WO2019219029A1 - 基因工程化的细胞及应用 - Google Patents

基因工程化的细胞及应用 Download PDF

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Publication number
WO2019219029A1
WO2019219029A1 PCT/CN2019/087077 CN2019087077W WO2019219029A1 WO 2019219029 A1 WO2019219029 A1 WO 2019219029A1 CN 2019087077 W CN2019087077 W CN 2019087077W WO 2019219029 A1 WO2019219029 A1 WO 2019219029A1
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Prior art keywords
cell
exogenous
cells
seq
amino acid
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PCT/CN2019/087077
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English (en)
French (fr)
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李宗海
骆红
蒋华
王华茂
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科济生物医药（上海）有限公司
上海市肿瘤研究所
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Application filed by 科济生物医药（上海）有限公司, 上海市肿瘤研究所 filed Critical 科济生物医药（上海）有限公司
Priority to EP19803938.0A priority Critical patent/EP3822345B1/en
Priority to JP2021514465A priority patent/JP2021523743A/ja
Priority to CN201980032482.XA priority patent/CN112154204A/zh
Priority to AU2019271819A priority patent/AU2019271819A1/en
Priority to SG11202011392VA priority patent/SG11202011392VA/en
Priority to KR1020207036137A priority patent/KR20210018838A/ko
Priority to CA3100446A priority patent/CA3100446A1/en
Priority to US17/055,470 priority patent/US20210213061A1/en
Publication of WO2019219029A1 publication Critical patent/WO2019219029A1/zh

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Definitions

  • the 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 an exogenous receptor of a target antigen and exogenous CCL21.
  • CAR-T cells can perform tumor-specific killing in a non-restrictive manner by MHC, and have shown good application prospects in tumor immunotherapy, but there are still many limitations, such as poor efficacy against solid tumors.
  • Candidate drugs that exhibit excellent effects in vitro often fail to exhibit corresponding effects in the body.
  • Adachi et al. attempted to use CAR-T cells expressing IL7 and CCL19 (IL-7 and CCL19 expression in CAR-T cells improved immune cell infiltration and CAR-T cell survival in the tumor. Nature Biotechnology, 2018, 36(4), 346 –351) to increase the anti-tumor ability of CAR-T cells.
  • a genetically engineered cell comprising an exogenous receptor that specifically binds to a target antigen and exogenous CCL21.
  • the cell expresses an exogenous receptor that specifically binds to a target antigen, exogenous CCL21, and a protein that promotes proliferation of the cell.
  • the protein that promotes proliferation of the cell is an IL-7R binding protein or exogenous IL-7.
  • the IL-7R binding protein is an exogenous IL-7R binding protein, ie, the cell comprises an exogenous receptor that specifically binds to a target antigen, exogenous CCL21, and exogenous IL- 7R binding protein.
  • the exogenous IL-7R binding protein is capable of specifically binding to IL-7R and enhancing IL-7R activity.
  • the exogenous IL-7R binding protein is selected from the group consisting of antibodies to IL-7R.
  • the amino acid sequence of said exogenous IL-7R is set forth in SEQ ID NO: 19.
  • the exogenous CCL21 is native CCL21, or a truncated fragment of native CCL21 that has the same function as native CCL21 or a mutant of native CCL21.
  • the native CCL21 has at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO: 21, or is SEQ ID a truncated fragment of the amino acid sequence shown by NO: 21; or at least 80%, 85%, 90%, 95%, 98% or the amino acid sequence encoded by the nucleotide represented by SEQ ID NO: 14 or 15. 99% sequence identity, or a truncated fragment of the amino acid sequence encoded by the nucleotide set forth in SEQ ID NO: 14 or 15.
  • the native CCL21 is human CCL21 having the amino acid sequence set forth in SEQ ID NO: 21; or the amino acid sequence thereof is encoded by the nucleotide set forth in SEQ ID NO: 14 or 15.
  • the exogenous CCL21 is constitutively expressed.
  • the exogenous CCL21 is inducible expression.
  • the inducible expression is initiated by an immune cell-inducible promoter.
  • the immune cell-inducible promoter is the NFAT promoter.
  • the exogenous IL-7 is native IL-7, or a truncated fragment of native IL-7 that has the same function as native IL-7 or a mutant of native IL-7.
  • the amino acid sequence of native IL-7 has at least 90% identity to the sequence set forth in SEQ ID NO: 18, or a truncated fragment of the amino acid sequence set forth in SEQ ID NO: 18. Or a truncated fragment of the amino acid sequence which is at least 90% identical to the amino acid sequence encoded by the nucleotide set forth in SEQ ID NO: 13 or which is encoded by the nucleotide set forth in SEQ ID NO: 13.
  • the exogenous IL-7R binding protein or exogenous IL-7 is constitutively expressed.
  • the exogenous IL-7R binding protein or exogenous IL-7 is inducible.
  • the inducible expression is initiated by an immune cell-inducible promoter.
  • the immune cell-inducible promoter is the NFAT promoter.
  • the cell is an immune effector cell.
  • the immune effector cell is selected from the group consisting of a T cell, a B cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a mast cell or a bone marrow-derived phagocytic cell, or a combination thereof; preferably, The immune effector cells are selected from the group consisting of T cells and NK cells; more preferably, the immune effector cells are T cells.
  • the cells are derived from autologous cells; preferably, are autologous T cells, autologous NK cells; more preferably, are autologous T cells.
  • the cells are from allogeneic cells; preferably, allogeneic T cells or allogeneic NK cells (cell lines also comprising NK cells, such as NK92 cells).
  • the target antigen is a tumor antigen or a pathogen antigen.
  • the target antigen is a tumor antigen.
  • the tumor antigen is selected from the group consisting of: 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- 13R ⁇ ); interleukin 11 receptor alpha (IL-11R ⁇ ); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; 1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endot
  • TSHR thyroid stimulating hormone receptor
  • the tumor antigen is GPC3, EGFR, EGFRvIII or Claudin 18.2.
  • the target antigen is a pathogen antigen.
  • the pathogen antigen is selected from the group consisting of: a virus, a bacterium, a fungus, a protozoa, or an antigen of a parasite.
  • the viral antigen is selected from the group consisting of: a cytomegalovirus antigen, an Epstein-Barr virus antigen, a human immunodeficiency virus antigen, or an influenza virus antigen.
  • the exogenous receptor is a chimeric receptor comprising an antigen binding domain, a transmembrane domain and an intracellular domain.
  • the exogenous receptor is a chimeric receptor selected from the group consisting of a chimeric antigen receptor (CAR), a modified T cell (antigen) receptor (TCR), T Cell fusion protein (TFP), T cell antigen coupler (TAC), or a combination thereof.
  • CAR chimeric antigen receptor
  • TCR modified T cell
  • TCP T Cell fusion protein
  • TAC T cell antigen coupler
  • the exogenous receptor is a chimeric antigen receptor
  • the antigen binding domain of the chimeric antigen receptor comprises: antibody, 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 a corresponding antigen, or a combination thereof.
  • the exogenous receptor is a chimeric antigen receptor
  • the transmembrane domain of the chimeric antigen receptor comprises a transmembrane domain of a protein selected from the group consisting of a T cell receptor ⁇ , ⁇ or ⁇ chain, CD28, CD3 ⁇ , 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, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD
  • a protein
  • the exogenous receptor is a chimeric antigen receptor
  • the intracellular domain of the chimeric antigen receptor comprises: a primary signaling domain and/or a costimulatory signaling domain
  • the primary signaling domain comprises a protein selected from the group consisting of: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , common FcR ⁇ (FCER1G), FcR ⁇ (Fc ⁇ R1b), CD79a, CD79b, Fc ⁇ RIIa, DAP10, and DAP12 a signaling domain, or a combination thereof
  • the costimulatory signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30 , CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83,
  • the chimeric antigen receptor comprises: (i) an antibody or fragment thereof that specifically binds to a target antigen, a transmembrane domain of CD28 or CD8, a costimulatory signal domain of CD28, and CD3 ⁇ ; or Ii) an antibody or fragment thereof that specifically binds to a target antigen, a transmembrane domain of CD28 or CD8, a costimulatory signal domain of 4-1BB, and CD3 ⁇ ; or (iii) an antibody or fragment thereof that specifically binds to a target antigen, CD28 or The transmembrane domain of CD8, the costimulatory signal domain of CD28, the costimulatory signal domain of 4-1BB, and CD3 ⁇ .
  • amino acid sequence of the antigen binding domain of the exogenous receptor is at least 90% identical to the sequence set forth in SEQ ID NO:2.
  • amino acid sequence of the exogenous receptor is at least 90% identical to the sequence set forth in SEQ ID NO: 26, 27 or 35.
  • the exogenous receptor, and/or exogenous IL-7R binding protein, and/or exogenous CCL21 are expressed using a viral vector.
  • the viral vector comprises a lentiviral vector, a retroviral vector or an adenoviral vector.
  • an expression construct comprising an expression cassette 1 comprising an exogenous receptor that specifically binds to a target antigen, an exogenous IL-7R binding protein or Expression cassette 2 of exogenous IL-7, expression cassette 3 of exogenous CCL21; preferably, the expression cassettes are joined by a tandem fragment selected from F2A, PA2, T2A, and/or E2A.
  • the nucleic acid sequences of the F2A and P2A are shown in SEQ ID NO: 11 and SEQ ID NO: 16, respectively.
  • an expression vector comprising the expression construct of the second aspect of the invention is provided.
  • a virus comprising the expression vector of the third aspect of the invention is provided.
  • a method for increasing the viability of an immune response cell comprising co-expression in an immune response cell comprising: a chimeric antigen specific for binding to a target antigen according to the first aspect of the invention Receptor, exogenous IL-7R binding protein or exogenous IL-7, exogenous CCL21.
  • it is used to prepare a medicament for inhibiting tumors.
  • the preparation of a tumor suppressing drug is combined with a chemotherapeutic agent.
  • the tumor is a blood tumor.
  • the tumor is a solid tumor.
  • the tumor is selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell carcinoma of the lung, 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, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumor of children, bladder cancer, kidney or ureteral cancer, renal pelvis Cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor angiogenesis, spinal tumor, brain stem glioma
  • CNS central
  • the solid tumor is selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel 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, vulvar cancer, endocrine Systemic cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, kidney or ureteral cancer, renal pelvic cancer, central nervous system (CNS) tumor, primary CNS lymphoma, tumor Angiogenesis, spinal tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma.
  • CNS central nervous system
  • the solid tumor is selected from the group consisting of colon cancer, rectal cancer, liver cancer, non-small cell lung cancer, small bowel cancer, esophageal cancer, pancreatic cancer, head and neck cancer, skin or intraocular malignant melanoma, uterine cancer, ovarian cancer. , rectal cancer, anal cancer, stomach cancer. More preferably, the solid tumor is gastric cancer, pancreatic cancer, or esophageal cancer.
  • a pharmaceutical composition comprising the cell of the first aspect of the invention and a pharmaceutically acceptable carrier or excipient is provided.
  • kits comprising a kit A and a kit B, the kit A comprising genetically engineered cells, the cells comprising the first aspect of the invention An exogenous receptor that specifically binds to a target antigen is expressed; the kit B includes CCL21, and/or a protein that promotes proliferation of the cell.
  • the protein that promotes cell proliferation comprises the IL-7R binding protein or IL-7 of the first aspect of the invention. More preferably, the administration of the kit A and the kit B is in no particular order.
  • the kit A comprises a chimeric receptor-modified immune effector cell.
  • the chimeric receptor is a chimeric antigen receptor.
  • the immune effector cells are T cells, NK cells or NKT cells.
  • a method for inhibiting or inhibiting a pathogen or enhancing immune tolerance of a subject comprising administering the cell of the first aspect of the invention, or the seventh aspect of the invention.
  • the pharmaceutical composition, or the kit of the eighth aspect of the invention Preferably, the administration of a chemotherapeutic drug is also included.
  • the cell provided by the present invention can enhance the cell 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. Survivability, ability to accumulate.
  • the immune effector cells prepared by the technical scheme of the invention have excellent tumor cell killing ability.
  • the cells prepared by the technique of the present invention are capable of combating immunosuppression in a 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 cancer.
  • Figure 1A is a plasmid map of MSCV-hu8E5(2I)-m28Z;
  • Figure 1B is a plasmid map of MSCV-hu8E5(2I)-m28Z-F2A-mIL-7-P2A-mCCL21a;
  • Figure 1C is a plasmid map of MSCV-hu8E5(2I)-m28Z-F2A-mIL7-P2A-mCCL21b;
  • Figure 1D is a MSCV-hu8E5(2I)-mBBZ plasmid map
  • Figure 1E is a plasmid map of MSCV-hu8E5(2I)-mBBZ-F2A-mIL-7-P2A-mCCL21a;
  • Figure 1F is a plasmid map of MSCV-hu8E5(2I)-m28Z-F2A-mIL7-P2A-mCCL21b;
  • Figure 2 shows the results of in vitro detection of cytokines IL7 and CCL21.
  • Figures 3A and 3B show the secretion of PD-1 by different groups of cells;
  • Figures 3C and 3D show the secretion of LAG-3 by different groups of cells;
  • Figures 3E and 3F show the secretion of TIM-3 by different groups of cells ;
  • Figure 4A shows the in vitro killing results of 28Z; 4B shows the in vitro killing results of BBZ;
  • Figure 5 shows the results of the in vitro proliferation assay
  • Figure 6 shows the results of a tumor treatment experiment in mice
  • Figure 7 is a plasmid map of mBBZ-7*19
  • Figure 8A shows the results of in vivo killing of CAR-T cells expressing IL7 and CCL21 with CAR-T cells expressing IL7 and CCL19;
  • Figure 8B shows changes in body weight of mice;
  • Figure 8C shows results of tumor weight comparison;
  • Figure 8D The CAR-T cell copy number after treatment of pancreatic cancer PANC02-A2 in mice is shown;
  • Figure 8E shows the results of immunohistochemical detection of mouse pancreatic cancer CD8+ cells;
  • Figure 9A shows the change in tumor volume of mouse breast cancer E0771-A2 orthotopic xenografts after treatment with CAR-T cells
  • Figure 9B shows tumor weight after treatment of mouse breast cancer E0771-A2 orthotopic transplantation tumor
  • Figure 9C shows CAR-T cell copy number after treatment of breast cancer in mice
  • Figure 9D shows the results of immunohistochemistry of mouse breast cancer CD8+ cells
  • Figure 10A shows changes in tumor volume after treatment of mouse liver cancer Hepal-6-A2 xenografts
  • Figure 10B shows the tumor weight after treatment of mouse liver cancer Hepa 1-6-A2 xenografts
  • Figure 10C shows the number of CAR-T cell copies after treatment of liver cancer in mice
  • Figure 10D shows the results of immunohistochemistry of mouse liver cancer CD8+ cells
  • FIG. 11 shows the results of detection of IFN- ⁇ in vitro
  • Fig. 12A shows changes in tumor volume after treatment with CAR-T cells in a mouse pancreatic cancer subcutaneous tumor clearing model
  • Fig. 12B shows tumor weight after treatment of a mouse pancreatic cancer subcutaneous tumor clearing model
  • Fig. 12C shows CAR-T cell copy number after treatment of mouse pancreatic cancer subcutaneous tumor clearing model
  • Figure 12D shows immunohistochemical results of mouse pancreatic cancer subcutaneous tumor clearing model CD8+ cells.
  • FIG. 13A shows the detection of Tcm in the spleen of the mouse pancreatic cancer PANC02-A2 subcutaneous tumor model CAR-T for d10 days;
  • Figure 13B shows the detection of Tcm in the spleen on day d20;
  • Figure 14A shows the content of Tcm in the d10-day bone marrow of the mouse pancreatic cancer PANC02-A2 subcutaneous tumor model CAR-T treatment;
  • Figure 14B shows the detection of Tcm in the spleen on the d20 day;
  • Figure 15 shows that the mouse pancreatic cancer PANC02-A2 subcutaneous tumor model CAR-T treatment has more DC cell infiltration in the tumor tissues of d10 days mice;
  • Figure 16 shows the content of MDSC in tumor tissues of mouse pancreatic cancer PANC02-A2 subcutaneous tumor model CAR-T treated d10 day mice.
