancreatic cancer mice model is shown; Fig.12D Shown are the results of immunohistochemical detection of CD8+ cells in the mouse pancreatic cancer subcutaneous tumor ablation model.

[0078] Fig.13A The results show the detection of Tcm in the spleen of the mouse pancreatic cancer PANC02-A2 subcutaneous tumor model on day 10 of CAR-T treatment; Fig. 13B The detection of Tcm in spleen on d20 is shown;

[0079] Fig.14A The content of Tcm in the bone marrow of the mouse pancreatic cancer PANC02-A2 subcutaneous tumor model on day 10 of CAR-T treatment is shown; Fig. 14B The detection of Tcm in the bone marrow on day d20 is shown;

[0080] Fig.15 It shows that there are more DC cell infiltration in the mouse tumor tissue of the mouse pancreatic cancer PANC02-A2 subcutaneous tumor model after CAR-T treatment on day 10;

[0081] Fig.16 The figure shows the content of MDSC in the tumor tissue of mice treated with CAR-T for d10 in the mouse pancreatic cancer PANC02-A2 subcutaneous tumor model. DETAILED DESCRIPTION

[0082] The present invention finds that immune effector cells expressing exogenous receptors targeting tumor antigens and CCL21 not only have a more excellent killing effect on tumors, but also improve the survival of immune effector cells in tumor tissues, and show more excellent anti-tumor ability even for refractory solid tumors.

[0083] According to the present disclosure, those skilled in the art will appreciate that many changes or modifications can be made in the disclosed specific embodiments, and still obtain the same or similar results, without departing from the spirit and scope described herein. The present invention is not limited in scope to the specific embodiments described herein (which are only intended to be illustrative of the various aspects described herein), and it should be understood that functionally equivalent methods and components are still included in the scope described herein.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art in the fields of gene therapy, biochemistry, genetics and molecular biology. All methods and materials similar or equivalent to those described herein can be used in the practice or testing described herein. These techniques, such as methods and materials, are fully described in the literature, see, for example, unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and son Inc, Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al, 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJ Gaited., 1984); Mullis et al. US Pat. No. 4,683,195; Nucleic Acid Hybridization (BD Harries & S. J. Higgins eds. 1984); Transcription And Translation (BDHames&S.J.Higginseds.1984); Culture OfAnimal Cells (RIFreshney, Alan R.Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols. 154 and 155 (Wuetal. eds.) and Vol. 185, "GeneExpression Technology" (D. Goeddel, ed.); Gene Transfer Vectors For Mammalian Cells (JHMiller and MPCaloseds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Hand book Of Experimental Immunology, Volumes I-IV (DMWeir and CC Blackwell, eds., 1986); Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). .

[0085] All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification shall prevail. In addition, unless otherwise specified, the materials, methods and embodiments listed in this specification are illustrative only and are not intended to be limiting.

[0086] The term "engineering" as used herein and its grammatical other forms can refer to one or more changes in nucleic acids, such as nucleic acids in the genome of an organism. The term "engineering" can refer to changes, additions and / or deletions of genes. Engineered cells can also refer to cells with added, deleted and / or altered genes.

[0087] The term "genetically engineered cells" as used herein refers to cells transformed by means of genetic engineering. In some embodiments, the cells are immune effector cells. In some embodiments, the cells are T cells. In some embodiments, the genetically engineered cells described herein refer to cells expressing exogenous receptors that specifically bind to target antigens. In some embodiments, the genetically engineered cells described herein refer to cells that express exogenous receptors that specifically bind to target antigens and express exogenous CLL21. In some embodiments, the genetically engineered cells described herein may also be T cells that co-express chimeric antigen receptors that specifically bind to tumor antigens, CLL21, and proteins that promote T cell proliferation. In some embodiments, the genetically engineered cells described herein may also be T cells that co-express chimeric antigen receptors that specifically bind to tumor antigens, CLL21, and IL-7R binding proteins or exogenous IL-7.

[0088] The term "immune effector cell" refers to cells that participate in immune responses and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes. In some embodiments, the immune effector cells are T cells, NK cells, and NKT cells. In some embodiments, the T cells can be autologous T cells, xenogeneic T cells, and allogeneic T cells. In some embodiments, the NK cells can be allogeneic NK cells.

[0089] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds consisting of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can comprise the sequence of a protein or peptide. Polypeptides include any peptide or protein containing two or more amino acids bound to each other by peptide bonds. As used herein, the term refers to short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides and oligomers) as well as longer chains (which are also commonly referred to in the art as proteins, which exist in many types). "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.

[0090] The term "IL-7" refers to a protein (preferably from a mammal, such as a mouse or a human) that can interact with (e.g., bind to) IL-7R (preferably from a mammal, such as a mouse or a human IL-7R), and has one of the following characteristics: (i) an amino acid sequence of a naturally occurring mammalian IL-7 or a fragment thereof, such as an amino acid sequence or a fragment thereof as shown in SEQ ID NO: 18 (human); (ii) an amino acid sequence that is substantially homologous to the amino acid sequence shown in SEQ ID NO: 18 (human) or a fragment thereof, such as at least 85%, 90%, 95%, 96%, 97%, 98%, 99%; (iii) an amino acid sequence encoded by a naturally occurring mammalian IL-7 nucleotide sequence or a fragment thereof (e.g., SEQ ID NO: 17 (human) or a fragment thereof); (iv) an amino acid sequence encoded by a naturally occurring mammalian IL-7 nucleotide sequence or a fragment thereof (e.g., SEQ ID NO: 18 (human) or a fragment thereof); The nucleotide sequence shown in NO:17 (human) or a fragment thereof has, for example, an amino acid sequence encoded by a nucleotide sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology; (v) an amino acid sequence encoded by a nucleotide sequence that is degenerate to a naturally occurring IL-7 nucleotide sequence or a fragment thereof (e.g., SEQ ID NO:17 (human) or a fragment thereof); or (vi) a nucleotide sequence that hybridizes to one of the aforementioned nucleotide sequences under stringent conditions.

[0091] "Exogenous IL-7R binding protein" refers to all proteins that can specifically bind to IL-7R and enhance IL-7R activity. "Enhancing IL-7R activity" should be understood to mean that the IL-7R binding protein can enhance any one or more activities of naturally occurring IL-7R, including but not limited to stimulating the proliferation, cytotoxicity or maturation of NK cells; stimulating the proliferation or differentiation of B cells and T cells; stimulating antibody production and affinity maturation in B cells; stimulating the cytotoxicity of CD8+T cells; stimulating interferon gamma production in T cells and NK cells; inhibiting dendritic cell (DC) activation and maturation; inhibiting the release of inflammatory mediators from mast cells; enhancing the phagocytosis of macrophages; inhibiting the generation or survival of TReg cells; and stimulating the proliferation of bone marrow progenitor cells.

[0092] "CCL21 (Chemokine (CC motif) ligand 21)" is one of the major immune chemokines, expressed in the T cell zone of secondary lymphoid tissues in the spleen and lymph nodes, and is responsible for the recruitment of antigen-activated (mature) dendritic cells (DCs), immature DCs and naive T cells. The CCL21 of the present invention has one of the following characteristics: (i) an amino acid sequence of a naturally occurring mammalian CCL21 or a fragment thereof, such as an amino acid sequence or a fragment thereof as shown in SEQ ID NO: 21 (human); (ii) an amino acid sequence having, for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the amino acid sequence or a fragment thereof as shown in SEQ ID NO: 21 (human); (iii) an amino acid sequence encoded by a naturally occurring mammalian CCL21 nucleotide sequence or a fragment thereof (such as SEQ ID NO: 20 (human) or a fragment thereof); (iv) an amino acid sequence encoded by a nucleotide sequence having, for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% homology to the nucleotide sequence or a fragment thereof as shown in SEQ ID NO: 20 (human); (v) an amino acid sequence encoded by a nucleotide sequence degenerate to a naturally occurring CCL21 nucleotide sequence or a fragment thereof (such as SEQ ID NO: NO:20 (human) or a fragment thereof); or (vi) a nucleotide sequence that hybridizes with one of the aforementioned nucleotide sequences under stringent conditions.

[0093] The term "amino acid modification" includes amino acid substitutions, additions and / or deletions, and "amino acid substitution" means replacing an amino acid at a specific position in a parent polypeptide sequence with another amino acid. "Amino acid insertion" as used herein means adding an amino acid at a specific position in a parent polypeptide sequence. "Amino acid deletion" or "deletion" as used herein means removing an amino acid at a specific position in a parent polypeptide sequence. The term "conservative modification" as used herein means an amino acid modification that does not significantly affect or change the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions. Modifications can be introduced into the antibodies of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions that replace amino acid residues with amino acid residues having similar side chains. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged acute side chains (e.g., glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0094] The terms "wild type", "parent", and "natural" used in this article have the same meaning when referring to proteins and DNA. The terms "mutation", "variant" or "mutant" have the same or better biological activity as natural protein or natural DNA, and one or more amino acids are substituted, added or deleted in the amino acid sequence of natural protein; or one or more nucleotides are substituted, added or deleted in the nucleic acid sequence of natural DNA. In a specific embodiment, the sequence of the mutant in this article has at least about 80%, preferably at least about 90%, more preferably at least about 95%, more preferably at least about 97%, more preferably at least about 98%, and most preferably at least about 99% identity with the amino acid sequence of natural protein or the nucleic acid sequence of natural DNA. For example, "variant of IL-7" generally refers to a polypeptide having similar biological activity or better biological activity as IL-7 obtained by amino acid modification of wild-type IL-7. The term "truncated fragment" refers to a non-full-length form of a natural protein or natural DNA, which has a continuous or non-continuous deletion of multiple amino acid residues or nucleotides in the natural amino acid sequence or nucleic acid sequence, and the deletion occurs at any position of the sequence, such as the head, middle, tail or a combination thereof. In the present invention, the truncated fragment of the protein still retains the same function as the natural protein from which it is derived.

[0095] "Constitutive expression" is also called continuous expression, which means that the gene can be continuously expressed in the cell under almost all physiological conditions. "Inducible expression" refers to expression under certain conditions, such as when T cells bind to antigens.

[0096] The term "effective amount" refers to the amount of a compound, preparation, substance or composition that is effective to achieve a specific biological result, such as but not limited to an amount or dosage sufficient to promote a T cell response. When indicating "immunologically effective amount", "anti-tumor effective amount", "tumor inhibition effective amount" or "therapeutically effective amount", the precise dosage of the immune effector cells or therapeutic agents of the present invention can be determined by a physician taking into account the individual's age, weight, tumor size, or degree of metastasis and the condition of the patient (subject). An effective amount of immune effector cells refers to but is not limited to an increase, enhancement or extension of the anti-tumor activity of immune effector cells; an increase in the number of anti-tumor immune effector cells or activated immune effector cells; promotion of IFN-γ secretion; the number of immune effector cells that cause tumor regression, tumor shrinkage, and tumor necrosis.

[0097] The term "promoter" as used herein is a DNA sequence recognized by the synthetic machinery of the cell or introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence.

[0098] A typical eukaryotic promoter consists of a minimal promoter and other cis elements. The minimal promoter is essentially a TATA box region, where RNA polymerase II (polII), TATA binding protein (TBP) and TBP-associated factor (TAF) can bind to initiate transcription. It has been found that such sequence elements (e.g., enhancers) increase the overall expression level of nearby genes, often in a manner that is independent of position and / or orientation.

[0099] "NFAT" (Nuclear factor of activated T cells) is a nuclear factor of activated T cells. In some embodiments, NFAT plays an important role in the transcriptional expression of cytokines during T cell activation. In some embodiments, CCL21 or IL-7 is inducibly expressed using an inducible promoter. In some embodiments, the inducible promoter is a NFAT promoter. In some embodiments, the CCL21 or IL-7 coding sequence is placed under the regulation of a minimal promoter containing a NFAT binding motif. In some embodiments, the IL2 minimal promoter containing 6 NFAT binding motifs is a promoter composed of 6 NFAT binding sites and the IL2 minimal promoter (minimal promoter) in series.