  • an immune effector cell expressing an exogenous receptor targeting a tumor antigen and CCL21 not only has a superior killing effect on tumors, but also enhances the survival of immune effector cells in tumor tissues, even for refractory Solid tumors have also shown superior anti-tumor capabilities.
  • engineered and its grammatical other forms as used herein may refer to one or more changes in a nucleic acid, such as a nucleic acid within the genome of an organism.
  • engineered can refer to alterations, additions, and/or deletions of genes.
  • Engineered cells can also refer to cells having genes that are added, deleted, and/or altered.
  • genetically engineered cells refers to cells engineered by genetic engineering means.
  • the cell is an immune effector cell.
  • the cell is a T cell.
  • a genetically engineered cell described herein refers to a cell that expresses an exogenous receptor that specifically binds to a target antigen.
  • a genetically engineered cell described herein refers to a cell that expresses an exogenous receptor that specifically binds to a target antigen and expresses exogenous CLL21.
  • the genetically engineered cells described herein can also be T cells that co-express a chimeric antigen receptor that specifically binds to a tumor antigen, CLL21, and a protein that promotes T cell proliferation.
  • the genetically engineered cells described herein may also be T that co-express a chimeric antigen receptor that specifically binds to a tumor antigen, a CLL21, and an IL-7R binding protein or an exogenous IL-7. cell.
  • immune effector cells refers to cells involved in an immune response that produce an immune effect, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytic cells.
  • the immune effector cells are T cells, NK cells, NKT cells.
  • the T cell can be an autologous T cell, a heterologous T cell, an allogeneic T cell.
  • the NK cells can be allogeneic NK cells.
  • peptide refers to a compound consisting of amino acid residues covalently linked by a peptide bond.
  • the protein or peptide must contain at least two amino acids and there is no limit to the maximum number of amino acids that can include the sequence of the protein or peptide.
  • a polypeptide includes any peptide or protein comprising two or more amino acids that are bonded to each other by a peptide bond.
  • the term refers to short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (which are also commonly referred to in the art as proteins, which are present in many Type).
  • Polypeptide includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, or a combination thereof.
  • IL-7 Interleukin7, interleukin 7 or IL7 refers to a protein (e.g., from lactation) capable of interacting (e.g., binding) with IL-7R, preferably from a mammal, such as a murine or human IL-7R.
  • An animal such as a mouse or a human, and having one of the following characteristics: (i) is the amino acid sequence of a naturally occurring mammalian IL-7 or a fragment thereof, such as the amino acid sequence set forth in SEQ ID NO: 18 (human) or a fragment; (ii) having substantially the same amino acid sequence as SEQ ID NO: 18 (human) or a fragment thereof, for example, at least 85%, 90%, 95%, 96%, 97%, 98%, 99% homology Amino acid sequence; (iii) an amino acid sequence encoded by a naturally occurring mammalian IL-7 nucleotide sequence or a fragment thereof (eg, SEQ ID NO: 17 (human) or a fragment thereof; (iv) by and SEQ ID NO: a nucleotide sequence represented by 17 (human) or a fragment thereof having an amino acid sequence encoded by, for example, a nucleotide sequence of at least 85%, 90%, 95%, 96%, 97%, 98%, 99% homo
  • Exogenous IL-7R binding protein refers to all proteins that specifically bind to IL-7R and enhance IL-7R activity.
  • “Enhancing IL-7R activity” is understood to mean that the IL-7R binding protein is capable of enhancing any one or more of the activities of naturally occurring IL-7R, including but not limited to stimulating proliferation, cytotoxicity or maturation of NK cells; Proliferation or differentiation of B cells and T cells; stimulation of antibody production and affinity maturation in B cells; stimulation of cytotoxicity of CD8+ T cells; stimulation of interferon gamma production in T cells and NK cells; inhibition of dendritic cells (DC) Activation and maturation; inhibition of inflammatory mediator release from mast cells; enhancement of phagocytosis by macrophages; inhibition of TReg cell production or survival; and stimulation of proliferation of myeloid progenitor cells.
  • DC dendritic cells
  • CTL21 (Chemokine (CC motif) ligand 21) is one of the major immunochemokines expressed in the T cell region of secondary lymphoid tissues of the spleen and lymph nodes, and is responsible for antigen-activated (mature) dendritic cells (DC). ), recruitment of immature DCs and naive T cells.
  • CCL21 has one of the following characteristics: (i) an amino acid sequence of a naturally occurring mammalian CCL21 or a fragment thereof, for example, an amino acid sequence represented by SEQ ID NO: 21 (human) or a fragment thereof; (ii) The amino acid sequence of SEQ ID NO: 21 (human) or a fragment thereof has, for example, an amino acid sequence of at least 85%, 90%, 95%, 96%, 97%, 98%, 99% homology; (iii) by natural The mammalian CCL21 nucleotide sequence or fragment thereof (for example, the amino acid sequence encoded by SEQ ID NO: 20 (human) or a fragment thereof; (iv) the nucleotide sequence shown by SEQ ID NO: 20 (human) Or a fragment thereof having, for example, an amino acid sequence encoded by a nucleotide sequence of at least 85%, 90%, 95%, 96%, 97%, 98%, 99% homology; (v) by a naturally occurring CCL21 nucleotide
  • amino acid modification includes amino acid substitutions, additions and/or deletions, and "amino acid substitution” means the replacement of an amino acid at a particular position in the parent polypeptide sequence with another amino acid.
  • amino acid insertion means the addition of an amino acid at a particular position in the parent polypeptide sequence.
  • amino acid deletion or “deletion” means removal of an amino acid at a particular position in the parent polypeptide sequence.
  • conservatively modified as used herein means an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody comprising the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions, and deletions.
  • Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are substitutions in which amino acid residues are replaced with amino acid residues having similar side chains.
  • a family of amino acid residues having similar side chains has been defined in the art.
  • amino acids containing basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged acute side chains eg, , glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan
  • non-polar side chains eg, alanine, valine, leucine, isoleucine
  • Acid, proline, phenylalanine, methionine ⁇ -branched side chains (eg, threonine, valine, isoleucine)
  • aromatic side chains eg, tyrosine, benzene
  • wild type means the same meaning when referring to proteins and DNA.
  • mutation has the same or superior biological activity as a native protein or natural DNA, which undergoes substitution, addition or addition of one or more amino acids in the amino acid sequence of the native protein. Deletion; or substitution, addition or deletion of one or more nucleotides on the nucleic acid sequence of the native DNA.
  • sequence of the mutant herein has at least about 80%, preferably at least about 90%, more preferably at least about 95%, more preferably at least about the amino acid sequence of the native protein or the nucleic acid sequence of the native DNA.
  • variant of IL-7 generally refers to a polypeptide obtained by amino acid modification of wild-type IL-7 having similar biological activity or superior biological activity as IL-7.
  • truncated fragment refers to a non-full length version of a native protein or natural DNA that has a continuous or non-contiguous deletion of multiple amino acid residues or nucleotides in the native amino acid sequence or nucleic acid sequence that occurs in the sequence Any position, such as the head, middle, tail or a combination of them. In the present invention, the truncated fragments of the protein still retain the same function as the native protein from which they are derived.
  • Constant expression also known as sustained expression, means that a gene can be continuously expressed in a cell under almost all physiological conditions.
  • “Inducible expression” refers to expression under certain conditions, such as when a T cell binds to an antigen.
  • an effective amount refers to an amount of a compound, formulation, substance, or composition that is effective to achieve a particular biological result, such as, but not limited to, an amount or dose sufficient to promote a T cell response.
  • an amount or dose sufficient to promote a T cell response.
  • the precise administration dose of the immune effector cell or therapeutic agent of the present invention may be administered by a physician.
  • the individual is considered to be determined in terms of age, body weight, tumor size, or degree of metastasis, and the condition of the patient (subject).
  • An effective amount of immune effector cells means, but is not limited to, an increase, increase or prolongation of 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- ⁇ secretion; tumor regression, tumor shrinkage The number of immune effector cells for tumor necrosis.
  • promoter is a DNA sequence that is recognized by the synthetic machinery or the introduced synthetic machinery required to initiate specific transcription of a polynucleotide sequence.
  • 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.
  • polyII RNA polymerase II
  • TBP TATA binding protein
  • TAF TBP-associated factor
  • sequence elements e.g., enhancers
  • enhancers have been found to increase the overall level of expression of adjacent genes, often in a manner that is independent of position and/or orientation.
  • NFAT Natural factor of activated T cells
  • RUNX3 is Inducible expression using an inducible promoter.
  • the inducible promoter is a NFAT promoter.
  • the coding sequence of RUNX3 is placed in a minimal promoter with a NFAT-binding motif
  • the IL2 minimal promoter comprising six NFAT binding motifs is a promoter consisting of a binding partner of six NFATs in tandem with the minimal promoter of IL2.
  • an antigen binding receptor described herein refers to a chimeric receptor.
  • chimeric receptor refers to a fusion molecule formed by linking DNA fragments of different origins or corresponding cDNAs of proteins by genetic recombination techniques. Chimeric receptors typically include extracellular domains, transmembrane domains, and intracellular domains. Chimeric receptors useful in the present invention include, but are not limited to, chimeric antigen receptor (CAR), modified T cell (antigen) receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler ( TAC).
  • CAR chimeric antigen receptor
  • TCR modified T cell
  • T cell fusion protein T cell fusion protein
  • TAC T cell antigen coupler
  • ORF Open Reading Frame
  • chimeric antigen receptor refers to a group of polypeptides that, when administered in an immune effector cell, provide said cells with specificity for a target cell, typically a cancer cell, and have Intracellular signal production.
  • CAR typically includes at least one extracellular antigen binding domain (also known as the extracellular domain), a transmembrane domain (also known as a transmembrane domain), and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling structure”).
  • Domain “or “intracellular region”) comprising a functional signaling domain derived from a stimulatory molecule and/or a costimulatory molecule as defined below.
  • the polypeptide groups are contiguous with each other.
  • a polypeptide group includes a dimerization switch that can couple the 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.
  • the stimulatory molecule is an ⁇ chain that binds to a T cell receptor complex.
  • the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below.
  • the costimulatory molecule is selected from a costimulatory molecule described herein, such as 4-1BB (ie, CD137), CD27, and/or CD28.
  • a 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 stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and a functional signaling domain comprising a costimulatory molecule and a functionality derived from a stimulatory molecule The intracellular signaling domain of the signaling domain.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and two functional signaling comprising one or more costimulatory molecules.
  • the invention contemplates the modification of the amino acid sequence of a starting antibody or fragment (eg, scFv) that produces a functionally equivalent molecule.
  • a VH or VL of an antigen binding domain of a cancer associated antigen described herein, such as an scFv contained in a CAR can be modified to retain the initial VH or VL framework of the antigen binding domain of a cancer associated antigen described herein.
  • the invention contemplates modifications of the entire CAR construct, such as modification of one or more amino acid sequences of multiple domains of a CAR construct to produce a functionally equivalent molecule.
  • the CAR construct can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 of the starting CAR construct. %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • a transmembrane domain may include one or more additional amino acids adjacent to a transmembrane region, such as one or more proteins derived from the transmembrane protein.
  • Amino acids associated with the extracellular region eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids in the extracellular region
  • One or more additional amino acids associated with the extracellular region of the protein eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids in the intracellular region).
  • the transmembrane domain is a domain associated with one of the other domains of the chimeric receptor, eg, in one embodiment, the transmembrane domain can be derived from a signaling domain, costimulatory The same protein from which the domain or hinge domain is derived. In certain instances, the transmembrane domain can be selected or substituted by amino acids to avoid binding of such domains to the transmembrane domain of the same or different surface membrane proteins, for example, to interact with other members of the receptor complex. Minimize the effect. In one aspect, the transmembrane domain is capable of homodimerization with another chimeric receptor on the cell surface of a cell expressing the chimeric receptor.
  • the amino acid sequence of the transmembrane domain can be modified or substituted to minimize interaction with the binding domain of the native binding partner present in cells expressing the same chimeric receptor.
  • Transmembrane domains can be derived from natural or recombinant sources. When the source is native, the domain may be derived from any membrane-bound protein or transmembrane protein. In one aspect, a transmembrane domain is capable of transmitting a signal to an intracellular domain as long as the chimeric receptor binds to the target antigen.
  • Transmembrane domains specifically used in the present invention may include at least the following transmembrane domains: for example, alpha, beta or scorpion chains of T-cell receptors, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • transmembrane domains for example, alpha, beta or scorpion chains of T-cell receptors, CD28, CD27, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • the transmembrane domain can comprise at least the following transmembrane regions: eg, 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, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6.
  • transmembrane regions eg, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44,
  • 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.
  • a transmembrane domain can be linked to an extracellular region of a CAR, such as an antigen binding domain of a CAR, via a hinge (eg, a hinge from a human protein).
  • a hinge eg, a hinge from a human protein.
  • the hinge can be a human Ig (immunoglobulin) hinge (eg, an IgG4 hinge, an IgD hinge), a GS linker (eg, a GS linker as described herein), a KIR2DS2 hinge, or a CD8a hinge.
  • the transmembrane domain can be recombinant, in which case it will primarily comprise hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan, and valine can be found at each end of the recombinant transmembrane domain.
  • a short oligopeptide or polypeptide linker between 2 and 10 amino acids in length can form a bond between the transmembrane domain of the CAR and the cytoplasmic region.
  • the glycine-serine duplex provides a particularly suitable linker.
  • an "intracellular domain” (also referred to as an intracellular domain), includes an intracellular signaling domain.
  • the intracellular signaling domain is typically responsible for the activation of at least one of the normal effector functions of immune cells into which the chimeric receptor has been introduced.
  • effector function refers to the specialized function of a cell.
  • the effector function of a T cell can be, for example, a cytolytic activity or a helper activity, including secretion of a cytokine.
  • 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 particular function.
  • intracellular signaling domain Although it is generally possible to apply all intracellular signaling domains, in many cases it is not necessary to use the entire chain. In the case of a truncated portion of the intracellular signaling domain, such a truncated portion can be used in place of the entire strand as long as it transduces an effector function signal. Thus, the term intracellular signaling domain is meant to include a truncated portion of an intracellular signaling domain sufficient to transduce an effector function signal.
  • T cell activation can be said to be mediated by two different classes of cytoplasmic signaling sequences: those that trigger antigen-dependent primary activation by TCR (primary intracellular signaling domain) and antigen-independent manner Those that function to provide secondary or costimulatory signals (secondary cytoplasmic domains, such as costimulatory domains).
  • stimulation refers to the binding of a stimulatory molecule (eg, a TCR/CD3 complex or CAR) to its cognate ligand (or a tumor antigen in the case of a CAR), thereby mediating signal transduction events (eg, However, it is not limited to the initial response induced via signal transduction of the TCR/CD3 complex or via signal transduction of a suitable NK receptor or CAR signaling domain. Stimulation can mediate altered expression of certain molecules.
  • a stimulatory molecule eg, a TCR/CD3 complex or CAR
  • the term "irritating molecule” refers to a molecule that provides a cytoplasmic signaling sequence expressed by immune cells (eg, T cells, NK cells, B cells) that modulate the signaling pathway for immune cells in an irritating manner. At least some aspects of activation of immune cells.
  • the signal is a primary signal initiated by binding of, for example, a TCR/CD3 complex to a peptide-loaded MHC molecule, and which results in a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • a primary cytoplasmic signaling sequence (also referred to as a "primary signaling domain") that acts in a stimulatory manner may contain an immunoreceptor tyrosine-based activation motif (ITAM).