[0100] In some embodiments, the antigen-binding receptor described herein refers to a chimeric receptor. "Chimeric receptor" as used herein refers to a fusion molecule formed by connecting DNA fragments or cDNAs corresponding to proteins from different sources using genetic recombination technology. Chimeric receptors generally include an extracellular domain, a transmembrane domain, and an intracellular domain. Chimeric receptors that can be used in the present invention include, but are not limited to, chimeric antigen receptors (CAR), modified T cell (antigen) receptors (TCR), T cell fusion proteins (TFP), and T cell antigen couplers (TAC).

[0101] The term "open reading frame (ORF)" refers to the normal nucleotide sequence of a structural gene. The reading frame from the start codon to the stop codon can encode a complete polypeptide chain without the presence of a stop codon that interrupts translation.

[0102] "Chimeric antigen receptor" or "CAR" as used herein refers to a group of polypeptides that, when in immune effector cells, provide the cells with specificity for target cells (usually cancer cells) and have intracellular signal generation. CAR generally includes at least one extracellular antigen binding domain (also referred to as an extracellular region), a transmembrane domain (also referred to as a transmembrane region), and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain" or "intracellular region"), which includes a functional signaling domain derived from a stimulatory molecule and / or a co-stimulatory molecule defined as follows. In some aspects, the polypeptide groups are adjacent to each other. The polypeptide group includes a dimerization switch that can couple polypeptides to each other in the presence of a dimerization molecule, for example, an antigen binding domain can be coupled to an intracellular signaling domain. In one aspect, the stimulatory molecule is a ζ chain bound to a T cell receptor complex. In one aspect, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one co-stimulatory molecule defined as follows. In one aspect, the costimulatory molecule is selected from the costimulatory molecules described herein, such as 4-1BB (ie, CD137), CD27 and / or CD28. In one aspect, CAR includes a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and two functional signaling domains derived from one or more costimulatory molecules.

[0103] In one aspect, the present invention contemplates the modification of the amino acid sequence of the starting antibody or fragment (e.g., scFv) of the functional equivalent molecule.For example, the VH or VL of the antigen binding domain of the cancer-associated antigen described herein, such as the scFv contained in the CAR, can be modified to retain the starting VH or VL framework region (e.g., scFv) of the antigen binding domain of the cancer-associated antigen described herein. At least about 70%, 71%, 72%. 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. The present invention contemplates the modification of the entire CAR construct, such as the modification of one or more amino acid sequences of multiple domains of the CAR construct, to produce functional equivalent molecules. The CAR construct can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity of the starting CAR construct.

[0104] As used herein, a "transmembrane domain" (also referred to as a transmembrane region) may include one or more additional amino acids adjacent to the transmembrane region, such as one or more amino acids associated with the extracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids in the extracellular region) and / or one or more additional amino acids associated with the extracellular region of the protein from which the transmembrane protein is derived (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 to 15 amino acids in the intracellular region). In one aspect, the transmembrane domain is a domain associated with one of the other domains of the chimeric receptor, for example, in one embodiment, the transmembrane domain may be from the same protein from which the signaling domain, the costimulatory domain, or the hinge domain is derived. In some cases, the transmembrane domain may be selected or replaced by amino acids to avoid binding of such a domain to the transmembrane domain of the same or different surface membrane protein, for example, to minimize interactions with other members of the receptor complex. In one aspect, the transmembrane domain is capable of homodimerizing with another chimeric receptor on the cell surface of a cell expressing a chimeric receptor. In a different aspect, the amino acid sequence of the transmembrane domain can be modified or substituted to minimize the interaction with the binding domain of a natural binding partner present in a cell expressing the same chimeric receptor. The transmembrane domain can be derived from a natural or recombinant source. When the source is natural, the domain can be derived from any membrane-bound protein or transmembrane protein. In one aspect, as long as the chimeric receptor is bound to the target antigen, the transmembrane domain can conduct a signal to the intracellular domain. The transmembrane domain particularly used in the present invention can include at least the following transmembrane domains: for example, α, β or ζ chains of T-cell receptors, CD28, CD27, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.In certain embodiments, the transmembrane domain may include at least the following transmembrane regions: for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2Rβ, IL2Rγ, IL7Rα, ITGA1, 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, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160(BY55), PSGL1, CD100(SEMA4D), SLAMF6(NTB-A, Ly108), SLAM(SLAMF1, CD150, IPO-3), BLAME(SLAMF8), SELPLG(CD162), LTBR, ​​PAG / Cbp, NKG2D, NKG2C.

[0105] In some cases, the transmembrane domain can be connected to the extracellular region of CAR via a hinge (e.g., a hinge from a human protein), such as the antigen binding domain of CAR. For example, in one embodiment, the hinge can be a human Ig (immunoglobulin) hinge (e.g., an IgG4 hinge, an IgD hinge), a GS joint (e.g., a GS joint as described herein), a KIR2DS2 hinge or a CD8a hinge. In one aspect, the transmembrane domain can be recombinant, in which case it will mainly include hydrophobic residues, such as leucine and valine. In one aspect, a triplet of phenylalanine, tryptophan and valine can be found at each end of the recombinant transmembrane domain. Optionally, a short oligopeptide or polypeptide linker with a length between 2 and 10 amino acids can form a key between the transmembrane domain of CAR and the cytoplasmic region. Glycine-serine dimers provide a particularly suitable linker.

[0106] As used herein, "intracellular domains" (also referred to as intracellular domains) include intracellular signaling domains. The intracellular signaling domain is generally responsible for the activation of at least one of the normal effector functions of the immune cell into which the chimeric receptor has been introduced. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell may be, for example, a cytolytic activity or an auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and directs the cell to perform a specific function. Although the entire intracellular signaling domain can generally be applied, in many cases it is not necessary to use the entire chain. In the case of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used instead of a complete chain as long as it transduces an effector function signal. Therefore, the term intracellular signaling domain means a truncated portion of an intracellular signaling domain that is sufficient to transduce an effector function signal.

[0107] It is well known that the signal generated by the TCR alone is insufficient to fully activate T cells, and secondary and / or co-stimulatory signals are also required. Thus, T cell activation can be referred to as being mediated by two different kinds of cytoplasmic signaling sequences: those that trigger antigen-dependent primary activation through the TCR (primary intracellular signaling domains) and those that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic domains, such as co-stimulatory domains).

[0108] The term "stimulation" refers to the primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex or CAR) to its cognate ligand (or a tumor antigen in the case of a CAR), thereby mediating a signal transduction event (such as, but not limited to, signal transduction via a TCR / CD3 complex or signal transduction via a suitable NK receptor or signaling domain of a CAR). Stimulation can mediate altered expression of certain molecules.

[0109] The term "stimulatory molecule" refers to a molecule that provides a cytoplasmic signaling sequence expressed by an immune cell (e.g., T cell, NK cell, B cell) that regulates the activation of immune cells for at least some aspects of the immune cell signaling pathway in a stimulatory manner. In one aspect, the signal is a primary signal initiated by the binding of, for example, a TCR / CD3 complex to an MHC molecule loaded with a peptide, and it results in mediating T cell responses, including, but not limited to, proliferation, activation, differentiation, etc. The primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Examples of cytoplasmic signaling sequences containing ITAMs particularly useful in the present invention include, but are not limited to, those derived from: CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcEpsilon R1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. In the specific CAR of the present invention, the intracellular signaling domain in any one or more CARs of the present invention includes an intracellular signaling sequence, such as a primary signaling sequence of CD3-ζ. In the specific CAR of the present invention, the primary signaling sequence of CD3-ζ is an equivalent residue from a human or non-human species such as a mouse, a rodent, a monkey, an ape, etc.

[0110] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of a T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that promote an effective immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Further examples of such co-stimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, 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, NKG2D, NKG2C, 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, CD19a, and ligands that specifically bind to CD83.

[0111] The co-stimulatory intracellular signaling domain can be the intracellular part of co-stimulatory molecules.Co-stimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins) and NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, antigen-1 (LFA-1) associated with lymphocyte function, CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3 and specifically bind to the ligand of CD83, etc.

[0112] The intracellular signaling domain may include the entire intracellular portion of a molecule or the entire native intracellular signaling domain, or a functional fragment or derivative thereof.

[0113] The term "4-1BB" refers to a member of the TNFR superfamily having an amino acid sequence as provided in GenBank Accession No.AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.; and a "4-1BB co-stimulatory domain" is defined as amino acid residues 214 to 255 of GenBank Accession No.AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc. In one aspect, a "4-1BB co-stimulatory domain" is an equivalent residue from a human or from a non-human species such as a mouse, rodent, monkey, ape, etc.

[0114] The term "T cell (antigen) receptor (T cell receptor, TCR)" is a characteristic marker on the surface of all T cells, which binds to CD3 with non-covalent bonds to form a TCR-CD3 complex. TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is a heterodimer composed of two different peptide chains, consisting of two peptide chains, α and β. Each peptide chain can be divided into several parts, such as the variable region (V region), the constant region (C region), the transmembrane region and the cytoplasmic region; its characteristic is that the cytoplasmic region is very short. TCR molecules belong to the immunoglobulin superfamily, and their antigen specificity exists in the V region; the V region (Vα, Vβ) each has three hypervariable regions CDR1, CDR2, and CDR3, among which CDR3 has the largest variation and directly determines the antigen binding specificity of TCR. When TCR recognizes the MHC-antigen peptide complex, CDR1 and CDR2 recognize and bind to the side walls of the antigen binding groove of the MHC molecule, while CDR3 directly binds to the antigen peptide. TCR is divided into two categories: TCR1 and TCR2; TCR1 is composed of two chains, γ and δ, and TCR2 is composed of two chains, α and β.

[0115] The term "T cell fusion protein (TFP)" includes recombinant polypeptides derived from various polypeptides constituting TCR, which can bind to surface antigens on target cells and interact with other polypeptides of the complete TCR complex, usually co-localized on the surface of T cells. TFP consists of a TCR subunit and an antigen binding domain composed of a human or humanized antibody domain, wherein the TCR subunit includes at least part of the TCR extracellular domain, the transmembrane domain, and the stimulatory domain of the intracellular signaling domain of the TCR intracellular domain; the TCR subunit and the antibody domain are effectively connected, wherein the extracellular, transmembrane, and intracellular signaling domains of the TCR subunit are derived from CD3ε or CD3γ, and the TFP is integrated into the TCR expressed on T cells.

[0116] The term "T cell antigen coupler (TAC)" includes three functional domains: 1. Tumor targeting domain, including single-chain antibody, designed ankyrin repeat protein (DARPin) or other targeting groups; 2. Extracellular domain, single-chain antibody that binds to CD3, thereby bringing the TAC receptor into close proximity with the TCR receptor; 3. Transmembrane region and intracellular region of CD4 co-receptor, wherein the intracellular region is connected to protein kinase LCK, catalyzing the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as the initial step of T cell activation.

[0117] The term "antibody" refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen. Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or complete immunoglobulins, and can be derived from natural or recombinant sources. Antibodies can be tetramers of immunoglobulin molecules.

[0118] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), 2 , Fv fragments, scFv, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies (such as sdAb), multispecific antibodies formed by antibody fragments (e.g., a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region), and isolated CDRs or other epitope-binding fragments of antibodies.

[0119] The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguous (e.g., via a synthetic linker such as a short flexible polypeptide linker) and can be expressed in a single-chain polypeptide form, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless otherwise specified, as used herein, scFv can have the VL and VH variable regions in any order (e.g., relative to the N-terminus and C-terminus of the polypeptide), and scFv can include VL-linker-VH or can include VH-linker-VL.

[0120] The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in an antibody molecule in its naturally occurring configuration and which generally determines the class to which the antibody belongs.

[0121] The term "antibody light chain" refers to the smaller of the two polypeptide chains present in an antibody molecule in its naturally occurring configuration. Kappa (k) and lambda (l) light chains refer to the two major isotypes of antibody light chains.

[0122] The term "recombinant antibody" refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or yeast expression system. The term should also be interpreted as referring to an antibody that has been produced by synthesizing a DNA molecule encoding the antibody (and wherein the DNA molecule expresses the antibody protein) or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or amino acid sequence technology available and well known in the art.

[0123] The term "antigen" refers to a molecule that causes an immune response. The immune response may involve antibody production or activation of cells with specific immune competence or both. It will be understood by those skilled in the art that any macromolecule including virtually all proteins or peptides can serve as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. When the term is used herein, it will be understood by those skilled in the art that any DNA, protein or peptide encoded including a nucleotide sequence or partial nucleotide sequence encoding a protein that causes an immune response. In addition, it will be understood by those skilled in the art that antigens do not need to be encoded only by the full-length nucleotide sequence of a gene. It is obvious that the present invention includes but is not limited to using partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to encode polypeptides that induce a desired immune response. Moreover, it will be understood by those skilled in the art that antigens do not need to be encoded by a "gene" at all. It is obvious that antigens can be produced synthetically, or can be derived from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells or fluids with other biological components.

[0124] "Tumor antigen" refers to an antigen that appears or is overexpressed during the development and progression of a hyperproliferative disease. In certain aspects, the hyperproliferative disorder of the present invention refers to cancer.

[0125] The tumor antigen described in the present invention may be a solid tumor antigen or a hematological tumor antigen.

[0126] The tumor antigens of the present invention include, but are not limited to, thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit alpha (IL-13Rα); interleukin 11 receptor alpha (IL-11Rα); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; proteinase serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6 ; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin type A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3; TGS5; high molecular weight melanoma associated antigen (HMWMAA); o-acetyl GD2 ganglioside (OAcGD2); folate receptor; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7 related (TEM7R); Claudin6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; kappa light chain (kappa light chain); CSPG4; EGP2, EGP40; FAP; FAR; FBP; embryonic AchR; HLA-A1, HLA-A2; MAGEA1, MAGE3; KDR; MCSP; NKG2D ligand; PSC1; ROR1; Sp17; SURVIVIN; TAG72; TEM1; fibronectin; tenascin; oncofetal variant of tumor necrosis zone; G protein-coupled receptor class C group 5 member D (GPRC5D); X chromosome open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1);The hexose moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 ( MAD-CT-2); Fos-related antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocytic virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (Rho C); cytochrome P4501B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); synovial sarcoma X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAI R1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1). ;

[0127] The pathogen antigen is selected from: antigens of viruses, bacteria, fungi, protozoa, or parasites; the viral antigen is selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen.

[0128] The term "tumor" refers to a broad category of disorders of hyperproliferative cell growth in vitro (e.g., transformed cells) or in vivo. Conditions that can be treated or prevented by the methods of the present invention include, for example, various neoplasms, including benign or malignant tumors, various hyperplasias, and the like. Specific examples of cancer include, but are not limited to, breast cancer, prostate cancer, leukemia, lymphoma, nasopharyngeal cancer, glioma, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumors, hemangiomas, spinal tumors, gliomas, astrocytomas, pituitary adenomas, combinations of the cancers, and metastatic lesions of the cancers.

[0129] The term "transfected" or "transformed" or "transduced" refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0130] The term "specifically binds" means that the antibody or ligand binds to a binding partner (eg, a tumor antigen) present in a sample, but does not substantially recognize or bind to other molecules in the sample.

[0131] As used herein, "refractory" refers to a disease, e.g., a tumor, that does not respond to treatment. In embodiments, a refractory tumor may be resistant to treatment before or at the start of treatment. In other embodiments, a refractory tumor may become resistant during treatment. In the present invention, refractory tumors include, but are not limited to, tumors that are insensitive to radiotherapy, relapse after radiotherapy, insensitive to chemotherapy, relapse after chemotherapy, insensitive to CAR-T therapy, or relapse after treatment. Refractory or recurrent malignancies may use the treatment regimens described herein.

[0132] As used herein, "relapsed" means that after a period of improvement, such as after a previous effective tumor treatment, the patient reappears the signs and symptoms that existed prior to the effective treatment.

[0133] The terms "individual" and "subject" have equivalent meanings herein and may refer to humans and animals from other species.

[0134] The term "enhancing" refers to allowing a subject or tumor cell to improve its ability to respond to treatment disclosed herein. For example, the enhanced response can include 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more increase in responsiveness. As used herein, "enhancing" can also refer to increasing the number of subjects of response treatment such as immune effector cell therapy. For example, the enhanced response can refer to the total percentage of subjects responding to treatment, wherein the percentage is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% more.

[0135] In one aspect, treatment is determined by clinical outcome; increased, enhanced or prolonged anti-tumor activity by T cells; an increase in the number of anti-tumor T cells or activated T cells compared to the number before treatment, promoting IFN-γ secretion, or a combination thereof. In another aspect, the clinical outcome is tumor regression; tumor shrinkage; tumor necrosis; anti-tumor response by the immune system; tumor expansion, recurrence or spread or a combination thereof. In another aspect, the therapeutic effect is predicted by the presence of T cells, the presence of gene markers indicating T cell inflammation, promoting IFN-γ secretion, or a combination thereof.

[0136] Immune effector cells as disclosed herein can be administered to an individual by a variety of routes, including, for example, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intrarectally, intracisternal, intratumorally, intravasally, intradermally, or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively.

[0137] In practicing the method of the present invention, the total amount of the agent to be administered can be administered to the subject as a single dose with a bolus or by infusion in a relatively short period of time, or a graded treatment regimen can be used for administration, wherein multiple doses are administered over an extended period of time. Those skilled in the art will know that the amount of the composition for the treatment of the pathological condition in the subject depends on many factors, including the age and general health of the subject, as well as the route of administration and the number of treatments to be administered. Taking these factors into account, the technician will adjust the specific dosage as needed. Generally speaking, initially, the preparation of the composition and the route of administration and frequency are determined using Phase I and Phase II clinical trials.

[0138] Range: Throughout the disclosure, various aspects of the invention may be presented in the form of ranges. It should be understood that the description in the form of ranges is only for convenience and brevity, and should not be regarded as an immutable limitation on the scope of the invention. Therefore, the description of the range should be considered to specifically disclose all possible sub-ranges and individual values ​​within the range. For example, the description of a range such as from 1 to 6 should be considered to specifically disclose sub-ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual values ​​within the range, such as 1, 2, 2.7, 3, 4, 5, 5.3 and 6. As another example, a range such as 95-99% identity includes a range with 95%, 96%, 97%, 98% or 99% identity, and includes sub-ranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the width of the range.

[0139] According to the present disclosure, it will be appreciated by those skilled in the art that many changes or modifications may be made in the disclosed specific embodiments, and still the same or similar results may be obtained without departing from the spirit and scope of the present invention. The present invention is not limited in scope to the specific embodiments described herein (which are only intended to be illustrative of various aspects of the present invention), and functionally equivalent methods and components are within the scope of the present invention. In fact, various modifications of the present invention, plus those shown and described herein, will become apparent to those skilled in the art according to the foregoing description.

[0140] When the CAR-T cells co-expressing IL7 and CCL21 are used in subjects, the corresponding species can be selected, such as when used in mice, mouse-derived IL7 and CCL21 are used, and the elements of CAR construction such as transmembrane domains, intracellular domains, etc. can also be selected from mice. When the subject is human, human-derived IL7 and CCL21 and human-derived CAR elements are preferred. In some embodiments, the sequence of the CAR used can be shown in SEQ ID NO: 26, 27, or 35.

[0141] In some embodiments, the cells of the present invention can be used in combination with chemotherapeutic drugs when used for tumor treatment.

[0142] The term "CLD18 (claudin 18)" refers to claudin-18, and includes any variants of CLD18 (including CLD18A1 (claudin 18.1) and CLD18A2 (claudin18.2)), conformations, isoforms and species homologs expressed by cells naturally expressed or cells transfected with the CLD18 gene. Preferably, "CLD18" refers to human CLD18, particularly CLD18A2 (SEQ ID NO: 22) and / or CLD18A1 (SEQ ID NO: 23), more preferably CLD18A2.

[0143] The term "CLD18A1" includes any post-translationally modified variants, isoforms and interspecies homologs of human CLD18A1 expressed naturally by a cell or by a cell transfected with the CLD18A1 gene.

[0144] The term "CLD18A2" includes any post-translationally modified variants, isoforms and interspecies homologs of human CLD18A2 expressed naturally by a cell or by a cell transfected with the CLD18A2 gene.

[0145] The term "CLD18 variant" shall include (i) CLD18 splice variants, (ii) CLD18 post-translational modification variants, in particular including variants with different N-glycosylation states, (iii) CLD18 conformational variants, in particular including CLD18-conformation-1, CLD18-conformation-2 and CLD18-conformation-3, (iv) free CLD18 and homotypic / heterotypic associated variants located in intercellular tight junctions, (v) CLD18 cancer-associated variants and CLD18 non-cancer-associated variants.

[0146] The chimeric antigen receptor polypeptide of the present invention can be selected from the following sequence:

[0147] Extracellular antigen binding region-CD8 transmembrane region-4-1BB-CD3ζ,

[0148] Extracellular antigen binding region-CD8 transmembrane region-CD28b-CD3ζ,

[0149] Extracellular antigen binding region-CD28a-CD28b-CD3ζ,

[0150] Extracellular antigen binding region-CD28a-CD28b-4-1BB-CD3ζ,

[0151] and combinations thereof, wherein CD28a in the relevant chimeric antigen receptor protein represents the transmembrane region of the CD28 molecule, and CD28b represents the intracellular signaling region of the CD28 molecule. The present invention also includes nucleic acids encoding the chimeric antigen receptor. The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention.

[0152] The present invention also provides a vector comprising the nucleic acid of the chimeric antigen receptor. The present invention also includes a virus comprising the vector. The virus of the present invention includes a virus with infectivity after packaging, and also includes a virus to be packaged that contains the necessary components for packaging into an infectivity virus. Other viruses known in the art that can be used to transduce foreign genes into immune effector cells and their corresponding plasmid vectors can also be used in the present invention.

[0153] The present invention also provides chimeric antigen modified immune effector cells, which are transduced with nucleic acid encoding the chimeric antigen receptor or transduced with the above-mentioned recombinant plasmid containing the nucleic acid, or a virus containing the plasmid. Conventional nucleic acid transduction methods in the art, including non-viral and viral transduction methods, can be used in the present invention. Non-viral transduction methods include electroporation and transposon methods. The Nucleofector nuclear transfection instrument recently developed by Amaxa can directly introduce exogenous genes into the cell nucleus to obtain efficient transduction of the target gene. In addition, the transduction efficiency of transposon systems such as Sleeping Beauty transposon or PiggyBac transposon is greatly improved compared with ordinary electroporation. The combined application of nucleofector transfection instrument and Sleeping Beauty transposon system has been reported [Davies JK., et al. Combining CD19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies. Cancer Res, 2010, 70 (10): OF1-10.], which has both high transduction efficiency and can achieve site-specific integration of target genes. In one embodiment of the present invention, the transduction method for immune effector cells modified with chimeric antigen receptor genes is a transduction method based on viruses such as retroviruses or lentiviruses. This method has the advantages of high transduction efficiency, stable expression of exogenous genes, and can shorten the time for in vitro cultured immune effector cells to reach clinical-level quantities. On the surface of the transgenic immune effector cells, the transduced nucleic acid is expressed on its surface through transcription and translation. By performing in vitro cytotoxic experiments on various cultured tumor cells, it is proved that the chimeric antigen-modified immune effector cells of the present invention have a highly specific tumor cell killing effect (also known as cytotoxicity) and can effectively survive in tumor tissue. Therefore, the nucleic acid encoding the chimeric antigen receptor of the present invention, the plasmid containing the nucleic acid, the virus containing the plasmid and the transgenic immune effector cells transduced with the above nucleic acid, plasmid or virus can be effectively used for tumor immunotherapy.

[0154] The chimeric antigen-modified immune effector cells of the present invention may also express another chimeric receptor in addition to the above-mentioned chimeric receptor, which receptor does not contain CD3ζ but contains the intracellular signaling domain of CD28, the intracellular signaling domain of CD137, or a combination of the two.