  • ITAM immunoreceptor tyrosine-based activation motif
  • ITAM-containing cytoplasmic signaling sequences specifically for use in the present invention include, but are not limited to, those derived from CD3 ⁇ , common FcR ⁇ (FCER1G), Fc ⁇ RIIa, FcR ⁇ (FcEpsilon R1b), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, DAP10 and DAP12.
  • the intracellular signaling domain in any one or more of the CARs of the invention comprises an intracellular signaling sequence, such as a CD3- ⁇ primary signaling sequence.
  • the primary signaling sequence of CD3- ⁇ is an equivalent residue derived from a human or non-human species such as mouse, rodent, monkey, donkey, and the like.
  • costimulatory molecule refers to a homologous 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.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that promotes an effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) and 4- 1BB (CD137).
  • costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1 CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD22), CD19
  • the costimulatory intracellular signaling domain can be an intracellular portion of a costimulatory molecule.
  • Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating 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-related antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and a ligand which specifically binds to CD83.
  • the intracellular signaling domain may comprise part or all of the native intracellular signaling domain, or a functional fragment or derivative thereof, of all cells within the molecule.
  • 4-1BB refers to a member of the TNFR superfamily having an amino acid sequence as provided by GenBank Accession No. AAA62478.2, or an equivalent residue from a non-human species such as a mouse, rodent, monkey, donkey, and the like;
  • the "4-1BB costimulatory domain” is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, ticks, and the like.
  • the "4-1BB costimulatory domain” is an equivalent residue from a human or from a non-human species such as a mouse, rodent, monkey, donkey, and the like.
  • T cell receptor a characteristic marker on the surface of all T cells, binds to CD3 with a non-covalent bond to form a TCR-CD3 complex.
  • the TCR is responsible for identifying antigens that bind to major histocompatibility complex molecules.
  • TCR is a heterodimer composed of two different peptide chains, consisting of two peptide chains, ⁇ and ⁇ . Each peptide chain can be further divided into variable region (V region), constant region (C region), transmembrane. The region and the cytoplasmic region are several parts; it is characterized by a short cytoplasmic region.
  • the TCR molecule belongs to the immunoglobulin superfamily, and its antigen specificity exists in the V region; the V region (V ⁇ , V ⁇ ) has three hypervariable regions CDR1, CDR2, and CDR3, among which the CDR3 mutation is the largest, which directly determines the TCR antigen. Binding specificity. When the TCR recognizes the MHC-antigen peptide complex, CDR1, CDR2 recognizes and binds to the side wall of the MHC molecule antigen binding groove, and CDR3 binds directly to the antigen peptide.
  • TCR is divided into two categories: TCR1 and TCR2; TCR1 consists of two chains of ⁇ and ⁇ , and TCR2 consists of two chains of ⁇ and ⁇ .
  • T cell fusion protein includes various polypeptide-derived recombinant polypeptides constituting a TCR, which are capable of binding to a surface antigen on a target cell, and to other polypeptides of the intact TCR complex. The effect is usually located on the surface of the T cell.
  • TFP consists of an antigen binding domain consisting of a TCR subunit and a human or humanized antibody domain, wherein the TCR subunit comprises at least a portion of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular domain.
  • the TCR subunit is operably linked to the antibody domain, wherein the extracellular, transmembrane, and intracellular signal domains of the TCR subunit are derived from CD3 epsilon or CD3 gamma, and the TFP is integrated TCR expressed on T cells.
  • T cell antigen coupler includes three functional domains: 1. Tumor targeting domain, including single-chain antibody, designed ankyrin repeat protein (DARPin). Or other targeting group; 2, an extracellular domain domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor closer to the TCR receptor; 3. the transmembrane region and the intracellular region of the CD4 co-receptor, Among them, the intracellular domain is linked to protein kinase LCK, which catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of TCR complexes as an initial step in T cell activation.
  • ITAMs immunoreceptor tyrosine activation motifs
  • 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-stranded or single-stranded, or intact immunoglobulins, and can be derived from natural or recombinant sources.
  • the antibody can be a tetramer of immunoglobulin molecules.
  • antibody fragment refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (eg, by binding, steric hindrance, stabilization/destabilization, spatial distribution).
  • 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 antibody (eg, sdAb), isolated CDR or other epitope binding of multispecific antibodies and antibodies formed by antibody fragments (eg, bivalent fragments comprising two Fab fragments joined by a disulfide bond in the hinge region) Fragment.
  • scFv refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein said light and heavy chain variable regions are contiguous (for example, via a synthetic linker such as a short flexible polypeptide linker), and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker such as a short flexible polypeptide linker
  • an scFv can have the VL and VH variable regions in any order (eg, relative to the N-terminus and C-terminus of the polypeptide), and the scFv can include a VL-linker-VH or A VH-linker-VL can be included.
  • antibody heavy chain refers to the larger of the two polypeptide chains that are present in the antibody molecule in their naturally occurring configuration and which typically determine the type to which the antibody belongs.
  • antibody light chain refers to the smaller of the two polypeptide chains present in the antibody molecule in their naturally occurring configuration.
  • the ⁇ (k) and ⁇ (l) light chains refer to the isoforms of the two major antibody light chains.
  • recombinant antibody refers to an antibody produced using recombinant DNA techniques, such as, for example, an antibody expressed by a bacteriophage or yeast expression system.
  • the term should also be interpreted to mean an antibody that has been produced by synthesizing a DNA molecule encoding an antibody (and wherein the DNA molecule expresses the antibody protein) or an amino acid sequence of a specified antibody, wherein the DNA or amino acid sequence has been obtained using recombinant DNA or is available in the art. And well known amino acid sequence techniques are available.
  • antigen refers to a molecule that elicits an immune response.
  • the immune response can involve activation of the antibody-producing or cells with specific immunity or both.
  • any macromolecule comprising virtually all proteins or peptides can serve as an antigen.
  • the antigen can be derived from recombinant or genomic DNA.
  • any DNA, encoded protein or peptide comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that causes an immune response.
  • the antigen need not be encoded only by the full length nucleotide sequence of the gene.
  • the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to encode a polypeptide that elicits a desired immune response.
  • the antigen does not need to be encoded by a "gene” at all.
  • the antigen may be produced synthetically, or may be derived from a biological sample, or may be a macromolecule other than a polypeptide.
  • biological samples can include, but are not limited to, tissue samples, tumor samples, cells or liquids with other biological components.
  • Tumor antigen refers to an antigen that is newly occurring or overexpressed during the development and progression of a hyperproliferative disease.
  • the hyperproliferative disorder of the invention refers to cancer.
  • the tumor antigen of the present invention may be a solid tumor antigen or a hematoma antigen.
  • the tumor antigen of the present invention includes, but is 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 ⁇ (IL-13R ⁇ ); 11 receptor alpha (IL-11R ⁇ ); 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; protease serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endothelial growth factor receptor 2 (VEGFR2);
  • the pathogen antigen is selected from the group consisting of: an antigen of a virus, a bacterium, a fungus, a protozoa, or a parasite;
  • the viral antigen is selected from the group consisting of: a cytomegalovirus antigen, an Epstein-Barr virus antigen, a human immunodeficiency virus antigen, or an influenza virus antigen.
  • tumor refers to a broad range of disorders in which hyperproliferative cells grow in vitro (eg, transformed cells) or in vivo.
  • Conditions which may be treated or prevented by the methods of the invention include, for example, various neoplasms, including benign or malignant tumors, various hyperplasias and the like.
  • cancer examples include, but are not limited to, breast cancer, prostate cancer, leukemia, lymphoma, nasopharyngeal carcinoma, glioma, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel cancer, esophagus Cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, stomach 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) tumor, hemangioma spinal tumor, glioma, astrocytoma, pituitary adenoma Combinations and metastatic lesions of the cancer.
  • CNS central nervous system
  • transfected or “transformed” or “transduced” refers to the process by which an exogenous nucleic acid is transferred or introduced into a host cell.
  • a “transfected” or “transformed” or “transduced” cell is one that has been transfected, transformed or transduced with an exogenous nucleic acid.
  • the cells include primary subject cells and their progeny.
  • binding partner eg, a tumor antigen
  • refractory refers to a disease, such as a tumor, which does not respond to treatment.
  • the refractory tumor can be resistant to treatment prior to or at the beginning of treatment.
  • the refractory tumor can be resistant during treatment.
  • 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.
  • the treatment regimens described herein can be used for refractory or recurrent malignancies.
  • relapsed means that after a period of improvement, such as prior effective tumor treatment, the patient reappears with signs and symptoms prior to the effective treatment.
  • an enhanced response refers to allowing a subject or tumor cell to improve its ability to respond to the treatments disclosed herein.
  • an enhanced response may include 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70 in responsiveness.
  • “enhanced” may also refer to increasing the number of subjects in response to treatment, such as immune effector cell therapy.
  • an enhanced response can refer to the total percentage of subjects responding to treatment, with percentages being 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55. %, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% more.
  • the treatment is by clinical outcome; the anti-tumor activity of the T cell is increased, enhanced or prolonged; the increase in the number of anti-tumor T cells or activated T cells, promotes IFN- ⁇ secretion, or Combination decision.
  • the clinical outcome is tumor regression; tumor shrinkage; tumor necrosis; anti-tumor response through the immune system; tumor enlargement, recurrence or spread, or a combination thereof.
  • the therapeutic effect is predicted by the presence of T cells, the presence of a genetic marker indicative of T cell inflammation, promotion of IFN-[gamma] secretion, or a combination thereof.
  • the immune effector cells as disclosed herein can be administered to an individual by various routes including, for example, oral or parenteral, such as intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intrarectal, intracisternal, intratumoral. Passive or accelerated absorption through the skin, intravasally, intradermally or separately using, for example, a skin patch or transdermal iontophoresis.
  • the total amount of agent to be administered in practicing the methods of the invention may be administered as a single dose by bolus or by infusion over a relatively short period of time, or may be administered using a fractionated treatment regimen, wherein over extended periods of time Multiple doses are administered in segments.
  • One skilled in the art will recognize that the amount of composition that treats a pathological condition in a subject depends on a number of 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. With these factors in mind, the technician will adjust the specific dose as needed. In general, initially, Phase I and Phase II clinical trials are used to determine the formulation of the composition as well as the route and frequency of administration.
  • a range such as 95-99% identity includes a range having 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96-99%, 96-98%, 96 to 97%, 97 to 99%, 97 to 98%, and 98 to 99% identity. This does not apply regardless of the width of the range.
  • CAR-T cells co-expressing IL7 and CCL21 When CAR-T cells co-expressing IL7 and CCL21 are used in a subject, the corresponding species can be selected, such as when used in a mouse, using a murine source of IL7 and CCL21 to construct a CAR element such as a transmembrane domain, The intracellular domain and the like can also be selected from the mouse source.
  • the subject is a human
  • components of human IL7 and CCL21 and human CAR are preferred.
  • the sequence of the CAR used can be as set forth in SEQ ID NO: 26, 27, or 34.
  • the cells of the invention can be used in combination with a chemotherapeutic agent when used in the treatment of tumors.
  • CLD18 refers to claudin-18 and includes any variant of CLD18 expressed by cells naturally expressed by the cell or transfected with the CLD18 gene (including CLD18A1 (claudin 18.1) and CLD18A2 (claudin 18.2)) , conformation, isoform and species homologs.
  • CLD18 refers to human CLD18, particularly CLD18A2 (SEQ ID NO: 22) and/or CLD18A1 (SEQ ID NO: 23), more preferably CLD18A2.
  • CLD18A1 includes post-translationally modified variants, isoforms and interspecies homologs of any human CLD18A1 expressed by cells which are naturally expressed by the cell or transfected with the CLD18A1 gene.
  • CLD18A2 includes post-translationally modified variants, isoforms and interspecies homologs of any human CLD18A2 expressed by cells which are naturally expressed by the cell or transfected with the CLD18A2 gene.
  • CLD18 variant shall include (i) a CLD18 splice variant, (ii) a CLD18 post-translational modification variant, in particular a variant comprising a different N glycosylation state, (iii) a CLD18 conformational variant, in particular comprising CLD18-conformation-1, CLD18-conformation-2 and CLD18-conformation-3, (iv) free CLD18 and homo/isotype-associated variants located at the intercellular junction, (v) CLD18 cancer-associated variant and CLD18 non- Cancer related variants.
  • the chimeric antigen receptor polypeptides of the invention may be selected from the group consisting of:
  • CD28a in the relevant chimeric antigen receptor protein represents the transmembrane region of the CD28 molecule
  • CD28b represents the intracellular signal region of the CD28 molecule.
  • the invention also includes nucleic acids encoding the chimeric antigen receptors.
  • the invention also relates to variants of the above polynucleotides which encode fragments, analogs and derivatives of polypeptides or polypeptides having the same amino acid sequence as the invention.
  • the invention also provides a vector comprising a nucleic acid of the above chimeric antigen receptor.
  • the invention also includes viruses comprising the vectors described above.
  • the virus of the present invention includes a packaged infectious virus, and also includes a virus to be packaged containing components necessary for packaging as an infectious 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.
  • the present invention also provides a chimeric antigen-modified immune effector cell transduced with a nucleic acid encoding the chimeric antigen receptor or transduced with the above-described recombinant plasmid containing the nucleic acid, or comprising the plasmid Virus.
  • Conventional nucleic acid transduction methods including non-viral and viral transduction methods, can be used in the present invention.
  • Non-viral based transduction methods include electroporation and transposon methods.
  • the Nucleofector nuclear transfection device developed by Amaxa can directly introduce foreign genes into the nucleus to obtain efficient transduction of the target gene.
  • the transduction efficiency of the transposon system based on Sleeping Beauty system or PiggyBac transposon is much higher than that of ordinary electroporation, and the nucleofector transfection apparatus is combined with the Sleeping Beauty transposon system. It 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.], The method has high transduction efficiency and can achieve targeted integration of the target gene.
  • the transduction method for the immune effector cells that effect the chimeric antigen receptor gene modification is based on a transduction method of a virus such as a retrovirus or a lentivirus.
  • the method has the advantages of high transduction efficiency, stable expression of the exogenous gene, and shortening the time for the cultured immune effector cells to reach the clinical level in vitro.
  • the transduced nucleic acid is expressed on its surface by transcription and translation.
  • the in vitro cytotoxicity assay of various cultured tumor cells demonstrates 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 be in tumor tissues. Effectively survive.
  • the nucleic acid encoding the chimeric antigen receptor of the present invention the plasmid containing the nucleic acid, the virus comprising the plasmid, and the transgenic immune effector cell transduced with the above nucleic acid, plasmid or virus can be effectively used for immunotherapy of tumors.
  • the chimeric antigen-modified immune effector cells of the present invention may also express another chimeric receptor other than the above chimeric receptor, which does not contain CD3 ⁇ , but contains the intracellular signal domain of CD28, CD137 Intracellular signal domain or a combination of the two.
  • the chimeric antigen receptor-modified immune effector cells of the present invention can be applied to the preparation of a pharmaceutical composition or a diagnostic reagent.
  • the composition may comprise a pharmaceutically acceptable carrier in addition to an effective amount of the immune cells.
  • pharmaceutically acceptable means that when the molecular body and composition are suitably administered to an animal or a human, they do not produce an adverse, allergic or other untoward reaction.
  • sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and derivatives thereof such as carboxymethyl fibers Sodium, ethyl cellulose and methyl cellulose; western yellow gum 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, glycerin, sorbitol, mannitol and polyethylene glycol; alginic acid; emulsifiers, such as Wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; compressed tablets, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions and phosphate buffers.
  • sugars such as lactose, glucose and sucrose
  • composition of the present invention can be formulated into various dosage forms as needed, and can be administered by a physician in accordance with factors such as patient type, age, body weight, and general disease condition, mode of administration, and the like.
  • the mode of administration can be by injection or other treatment.