[0155] The chimeric antigen receptor modified immune effector cells of the present invention can be used to prepare pharmaceutical compositions or diagnostic reagents. In addition to an effective amount of the immune cells, the composition may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that when the molecular entities and compositions are appropriately administered to animals or humans, they do not produce adverse, allergic or other adverse reactions.

[0156] Specific examples of some substances that can serve as pharmaceutically acceptable carriers or components thereof are sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethylcellulose, ethylcellulose and methylcellulose; tragacanth powder; malt; gelatin; talc; solid lubricants, such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa butter; polyols, such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions and phosphate buffers, etc.

[0157] The composition of the present invention can be prepared into various dosage forms as required, and the physician can determine the dosage that is beneficial to the patient according to factors such as the patient type, age, weight, general disease condition, and administration method. The administration method can be injection or other treatment methods.

[0158] Advantages of the present invention:

[0159] 1. The immune effector cells provided in this article can effectively increase the proliferation, survival and function of the immune effector cells in tumors; reduce the expression of inhibitory immune checkpoints, thereby alleviating the exhaustion of T cells.

[0160] 2. The immune effector cells provided in this article have better killing effects and in vitro expansion performance on solid tumor cells.

[0161] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0162] Exemplary antigen receptors of the present invention, including CARs, and methods for engineering and introducing receptors into cells, refer to, for example, those disclosed in Chinese Patent Application Publication Nos. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, CN104140974A, International Patent Application Publication Nos. WO2017186121A1, WO2018006882A1, WO2015172339A8, and WO2018 / 018958A1.

[0163] Example 1. Construction of T cells expressing chimeric antigen receptors

[0164] In this example, Claudin18.2 was selected as the target of CAR-T cells. In order to more accurately verify the anti-tumor effect in mice, the selected signal peptide, transmembrane region, intracellular region, etc. were of mouse origin. The preparation method was performed according to the conventional CAR-T cell preparation method in the art.

[0165] 1. Plasmid construction

[0166] Conventional molecular biological methods in the art are used. The scFv used in this embodiment is an antibody targeting human Claudin18.2, and the nucleic acid sequence is shown in SEQ ID NO: 1. The chimeric antigen receptor used is a second-generation chimeric antigen receptor, which has the transmembrane domain of mCD8, the intracellular domain of mCD28 and / or the intracellular domain of m4-1BB, and mCD3ζ.

[0167] 1. Using MSCV.pBABE 5 (purchased from Addgene) as a vector, a retroviral plasmid MSCV-hu8E5(2I)-28Z expressing the second-generation chimeric antigen receptor was constructed. The nucleic acid sequence of hu8E5(2I)-28Z includes CD8α signal peptide (SEQID NO:3), scFv (SEQ ID NO:1), mCD8 hinge region and transmembrane region (SEQ ID NO:5), mCD28 intracellular signaling domain (SEQ ID NO:7), and mCD3ζ (SEQ ID NO:9), the intracellular segment of mCD3. The plasmid construction diagram of hu8E5(2I)-28Z is shown in the figure below. Figure 1A shown.

[0168] The gene of F2A-mIL7-P2A-mCCL21a or F2A-mIL7-P2A-mCCL21b was inserted into the MSCV-hu8E5(2I)-m28Z plasmid to construct the retroviral plasmid MSCV-hu8E5(2I)-m28Z-F2A-mIL7-P2A-mCCL21a expressing CAR, IL7 and CCL21 (plasmid Figure 2A). Figure 1B ) and MSCV-hu8E5(2I)-m28Z-F2A-mIL7-P2A-mCCL21b (plasmids shown in Figure 2 Figure 1C shown).

[0169] F2A-mIL7-P2A-mCCL21a consists of F2A (SEQ ID NO: 11), mouse IL7 (SEQ ID NO: 13), P2A (SEQ ID NO: 16), and mouse CCL21a (SEQ ID NO: 14); F2A-mIL7-P2A-mCCL21b consists of F2A (SEQ ID NO: 11), mouse IL7 (SEQ ID NO: 13), P2A (SEQ ID NO: 16), and mouse CCL21b (SEQID NO: 15).

[0170] Using MSCV.pBABE 5 as a vector, a retroviral plasmid MSCV-hu8E5(2I)-mBBZ expressing the second-generation chimeric antigen receptor was constructed. The hu8E5(2I)-mBBZ sequence consists of CD8α signal peptide (SEQ ID NO:3), scFv (SEQ ID NO:1), mCD8 hinge and transmembrane region (SEQ ID NO:5), m4-1BB intracellular signaling domain (SEQ ID NO:24), and the intracellular segment CD3ζ of mCD3 (SEQ ID NO:9). The plasmid is shown in Figure 1. Figure 1D shown.

[0171] Based on the MSCV-hu8E5(2I)-mBBZ plasmid, F2A-mIL-7-P2A-mCCL21a and F2A-mIL7-P2A-mCCL21b were inserted respectively to construct the retroviral plasmid MSCV-hu8E5(2I)-mBBZ-F2A-mIL-7-P2A-mCCL21a expressing CAR, IL7 and CCL21 (plasmid Figure 2A). Figure 1E As shown), MSCV-hu8E5(2I)-mBBZ-F2A-mIL7-P2A-mCCL21b (plasmid as shown Figure 1F shown).

[0172] 2. Combine MSCV-hu8E5(2I)-m28Z, MSCV-hu8E5(2I)-m28Z-F2A-mIL-7-P2A-mCCL21a, MSCV-hu8E5(2I )-m28Z-F2A-mIL7-P2A-mCCL21b, MSCV-hu8E5(2I)-mBBZ, MSCV-hu8E5(2I)-mBBZ-F2A-mIL-7-P 2A-mCCL21a and MSCV-hu8E5(2I)-mBBZ-F2A-mIL7-P2A-mCCL21b were transfected into 293T cells respectively to obtain retroviruses hu8E5(2I)-28Z, IL7-CCL21a-28Z, IL7-CCL21b-28Z, hu8E5(2I)-BBZ, IL7-CCL21a-BBZ, and IL7-CCL21b-BBZ.

[0173] 3. Mouse T cell extraction and activation: The spleen of C57BL / 6 mice was stripped to extract mouse T cells. After culture and activation, the obtained retroviruses hu8E5(2I)-28Z, IL7-CCL21a-28Z, IL7-CCL21b-28Z, hu8E5(2I)-BBZ, IL7-CCL21a-BBZ, and IL7-CCL21b-BBZ were used to infect T cells, respectively, to obtain m28Z CAR-T cells, m28Z-7*21A CAR-T cells, m28Z-7*21B CAR-T cells, mBBZ CAR-T cells, mBBZ-7*21A CAR-T cells, and mBBZ-7*21B CAR-T cells.

[0174] Example 2. In vitro cytokine detection

[0175] First, mouse pancreatic cancer cells PANC02 (claudin18.2 expression negative, purchased from ATCC) and PANC02-A2 (claudin18.2 expression positive) were pretreated with mitomycin C (40 μg / ml, 37° C., 2-3 h).

[0176] Press 2×10 5 Cells / 400ul were inoculated in 24-well plates, and Untransduced T cells (UTD), mBBZ CAR-T cells, mBBZ-7*21A CAR-T cells, and mBBZ-7*21B CAR-T cells were inoculated in 24-well plates at an effector-target ratio of 1:1. A control group without target cells was set up. After the third day, the cell supernatant was collected and the secretion of each cytokine IL7 and CCL21 was detected using an ELISA kit. The results are shown in Figure 2 shown.

[0177] Among them, PANC02-A2 cells were constructed by infecting PANC02 cells with pwpt-mclaudin18.2 lentivirus. The construction process of pWPT-mclaudin18.2 plasmid is as follows: the mouse claudin18.2 gene (GeneBank reference sequence number: NM_001194921) was synthesized in vitro, and it was loaded into the lentiviral expression vector pWPT by enzyme digestion and ligation to construct the pwpt-mclaudin18.2 plasmid.

[0178] Example 3. In vitro CAR-T cell phenotype detection

[0179] The UTD, mBBZ CAR-T cells, mBBZ-7*21A CAR-T cells, and mBBZ-7*21B CAR-T cells were used to detect cell surface immune checkpoints: PD-1, LAG-3, and TIM-3. First, different CAR-T cells were collected in EP tubes, and each cell was divided into 3 tubes. They were washed twice with pre-ice-bathed flow washing buffer (1% NCS plus PBS). BV421-labeled anti-PD-1 antibody, APC-labeled anti-LAG-3 antibody, and APC-labeled anti-TIM-3 antibody were added to different detection tubes at a dilution ratio of 1:50, incubated on ice for 45 minutes, washed 3 times, and transferred to flow tube detection. The results are as follows Figures 3A to 3F shown.

[0180] Figure 3A The results showed that the expression of PD-1 in cells of different groups reached 30.2% in the mBBZ group, only 11.7% in the mBBZ-7*21A group, and only 9.4% in the mBBZ-7*21B group. Figure 3B The expression intensity of PD-1 is shown. Figure 3B It can be seen that the expression of PD-1 in the mBBZ group was higher than that in the mBBZ-7*21A group and the mBBZ-7*21B group.

[0181] Figure 3C The expression of LAG-3 in cells of different groups was shown. The results showed that the secretion of LAG-3 in the mBBZ group reached 80.7%, the secretion of LAG-3 in the mBBZ-7*21A group was 53.4%, and the secretion of LAG-3 in the mBBZ-7*21B group was 13.7%. Figure 3D The expression intensity of LAG-3 is shown in Figure 3D It can be seen that the expression of LAG-3 in the mBBZ group was higher than that in the mBBZ-7*21A group and the mBBZ-7*21B group.

[0182] Figure 3EThe expression of TIM-3 in cells of different groups was shown. The results showed that the secretion of TIM-3 in the mBBZ group reached 41.3%, the secretion of TIM-3 in the mBBZ-7*21A group was 16.2%, and the secretion of TIM-3 in the mBBZ-7*21B group was 13.2%. Figure 3F The expression intensity of TIM-3 is shown in Figure 3F It can be seen that the expression of TIM-3 in the mBBZ group was higher than that in the mBBZ-7*21A group and mBBZ-7*21B group.

[0183] In summary, the expression of PD-1, LAG-3, and TIM-3 in mBBZ-7*21A CAR-T cells and mBBZ-7*21B CAR-T cells was lower than that in mBBZ-CAR-T cells, indicating that overexpression of cytokines IL7 and CCL21 could reduce the expression of these inhibitory immune checkpoints, thereby alleviating T cell exhaustion.

[0184] Example 4. In vitro killing toxicity test

[0185] CytoTox 96 non-radioactive cytotoxicity detection kit (Promega) was used for the assay, and the specific method was referred to the instruction manual of CytoTox 96 non-radioactive cytotoxicity detection kit.

[0186] Effector cells: UTD cells, m28Z CAR-T cells, m28Z-7*21A CAR-T cells, m28Z-7*21B CAR-T, mBBZ CAR-T cells, mBBZ-7*21A CAR-T cells, and mBBZ-7*21BCAR-T cells were inoculated in 96-well plates at effector-target ratios of 3:1, 1:1, or 1:3, respectively.

[0187] Target cells: 50 μL of 2×10 5 / mL of mouse pancreatic cancer cell line PANC02-A2 and PANC02 cells were added to corresponding 96-well plates.

[0188] Each group was set up with 5 replicate wells. The culture plate was placed in a cell culture incubator and incubated for 18 h.

[0189] The experimental groups and control groups were set up as follows: experimental group: each target cell + different CAR-T cells; control group 1: target cell maximum release of LDH; control group 2: target cell spontaneous release of LDH; control group 3: effector cell spontaneous release of LDH. The calculation formula is: % cytotoxicity = [(experimental group - effector cell spontaneous group - target cell spontaneous group) / (target cell maximum - target cell spontaneous)] * 100. The experimental results are as follows Figure 4A and 4B shown.

[0190] Figure 4A It can be seen that compared with the control group UTD, m28Z CAR-T cells, m28Z-7*21A CAR-T cells, or m28Z-7*21B CAR-T cells had significant toxic and killing effects on PANC02-A2 at effector-target ratios of 3:1 and 1:1, but had no killing effect on PANC02 cells.

[0191] Figure 4B It can be seen that compared with the control group UTD, mBBZ CAR-T cells, mBBZ-7*21A CAR-T cells, and mBBZ-7*21B CAR-T cells had significant toxic and killing effects on PANC02-A2 at effector-target ratios of 3:1 and 1:1, but had no killing effect on PANC02 cells.

[0192] Example 5. In vitro proliferation assay

[0193] The target cells PANC02-A2 cells were treated with mitomycin C (40 μg / ml, 37 degrees, 2-3 h), and the effector cells UTD cells, mBBZ CAR-T cells, mBBZ-7*21A CAR-T cells, and mBBZ-7*21B CAR-T cells were stained with CFSE, and then the effector-target ratio (1×10 6 cells / ml) 1:1 co-incubated for 2 days,

[0194] Flow cytometry was used to detect the proliferation of CAR-T cells. The results were as follows: Figure 5 As shown in the figure, mBBZ-7*21A CAR-T cells and mBBZ-7*21B CAR-T cells can proliferate faster than mBBZ CAR-T cells.

[0195] Example 6. Tumor treatment in PANC02-A2 pancreatic cancer subcutaneous transplant model

[0196] 1) Experimental groups: C57BL / 6 mice (purchased from Shanghai Xipu-Bikai Experimental Animal Co., Ltd.) were randomly divided into groups at 6-8 weeks of age, with 5-6 mice in each group, namely, the UTD cell, mBBZ CAR-T cell, mBBZ-7*21A CAR-T cell, and mBBZ-7*21B CAR-T cell treatment groups.

[0197] 2) Inoculation of subcutaneous transplanted tumors: PANC02-A2 cells in the logarithmic growth phase and in good growth condition were collected by trypsin digestion, washed once with PBS, and the cell density was adjusted to 6×10 6 / mL, 200 μL of cell suspension was injected subcutaneously into the right abdomen of C57BL / 6 mice, that is, 1.2×10 6 The inoculation date of tumor cells is day 0.

[0198] 3) CAR-T cell infusion: D11 days after subcutaneous inoculation of tumor cells, the average tumor volume is about 60mm 3 Injection of untreated T cells or CAR-T cells, injection dose: 2.5×10 6 / Only.

[0199] The results are as follows Figure 6 As shown in the figure, 20 days after CAR-T injection, the tumor inhibition rates were: mBBZ CAR-T group: 35.5%, mBBZ-7*21A CAR-T group: 63%, mBBZ-7*21B CAR-T group: 62.4%, indicating that the anti-tumor effects of the mBBZ-7*21A CAR-T cell and mBBZ-7*21B CAR-T cell treatment groups were better than those of the mBBZ CAR-T cells (P<0.05).

[0200] Example 7. Comparison of tumor killing by expressing different chemokines

[0201] In this example, CAR-T cells expressing IL7 and CCL19 (mBBZ-7*19CAR-T cells) were selected as controls. The preparation of mBBZ-7*19CAR-T cells was carried out with reference to Example 1. F2A-mIL7-P2A-mCCL19 was inserted into the MSCV-hu8E5(2I)-mBBZ plasmid to construct a retroviral plasmid expressing CAR, IL7 and CCL19. The plasmid is shown in the figure. Figure 7 The nucleic acid sequence of mCCL19 is shown in SEQ ID NO: 34. The plasmid was used to infect T cells of mice to obtain mBBZ-7*19CAR-T cells.

[0202] 1) A pancreatic cancer subcutaneous transplanted tumor model was prepared in mice according to the procedure of Example 6. 3 At the same time, the mBBZ CAR-T cell, mBBZ-7*19CAR-T cell, and mBBZ-7*21B CAR-T cell treatment groups were injected respectively, and the injection dose was 2.5×10 6 Cells / cell, tumor killing results are as follows Fig. 8A As shown, the tumor inhibition rates were: mBBZ CAR-T group: 22.8%, mBBZ-7*19CAR-T group: 32.7%, mBBZ-7*21B CAR-T group: 76.6%. The anti-tumor effect of the mBBZ-7*21B CAR-T cell treatment group was better than that of the mBBZ CAR-T cell and mBBZ-7*19CAR-T cell groups.

[0203] At the same time, the weight changes of mice in each group were detected (such as Figure 8BThe results showed that there was no significant difference in body weight among the mice in each group (ns), suggesting that the secretion of cytokines had no toxic effect on mice.

[0204] 2) On the 31st day after tumor inoculation, the mice were euthanized, the tumors were removed and the tumor weights were weighed. The specific statistical results are as follows: Figure 8C The results showed that the tumor weight in the mBBZ-7*21B CAR-T treatment group was significantly smaller than that in the mBBZ group (P<0.05), indicating that the co-expression of IL7 and CCL21 by T cells modified with chimeric antigen receptors can significantly increase the inhibition of T cells on tumors in vivo.

[0205] 3) After the CAR-T cells were reinfused, the tumor tissue was removed at the end of the experiment on day 31 to detect the number of CAR-T copies in the tumor tissue.

[0206] 1 mg of tumor tissue was mechanically ground and then used Genomic DNA kits were used to extract DNA from tumors and measure the concentration of each sample. Real-time quantitative PCR (qPCR) was used to detect the copy number of CAR. A standard curve was prepared based on the template plasmid, and finally the copy number of CAR in each sample was calculated.

[0207] The results are as follows Fig.8D As shown, the CAR-T copy number of mBBZ-7*21B CAR-T is higher

[0208] 4) Immunohistochemical detection of CD8 + Tumor infiltration of cells

[0209] Tumor tissues of mice euthanized in step 2) were taken to prepare paraffin tissue sections. After conventional dewaxing, the specimens were hydrated. After hydration, the sections were placed on a shaker and washed with PBS for 3 times. After boiling the citric acid buffer, the tissue sections were placed in the citric acid buffer for antigen heat repair. After repair, 1% BSA was used for blocking.

[0210] The blocked sections were added with corresponding CD8a antibodies (anti-mouse CD8αantibody, purchased from Cell Signaling) or blank control reagents, incubated at 4°C overnight; washed with 0.5% PBST buffer; and then washed with PBS buffer.

[0211] The washed sections were added with secondary antibody goat anti-rabbit-HRP and incubated at 37°C for 1 h. After washing twice with 0.5% PBST buffer, washed once with PBS buffer, and DAB (Dako REAL TM EnVision TMDetection System, Peroxidase / DAB+, 1:50 dilution) for color development.

[0212] Counterstain with hematoxylin until the nucleus is stained dark red. Place the counterstained tissue sections in 1% hydrochloric acid ethanol differentiation solution for 3 to 5 seconds; rinse with tap water for 20 minutes and dehydrate to make them transparent; soak in 90% ethanol for 1 minute; soak in 100% ethanol I for 1 minute; soak in 100% ethanol II for 1 minute; soak in xylene for 3 minutes; seal with neutral gum and air-dry.

[0213] Observation under a microscope revealed Fig. 8E As shown, although there were obvious CD8 + T cell infiltration, but the mBBZ-7*21B CAR-T group CD8 + T cell infiltration is more frequent.

[0214] Example 8. Orthotopic breast cancer transplantation model in mice

[0215] A mouse breast cancer subcutaneous transplant tumor model was established. E0771-A2 cells in the logarithmic growth phase and in good growth condition were collected by trypsin digestion (preparation method: E0771 cells were infected with lentivirus packaged with pwpt-mclaudin18.2 plasmid), washed once with PBS, and the cell density was adjusted to 2×10 7 / mL, 50 μL of cell suspension was injected subcutaneously into the fourth pair of mammary glands on the right side of the abdomen of C57BL / 6 mice, that is, 1×10 6 The E0771-A2 cells were seeded on day 0.

[0216] CAR-T cell infusion: D12 days after subcutaneous inoculation of tumor cells, the average tumor volume is about 150mm 3 Injection of untreated T cells or CAR-T cells, injection dose: 2.5×10 6 / Only.

[0217] The size of the E0771-A2 transplanted tumor was measured every 3-4 days, and the changes in tumor volume of each group of mice were recorded. Fig.9A As shown, compared with the mBBZ-7*19CAR-T group, the tumor killing ability of the mBBZ-7*21B CAR-T treatment group was significantly enhanced.

[0218] On the 31st day after tumor inoculation, the mice were euthanized, the tumors were removed and the tumor weights were weighed. The specific statistical results are as follows: Fig. 9BThe results showed that the tumor weight in the mBBZ-7*21B CAR-T treatment group was significantly lower than that in the mBBZ-7*19CAR-T group (P<0.05) and the mBBZ CAR-T group (P<0.001), indicating that the co-expression of IL7 and CCL21 by T cells modified with chimeric antigen receptors can significantly increase the inhibition of T cells on tumors in vivo.

[0219] Referring to the operation of step 3) of Example 7, the cell copies of CAR-T in the subcutaneous transplanted tumor model of breast cancer were detected. The results are as follows: Fig. 9C As shown, the CAR-T copy numbers in the mBBZ-7*19CAR-T and mBBZ-7*21B CAR-T groups were higher than those in the UTD and BBZ groups.

[0220] Referring to the operation of step 4) of Example 7, CD8 + The tumor infiltration of cells, the results are as follows Fig.9D As shown in the figure, there was obvious infiltration of CD8 T cells in the tumor tissues of the mBBZ-7*19CAR-T group and the mBBZ-7*21B CAR-T group, among which CD8 + T cell infiltration is more frequent.

[0221] Example 9. Subcutaneous transplantation model of liver cancer in mice

[0222] To establish a mouse liver cancer transplant model, Hepa1-6-A2 cells in the logarithmic growth phase and in good growth condition were collected by trypsin digestion (Hepa1-6 cells were infected with lentivirus packaged with pwpt-mclaudin18.2 plasmid), washed once with PBS, and the cell density was adjusted to 5×10 7 / mL, 200 μL of cell suspension was injected subcutaneously into the right abdomen of C57BL / 6 mice, that is, 1×10 7 The inoculation date of Hepal1-6-A2 liver cancer cells is day 0.

[0223] CAR-T cell infusion: D7 days after subcutaneous inoculation of tumor cells, the average tumor volume is about 300mm 3 Injection of untreated T cells or CAR-T cells, injection dose: 1×10 6 / Only.

[0224] The size of the Hepal1-6-A2 transplanted tumor was measured every 3-4 days, and the changes in tumor volume of each group of mice were recorded. Fig. 10A As shown, compared with the mBBZ-7*19CAR-T group, the tumor killing ability of the mBBZ-7*21B CAR-T treatment group was significantly enhanced.

[0225] On the 31st day after tumor inoculation, the mice were euthanized, the tumors were removed and the tumor weights were weighed. The specific statistical results are as follows: Fig. 10B The results showed that the tumor weight in the mBBZ-7*21B CAR-T treatment group was significantly lower than that in the mBBZ-7*19CAR-T group (p<0.01) and the 8E5-2I-mBBZ CAR-T group (p<0.05), indicating that the co-expression of IL7 and CCL21 by T cells modified with chimeric antigen receptors can significantly increase the inhibition of T cells on tumors in vivo.

[0226] Referring to the operation of step 3) of Example 7, the copy number of CAR-T cells was detected, and the result was as follows Fig. 10C As shown, the CAR-T copy number was higher in the mBBZ-7*21B CAR-T group.

[0227] Referring to the operation of step 4) of Example 7, CD8 + The tumor infiltration of cells, the results are as follows Fig. 10D As shown in the figure, there was obvious CD8 + T cell infiltration, including CD8 + T cell infiltration is more frequent.

[0228] Example 10. In vitro IFN-γ detection

[0229] UTD, 8E5-2I-mBBZ-CAR, mBBZ-7*21B CAR-T, and mBBZ-7*19CAR-T were incubated with target cells PANC02-A2 at a ratio of 1:1 for 24 hours, and the supernatant was collected and the secretion level of IFN-γ in the supernatant was detected by ELISA. The ELISA kit used was the Lianke Bio mouse IFN-γ detection kit.