  • the immune effector cells provided herein can effectively increase the proliferation, survival and function of the immune effector cells in the tumor; reduce the expression of the inhibitory immune checkpoint, thereby alleviating the depletion of T cells.
  • the immune effector cells provided in this paper have better killing effect and in vitro expansion performance on solid tumor cells.
  • Exemplary antigen receptors of the present invention including CAR, and methods for engineering and introducing a receptor into a cell, are described, for example, in Chinese Patent Application Publication No. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A. , CN105331585A, CN106397593A, CN106467573A, CN104140974A, International Patent Application Publication No. WO2017186121A1, WO2018006882A1, WO2015172339A8, WO2018/018958A1.
  • Claudin 18.2 was selected as a target of CAR-T cells, and in order to more accurately verify the anti-tumor effect in mice, the selected signal peptide, transmembrane region, intracellular region and the like were mouse-derived.
  • the method of preparation is operated according to conventional CAR-T cell preparation methods in the art.
  • the scFv used in this embodiment is a human Claudin 18.2 antibody
  • the nucleic acid sequence is shown in SEQ ID NO: 1
  • the chimeric antigen receptor used is a second generation chimeric antigen.
  • the receptor has a transmembrane domain of mCD8, an intracellular domain of mCD28 and/or an intracellular domain of m4-1BB, and mCD3 ⁇ .
  • a retroviral plasmid MSCV-hu8E5(2I)-28Z expressing a second-generation chimeric antigen receptor was constructed using MSCV.pBABE 5 (purchased from addgene) as a vector.
  • the nucleic acid sequence of hu8E5(2I)-28Z comprises the CD8 ⁇ signal peptide (SEQ ID NO: 3), the scFv (SEQ ID NO: 1), the mCD8 hinge region and the transmembrane region (SEQ ID NO: 5), and the mCD28 intracellular signal.
  • the conduction domain (SEQ ID NO: 7) and the intracellular domain mCD3 ⁇ (SEQ ID NO: 9) of mCD3 are composed.
  • the plasmid construction map of hu8E5(2I)-28Z is shown in Figure 1A.
  • 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) expressing CAR, IL7 and CCL21.
  • MSCV-hu8E5(2I)-m28Z-F2A-mIL7-P2A-mCCL21a (the plasmid map is shown in Figure 1B)
  • MSCV-hu8E5(2I)-m28Z-F2A-mIL7-P2A-mCCL21b the plasmid map is shown in Figure 1C).
  • F2A-mIL7-P2A-mCCL21a consists of F2A (SEQ ID NO: 11), mouse IL7 (SEQ ID NO: 13), P2A (SEQ ID NO: 16), 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 (SEQ ID NO: 15).
  • the retroviral plasmid MSCV-hu8E5(2I)-mBBZ expressing the second-generation chimeric antigen receptor was constructed using MSCV.pBABE 5 as a vector.
  • the hu8E5(2I)-mBBZ sequence consists of the CD8 ⁇ signal peptide (SEQ ID NO: 3), the scFv (SEQ ID NO: 1), the mCD8hinge and the transmembrane region (SEQ ID NO: 5), and the m4-1BB intracellular signaling domain. (SEQ ID NO: 24) and the intracellular portion of mCD3, CD3 ⁇ (SEQ ID NO: 9).
  • the plasmid map is shown in Figure 1D.
  • F2A-mIL-7-P2A-mCCL21a and F2A-mIL7-P2A-mCCL21b were inserted into the MSCV-hu8E5(2I)-mBBZ plasmid, and the retroviral plasmid MSCV-hu8E5 expressing CAR, IL7 and CCL21 was constructed.
  • (2I)-mBBZ-F2A-mIL-7-P2A-mCCL21a (the plasmid map is shown in Figure 1E)
  • MSCV-hu8E5(2I)-mBBZ-F2A-mIL7-P2A-mCCL21b the plasmid map is shown in Figure 1F) .
  • viruses hu8E5(2I)-28Z, IL7-CCL21a-28Z, IL7-CCL21b-28Z, hu8E5(2I)-BBZ, IL7-CCL21a-BBZ, IL7-CCL21b-BBZ were recorded.
  • Mouse T cell extraction and activation The mouse T cells were extracted from the spleen of C57BL/6 mice. After culture and activation, the obtained retroviruses hu8E5(2I)-28Z, IL7-CCL21a-28Z, IL7- were obtained.
  • CCL21b-28Z, hu8E5(2I)-BBZ, IL7-CCL21a-BBZ, IL7-CCL21b-BBZ were infected with 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.
  • mitomycin C was used to pre-treat mouse pancreatic cancer cell line PANC02 (cludin18.2 expression negative, purchased from ATCC) and PANC02-A2 (claudin18.2 expression positive) (40 ⁇ g/ml, 37°C, 2-3h) ).
  • PANC02-A2 cells were constructed by infecting PANC02 cells with pwpt-mclaudin 18.2 lentivirus.
  • the pWPT-mclaudin18.2 plasmid was constructed by in vitro synthesis of the mouse claudin18.2 gene (GeneBank reference sequence: NM_001194921), which was inserted into the lentiviral expression vector pWPT by restriction enzyme digestion and ligation to construct pwpt-mclaudin18. 2 plasmid.
  • Cell surface immune checkpoints were detected from UTD, mBBZ CAR-T cells, mBBZ-7*21A CAR-T cells, and mBBZ-7*21B CAR-T cells: PD-1, LAG-3, and TIM-3.
  • PD-1, LAG-3, and TIM-3 were collected in EP tubes. Each cell was divided into 3 tubes and washed twice with a pre-ice bath flow solution (1% NCS plus PBS). The different tubes were labeled 1: BV421-labeled anti-PD-1 antibody, APC-labeled anti-LAG-3 antibody, and APC-labeled anti-TIM-3 antibody were added in 50-fold dilution, incubated on ice for 45 min, washed 3 times and then transferred to flow tube assay. The results are shown in Figures 3A to 3F.
  • Figure 3A shows the expression of PD-1 in different groups of cells. The results showed that the secretion of PD-1 in the mBBZ group reached 30.2%, and the secretion of PD-1 in the mBBZ-7*21A group was only 11.7%, mBBZ-7* The secretion of PD-1 in group 21B was only 9.4%.
  • Figure 3B shows the expression intensity of PD-1. From Figure 3B, the expression of PD-1 in mBBZ group was higher than that in mBBZ-7*21A group and mBBZ-7*21B group. .
  • Figure 3C shows the expression of LAG-3 by different groups of cells. The results showed that the secretion of LAG-3 in the mBBZ group reached 80.7%, and the secretion of LAG-3 in the mBBZ-7*21A group was 53.4%, mBBZ-7*21B. The secretion was 13.7%.
  • Figure 3D shows the expression intensity of LAG-3. From Figure 3D, 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.
  • Figure 3E shows the expression of TIM-3 in different groups of cells. The results showed that the secretion of TIM-3 in the mBBZ group reached 41.3%, and the secretion of TIM-3 in the mBBZ-7*21A group was 16.2%, mBBZ-7*21B. The secretion was 13.2%.
  • Figure 3F shows the expression intensity of TIM-3. From Figure 3F, the expression of TIM-3 in the mBBZ group was higher than that in the mBBZ-7*21A group and mBBZ-7*21B.
  • CytoTox 96 non-radioactive cytotoxicity assay kit (Promega) was used. The specific method refers to the instruction manual of CytoTox 96 non-radioactive cytotoxicity test kit.
  • Effector cells UTD cells, m28Z CAR-T cells, 28Z-7*21A CAR-T cells, m28Z-7*21B CAR-T, mBBZ CAR were inoculated at a specific target ratio of 3:1, 1:1 or 1:3. -T cells, mBBZ-7*21A CAR-T cells, mBBZ-7*21B CAR-T cells in 96-well plates.
  • Target cells 50 ⁇ L of 2 ⁇ 10 5 /mL mouse pancreatic cancer cell lines PANC02-A2 and PANC02 cells were inoculated separately to the corresponding 96-well plates.
  • the experimental groups and the control groups were set as follows: experimental group: each target cell + different CAR-T cells; control group 1: maximum release of LDH from target cells; control group 2: spontaneous release of LDH from target cells; control group 3: effect The cells spontaneously release LDH.
  • the experimental results are shown in Figures 4A and 4B.
  • m28Z CAR-T cells, m28Z-7*21A CAR-T cells, or m28Z-7*21B CAR-T have a target ratio of 3:1 and 1 to PANC02-A2 compared to the control UTD. :1 has significant toxic killing effect and has no killing effect on PANC02 cells.
  • mBBZ CAR-T cells As can be seen from Figure 4B, mBBZ CAR-T cells, mBBZ-7*21A CAR-T cells, and mBBZ-7*21B CAR-T have a target ratio of 3:1 and 1 to PANC02-A2 compared to the control UTD: 1 has significant toxic killing effect, no killing effect on PANC02 cells.
  • Target cells PANC02-A2 cells (40 ⁇ g/ml, 37 °, 2-3 h) were treated with mitomycin C, and effector cells UTD cells, mBBZ CAR-T cells, mBBZ-7*21A CAR-T cells, mBBZ- 7*21B CAR-T cells were stained with CFSE, respectively, and then incubated for 1-2 days according to the target ratio (1 ⁇ 10 6 cells/ml).
  • Flow cytometry was used to detect the proliferation of CAR-T cells. As shown in Figure 5, mBBZ-7*21A CAR-T cells and mBBZ-7*21B CAR-T cells were able to proliferate faster than mBBZ CAR-T cells. .
  • Example 6 PANC02-A2 pancreatic cancer subcutaneous xenograft model tumor treatment
  • mice purchased from Shanghai Xipuer-Beikai Experimental Animal Co., Ltd.
  • groups of 6-8 weeks each group of 5-6, respectively UTD cells, mBBZ CAR-T cells, mBBZ-7*21A CAR-T cells, mBBZ-7*21B CAR-T cell treatment group.
  • CAR-T cell reinfusion D11 days after subcutaneous inoculation of tumor cells, the average tumor volume was about 60 mm 3 . Untreated T cells or CAR-T cells were injected at a dose of 2.5 x 10 6 /piece.
  • the results are shown in Fig. 6.
  • the tumor inhibition rates were 20 days after CAR-T injection: mBBZ CAR-T group: 35.5%, mBBZ-7*21A CAR-T group: 63%, mBBZ-7*21B CAR- Group T: 62.4%, indicating that the anti-tumor effect of mBBZ-7*21A CAR-T cells and mBBZ-7*21B CAR-T cells was better than that of mBBZ CAR-T cells (P ⁇ 0.05).
  • CAR-T cells mBBZ-7*19 CAR-T cells
  • IL7 and CCL19 were selected as controls.
  • Preparation of mBBZ-7*19 CAR-T cells was carried out as described in Example 1.
  • the F2A-mIL7-P2A-mCCL19 was inserted into the MSCV-hu8E5(2I)-mBBZ plasmid to construct reverse transcription of CAR, IL7 and CCL19.
  • the viral plasmid, plasmid map is shown in Figure 7, and the nucleic acid sequence of mCCL19 is shown in SEQ ID NO:34.
  • the plasmid infects mouse T cells to obtain mBBZ-7*19 CAR-T cells.
  • a mouse pancreatic cancer subcutaneous xenograft model was prepared according to the procedure of Example 6.
  • the average tumor volume was about 65 mm 3
  • mBBZ CAR-T cells, mBBZ-7*19 CAR-T cells, and mBBZ-7 were injected, respectively.
  • mBBZ CAR-T group 22.8%
  • mBBZ-7*19 CAR -T group 32.7%
  • mBBZ-7*21B CAR-T group 76.6%
  • mBBZ-7*21B CAR-T cell treatment group had better antitumor effect than mBBZ CAR-T cells and mBBZ-7*19 CAR- T cell group.
  • mice On the 31st day after tumor inoculation, the mice were euthanized, the tumors of the mice were excised and the tumor weight was weighed, and the specific statistical results are shown in Fig. 8C.
  • the results showed that the tumor weight of the mBBZ-7*21B CAR-T treatment group was significantly lower than that of the mBBZ group (P ⁇ 0.05), indicating that the co-expression of IL7 and CCL21 by chimeric antigen receptor-modified T cells can significantly increase the inhibition of T cells on tumors in vivo.
  • Genomic DNA kits were used to extract DNA from tumors and each sample concentration was measured. CAR copy number was detected using real-time quantitative PCR (qPCR). The standard curve was prepared based on the template plasmid, and finally the copy number of CAR in each sample was calculated.
  • Step 2 After euthanasia, the tumor tissue of the mouse is prepared, and the paraffin tissue section is prepared. After the conventional dewaxing, the specimen is hydrated; after the hydration is completed, the slice is placed on a shaker PBS for 3 times, and the citrate buffer is contained. After boiling, the tissue sections were placed in citrate buffer for antigen thermal repair; after repair, they were blocked with 1% BSA.
  • the blocked sections were added to the corresponding CD8a antibody (anti-mouse CD8 ⁇ antibody, purchased from Cell Signaling) or blank control reagent, incubated overnight at 4 ° C; washed with 0.5% PBST buffer; then washed with PBS buffer.
  • CD8a antibody anti-mouse CD8 ⁇ antibody, purchased from Cell Signaling
  • blank control reagent incubated overnight at 4 ° C; washed with 0.5% PBST buffer; then washed with PBS buffer.
  • the washed sections were added to secondary anti-goat anti-rabbit-HRP and incubated for 1 h at 37 °C. Washed with 0.5% PBST buffer twice, washed once with PBS buffer, DAB (1 Dako REAL TM EnVision TM Detection System, Peroxidase / DAB +,: 50 dilution) color.
  • Fig. 8E Observed under the microscope, the results are shown in Fig. 8E. Although there were obvious CD8 + T cell infiltration in the tumor tissues of the mBBZ-7*19 group and the mBBZ-7*21B group, the mBBZ-7*21B CAR-T group was included. CD8 + T cells infiltrate more.
  • a mouse model of subcutaneous xenograft of breast cancer was prepared, and E0771-A2 cells in a logarithmic growth phase and in good growth state were collected by trypsinization (preparation method: E0771 cells were infected with pwpt-mclaudin18.2 plasmid packaging lentivirus), After washing once with PBS, the cell density was adjusted to 2 ⁇ 10 7 /mL, and 50 ⁇ L of the cell suspension was subcutaneously injected into the fourth pair of breasts of the right abdomen of C57BL/6 mice, that is, each mouse was inoculated with 1 ⁇ 10 6 of E0771- A2 cells, the inoculation diary is the 0th day.
  • CAR-T cell reinfusion D12 days after subcutaneous inoculation of tumor cells, the average tumor volume was about 150 mm 3 . Untreated T cells or CAR-T cells were injected at a dose of 2.5 x 10 6 /piece.
  • the volume of E0771-A2 xenografts was measured every 3-4 days, and the tumor volume of each group was recorded. The results are shown in Fig. 9A, compared with mBBZ-7*19 CAR-T group, mBBZ-7*21B CAR The tumor killing ability of the -T treatment group was significantly enhanced.
  • Fig. 9B The results showed that the tumor weight of the mBBZ-7*21B CAR-T treatment group was significantly smaller than that of the mBBZ-7*19 CAR-T group (P ⁇ 0.05) and the mBBZ CAR-T group (P ⁇ 0.001), indicating that the chimeric antigen receptor was modified.
  • Co-expression of IL7 and CCL21 by T cells can significantly increase the inhibition of T cells on tumors in vivo.
  • step 3) of Example 7 the cell copy of CAR-T of the breast cancer subcutaneous xenograft model was examined, and the results are shown in Fig. 9C, mBBZ-7*19 CAR-T and mBBZ-7*21B CAR-T group.
  • the number of CAR-T copies is higher than that of the UTD and BBZ groups.
  • step 4) of Example 7 the tumor infiltration of CD8 + cells was examined, and the results are shown in Fig. 9D, and the tumor tissues of the mBBZ-7*19 CAR-T group and the mBBZ-7*21B CAR-T group were significantly observed.
  • CD8 T cells in which mBBZ-7*21B CAR-T group CD8 + T cells infiltrated more.
  • Example 9 Mouse liver cancer subcutaneous xenograft model
  • a mouse liver cancer xenograft model was prepared, and Hepa1-6-A2 cells in a logarithmic growth phase and in good growth state were collected by trypsinization (Hepa1-6 cells were infected with pwpt-mclaudin18.2 plasmid-packed lentivirus), and PBS was used. After washing once, the cell density was adjusted to 5 ⁇ 10 7 /mL, and 200 ⁇ L of the cell suspension was subcutaneously injected into the right abdomen of C57BL/6 mice, that is, 1 ⁇ 10 7 Hepal 1-6-A2 liver cancer cells were inoculated into each mouse. The vaccination diary is the 0th day.
  • CAR-T cell reinfusion D7 days after subcutaneous inoculation of tumor cells, the average tumor volume was about 300 mm 3 . Untreated T cells or CAR-T cells were injected at a dose of 1 ⁇ 10 6 /piece.
  • the volume of Hepal 1-6-A2 tumor was measured every 3-4 days, and the tumor volume of each group was recorded. The results are shown in Fig. 10A, compared with mBBZ-7*19 CAR-T group, mBBZ-7* The tumor killing ability of the 21B CAR-T treatment group was significantly enhanced.
  • Fig. 10B On the 31st day after tumor inoculation, the mice were euthanized, the tumors of the mice were excised and the tumor weight was weighed, and the specific statistical results are shown in Fig. 10B.
  • the results showed that the tumor weight of mBBZ-7*21B CAR-T treatment group was significantly smaller than that of mBBZ-7*19 CAR-T group (p ⁇ 0.01) and 8E5-2I-mBBZ CAR-T group (p ⁇ 0.05), indicating chimeric antigen.
  • Co-expression of IL7 and CCL21 by receptor-modified T cells can significantly increase the inhibition of T cells on tumors in vivo.
  • the CAR-T cell copy number was examined by the procedure of Example 3, step 3), and as a result, as shown in Fig. 10C, the number of CAR-T copies of the mBBZ-7*21B CAR-T group was high.
  • step 4) of Example 7 the tumor infiltration of CD8 + cells was examined, and the results are shown in Fig. 10D, and the tumor tissues of the mBBZ-7*19 CAR-T group and the mBBZ-7*21B CAR-T group were significantly observed.
  • CD8 + T cells in which mBBZ-7*21B CAR-T group CD8 + T cells infiltrated more.
  • UTD, 8E5-2I-mBBZ-CAR, mBBZ-7*21B CAR-T, and mBBZ-7*19 CAR-T were separately incubated with target cell PANC02-A2 for 1:1, and then ELISA was performed. The secretion level of IFN- ⁇ in the supernatant was examined.
  • the ELISA kit used was a Linkage Bio-mouse IFN- ⁇ test kit.
  • mBBZ-7*21B CAR-T cells were more IFN- ⁇ secreted after co-incubation with claudin 18.2 positive tumor cells.
  • Example 11 Treatment of mouse PANC02-A2 pancreatic cancer subcutaneous tumor clearing model
  • the PANC02-A2 subcutaneous xenograft model of C57BL/6 mice was prepared according to the procedure of Example 6.
  • the inoculation diary was on day 0, and on the 14th day after tumor inoculation, the average tumor volume was about 60 mm 3 , according to 100 mg/kg, tail vein.
  • Cyclophosphamide was injected, and untreated T cells or CAR-T cells were injected on the 15th day after tumor inoculation at a dose of 2.5 ⁇ 10 6 /piece.
  • the volume of PANC02-A2 tumor was measured every 3-4 days, and the tumor volume of each group was recorded. The results are shown in Fig. 12A. On the 38th day after tumor inoculation, the mice were euthanized and the mice were exfoliated. The tumor was weighed and the tumor was weighed. The specific statistical results are shown in Fig. 12B.
  • the CAR-T cell copy number was detected by the operation of the step 3) of Example 7, and as a result, as shown in Fig. 12C, the CAR-T copy number of the mBBZ-7*19 CAR-T and mBBZ-7*21B CAR-T groups was high. In the UTD and BBZ groups.
  • Fig. 12D Tumor infiltration of CD8 + cells was examined by following the procedure of Example 4, step 4), and the results are shown in Fig. 12D. There were obvious CD8 T cell infiltration in the tumor tissues of mBBZ-7*19 CAR-T group and mBBZ-7*21B CAR-T group, and the infiltration of CD8 T cells in mBBZ-7*21B CAR-T group was more.
  • Example 12 Detection of CAR-T cells in mouse PANC02-A2 pancreatic cancer subcutaneous tumor model
  • Example 6 a subcutaneous xenograft model of mouse pancreatic cancer was prepared, and 2 ⁇ 10 6 PANC02-A2 pancreatic cancer cells were subcutaneously injected into the right abdomen of C57BL/6 mice, and tumor cells were inoculated subcutaneously for 14 days.
  • the volume of about 60 mm 3 was injected into untreated T cells or CAR-T cells (mBBZ CAR-T cells, mBBZ-7*21A CAR-T cells, and mBBZ-7*19 CAR-T cells) at a dose of 4 ⁇ . 10 6 / only.
  • Tcm central memory T cells
  • Spleen cell extraction The mice were sacrificed by cervical dislocation, the spleen was taken, placed in a clean 2 mL EP tube, and the blood was washed away with PBS. The spleen cells were obtained by grinding using a 40 um filter. The spleen cell mixture was centrifuged at 400 g for 5 min, the supernatant was removed, 400 ⁇ L of mouse erythrocyte lysate (1 ⁇ ) was added, and the cells were allowed to stand for 5 min. The reaction was neutralized by adding 1.5 mL of PBS, centrifuged, resuspended in PBS, and subjected to antibody incubation, antibody labeling. It is CD8 (PerCP), CD44 (BV510), CD62L (APC).
  • Bone marrow cell extraction The mice were sacrificed by cervical dislocation, the mouse femur and tibia were taken, the muscles were removed, placed in a clean 2 mL EP tube, and the blood was washed away with PBS. Take a 2mL syringe, aspirate 2mL of PBS, puncture the needle along one end of the femur or tibia, hold it with a small forceps, squeeze the plunger, and rinse the bone marrow cells.
  • the detection of Tcm in the spleen on day d10 is shown in Fig. 13A
  • the detection of Tcm in the spleen on day d20 is shown in Fig. 13B
  • the mBBZ-CAR-T cell group Tcm expressing IL7 and CCL21 is compared with the conventional CAR-T. The content has increased significantly.
  • the content of Tcm in d10 day bone marrow is shown in Fig. 14A, and the detection of Tcm in spleen on d20 day is shown in Fig. 14B, compared with conventional CAR-T, mBBZ-7*21BCAR-T treatment, Tcm in bone marrow.
  • the content has increased significantly.
  • the tumor tissues of the mice were frozen and sectioned to detect DC infiltration.
  • the results are shown in Fig. 15.
  • the mBBZ-7*21B cell group was tumor-bearing. There is more DC cell infiltration in the tissue.
  • MDSC bone marrow-derived inhibitory cells
  • the tumor tissues of the UTD group, the mBBZ group, the mBBZ-7*21B group, and the mBBZ-7*19 CAR-T group were extracted from the 10th day after the treatment of CAR-T cells (d10), and the tumor surface fat was removed.
  • Enzyme collagenase type I (0.05mg/ml), collagenase type IV (0.05mg/ml, hyaluronidase (0.025mg/ml), DNase I (0.01mg/ml) shaken at 37°C for 30min (intermediate removal to observe digestion)
  • the digested suspension was passed through a 70 um cell sieve to a 50 mL tube (operating on ice), the undigested tissue was tapped with a syringe pusher, the sieve was washed with a large amount (to 20 mL) of the culture solution, and the digestion was terminated while 400 g was centrifuged.
  • the above examples select CAR-T cells that target CLD18A2, it being understood that selection of CAR-T cells that act on other targets also have the same effect, such as GPC3, EGFR, EGFRvIII, CD19, BCMA, and the like.
  • the antibody to be used may be mouse anti-human or humanized, and the transmembrane domain and the intracellular domain may be different species depending on the purpose, such as human.
  • the T cells can also express other cytokines that enhance CAR-T cell function, such as CAR-T cells co-expressed by CAR and type I interferons. CAR-T cells co-expressed by CAR and PD-1.
  • CAR-T cells co-expressed by CAR and PD-1.