[0230] The results are as follows Fig.11 As shown, mBBZ-7*21B CAR-T cells secreted more IFN-γ after co-incubation with claudin18.2-positive tumor cells.

[0231] Example 11. Treatment of the mouse PANC02-A2 pancreatic cancer subcutaneous tumor-clearing model

[0232] Referring to the procedure of Example 6, a PANC02-A2 subcutaneous transplant tumor model of C57BL / 6 mice was prepared. The inoculation date was day 0. On the 14th day after tumor inoculation, the average tumor volume was about 60 mm 3Cyclophosphamide was injected into the tail vein at 100 mg / kg. On the 15th day after tumor inoculation, untreated T cells or CAR-T cells were injected. The injection dose was 2.5×10 6 / Only.

[0233] The size of PANC02-A2 transplanted tumors was measured every 3-4 days, and the changes in tumor volume of each group of mice were recorded. Fig. 12A As shown, on the 38th day after tumor inoculation, the mice were euthanized, the tumors were removed and the tumor weights were weighed. The specific statistical results are shown in Fig. 12B shown.

[0234] Referring to the operation of step 3) of Example 7, the copy number of CAR-T cells was detected, and the result was as follows Fig. 12C As shown, the CAR-T copy numbers in the mBBZ-7*19CAR-T and mBBZ-7*21B CAR-T groups were higher than those in the UTD and BBZ groups.

[0235] Referring to the operation of step 4) of Example 7, CD8 + The tumor infiltration of cells, the results are as follows Fig.12D As shown in the figure, there was obvious infiltration of CD8 T cells in the tumor tissues of the mBBZ-7*19CAR-T group and the mBBZ-7*21B CAR-T group, and the CD8 T cell infiltration was more in the mBBZ-7*21B CAR-T group.

[0236] Example 12. CAR-T cell detection and analysis of mouse PANC02-A2 pancreatic cancer subcutaneous tumor model

[0237] Referring to the procedure of Example 6, a mouse pancreatic cancer subcutaneous transplant tumor model was prepared. 2×10 6 The average tumor volume of PANC02-A2 pancreatic cancer cells was about 60 mm on D14 days after subcutaneous inoculation of tumor cells. 3 , injected with untreated T cells or CAR-T cells (mBBZ CAR-T cells, mBBZ-7*21A CAR-T cells, and mBBZ-7*19CAR-T cells), injection dose: 4×10 6 / Only.

[0238] 1. The spleen and bone marrow of mice were extracted on the 10th day (d10) and 20th day (d20) after CAR-T cell treatment to detect the content of Tcm (central memory T cells) (two mice were taken from each treatment group). The experimental method is as follows:

[0239] 1) Spleen cell extraction: Kill the mice by cervical dislocation, take the spleen, put it in a clean 2mL EP tube, and wash off the blood with PBS. Grind and take spleen cells using a 40um filter. Centrifuge the spleen cell mixture at 400g for 5min, remove the supernatant, add 400μL mouse red blood cell lysis buffer (1×), let stand for 5min, add 1.5mL PBS to neutralize the reaction, centrifuge, resuspend in PBS, and incubate with antibodies in separate tubes. The antibodies are labeled with CD8 (PerCP), CD44 (BV510), and CD62L (APC).

[0240] 2) Extraction of bone marrow cells: Kill mice by cervical dislocation, remove the femur and tibia, remove the muscle, place in a clean 2mL EP tube, and wash away the blood with PBS. Take a 2mL syringe, draw 2mL of PBS, insert the needle along one end of the femur or tibia, fix it with tweezers, squeeze the piston, and rinse to obtain bone marrow cells. Centrifuge the cell mixture at 400g for 5min, remove the supernatant, add 400μL mouse erythrocyte lysis buffer (1×), let it stand for 5min, add 400μL mouse erythrocyte lysis buffer (1×), let it stand for 5min, add 1.5mL PBS to neutralize the reaction, centrifuge, resuspend in PBS, and incubate with antibodies in separate tubes. The antibodies are labeled with CD8 (PerCP), CD44 (BV510), and CD62L (APC).

[0241] The detection of Tcm in spleen on d10 is as follows Fig.13A As shown in Figure 2, the detection of Tcm in the spleen on day 20 is as follows Fig. 13B As shown, compared with conventional CAR-T, the Tcm content in the mBBZ-CAR-T cell group expressing IL7 and CCL21 was significantly increased.

[0242] The content of Tcm in the bone marrow on day 10 is as follows Fig.14A As shown in Figure 2, the detection of Tcm in the bone marrow on day 20 is as follows Fig. 14B As shown, compared with conventional CAR-T, the content of Tcm in the bone marrow increased significantly after mBBZ-7*21BCAR-T treatment.

[0243] 2. DC infiltration detection

[0244] On the 10th day (d10) after CAR-T cell treatment, frozen sections of the tumor tissues of mice were made to detect DC infiltration. The results are as follows Fig.15 As shown, compared with the mBBZ cell group, there were more DC cell infiltrations in the tumor tissues of mice in the mBBZ-7*21B cell group.

[0245] 3. Detection of MDSC (myeloid-derived suppressor cells) content

[0246] The tumor tissues of mice in the UTD group, mBBZ group, mBBZ-7*21B group, and mBBZ-7*19CAR-T group on the 10th day (d10) after CAR-T cell treatment were extracted, and the fat, blood vessels, capsule and internal necrotic tissue on the tumor surface were removed. The tumors were rinsed with PBS and transferred to 5 mL centrifuge tubes and 2 mL of culture medium was added. The tumors were cut into pieces of about 1×1 mm, and the culture medium was added to 4.7 mL. Digestive enzymes were added in proportion (digestive enzymes used for tissue separation: collagenase type I (0.05 mg / ml), collagenase type IV (0.05 mg / ml, hyaluronidase (0.025 mg / ml), DNase I (0.01 mg / ml) was shaken at 37°C for about 30 minutes (taken out in the middle to observe the digestion status); after digestion, the suspension was passed through a 70um cell sieve into a 50mL tube (operated on ice), and the undigested tissue was gently pushed out with a syringe push rod, and the sieve was rinsed and collected with a large amount of culture medium (up to 20mL), and the digestion was terminated at the same time, centrifuged at 400g for 8 minutes at 4°C, and the supernatant was removed and washed with PBS, and then the tubes were incubated with antibodies to detect CD45 + 、CD11b + (FITC) and Gr-1 + The content of (PE) cells, that is, the content of MDSC. The experimental results are as follows Fig.16 As shown: Compared with the mBBZ group, the content of MDSCs in tumor tissue was lower after mBBZ-7*21B CAR-T treatment.

[0247] As an example, the above embodiment selects CAR-T cells targeting CLD18A2, and it should be understood that the selection of CAR-T cells acting on other targets also has the same effect, such as GPC3, EGFR, EGFRvIII, CD19, BCMA, etc. The antibodies used can be mouse antibodies or humanized antibodies, and the transmembrane domains and intracellular domains used can also be of different species according to different purposes, such as human antibodies.

[0248] As an example, although the above embodiment uses CAR-T cells, the T cells can also express other cytokines that enhance the function of CAR-T cells, such as CAR-T cells co-expressing CAR and type I interferon, CAR-T cells co-expressing CAR and PD-1, etc. As an example, although the above embodiment uses CAR-T cells, other immune cells can also be selected, such as NK cells, NK-T cells, and specific subtypes of immune cells can also be selected, such as γ / δT cells, etc.

[0249] The sequences used in the present invention are summarized in the following table:

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application. Sequence Listing <110> Cogen Biopharmaceuticals (Shanghai) Co., Ltd. Shanghai Institute of Oncology <120> Genetically engineered cells and their applications <130> P19401638C <150> CN201810463564.7 <151> 2018-05-15 <150> CN201811088090.9 <151> 2018-09-18 <150> CN201811552806.6 <151> 2018-12-19 <150> CN201910151930.X <151> 2019-02-28 <160> 35 <170> PatentIn version 3.5 <210> 1 <211> 741 <212> DNA <213> Artificial Sequence <400> 1 caggtgcagc tgcaggagag cggccccggc ctgatcaagc ccagccagac cctgagcctg 60 acctgcaccg tgagcggcgg cagcatcagc agcggctaca actggcactg gatccggcag 120 ccccccggca agggcctgga gtggatcggc tacatccact acaccggcag caccaactac 180 aaccccgccc tgcggagccg ggtgaccatc agcgtggaca ccagcaagaa ccagttcagc 240 ctgaagctga gcagcgtgac cgccgccgac accgccatct actactgcgc ccggatctac 300 aacggcaaca gcttccccta ctggggccag ggcaccaccg tgaccgtgag cagcggtgga 360 ggcggttcag gcggaggtgg ttctggcggt ggcggatcgg acatcgtgat gacccagagc 420 cccgacagcc tggccgtgag cctgggcgag cgggccacca tcaactgcaa gagcagccag 480 agcctgttca acagcggcaa ccagaagaac tacctgacct ggtaccagca gaagcccggc 540 cagcccccca agctgctgat ctactgggcc agcacccggg agagcggcgt gcccgaccgg 600 ttcagcggca gcggcagcgg caccgacttc accctgacca tcagcagcct gcaggccgag 660 gacgtggccg tgtactactg ccagaacgcc tacagcttcc cctacacctt cggcggcggc 720 accaagctgg agatcaagcg g 741 <210> 2 <211> 247 <212> PRT <213> Artificial Sequence <400> 2 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ile Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60 Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg 245 <210> 3 <211> 81 <212> DNA <213> Artificial Sequence <400> 3 atggcctcac cgttgacccg ctttctgtcg ctgaacctgc tgctgctggg tgagtcgatt 60 atcctgggga gtggagaagc t 81 <210> 4 <211> 27 <212> PRT <213> Artificial Sequence <400> 4 Met Ala Ser Pro Leu Thr Arg Phe Leu Ser Leu Asn Leu Leu Leu Leu 1 5 10 15 Gly Glu Ser Ile Ile Leu Gly Ser Gly Glu Ala 20 25 <210> 5 <211> 216 <212> DNA <213> Artificial Sequence <400> 5 actactacca agccagtgct gcgaactccc tcacctgtgc accctaccgg gacatctcag 60 ccccagagac cagaagattg tcggccccgt ggctcagtga aggggaccgg attggacttc 120 gcctgtgata tttacatctg ggcacccttg gccggaatct gcgtggccct tctgctgtcc 180 ttgatcatca ctctcatctg ctaccacagg agccga 216 <210> 6 <211> 72 <212> PRT <213> Artificial Sequence <400> 6 Thr Thr Thr Lys Pro Val Leu Arg Thr Pro Ser Pro Val His Pro Thr 1 5 10 15 Gly Thr Ser Gln Pro Gln Arg Pro Glu Asp Cys Arg Pro Arg Gly Ser 20 25 30 Val Lys Gly Thr Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala 35 40 45 Pro Leu Ala Gly Ile Cys Val Ala Leu Leu Leu Ser Leu Ile Ile Thr 50 55 60 Leu Ile Cys Tyr His Arg Ser Arg 65 70 <210> 7 <211> 123 <212> DNA <213> Artificial Sequence <400> 7 aatagtagaa ggaacagact ccttcaaagt gactacatga acatgactcc ccggaggcct 60 gggctcactc gaaagcctta ccagccctac gcccctgcca gagactttgc agcgtaccgc 120 ccc 123 <210> 8 <211> 41 <212> PRT <213> Artificial Sequence <400> 8 Asn Ser Arg Arg Asn Arg Leu Leu Gln Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Leu Thr Arg Lys Pro Tyr Gln Pro Tyr Ala Pro 20 25 30 Ala Arg Asp Phe Ala Ala Tyr Arg Pro 35 40 <210> 9 <211> 321 <212> DNA <213> Artificial Sequence <400> 9 agcaggagtg cagagactgc tgccaacctg caggacccca accagctcta caatgagctc 60 aatctagggc gaagagagga atatgacgtc ttggagaaga agcgggctcg ggatccagag 120 atgggaggca aacagcagag gaggaggaac ccccaggaag gcgtatacaa tgcactgcag 180 aaagacaaga tggcagaagc ctacagtgag atcggcacaa aaggcgagag gcggagaggc 240 aaggggcacg atggccttta ccagggtctc agcactgcca ccaaggacac ctatgatgcc 300 ctgcatatgc agaccctggc c 321 <210> 10 <211> 107 <212> PRT <213> Artificial Sequence <400> 10 Ser Arg Ser Ala Glu Thr Ala Ala Asn Leu Gln Asp Pro Asn Gln Leu 1 5 10 15 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Glu 20 25 30 Lys Lys Arg Ala Arg Asp Pro Glu Met Gly Gly Lys Gln Gln Arg Arg 35 40 45 Arg Asn Pro Gln Glu Gly Val Tyr Asn Ala Leu Gln Lys Asp Lys Met 50 55 60 Ala Glu Ala Tyr Ser Glu Ile Gly Thr Lys Gly Glu Arg Arg Arg Gly 65 70 75 80 Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp 85 90 95 Thr Tyr Asp Ala Leu His Met Gln Thr Leu Ala 100 105 <210> 11 <211> 66 <212> DNA <213> Artificial Sequence <400> 11 gtgaaacaga ctttgaattt tgaccttctg aagttggcag gagacgttga gtccaaccct 60 gggccc 66 <210> 12 <211> 22 <212> PRT <213> Artificial Sequence <400> 12 Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val 1 5 10 15 Glu Ser Asn Pro Gly Pro 20 <210> 13 <211> 462 <212> DNA <213> Artificial Sequence <400> 13 atgttccatg tttcttttag atatatcttt ggaattcctc cactgatcct tgttctgctg 60 cctgtcacat catctgagtg ccacattaaa gacaaagaag gtaaagcata tgagagtgta 120 ctgatgatca gcatcgatga attggacaaa atgacaggaa ctgatagtaa ttgcccgaat 180 aatgaaccaa acttttttag aaaacatgta tgtgatgata caaaggaagc tgcttttcta 240 aatcgtgctg ctcgcaagtt gaagcaattt cttaaaatga atatcagtga agaattcaat 300 gtccacttac taacagtatc acaaggcaca caaacactgg tgaactgcac aagtaaggaa 360 gaaaaaaacg taaaggaaca gaaaaagaat gatgcatgtt tcctaaagag actactgaga 420 gaaataaaaa cttgttggaa taaaattttg aagggcagta ta 462 <210> 14 <211> 399 <212> DNA <213> Artificial Sequence <400> 14 atggctcaga tgatgactct gagcctcctt agcctggtcc tggctctctg catcccctgg 60 acccaaggca gtgatggagg gggtcaggac tgctgcctta agtacagcca gaagaaaatt 120 ccctacagta ttgtccgagg ctataggaag caagaaccaa gtttaggctg tcccatcccg 180 gcaatcctgt tctcaccccg gaagcactct aagcctgagc tatgtgcaaa ccctgaggaa 240 ggctgggtgc agaacctgat gcgccgcctg gaccagcctc cagccccagg gaaacaaagc 300 cccggctgca ggaagaaccg gggaacctct aagtctggaa agaaaggaaa gggctccaag 360 ggctgcaaga gaactgaaca gacacagccc tcaagagga 399 <210> 15 <211> 399 <212> DNA <213> Artificial Sequence <400> 15 atggctcaga tgatgactct gagcctcctt agcctggtcc tggctctctg catcccctgg 60 acccaaggca gtgatggagg gggacaggac tgctgcctta agtacagcca gaagaaaatt 120 ccctacagta ttgtccgagg ctataggaag caagaaccaa gtttaggctg tcccatcccg 180 gcaatcctgt tcttaccccg gaagcactct aagcctgagc tatgtgcaaa ccctgaggaa 240 ggctgggtgc agaacctgat gcgccgcctg gaccagcctc cagccccagg gaaacaaagc 300 cccggctgca ggaagaaccg gggaacctct aagtctggaa agaaaggaaa gggctccaag 360 ggctgcaaga gaactgaaca gacacagccc tcaagagga 399 <210> 16 <211> 57 <212> DNA <213> Artificial Sequence <400> 16 gctactaact tcagcctgct gaagcaggct ggagacgtgg aggagaaccc tggacct 57 <210> 17 <211> 534 <212> DNA <213> Artificial Sequence <400> 17 atgttccatg tttcttttag gtatatcttt ggacttcctc ccctgatcct tgttctgttg 60 ccagtagcat catctgattg tgatattgaa ggtaaagatg gcaaacaata tgagagtgtt 120 ctaatggtca gcatcgatca attattggac agcatgaaag aaattggtag caattgcctg 180 aataatgaat ttaacttttt taaaagacat atctgtgatg ctaataagga aggtatgttt 240 ttattccgtg ctgctcgcaa gttgaggcaa tttcttaaaa tgaatagcac tggtgatttt 300 gatctccact tattaaaagt ttcagaaggc acaacaatac tgttgaactg cactggccag 360 gttaaaggaa gaaaaccagc tgccctgggt gaagcccaac caacaaagag tttggaagaa 420 aataaatctt taaaggaaca gaaaaaactg aatgacttgt gtttcctaaa gagactatta 480 caagagataa aaacttgttg gaataaaatt ttgatgggca ctaaagaaca ctga 534 <210> 18 <211> 177 <212> PRT <213> Artificial Sequence <400> 18 Met Phe His Val Ser Phe Arg Tyr Ile Phe Gly Leu Pro Pro Leu Ile 1 5 10 15 Leu Val Leu Leu Pro Val Ala Ser Ser Asp Cys Asp Ile Glu Gly Lys 20 25 30 Asp Gly Lys Gln Tyr Glu Ser Val Leu Met Val Ser Ile Asp Gln Leu 35 40 45 Leu Asp Ser Met Lys Glu Ile Gly Ser Asn Cys Leu Asn Asn Glu Phe 50 55 60 Asn Phe Phe Lys Arg His Ile Cys Asp Ala Asn Lys Glu Gly Met Phe 65 70 75 80 Leu Phe Arg Ala Ala Arg Lys Leu Arg Gln Phe Leu Lys Met Asn Ser 85 90 95 Thr Gly Asp Phe Asp Leu His Leu Leu Lys Val Ser Glu Gly Thr Thr 100 105 110 Ile Leu Leu Asn Cys Thr Gly Gln Val Lys Gly Arg Lys Pro Ala Ala 115 120 125 Leu Gly Glu Ala Gln Pro Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu 130 135 140 Lys Glu Gln Lys Lys Leu Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu 145 150 155 160 Gln Glu Ile Lys Thr Cys Trp Asn Lys Ile Leu Met Gly Thr Lys Glu 165 170 175 His <210> 19 <211> 459 <212> PRT <213> Artificial Sequence <400> 19 Met Thr Ile Leu Gly Thr Thr Phe Gly Met Val Phe Ser Leu Leu Gln 1 5 10 15 Val Val Ser Gly Glu Ser Gly Tyr Ala Gln Asn Gly Asp Leu Glu Asp 20 25 30 Ala Glu Leu Asp Asp Tyr Ser Phe Ser Cys Tyr Ser Gln Leu Glu Val 35 40 45 Asn Gly Ser Gln His Ser Leu Thr Cys Ala Phe Glu Asp Pro Asp Val 50 55 60 Asn Ile Thr Asn Leu Glu Phe Glu Ile Cys Gly Ala Leu Val Glu Val 65 70 75 80 Lys Cys Leu Asn Phe Arg Lys Leu Gln Glu Ile Tyr Phe Ile Glu Thr 85 90 95 Lys Lys Phe Leu Leu Ile Gly Lys Ser Asn Ile Cys Val Lys Val Gly 100 105 110 Glu Lys Ser Leu Thr Cys Lys Lys Ile Asp Leu Thr Thr Ile Val Lys 115 120 125 Pro Glu Ala Pro Phe Asp Leu Ser Val Val Tyr Arg Glu Gly Ala Asn 130 135 140 Asp Phe Val Val Thr Phe Asn Thr Ser His Leu Gln Lys Lys Tyr Val 145 150 155 160 Lys Val Leu Met His Asp Val Ala Tyr Arg Gln Glu Lys Asp Glu Asn 165 170 175 Lys Trp Thr His Val Asn Leu Ser Ser Thr Lys Leu Thr Leu Leu Gln 180 185 190 Arg Lys Leu Gln Pro Ala Ala Met Tyr Glu Ile Lys Val Arg Ser Ile 195 200 205 Pro Asp His Tyr Phe Lys Gly Phe Trp Ser Glu Trp Ser Pro Ser Tyr 210 215 220 Tyr Phe Arg Thr Pro Glu Ile Asn Asn Ser Ser Gly Glu Met Asp Pro 225 230 235 240 Ile Leu Leu Thr Ile Ser Ile Leu Ser Phe Phe Ser Val Ala Leu Leu 245 250 255 Val Ile Leu Ala Cys Val Leu Trp Lys Lys Arg Ile Lys Pro Ile Val 260 265 270 Trp Pro Ser Leu Pro Asp His Lys Lys Thr Leu Glu His Leu Cys Lys 275 280 285 Lys Pro Arg Lys Asn Leu Asn Val Ser Phe Asn Pro Glu Ser Phe Leu 290 295 300 Asp Cys Gln Ile His Arg Val Asp Asp Ile Gln Ala Arg Asp Glu Val 305 310 315 320 Glu Gly Phe Leu Gln Asp Thr Phe Pro Gln Gln Leu Glu Glu Ser Glu 325 330 335 Lys Gln Arg Leu Gly Gly Asp Val Gln Ser Pro Asn Cys Pro Ser Glu 340 345 350 Asp Val Val Ile Thr Pro Glu Ser Phe Gly Arg Asp Ser Ser Leu Thr 355 360 365 Cys Leu Ala Gly Asn Val Ser Ala Cys Asp Ala Pro Ile Leu Ser Ser 370 375 380 Ser Arg Ser Leu Asp Cys Arg Glu Ser Gly Lys Asn Gly Pro His Val 385 390 395 400 Tyr Gln Asp Leu Leu Leu Ser Leu Gly Thr Thr Asn Ser Thr Leu Pro 405 410 415 Pro Pro Phe Ser Leu Gln Ser Gly Ile Leu Thr Leu Asn Pro Val Ala 420 425 430 Gln Gly Gln Pro Ile Leu Thr Ser Leu Gly Ser Asn Gln Glu Glu Ala 435 440 445 Tyr Val Thr Met Ser Ser Phe Tyr Gln Asn Gln 450 455 <210> 20 <211> 405 <212> DNA <213> Artificial Sequence <400> 20 atggctcagt cactggctct gagcctcctt atcctggttc tggcctttgg catccccagg 60 acccaaggca gtgatggagg ggctcaggac tgttgcctca agtacagcca aaggaagatt 120 cccgccaagg ttgtccgcag ctaccggaag caggaaccaa gcttaggctg ctccatccca 180 gctatcctgt tcttgccccg caagcgctct caggcagagc tatgtgcaga cccaaaggag 240 ctctgggtgc agcagctgat gcagcatctg gacaagacac catccccaca gaaaccagcc 300 cagggctgca ggaaggacag gggggcctcc aagactggca agaaaggaaa gggctccaaa 360 ggctgcaaga ggactgagcg gtcacagacc cctaaagggc catag 405 <210> 21 <211> 134 <212> PRT <213> Artificial Sequence <400> 21 Met Ala Gln Ser Leu Ala Leu Ser Leu Leu Ile Leu Val Leu Ala Phe 1 5 10 15 Gly Ile Pro Arg Thr Gln Gly Ser Asp Gly Gly Ala Gln Asp Cys Cys 20 25 30 Leu Lys Tyr Ser Gln Arg Lys Ile Pro Ala Lys Val Val Arg Ser Tyr 35 40 45 Arg Lys Gln Glu Pro Ser Leu Gly Cys Ser Ile Pro Ala Ile Leu Phe 50 55 60 Leu Pro Arg Lys Arg Ser Gln Ala Glu Leu Cys Ala Asp Pro Lys Glu 65 70 75 80 Leu Trp Val Gln Gln Leu Met Gln His Leu Asp Lys Thr Pro Ser Pro 85 90 95 Gln Lys Pro Ala Gln Gly Cys Arg Lys Asp Arg Gly Ala Ser Lys Thr 100 105 110 Gly Lys Lys Gly Lys Gly Ser Lys Gly Cys Lys Arg Thr Glu Arg Ser 115 120 125 Gln Thr Pro Lys Gly Pro 130 <210> 22 <211> 261 <212> PRT <213> Artificial Sequence <400> 22 Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile 1 5 10 15 Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 23 <211> 261 <212> PRT <213> Artificial Sequence <400> 23 Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu 1 5 10 15 Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr 20 25 30 Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly 35 40 45 Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg 50 55 60 Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg 65 70 75 80 Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val 85 90 95 Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser 100 105 110 Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser 115 120 125 Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val 130 135 140 Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly 145 150 155 160 Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe 165 170 175 Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met 180 185 190 Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala 195 200 205 Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly 210 215 220 Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile 225 230 235 240 Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser 245 250 255 Lys His Asp Tyr Val 260 <210> 24 <211> 135 <212> DNA <213> Artificial Sequence <400> 24 aaatggatca ggaaaaaatt cccccacata ttcaagcaac catttaagaa gaccactgga 60 gcagctcaag aggaagatgc ttgtagctgc cgatgtccac aggaagaaga aggaggagga 120 ggaggctatg agctg 135 <210> 25 <211> 45 <212> PRT <213> Artificial Sequence <400> 25 Lys Trp Ile Arg Lys Lys Phe Pro His Ile Phe Lys Gln Pro Phe Lys 1 5 10 15 Lys Thr Thr Gly Ala Ala Gln Glu Glu Asp Ala Cys Ser Cys Arg Cys 20 25 30 Pro Gln Glu Glu Glu Gly Gly Gly Gly Gly Tyr Glu Leu 35 40 45 <210> 26 <211> 473 <212> PRT <213> Artificial Sequence <400> 26 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ile Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60 Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 290 295 300 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 305 310 315 320 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 325 330 335 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 340 345 350 Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala 355 360 365 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 370 375 380 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 385 390 395 400 Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln 405 410 415 Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 420 425 430 Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 435 440 445 Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 450 455 460 Leu His Met Gln Ala Leu Pro Pro Arg 465 470 <210> 27 <211> 471 <212> PRT <213> Artificial Sequence <400> 27 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ile Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60 Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly 290 295 300 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg 305 310 315 320 Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln 325 330 335 Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 340 345 350 Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 355 360 365 Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 370 375 380 Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 385 390 395 400 Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly 405 410 415 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 420 425 430 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 435 440 445 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 450 455 460 Met Gln Ala Leu Pro Pro Arg 465 470 <210> 28 <211> 21 <212> PRT <213> Artificial Sequence <400> 28 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 29 <211> 45 <212> PRT <213> Artificial Sequence <400> 29 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 30 <211> 27 <212> PRT <213> Artificial Sequence <400> 30 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 31 <211> 41 <212> PRT <213> Artificial Sequence <400> 31 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 32 <211> 42 <212> PRT <213> Artificial Sequence <400> 32 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 33 <211> 113 <212> PRT <213> Artificial Sequence <400> 33 Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly 1 5 10 15 Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45 Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln 50 55 60 Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu 65 70 75 80 Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr 85 90 95 Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro 100 105 110 Arg <210> 34 <211> 324 <212> DNA <213> Artificial Sequence <400> 34 atggcccccc gtgtgacccc actcctggcc ttcagcctgc tggttctctg gaccttccca 60 gccccaactc tggggggtgc taatgatgcg gaagactgct gcctgtctgt gacccagcgc 120 cccatccctg ggaacatcgt gaaagccttc cgctaccttc ttaatgaaga tggctgcagg 180 gtgcctgctg ttgtgttcac cacactaagg ggctatcagc tctgtgcacc tccagaccag 240 ccctgggtgg atcgcatcat ccgaagactg aagaagtctt ctgccaagaa caaaggcaac 300 agcaccagaa ggagccctgt gtct 324 <210> 35 <211> 515 <212> PRT <213> Artificial Sequence <400> 35 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ile Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Tyr Asn Trp His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile His Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ala Leu 50 55 60 Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ala Arg Ile Tyr Asn Gly Asn Ser Phe Pro Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu 130 135 140 Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln 145 150 155 160 Ser Leu Phe Asn Ser Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln 165 170 175 Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr 180 185 190 Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr 195 200 205 Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val 210 215 220 Tyr Tyr Cys Gln Asn Ala Tyr Ser Phe Pro Tyr Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Leu Glu Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255 Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260 265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp 275 280 285 Phe Ala Cys Asp Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala 290 295 300 Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg 305 310 315 320 Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro 325 330 335 Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 340 345 350 Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu 355 360 365 Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu 370 375 380 Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys 385 390 395 400 Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln 405 410 415 Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu 420 425 430 Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly 435 440 445 Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu 450 455 460 Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys 465 470 475 480 Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu 485 490 495 Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu 500 505 510 Pro Pro Arg 515