  • other immune cells such as NK cells and NK-T cells, may also be selected, and specific subtypes of immune cells, such as ⁇ / ⁇ T cells, may be specifically selected. Wait.

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Abstract

本发明公开一种基因工程化的细胞，其表达特异性结合靶抗原的外源性受体和外源性CCL21，还可以进一步表达促进细胞增殖的IL-7R结合蛋白或外源性IL-7。还公开了一种包括外源性CCL21表达盒的表达构建物与含其的载体、病毒以及包括上述细胞的药物组合物。还公开了所述细胞、表达构建物、载体和病毒在制备抑制肿瘤或抑制病原体的药物上的应用。

Description

基因工程化的细胞及应用
本申请要求申请日为2018年5月15日的中国专利申请CN201810463564.7、2018年9月18日的中国专利申请CN201811088090.9、2018年12月19日的中国专利申请CN201811552806.6、2019年2月28日的中国专利申请CN201910151930.X的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于细胞治疗领域，涉及基因工程化的细胞及应用。更具体地，本发明涉及特异性结合靶抗原的外源性受体和外源性CCL21的细胞。
背景技术
CAR-T细胞能够以MHC非限制性的方式进行肿瘤特异性地杀伤，在肿瘤免疫治疗中显示出较好的的应用前景，但其仍有较多局限性，如对实体瘤的疗效不佳、在体外表现出优良效果的候选药物，在体内往往无法表现出相应的效果。
Adachi等尝试采用表达IL7和CCL19的CAR-T细胞(IL-7 and CCL19 expression in CAR-T cells improves immune cell infiltration and CAR-T cell survival in the tumor.Nature Biotechnology,2018,36(4),346–351)，以提高CAR-T细胞的抗肿瘤能力。
发明内容
本发明的目的在于提供一种基因工程改造的细胞。
在本发明的第一方面，提供了一种基因工程化的细胞，所述细胞包含特异性结合靶抗原的外源性受体和外源性CCL21。
在具体实施方式中，所述细胞表达特异性结合靶抗原的外源性受体、外源性CCL21以及促进所述细胞增殖的蛋白。优选地，所述促进所述细胞增殖的蛋白为IL-7R结合蛋白或外源性IL-7。
在具体实施方式中，所述IL-7R结合蛋白为外源性IL-7R结合蛋白，即所述细胞包含特异性结合靶抗原的外源性受体、外源性CCL21以及外源性IL-7R结合蛋白。
在具体实施方式中，所述外源性IL-7R结合蛋白能特异性结合IL-7R且增强IL-7R活性。
在具体实施方式中，所述外源性IL-7R结合蛋白选自IL-7R的抗体。优选地，所 述外源性IL-7R的氨基酸序列如SEQ ID NO:19所示。
在具体实施方式中，所述外源性CCL21为天然CCL21、或与天然CCL21具有相同功能的天然CCL21的截短片段或天然CCL21的突变体。
在一具体实施方式中，所述天然CCL21与SEQ ID NO:21所示的氨基酸序列具有至少80％、85％、90％、95％、98％或99％的序列同一性、或是SEQ ID NO:21所示的氨基酸序列的截短片段；或者与由SEQ ID NO:14或15所示的核苷酸编码的氨基酸序列具有至少80％、85％、90％、95％、98％或99％的序列同一性、或是由SEQ ID NO:14或15所示的核苷酸编码的氨基酸序列的截短片段。在一优选例中，所述天然CCL21为人的CCL21，其氨基酸序列如SEQ ID NO:21所示；或者其氨基酸序列由SEQ ID NO:14或15所示的核苷酸编码。
在具体实施方式中，所述外源性CCL21是组成型表达。
在具体实施方式中，所述外源性CCL21是诱导型表达。在一优选例中，所述诱导型表达通过免疫细胞诱导型启动子启动。在一较佳优选例中，所述的免疫细胞诱导型启动子是NFAT启动子。
在具体实施方式中，所述外源性IL-7为天然IL-7、或与天然IL-7具有相同功能的天然IL-7的截短片段或天然IL-7的突变体。
在具体实施方式中，所述的天然IL-7的氨基酸序列与SEQ ID NO:18所示的序列具有至少90％的同一性、或是SEQ ID NO:18所示的氨基酸序列的截短片段；或者与由SEQ ID NO:13所示的核苷酸编码的氨基酸序列具有至少90％同一性、或是由SEQ ID NO:13所示的核苷酸编码的氨基酸序列的截短片段。
在具体实施方式中，所述外源性IL-7R结合蛋白或外源性IL-7是组成型表达。
在优选的实施方式中，所述外源性IL-7R结合蛋白或外源性IL-7是诱导性表达。
在优选的实施例中，所述诱导型表达通过免疫细胞诱导型启动子启动。在一较佳优选例中，所述的免疫细胞诱导型启动子是NFAT启动子。
在具体实施方式中，所述的细胞为免疫效应细胞。在具体实施例中，所述的免疫效应细胞选自T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞或骨髓源性吞噬细胞或其组合；优选地，所述免疫效应细胞选自T细胞、NK细胞；更优选地，所述免疫效应细胞为T细胞。
在具体实施方式中，所述的细胞来自自体细胞；优选地，为自体T细胞、自体NK细胞；更优选地，为自体T细胞。
在具体实施方式中，所述的细胞来自同种异体细胞；优选地，为同种异体的T 细胞或同种异体NK细胞(也包含NK细胞的细胞系，如NK92细胞)。
在具体实施方式中，所述靶抗原为肿瘤抗原或病原体抗原。
在具体实施方式中，所述靶抗原为肿瘤抗原。在优选的实施方式中，所述肿瘤抗原选自：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1 Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长20因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记25 1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin 6，Claudin18.2(CLD18A2)、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受5体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基 转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。
在一优选例中，所述肿瘤抗原为GPC3、EGFR、EGFRvIII或Claudin18.2。
在具体实施方式中，所述靶抗原为病原体抗原。在优选的实施方式中，所述病原体抗原选自：病毒、细菌、真菌、原生动物，或寄生虫的抗原。在一具体实施例中，所述病毒抗原选自：巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原或流感病毒抗原。
在具体实施方式中，所述外源性受体为嵌合受体，所述的嵌合受体包含抗原结合结构域，跨膜结构域和细胞内结构域。
在具体实施方式中，所述外源性受体为嵌合受体，该嵌合受体选自：嵌合抗原受体(CAR)、修饰的T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)或其组合。
在优选的实施方式中，所述外源性受体为嵌合抗原受体，该嵌合抗原受体的抗原结合结构域包含：抗体，抗体片段，scFv，Fv，Fab，(Fab')2，单结构域抗体(SDAB)，VH或VL结构域，或骆驼科VHH结构域，或相应抗原的天然配体，或其组合。
在优选的实施方式中，所述外源性受体为嵌合抗原受体，该嵌合抗原受体的跨膜结构域包含选自以下组成的蛋白质的跨膜结构域：T细胞受体的α、β或ζ链，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，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，NKp44，NKp30，NKp46，NKG2D，和NKG2C。
在优选的实施方式中，所述外源性受体为嵌合抗原受体，该嵌合抗原受体的细胞内结构域包括：一级信号传导结构域和/或共刺激信号传导结构域，其中：(1)所述一级信号传导结构域包含选自：CD3ζ，CD3γ，CD3δ，CD3ε，常见FcRγ(FCER1G)，FcRβ(FcεR1b)，CD79a，CD79b，FcγRIIa，DAP10，和DAP12的蛋白质的功能信号传导结构域，或其组合；和/或(2)所述共刺激信号传导结构域包含选自如下的蛋白质的功能信号传导结构域：CD27，CD28，4-1BB(CD137)，OX40，CD30，CD40，PD-1，ICOS，淋巴细胞功能相关的抗原-1(LFA-1)，CD2，CD7，LIGHT，NKG2C，B7-H3，特异性结合CD83的配体，CDS，ICAM-1，GITR，BAFFR，HVEM(LIGHTR)，SLAMF7，NKp80(KLRF1)，CD160，CD19，CD4，CD8α，CD8β，IL2Rβ，IL2Rγ，IL7Rα，ITGA4，VLA1，CD49a，ITGA4，IA4，CD49D，ITGA6，VLA-6，CD49f，ITGAD，CD11d，ITGAE，CD103，ITGAL，CD11a，LFA-1，ITGAM，CD11b，ITGAX，CD11c，ITGB1，CD29，ITGB2，CD18，LFA-1，ITGB7，TNFR2，TRANCE/RANKL，DNAM1(CD226)，SLAMF4(CD244，2B4)，CD84，CD96(Tactile)，CEACAM1，CRTAM，Ly9(CD229)，CD160(BY55)，PSGL1，CD100(SEMA4D)，CD69，SLAMF6(NTB-A，Ly108)，SLAM(SLAMF1，CD150，IPO-3)，BLAME(SLAMF8)，SELPLG(CD162)，LTBR，LAT，GADS，SLP-76，PAG/Cbp，NKp44，NKp30，NKp46，和NKG2D，或其组合。
在具体实施方式中，所述的嵌合抗原受体包括：(i)特异性结合靶抗原的抗体或其片段、CD28或CD8的跨膜域、CD28的共刺激信号结构域和CD3ζ；或(ii)特异性结合靶抗原的抗体或其片段、CD28或CD8的跨膜域、4-1BB的共刺激信号结构域和CD3ζ；或(iii)特异性结合靶抗原的抗体或其片段、CD28或CD8的跨膜域、CD28的共刺激信号结构域、4-1BB的共刺激信号结构域和CD3ζ。
在具体实施方式中，所述外源性受体的抗原结合结构域的氨基酸序列与SEQ ID NO:2所示的序列具有至少90％的同一性。
在具体实施方式中，所述外源性受体的氨基酸序列与SEQ ID NO:26、27或35所 示的序列具有至少90％的同一性。
在具体实施方式中，所述外源性受体、和/或外源性IL-7R结合蛋白、和/或外源性CCL21利用病毒载体表达。较佳地，所述的病毒载体包括：慢病毒载体，逆转录病毒载体或腺病毒载体。
在本发明的第二方面，提供了一种表达构建物，该表达构建物包括顺序连接的：特异性结合靶抗原的外源性受体的表达盒1，外源性IL-7R结合蛋白或外源性IL-7的表达盒2，外源性CCL21的表达盒3；优选地，所述表达盒之间由串联片段连接，选自F2A、PA2、T2A、和/或E2A。其中，所述F2A和P2A的核酸序列分别如SEQ ID NO:11和SEQ ID NO:16所示。
在本发明的第三方面，提供了一种表达载体，其包含本发明第二方面所述的表达构建物。
在本发明的第四方面，提供了一种病毒，所述的病毒包含本发明的第三方面所述的表达载体。
在本发明的第五方面，提供了一种提高免疫应答细胞活力的方法，所述方法包括在免疫应答细胞中共表达：包括本发明的第一方面所述的特异性结合靶抗原的嵌合抗原受体、外源性IL-7R结合蛋白或外源性IL-7、外源性CCL21。
在本发明的第六方面，提供了本发明的第一方面所述的细胞，或本发明的第二方面所述的表达构建物，或本发明的第三方面所述的表达载体，或本发明的第四方面所述的病毒的用途，用于制备抑制肿瘤、抑制病原体或加强受试者免疫耐受能力的药物。在具体的实施方式中，用于制备抑制肿瘤的药物。在优选的实施方式中，所述制备抑制肿瘤的药物与化疗药物联用。
在具体实施方式中，所述的肿瘤为血液肿瘤。
在具体实施方式中，所述的肿瘤为实体肿瘤。
在具体实施方式中，所述的肿瘤选自结肠癌，直肠癌，肾细胞癌，肝癌，肺的非小细胞癌，小肠癌，食道癌，黑素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，皮肤或眼内恶性黑素瘤，子宫癌，卵巢癌，直肠癌，肛区癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，阴户癌，霍奇金氏病，非霍奇金淋巴瘤，内分泌系统癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，阴茎癌，儿童实体瘤，膀胱癌，肾或输尿管癌，肾盂癌，中枢神经系统(CNS)瘤，原发性CNS淋巴瘤，肿瘤血管发生，脊椎肿瘤，脑干神经胶质瘤，垂体腺瘤，卡波西肉瘤，表皮样癌，鳞状细胞癌，T细胞淋巴瘤，环境诱发的癌症，所述癌症的组合和所述癌症 的转移性病灶。
在一优选例中，所述的实体肿瘤选自结肠癌，直肠癌，肾细胞癌，肝癌，非小细胞肺癌，小肠癌，食道癌，黑素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，皮肤或眼内恶性黑素瘤，子宫癌，卵巢癌，直肠癌，肛区癌，胃癌，睾丸癌，子宫癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，阴户癌，内分泌系统癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，阴茎癌，膀胱癌，肾或输尿管癌，肾盂癌，中枢神经系统(CNS)瘤，原发性CNS淋巴瘤，肿瘤血管发生，脊椎肿瘤，脑干神经胶质瘤，垂体腺瘤，卡波西肉瘤，表皮样癌，鳞状细胞癌。
更优选的，所述的实体肿瘤选自结肠癌，直肠癌，肝癌，非小细胞肺癌，小肠癌，食道癌，胰腺癌，头颈癌，皮肤或眼内恶性黑素瘤，子宫癌，卵巢癌，直肠癌，肛区癌，胃癌。更优选的，所述的实体肿瘤为胃癌、胰腺癌、或食道癌。
在本发明的第七方面，提供了一种药物组合物，所述的药物组合物包括本发明的第一方面所述的细胞和药学上可接受的载体或赋形剂。
在本发明的第八方面，提供了一种药盒，其包括药盒A和药盒B，所述药盒A包括基因工程化的细胞，所述细胞包括本发明的第一方面所述的表达特异性结合靶抗原的外源性受体；所述药盒B包括CCL21，和/或，促进所述细胞增殖的蛋白。优选地，所述促进细胞增殖的蛋白包括本发明的第一方面所述的IL-7R结合蛋白或IL-7。更优选地，所述药盒A和药盒B的施用不分先后。
在一优选例中，所述药盒A包括嵌合受体修饰的免疫效应细胞。优选地，所述嵌合受体为嵌合抗原受体。
在一优选例中，所述免疫效应细胞为T细胞、NK细胞或NKT细胞。
在本发明的第九方面，提供了一种抑制肿瘤或抑制病原体或加强受试者免疫耐受能力的方法，其包括施用本发明的第一方面所述的细胞，或本发明的第七方面所述的药物组合物，或本发明的第八方面所述的药盒。优选地，还包括施用化疗药物。
本发明的有益效果：
1、本发明所提供的细胞，由于共表达特异性结合靶抗原的外源性受体，外源性IL-7R结合蛋白或外源性IL-7、和外源性CCL21，能够提高细胞的存活能力、积蓄能力。
2、采用本发明的技方案制备得到的免疫效应细胞具有优异的肿瘤细胞杀伤能力。
3、采用本发明的技方案制备得到的细胞在治疗癌症中，能够对抗癌症微环境中的免疫抑制，从而对于实体瘤的作用显著增强。对于难治性和进行性的癌症也具有较好的效果。
附图说明
图1A为MSCV-hu8E5(2I)-m28Z的质粒图谱；
图1B为MSCV-hu8E5(2I)-m28Z-F2A-mIL-7-P2A-mCCL21a质粒图谱；
图1C为MSCV-hu8E5(2I)-m28Z-F2A-mIL7-P2A-mCCL21b质粒图谱；
图1D为MSCV-hu8E5(2I)-mBBZ质粒图谱；
图1E为MSCV-hu8E5(2I)-mBBZ-F2A-mIL-7-P2A-mCCL21a质粒图谱；
图1F为MSCV-hu8E5(2I)-m28Z-F2A-mIL7-P2A-mCCL21b质粒图谱；
图2显示了体外细胞因子IL7、CCL21检测的结果；
图3A和3B显示了不同组的细胞分泌PD-1的情况；图3C和3D显示了不同组的细胞分泌LAG-3的情况；图3E和3F显示了不同组的细胞分泌TIM-3的情况；
图4A显示了28Z的体外杀伤结果；4B显示了BBZ的体外杀伤结果；
图5显示了体外增殖检测的结果；
图6显示了小鼠体内肿瘤治疗实验的结果；
图7为mBBZ-7*19的质粒图；
图8A显示了表达IL7和CCL21的CAR-T细胞与表达IL7和CCL19的CAR-T细胞的体内杀伤对比结果；图8B显示了小鼠体重的变化；图8C显示了肿瘤重量比较结果；图8D显示了小鼠的胰腺癌PANC02-A2治疗后的CAR-T细胞拷贝数；图8E显示了小鼠胰腺癌CD8+细胞的免疫组化检测结果；
图9A显示了小鼠乳腺癌E0771-A2原位移植瘤经CAR-T细胞治疗后的肿瘤体积变化情况；
图9B显示了了小鼠乳腺癌E0771-A2原位移植瘤治疗后的肿瘤重量；
图9C显示了小鼠的乳腺癌治疗后的CAR-T细胞拷贝数；
图9D显示了小鼠乳腺癌CD8+细胞的免疫组化检测结果；
图10A显示了小鼠肝癌Hepal-6-A2移植瘤治疗后的肿瘤体积变化情况；
图10B显示了小鼠肝癌Hepa1-6-A2移植瘤治疗后的肿瘤重量；
图10C显示了小鼠的肝癌治疗后的CAR-T细胞拷贝数；
图10D显示了小鼠肝癌CD8+细胞的免疫组化检测结果；
图11显示了体外IFN-γ的检测结果；
图12A显示了小鼠胰腺癌皮下瘤清淋模型经CAR-T细胞治疗后的肿瘤体积变化情况；图12B显示了了小鼠胰腺癌皮下瘤清淋模型治疗后的肿瘤重量；图12C显示了 小鼠胰腺癌皮下瘤清淋模型治疗后的CAR-T细胞拷贝数；图12D显示了小鼠胰腺癌皮下瘤清淋模型CD8+细胞的免疫组化检测结果。
图13A显示了小鼠胰腺癌PANC02-A2皮下瘤模型CAR-T治疗d10天脾脏中Tcm的检测情况；图13B显示了d20天脾脏中Tcm的检测情况；
图14A显示了小鼠胰腺癌PANC02-A2皮下瘤模型CAR-T治疗d10天骨髓中Tcm的含量情况；图14B显示了d20天脾脏中Tcm的检测情况；
图15显示了小鼠胰腺癌PANC02-A2皮下瘤模型CAR-T治疗d10天小鼠肿瘤组织中有更多的DC细胞浸润情况；
图16显示了小鼠胰腺癌PANC02-A2皮下瘤模型CAR-T治疗d10天小鼠的肿瘤组织中MDSC的含量情况。
具体实施方式
本发明发现，表达有靶向肿瘤抗原的外源性受体和CCL21的免疫效应细胞不仅对肿瘤具有更优异的杀伤效果，并且也提高了免疫效应细胞在肿瘤组织中的存活，即使对于难治的实体瘤，也显示出更为优异的抗肿瘤能力。
根据本公开内容，本领域技术人员应了解在所公开的具体实施方案中可以作出许多变化或改变，并且仍获得相同或相似结果，而不背离本文所述的精神和范围。本发明在范围上并不受限于本文描述的具体实施方案(其仅预期作为本文所述的各方面的举例说明)，并且应当认为，在功能上等价的方法和组分仍包括在本文所述的范围内。
除非专门定义，本文所用的所有技术和科学术语具有在基因治疗，生物化学、遗传学和分子生物学领域内的技术人员通常理解的含义。类似或等效于本文中描述的所有方法和材料都可以在本文所述的实践或测试中使用。这些技术如方法和材料充分记载于文献中，参见，例如，除非另有说明，本发明的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领域的技术范围Current Protocols in Molecular Biology(FrederickM.AUSUBEL，2000，Wileyand sonInc，Library of Congress，USA)；Molecular Cloning:A Laboratory Manual，Third Edition，(Sambrooketal，2001，Cold Spring Harbor，NewYork:Cold Spring Harbor Laboratory Press)；Oligonucleotide Synthesis(M.J.Gaited.，1984)；Mullis et al.U.S.Pat.No.4,683,195；Nucleic Acid Hybridization(B.D.Harries&S.J.Higginseds.1984)；Transcription And Translation(B.D.Hames&S.J.Higginseds.1984)；Culture Of Animal Cells(R.I.Freshney，Alan R. Liss，Inc.，1987)；Immobilized Cells And Enzymes(IRL Press，1986)；B.Perbal，A Practical Guide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY(J.Abelson和M.Simon，eds.-in-chief，Academic Press，Inc.，New York)，尤其是Vols.154和155(Wuetal.eds.)和Vol.185，“Gene Expression Technology”(D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Caloseds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell And Molecular Biology(Mayer和Walker，eds.，Academic Press，London，1987)；Hand book Of Experimental Immunology，卷I-IV(D.M.Weir和C.C.Blackwell，eds.，1986)；和Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1986)。
本文提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容结合于本文中作为参考。在冲突的情况下，以本说明书为准。此外，除非另有规定，本说明书中所列举的材料、方法和实施例仅是说明性的，而并非旨在进行限制。
本文所用的术语“工程化”及其语法上的其他形式可以指核酸的一个或多个改变，例如生物体基因组内的核酸。术语“工程化”可以指基因的改变、添加和/或缺失。工程细胞还可以指具有加入、缺失和/或改变的基因的细胞。
本文所用的术语“基因工程化的细胞”是指通过基因工程的手段改造的细胞。在一些实施方案中，所述的细胞是免疫效应细胞。在一些实施方案中，所述的细胞是T细胞。在一些实施方案中，本文所述的基因工程化的细胞是指表达特异性结合靶抗原的外源性受体的细胞。在一些实施方案中，本文所述的基因工程化的细胞是指表达特异性结合靶抗原的外源性受体并且表达外源性的CLL21的细胞。在一些实施方案中，本文所述的基因工程化的细胞还可以是共表达特异性结合肿瘤抗原的嵌合抗原受体、CLL21、及促进T细胞增殖的蛋白的T细胞。在一些实施方案中，本文所述的基因工程化的细胞还可以是共表达特异性结合肿瘤抗原的嵌合抗原受体、CLL21、及IL-7R结合蛋白或外源性的IL-7的T细胞。
术语“免疫效应细胞”，是指参与免疫应答，产生免疫效应的细胞，如T细胞，、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和骨髓源性吞噬细胞。在一些实施方案中，所述的免疫效应细胞为T细胞、NK细胞、NKT细胞。在一些实施方案中，所述T细胞可以是自体T细胞、异种T细胞、同种异体T细胞。在一些实施方案中，所述的NK细胞可以是同种异体NK细胞。
术语“肽”、“多肽”和“蛋白质”可互换地使用，并且是指由肽键共价连接的氨基酸残基组成的化合物。蛋白质或肽必须含有至少两个氨基酸，并且对于可以包括蛋白 质或肽的序列的氨基酸的最大数量没限制。多肽包括含有彼此通过肽键结合的两个或多个氨基酸的任何肽或蛋白质。正如本文中使用的那样，该术语是指短链(其在本领域通常也称为例如肽、寡肽和寡聚物)以及较长链(其在本领域通常也称为蛋白质，其存在多种类型)。“多肽”包括例如生物学活性片段、基本上同源的多肽、寡肽、同型二聚体、异二聚体、多肽的变体、修饰的多肽、衍生物、类似物、融合蛋白等。多肽包括天然肽、重组肽或其组合。
本术语“IL-7(Interleukin7，白细胞介素7或IL7)”是指能够与IL-7R(优选来自哺乳动物，例如鼠或人IL-7R)相互作用(例如结合)的蛋白质(优选来自哺乳动物，例如鼠或人)，并且具有以下特征之一：(i)为天然存在的哺乳动物IL-7的氨基酸序列或其片段，例如SEQ ID NO：18(人)所示的氨基酸序列或其片段；(ii)基本上与SEQ ID NO：18(人)所示氨基酸序列或其片段具有例如至少85％、90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(iii)由天然存在的哺乳动物IL-7核苷酸序列或其片段(例如SEQ ID NO:17(人)或其片段所编码的氨基酸序列；(iv)由与SEQ ID NO:17(人)所示核苷酸序列或其片段具有例如至少85％、90％、95％、96％、97％、98％、99％同源性的核苷酸序列所编码的氨基酸序列；(v)由与天然存在的IL-7核苷酸序列或其片段简并的核苷酸序列(例如SEQ ID NO：17(人)或其片段)所编码的氨基酸序列；或(vi)在严格性条件与前述核苷酸序列之一杂交的核苷酸序列。
“外源性IL-7R结合蛋白”是指能特异性结合IL-7R且增强IL-7R活性的所有蛋白质。“增强IL-7R活性”应理解为意指IL-7R结合蛋白能够增强天然存在的IL-7R的任何一种或多种活性，包括但不限于刺激NK细胞的增殖、细胞毒性或成熟；刺激B细胞和T细胞的增殖或分化；刺激B细胞中的抗体产生和亲和力成熟；刺激CD8+T细胞的细胞毒性；刺激T细胞和NK细胞中干扰素γ产生；抑制树突状细胞(DC)活化和成熟；抑制炎性介质从肥大细胞释放；增强巨噬细胞的吞噬作用；抑制TReg细胞的产生或存活；和刺激骨髓祖细胞的增殖。
“CCL21(Chemokine(C-C motif)ligand 21)”是主要的免疫趋化因子之一，在脾脏和淋巴结的二级淋巴组织的T细胞区表达，负责抗原激活的(成熟的)树突细胞(DC)、非成熟的DC和幼稚T细胞的招募。本发明中CCL21具有以下特征之一：(i)为天然存在的哺乳动物CCL21的氨基酸序列或其片段，例如SEQ ID NO:21(人)所示的氨基酸序列或其片段；(ii)为与SEQ ID NO:21(人)所示氨基酸序列或其片段具有例如至少85％、90％、95％、96％、97％、98％、99％同源性的氨基酸序列；(iii)由天然存在的哺乳动物CCL21核苷酸序列或其片段(例如SEQ ID NO:20(人)或其片段所编码的氨 基酸序列；(iv)由与SEQ ID NO:20(人)所示核苷酸序列或其片段具有例如至少85％、90％、95％、96％、97％、98％、99％同源性的核苷酸序列所编码的氨基酸序列；(v)由与天然存在的CCL21核苷酸序列或其片段简并的核苷酸序列(例如SEQ ID NO:20(人)或其片段)所编码的氨基酸序列；或(vi)在严格性条件下与前述核苷酸序列之一杂交的核苷酸序列。
术语“氨基酸修饰”包括氨基酸取代、添加和/或缺失，“氨基酸取代”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。本文中使用的“氨基酸插入”意指在亲本多肽序列中的特定位置添加氨基酸。文中使用的“氨基酸缺失”或“缺失”意指去除亲本多肽序列中特定位置上的氨基酸。本文中使用的术语“保守修饰”意指不显着影响或改变含有所述氨基酸序列的抗体的结合特征的氨基酸修饰。此类保守修饰包括氨基酸取代、插入和缺失。可通过本领域已知的标准技术将修饰导入本发明的抗体中，例如定点诱变和PCR介导的诱变。保守的氨基酸取代是用具有相似侧链的氨基酸残基替换氨基酸残基的取代。本领域已经定义了具有相似侧链的氨基酸残基家族。这些家族包括含碱性侧链的氨基酸(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电的急性侧链(例如，甘氨酸、天冬酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
本文使用的术语“野生型”、“亲本”、“天然”在涉及蛋白质和DNA时，代表相同的意思。术语“突变”、“变体”或“突变体”具有与天然蛋白质或天然DNA具有相同或更优的生物活性，其在天然蛋白质的氨基酸序列上发生了一个或多个氨基酸的取代、添加或缺失；或在天然DNA的核酸序列上发生了一个或多个核苷酸的取代、添加或缺失。在具体的实施方式中，本文中的突变体的序列具有与天然蛋白质的氨基酸序列或天然DNA的核酸序列具有至少约80％、优选至少约90％、更优选至少约95％、更优选至少约97％、更优选至少约98％、最优选至少约99％的同一性。如“IL-7的变体”通常指将野生型的IL-7进行氨基酸修饰后得到的与IL-7具有类似生物活性或者更优的生物活性的多肽。术语“截短片段”指天然蛋白质或天然DNA的非全长形式，其在天然氨基酸序列或核酸序列上有连续或非连续的多个氨基酸残基或核苷酸的缺失，该缺失发生于序列的任何位置，例如头部、中部、尾部或他们的结合。在本发明中，蛋白质的截短片段仍然保留其来源的天然蛋白质相同的功能。
“组成型表达”(constitutive expression)又称持续表达，是指在几乎所有的生 理条件下，基因都能够在细胞中持续的表达。“诱导型表达(inducible expression)”是指在一定条件下的表达，所述的条件例如T细胞与抗原发生结合的时候。
术语“有效量”是指有效地实现特定生物学结果的化合物、制剂、物质或组合物的量，例如但不限于足以促进T细胞应答的量或剂量。当指示“免疫学上有效量”、“抗肿瘤有效量”、“抑制肿瘤有效量”或“治疗有效量”时，本发明的免疫效应细胞、或治疗剂的精确给药剂量可以由医师在考虑个体在年龄、体重、肿瘤大小、或转移的程度以及患者(受试者)的状况的情况下确定。有效量的免疫效应细胞是指但不限于能使免疫效应细胞抗肿瘤活性增加、增强或延长；抗肿瘤免疫效应细胞或活化免疫效应细胞数目的增加；促进IFN-γ分泌；肿瘤消退、肿瘤缩小、肿瘤坏死的免疫效应细胞的数量。
本文使用的术语“启动子”为由启动多核苷酸序列的特异性转录所需的细胞的合成机制或引入的合成机制识别的DNA序列。
典型的真核生物启动子由最小启动子和其他顺式元件组成。最小启动子实质上是一个TATA框区，RNA聚合酶II(polII)、TATA结合蛋白(TBP)和TBP相关因子(TAF)可在此结合而启动转录。已发现这类序列元件(例如增强子)提高临近的基因的总体表达水平，且往往是以不依赖位置和/或取向的方式。
NFAT”(Nuclear factor of activated T cells，)是活化T细胞核因子。在一些具体实施方案中，NFAT在T细胞活化过程中细胞因子的转录表达起着重要的作用。在一些实施方案中，RUNX3是采用诱导性启动子的诱导性表达。在一些实施方案中，所述诱导性启动子为NFAT启动子。在一些实施方案中，将RUNX3的编码序列置于与含NFAT结合基序的最小启动子的调控之下。在一些具体实施方案中，含6个NFAT结合基序的IL2最小启动子是利用6个NFAT的结合位子与IL2的最小启动子(minimal promoter)串联在一起组成的启动子。
在一些实施方案中，本文所述的结合抗原的受体是指嵌合受体。本文所用的“嵌合受体”，是指用基因重组技术将不同来源的DNA片段或蛋白质相应的cDNA连接而成的融合分子。嵌合受体通常包括胞外域、跨膜域和胞内域。可用于本发明的嵌合受体包括但不限于：嵌合抗原受体(CAR)、修饰的T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)。
术语“开放阅读框(Open Reading Frame,ORF)”是结构基因的正常核苷酸序列，从起始密码子到终止密码子的阅读框可编码完整的多肽链，其间不存在使翻译中断的终止密码子。
本文所用的“嵌合抗原受体”或“CAR”是指一组多肽，当其在免疫效应细胞中时， 给所述的细胞提供针对靶细胞(通常是癌细胞)的特异性，并且具有细胞内信号产生。CAR通常包括至少一个细胞外抗原结合结构域(也称为胞外区)、跨膜结构域(也称为跨膜区)和细胞质信号传导结构域(本文中也称为“胞内信号传导结构域”或“胞内区”)，其包括来源于如下定义的刺激性分子和/或共刺激性分子的功能信号传导结构域。在某些方面，多肽组彼此邻接。多肽组包括在存在二聚化分子时可以使多肽彼此偶联的二聚化开关，例如，可以使抗原结合结构域偶联至胞内信号传导结构域。在一个方面，刺激性分子为与T细胞受体复合体结合的ζ链。在一个方面，细胞质信号传导结构域进一步包括一种或多种来源于至少一个如下定义的共刺激性分子的功能性信号传导结构域。在一个方面，共刺激性分子选自本文所述共刺激性分子，例如4-1BB(即，CD137)、CD27和/或CD28。在一个方面，CAR包括嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于刺激性分子的功能性信号传导结构域的胞内信号传导结构域。在一个方面，CAR包含嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于共刺激性分子的功能性信号传导结构域和来源于刺激性分子的功能性信号传导结构域的胞内信号传导结构域。在一个方面中，CAR包含嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于一个或更多个共刺激性分子的两个功能性信号传导。
在一个方面，本发明设想产生功能等同分子的起始抗体或片段(例如scFv)氨基酸序列的修饰。例如，本文所述的癌症相关抗原的抗原结合结构域的VH或VL，例如CAR中包含的scFv，可以被修饰以保留本文所述的癌症相关抗原的抗原结合结构域的起始VH或VL构架区(例如scFv)的至少约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％的同一性。本发明设想整个CAR构建体的修饰，例如CAR构建体的多个结构域的一个或更多个氨基酸序列的修饰，以产生功能等同分子。CAR构建体可以被修饰以保留起始CAR构建体的至少约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％的同一性。
本文所用的“跨膜结构域”(也称为跨膜区)，可以包括一个或更多个邻接跨膜区域的另外氨基酸，例如一个或更多个与所述跨膜蛋白所源自的蛋白质的胞外区域相关联的氨基酸(例如，胞外区域的1、2、3、4、5、6、7、8、9、10直至15个氨基酸)和/或与所述跨膜蛋白所源自的蛋白质的胞外区域相关联的一个或更多个另外的氨基酸(例如，胞 内区域的1、2、3、4、5、6、7、8、9、10直至15个氨基酸)。在一个方面，跨膜结构域是与嵌合受体的其它结构域中的一个有关的结构域，例如，在一种实施方式中，所述跨膜结构域可以来自信号传导结构域、共刺激结构域或铰链结构域所源自的相同蛋白质。在某些情况下，跨膜结构域可以被选择或通过氨基酸取代以避免这样的结构域与相同或不同表面膜蛋白的跨膜结构域结合，例如以使与受体复合体的其它成员的相互作用最小化。在一个方面，跨膜结构域能够与表达嵌合受体的细胞的细胞表面上的另一个嵌合受体同型二聚化。在一个不同的方面，跨膜结构域的氨基酸序列可以被修饰或取代，以便使与存在于表达相同嵌合受体的细胞中的天然结合配偶体的结合结构域的相互作用最小化。跨膜结构域可以来源于天然或重组来源。当所述来源是天然的时，所述结构域可以来源于任何膜结合的蛋白质或跨膜蛋白质。在一个方面，只要所述嵌合受体与所述靶抗原结合时，跨膜结构域能够向胞内结构域传导信号。在本发明中特别使用的跨膜结构域可以包括至少以下的跨膜结构域：例如，T-细胞受体的α、β或ζ链、CD28、CD27、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154。在某些实施方式中，跨膜结构域可以包括至少下述跨膜区域：例如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。
在某些情况下，跨膜结构域可以经由铰链(例如，来自人蛋白质的铰链)连接至CAR的胞外区域，例如CAR的抗原结合结构域。例如，在一种实施方式中，铰链可以是人Ig(免疫球蛋白)铰链(例如，IgG4铰链、IgD铰链)、GS接头(例如，本文所述的GS接头)、KIR2DS2铰链或CD8a铰链。在一个方面，跨膜结构域可以是重组的，在这样情况下，其将会主要包含疏水性残基，比如亮氨酸和缬氨酸。在一个方面，在重组跨膜结构域的每个末端可以发现苯丙氨酸、色氨酸和缬氨酸的三联体。任选地，长度在2至10个氨基酸之间的短的寡肽或多肽接头可以在CAR的跨膜结构域与细胞质区之间形成键。甘氨酸-丝氨酸二联体提供一种特别合适的接头。