Claims

1. A genetically engineered cell, characterized in that: The cells express exogenous receptors that specifically bind to target antigens and exogenous CCL21; they also express exogenous IL-7; the cells are immune effector cells, and the cells do not express exogenous CCL19.

2. The cell according to claim 1, characterized in that The exogenous CCL21 is natural CCL21.

3. The cell according to claim 2, characterized in that The natural CCL21 has the amino acid sequence shown in SEQ ID NO: 21; or has the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 14 or 15.

4. The cell according to any one of claims 1 to 3, characterized in that The exogenous CCL21 is constitutively expressed.

5. The cell according to any one of claims 1 to 3, characterized in that The exogenous CCL21 is expressed in an inducible manner.

6. The cell according to claim 5, characterized in that The inducible expression is driven by an immune cell inducible promoter.

7. The cell according to claim 6, characterized in that The immune cell inducible promoter is the NFAT promoter.

8. The cell according to claim 1, wherein The exogenous IL-7 is natural IL-7.

9. The cell according to claim 8, characterized in that The natural IL-7 has the amino acid sequence shown in SEQ ID NO: 18; or has the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:

13.

10. The cell according to any one of claims 1, 8 or 9, wherein The exogenous IL-7 is constitutively expressed.

11. The cell according to any one of claims 1, 8 or 9, wherein The exogenous IL-7 is expressed in an inducible manner.

12. The cell according to claim 11, wherein The inducible expression is driven by an immune cell inducible promoter.

13. The cell according to claim 12, wherein The immune cell inducible promoter is the NFAT promoter.

14. The cell according to claim 1, wherein The immune effector cells are T cells, NK cells or NKT cells.

15. The cell according to claim 14, characterized in that The immune effector cells are T cells.

16. The cell according to claim 1, wherein The target antigens include tumor antigens and / or pathogen antigens.

17. The cell according to claim 16, wherein The target antigen is a tumor antigen.

18. The cell according to claim 16, wherein The target antigen is a solid tumor antigen.

19. The cell according to claim 18, characterized in that The solid tumor antigen is GPC3, EGFR or Claudin18.

2.

20. The cell according to claim 19, wherein The solid tumor antigen is Claudin18.

2.

21. The cell according to claim 1, wherein The exogenous receptor is a chimeric receptor, which comprises an antigen binding domain that specifically binds to a target antigen, a transmembrane domain, and an intracellular domain.

22. The cell according to claim 21, wherein The exogenous receptor is selected from a chimeric antigen receptor (CAR), a modified T cell (antigen) receptor (TCR), a T cell fusion protein (TFP), a T cell antigen coupler (TAC) or a combination thereof.

23. The cell according to claim 22, characterized in that The exogenous receptor is a chimeric antigen receptor.

24. The cell according to claim 23, wherein The chimeric antigen receptor comprises: (i) an antibody or fragment thereof that specifically binds to a target antigen, CD28 or a transmembrane domain of CD8, a co-stimulatory signaling domain of CD28, and CD3ζ; or (ii) an antibody or fragment thereof that specifically binds to a target antigen, a transmembrane domain of CD28 or CD8, a co-stimulatory signaling domain of 4-1BB, and CD3ζ; or (iii) an antibody or a fragment thereof that specifically binds to a target antigen, CD28 or a transmembrane domain of CD8, a costimulatory signaling domain of CD28, a costimulatory signaling domain of 4-1BB, and CD3ζ.

25. The cell according to claim 21, wherein The amino acid sequence of the antigen binding domain of the exogenous receptor has the amino acid sequence shown in SEQ ID NO:

2.

26. The cell according to claim 25, wherein The amino acid sequence of the exogenous receptor has the amino acid sequence shown in SEQ ID NO: 26, 27 or 35.

27. The cell according to claim 1, wherein The exogenous receptor, and / or exogenous IL-7, and / or exogenous CCL21 are expressed using a viral vector.

28. The cell according to claim 27, wherein The viral vector includes: a lentiviral vector, a retroviral vector or an adenoviral vector.

29. An expression construct, characterized in that The expression construct is composed of the following expression cassettes connected in sequence: an expression cassette 1 of an exogenous receptor that specifically binds to a target antigen, an expression cassette 2 of exogenous IL-7, and an expression cassette 3 of exogenous CCL21.

30. The expression construct of claim 29, wherein The expression cassettes are optionally connected by a tandem fragment selected from F2A, P2A, T2A, and / or E2A.

31. The expression construct of claim 29, wherein The expression cassette 2 contains the nucleic acid sequence shown in SEQ ID NO:

17.

32. The expression construct of claim 29, wherein The expression cassette 3 contains the nucleic acid sequence shown in SEQ ID NO:

20.

33. An expression vector comprising the expression construct of any one of claims 29-32.

34. A virus, characterized in that The virus comprises the expression vector of claim 33.

35. Use of an immune effector cell expressing a chimeric receptor in the preparation of a medicament for increasing the activity of an immune effector cell expressing a chimeric receptor in an individual, characterized in that: Exogenous CCL21 and exogenous IL-7 are co-expressed in the immune effector cells, and the cells do not express exogenous CCL19.

36. The use according to claim 35, characterized in that The chimeric receptor is a chimeric antigen receptor.

37. The use according to claim 35, characterized in that The exogenous CCL21 is natural CCL21.

38. The use according to claim 37, characterized in that The natural CCL21 has the amino acid sequence shown in SEQ ID NO: 21; or has the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO: 14 or 15.

39. The use according to claim 35, characterized in that The exogenous CCL21 is constitutively expressed.

40. The use according to claim 35, characterized in that The exogenous CCL21 is expressed in an inducible manner.

41. The use according to claim 40, characterized in that The inducible expression is driven by an immune cell inducible promoter.

42. The use according to claim 41, characterized in that The immune cell inducible promoter is the NFAT promoter.

43. The use according to claim 35, characterized in that The exogenous IL-7 is natural IL-7.

44. The use according to claim 43, characterized in that The IL-7 has the amino acid sequence shown in SEQ ID NO: 18; or has the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO:

13.

45. The use according to claim 35, characterized in that: The exogenous IL-7 is constitutively expressed.

46. ​​The use according to claim 35, characterized in that The exogenous IL-7 is expressed in an inducible manner.

47. The use according to claim 46, characterized in that The inducible expression is driven by an immune cell inducible promoter.

48. The use according to claim 47, characterized in that The immune cell inducible promoter is the NFAT promoter.

49. The use according to claim 35, characterized in that The immune effector cells are T cells, NK cells or NKT cells.

50. Use of the cell according to any one of claims 1-28, or the expression construct according to any one of claims 29-32, or the expression vector according to claim 33, or the virus according to claim 34 for preparing a drug for inhibiting tumors.

51. The use according to claim 50, characterized in that The prepared tumor-inhibiting drug is used in combination with a chemotherapeutic drug.

52. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the cell according to any one of claims 1 to 28 and a pharmaceutically acceptable carrier or excipient.

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