本文所用的“细胞内结构域”(也称为胞内域),包括胞内信号传导结构域。胞内信号传导结构域通常负责其中已经引入嵌合受体的免疫细胞的正常效应子功能中至少一个的活化。术语“效应子功能”是指细胞的特化功能。T细胞的效应子功能例如可以是细胞溶解活性或辅助活性，包括分泌细胞因子。因此，术语“胞内信号传导结构域”是指转导效应子功能信号且引导细胞执行特定功能的蛋白质的部分。虽然通常可以应用全部胞内信号传导结构域，但是在许多情况下不必使用整个链。就使用胞内信号传导结构域的截短部分来说，可以使用这样的截短部分代替完整的链，只要其转导效应子功能信号。因此，术语胞内信号传导结构域意味着包括足以转导效应子功能信号的胞内信号传导结构域的截短部分。
众所周知，通过单独的TCR产生的信号不足以完全活化T细胞，并且也需要二级和/或共刺激信号。因此，T细胞活化可以被称为是由两个不同种类的细胞质信号传导序列介导的:通过TCR引发抗原依赖性一级活化的那些(一级胞内信号传导结构域)以及以抗原独立方式起作用以提供二级或共刺激信号的那些(二级细胞质结构域，例如共刺激结构域)。
术语“刺激”是指由刺激性分子(例如，TCR/CD3复合体或CAR)与其同源配体(或在CAR的情况下为肿瘤抗原)的结合，由此介导信号转导事件(比如但不限于经由TCR/CD3复合体的信号转导或经由适合的NK受体或CAR的信号传导结构域的信号转导)而诱导的初次应答。刺激可以介导某些分子的改变的表达。
术语“刺激性分子”是指由免疫细胞(例如，T细胞、NK细胞、B细胞)表达的提供细胞质信号传导序列的分子，该信号传导序列以刺激性方式调节用于免疫细胞信号传导途径的至少一些方面的免疫细胞的活化。在一个方面，信号是通过例如TCR/CD3复合体与负载有肽的MHC分子的结合启动的初级信号，并且其导致介导T细胞应答，包括，但不限于增殖、活化、分化等。以刺激方式起作用的一级细胞质信号传导序列(也称为“一级信号传导结构域”)可以含有被称为基于免疫受体酪氨酸的活化基序(Immunoreceptor tyrosine-based activation motif，ITAM)的信号传导基序。特别地用于本发明的含有ITAM的细胞质信号传导序列的实例包括，但不限于来源于下述的那些：CD3ζ、常见的FcRγ(FCER1G)、FcγRIIa、FcRβ(FcEpsilon R1b)、CD3γ、CD3δ、CD3ε、CD79a、CD79b、DAP10和DAP12。在本发明的特异性CAR中，在本发明的任一个或更多个CAR中的胞内信号传导结构域包括细胞内信号传导序列，例如CD3-ζ的初级信号传导序列。在本发明的特异性CAR中，CD3-ζ的初级信号传导序列是来自人或非人类种类例如小鼠、啮齿类动物、猴、猿等的等同残基。
术语“共刺激性分子”是指T细胞上的同源结合配偶体，其特异性地结合共刺激配 体，从而介导T细胞的共刺激反应，比如但不限于增殖。共刺激性分子为除了抗原受体或其配体之外的细胞表面分子，其促进有效的免疫应答。共刺激性分子包括但不限于MHC I类分子，BTLA和Toll配体受体，以及OX40、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)和4-1BB(CD137)。这样的共刺激性分子的进一步实例包括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以及特异性地结合CD83的配体。
共刺激性胞内信号传导结构域可以为共刺激性分子的细胞内部分。共刺激性分子可以以下述蛋白质家族代表：TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、信号传导淋巴细胞性活化分子(SLAM蛋白质)、和NK细胞受体。这样的分子的实例包括CD27、CD28、4-1BB(CD137)、OX40、GITR、CD30、CD40、ICOS、BAFFR、HVEM、ICAM-1、与淋巴细胞功能相关的抗原-1(LFA-1)、CD2、CDS、CD7、CD287、LIGHT、NKG2C、NKG2D、SLAMF7、NKp80、NKp30、NKp44、NKp46、CD160、B7-H3以及特异性地结合CD83的配体等。
胞内信号传导结构域可以包括分子的全部细胞内部分或全部天然胞内信号传导结构域、或其功能片段或衍生物。
术语“4-1BB”是指具有如GenBank Accession No.AAA62478.2提供的氨基酸序列的TNFR超家族的成员，或来自非人类物种例如小鼠、啮齿类动物、猴子、猿等的等同残基；并且“4-1BB共刺激结构域”被定义为GenBank Accession No.AAA62478.2的氨基酸残基214～255，或来自非人类物种例如小鼠、啮齿类动物、猴子、猿等的等同残基。在一个方面，“4-1BB共刺激结构域”为来自人或者来自非人类物种例如小鼠、啮齿类动物、猴子、猿等的等同残基。
术语“T细胞(抗原)受体(T cell receptor，TCR)”，为所有T细胞表面的特征性标志，以非共价键与CD3结合，形成TCR-CD3复合物。TCR负责识别与主要组织相容性复合体分子结合的抗原。TCR是由两条不同肽链构成的异二聚体，由α、β两条肽链 组成，每条肽链又可分为可变区(V区)，恒定区(C区)，跨膜区和胞质区等几部分；其特点是胞质区很短。TCR分子属于免疫球蛋白超家族，其抗原特异性存在于V区；V区(Vα、Vβ)又各有三个高变区CDR1、CDR2、CDR3，其中以CDR3变异最大，直接决定了TCR的抗原结合特异性。在TCR识别MHC-抗原肽复合体时，CDR1，CDR2识别和结合MHC分子抗原结合槽的侧壁，而CDR3直接与抗原肽相结合。TCR分为两类：TCR1和TCR2；TCR1由γ和δ两条链组成，TCR2由α和β两条链组成。
术语“T细胞融合蛋白(T cell fusion protein，TFP)”，包括构成TCR的各种多肽衍生的重组多肽，其能够结合到靶细胞上的表面抗原，和与完整的TCR复合物的其他多肽相互作用，通常同定位在T细胞表面。TFP由一个TCR亚基与人或人源化抗体结构域组成的一个抗原结合结构域组成，其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内结构域的胞内信号结构域的刺激结构域；该TCR亚基和该抗体结构域有效连接，其中，TCR亚基的胞外、跨膜、胞内信号结构域来源于CD3ε或CD3γ，并且，该TFP整合进T细胞上表达的TCR。
术语“T细胞抗原耦合器(T cell antigen coupler，TAC)”，包括三个功能结构域：1、肿瘤靶向结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；2、胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；3、跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAMs)磷酸化作为T细胞活化的初始步骤。
术语“抗体”是指源于特异性地结合抗原的免疫球蛋白分子的蛋白质或多肽序列。抗体可以为多克隆的或单克隆的、多链或单链、或完整的免疫球蛋白，并且可以来源于天然来源或重组来源。抗体可以为免疫球蛋白分子的四聚体。
术语“抗体片段”是指保留与抗原的表位特异性地相互作用(例如，通过结合、空间位阻、稳定化/去稳定化、空间分布)的能力的抗体的至少一部分。抗体片段的实例包括，但不限于Fab，Fab'、F(ab') 2、Fv片段、scFv、二硫键-连接的Fvs(sdFv)、由VH和CH1结构域组成的Fd片段、线性抗体、单域抗体(如sdAb)、由抗体片段(例如包括在铰链区通过二硫键连接的两个Fab片段的二价片段)形成的多特异性抗体和抗体的分离的CDR或其它表位结合片段。
术语“scFv”是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非指定，否则如正如本文中使用的那样，scFv可以以任何顺序 (例如相对于多肽的N-末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
术语“抗体重链”是指以其天然存在的构型存在于抗体分子中且通常决定抗体所属类型的两种多肽链中较大者。
术语“抗体轻链”是指以其天然存在构型存在于抗体分子中的两种多肽链的较小者。κ(k)和λ(l)轻链是指两种主要的抗体轻链的同种型。
术语“重组抗体”是指使用重组DNA技术产生的抗体，比如例如由噬菌体或酵母菌表达系统表达的抗体。该术语也应当解释为指已经通过合成编码抗体的DNA分子(且其中DNA分子表达抗体蛋白质)或指定抗体的氨基酸序列产生的抗体，其中所述DNA或氨基酸序列已经使用重组DNA或本领域可获得且熟知的氨基酸序列技术获得。
术语“抗原”是指引起免疫应答的分子。该免疫应答可以涉及抗体产生或有特异性免疫能力的细胞的活化或两者。本领域技术人员应当理解包括实际上所有蛋白质或肽的任何大分子都可以充当抗原。此外，抗原可以来源于重组或基因组DNA。当在本文中使用该术语时，本领域技术人员应当理解包括编码引起免疫应答的蛋白质的核苷酸序列或部分核苷酸序列的任何DNA，所编码的蛋白质或肽。此外，本领域技术人员应当理解抗原无需仅通过基因的全长核苷酸序列编码。显而易见的是，本发明包括但不限于使用超过一个基因的部分核苷酸序列，并且这些核苷酸序列以不同组合排列以编码引发期望免疫应答的多肽。而且，本领域技术人员应当理解抗原根本无需由“基因”编码。显而易见的是，抗原可以合成产生，或者可以来源于生物样品，或者可以是除了多肽之外的大分子。这样的生物样品可以包括，但不限于组织样品、肿瘤样品、具有其它生物组分的细胞或液体。
“肿瘤抗原”指的是过度增生性疾病发生、发展过程中新出现的或过度表达的抗原。在某些方面，本发明的过度增生性病症是指癌症。
本发明所述的肿瘤抗原可以是实体瘤抗原，也可以是血液瘤抗原。
本发明的肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1 Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体，血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生 长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。
病原体抗原选自：病毒、细菌、真菌、原生动物，或寄生虫的抗原；病毒抗原选自： 巨细胞病毒抗原、爱泼斯坦-巴尔病毒抗原、人类免疫缺陷病毒抗原,或流感病毒抗原。
术语“肿瘤”指在体外(例如经转化的细胞)或体内的过度增殖性细胞生长的广泛病症类别。可以通过本发明的方法治疗或预防的病况包括例如各种赘生物，包括良性或恶性肿瘤，各种增生等等。癌症的具体例子包括但不限于：乳腺癌，前列腺癌，白血病，淋巴瘤，鼻咽癌，脑胶质瘤，结肠癌，直肠癌，肾细胞癌，肝癌，非小细胞肺癌，小肠癌，食道癌，黑色素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，子宫癌，卵巢癌，胃癌，睾丸癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，阴茎癌，膀胱癌，输尿管癌，肾盂癌，中枢神经系统(CNS)瘤，血管瘤脊椎肿瘤，胶质瘤，星性细胞瘤，垂体腺瘤所述癌症的组合和所述癌症的转移性病灶。
术语“转染的”或“转化的”或“转导的”是指外源性核酸通过其转移或引入到宿主细胞中的过程。“转染的”或“转化的”或“转导的”细胞是已经用外源性核酸转染、转化或转导的细胞。所述细胞包括原发性受试者细胞及其后代。
术语“特异性地结合”是指抗体或配体结合存在于样品中的结合配偶体(例如肿瘤抗原)，但基本上不识别或结合样品中的其它分子。
这里使用的“难治”指的是一种疾病，例如，肿瘤，其不应答治疗。在实施方案中，难治性肿瘤可以是对治疗开始前或开始时的治疗有抗性。在其他实施方案中，难治性肿瘤可以成为治疗期间抗性的。在本发明中，难治性肿瘤包括但不限于放疗不敏感、放疗后复发、化疗不敏感、化疗后复发、对CAR-T治疗不敏感或治疗后复发的肿瘤。难治性或复发性恶性肿瘤可以使用本文中描述的治疗方案。
如本文所用“复发的”是指在一段改进期，例如在先有效肿瘤治疗后，患者又再出现该有效治疗之前的体征和症状。
术语“个体”和“受试者”在本文中具有同等含义，可以是人和来自其他种属的动物。
术语“增强”指允许受试者或肿瘤细胞改善其响应本文公开的治疗的能力。例如，增强的应答可以包含应答性中5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％或更多的增加。如本文使用的，“增强”还可以指增加响应治疗例如免疫效应细胞疗法的受试者数目。例如，增强的应答可以指响应治疗的受试者总百分比，其中百分比是5％、10％、15％、20％、25％、30％、35％、40％、45％、50％、55％、60％、65％、70％、75％、80％、85％、90％、95％或98％更多。
在一个方面，治疗由临床结果；通过T细胞的抗肿瘤活性增加、增强或延长；与治疗 前的数目相比较，抗肿瘤T细胞或活化T细胞数目的增加，促进IFN-γ分泌，或其组合决定。在另一个方面，临床结果是肿瘤消退；肿瘤缩小；肿瘤坏死；通过免疫系统的抗肿瘤应答；肿瘤扩大，复发或扩散或其组合。在一个另外方面，治疗效应通过T细胞的存在、指示T细胞炎症的基因标记的存在，促进IFN-γ分泌，或其组合预测。
如本文公开的免疫效应细胞可以通过各种途径施用于个体，包括例如经口或肠胃外，例如静脉内、肌内、皮下、眶内、囊内、腹膜内、直肠内、脑池内、瘤内、鼻内(intravasally)、皮内或者分别使用例如皮肤贴片或透皮离子电渗疗法通过皮肤的被动或促进吸收。
在实践本发明的方法中待施用的试剂总量可以作为单一剂量以推注或通过在相对短时间段的输注，施用于受试者，或可以使用分级治疗方案进行施用，其中在延长时间段施用多个剂量。本领域技术人员将知道治疗受试者中的病理状况的组合物的量取决于许多因素，包括受试者的年龄和一般健康、以及施用途径和待施用的治疗次数。考虑到这些因素，技术人员将根据需要调整具体剂量。一般而言，最初，使用I期和II期临床试验测定组合物的配制以及施用途径和频率。
范围：在整个公开中，本发明的各个方面都可以以范围形式存在。应当理解，范围形式的描述仅仅为方便和简洁起见，而不应当被看作是对本发明的范围不可改变的限制。因此，范围的描述应当被认为特别地公开了所有可能的子范围以及该范围内的单独数值。例如，范围的描述比如从1至6就应当被认为具体地公开了子范围比如1至3、1至4、1至5、2至4、2至6、3至6等，以及该范围内的单独数值，例如1、2、2.7、3、4、5、5.3和6。作为另一个实例，范围比如95-99％的同一性包括具有95％、96％、97％、98％或99％同一性的范围，并且包括子范围比如96～99％、96～98％、96～97％、97～99％、97～98％和98～99％的同一性。不考虑范围的宽度，这均适用。
根据本公开内容，本领域技术人员应了解在所公开的具体实施方案中可以作出许多变化或改变，并且仍获得相同或相似结果，而不背离本发明的精神和范围。本发明在范围上并不受限于本文描述的具体实施方案(其仅预期作为本发明的各方面的举例说明)，并且功能等价的方法和组分在本发明的范围内。事实上，本发明的各种修饰加上本文显示且描述的那些，根据前述描述对于本领域技术人员将变得明显。
当采用的共表达IL7和CCL21的CAR-T细胞用于受试者时，可以选择相应种属，如当用于鼠时，采用鼠源的IL7和CCL21，构建CAR的元件如跨膜域、胞内域等也可以选择鼠源的。当受试者为人时，优选人源的IL7和CCL21及人源的CAR的元件。在一些实施方式中，使用的CAR的序列可以如SEQ ID NO：26、27、或34所示。
在一些实施方案中，本发明的细胞在用于肿瘤治疗时可以与化疗药物联合应用。
术语“CLD18(claudin 18)”指密蛋白-18，并包括细胞天然表达的或转染了CLD18基因的细胞所表达的任何CLD18的变体(包括CLD18A1(claudin 18.1)和CLD18A2(claudin 18.2))、构象、同工型(isoform)和种间同源物(specieshomologs)。优选地，“CLD18”指人CLD18，特别是CLD18A2(SEQ ID NO:22)和/或CLD18A1(SEQ ID NO:23)，更优选CLD18A2。
术语“CLD18A1”包括细胞天然表达的或转染了CLD18A1基因的细胞所表达的任何人CLD18A1的翻译后修饰变体、同工型和种间同源物。
术语“CLD18A2”包括细胞天然表达的或转染了CLD18A2基因的细胞所表达的任何人CLD18A2的翻译后修饰变体、同工型和种间同源物。
术语“CLD18变体”应包括(i)CLD18剪接变体，(ii)CLD18翻译后修饰变体，特别是包括N糖基化状态不同的变体，(iii)CLD18构象变体，特别是包括CLD18-构象-1、CLD18-构象-2和CLD18-构象-3，(iv)位于胞间紧密连接处的游离CLD18和同型/异型相关联变体，(v)CLD18癌症相关变体和CLD18非癌症相关变体。
本发明的嵌合抗原受体多肽可以选自按如下方式顺序连接：
胞外抗原结合区-CD8跨膜区-4-1BB-CD3ζ，
胞外抗原结合区-CD8跨膜区-CD28b-CD3ζ，
胞外抗原结合区-CD28a-CD28b-CD3ζ，
胞外抗原结合区-CD28a-CD28b-4-1BB-CD3ζ，
及其组合，其中相关嵌合抗原受体蛋白中CD28a代表CD28分子的跨膜区，CD28b代表CD28分子的胞内信号区。本发明也包括编码所述嵌合抗原受体的核酸。本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的多肽或多肽的片段、类似物和衍生物。
本发明还提供了包含上述嵌合抗原受体的核酸的载体。本发明还包括包含上述载体的病毒。本发明的病毒包括包装后的具有感染力的病毒，也包括包含包装为具有感染力的病毒所必需成分的待包装的病毒。本领域内已知的其它可用于将外源基因转导入免疫效应细胞的病毒及其对应的质粒载体也可用于本发明。
本发明还提供了嵌合抗原修饰的免疫效应细胞，其被转导有编码所述的嵌合抗原受体的核酸或被转导有上述包含所述含有该核酸的重组质粒，或包含该质粒的病毒。本领域常规的核酸转导方法，包括非病毒和病毒的转导方法都可以用于本发明。基于非病毒的转导方法包括电穿孔法和转座子法。近期Amaxa公司研发的Nucleofector核转染仪能够直接将外源基因导入细胞核获得目的基因的高效转导。另外，基于睡美人转座子(Sleeping  Beauty system)或PiggyBac转座子等转座子系统的转导效率较普通电穿孔有较大提高，将nucleofector转染仪与睡美人转座子系统联合应用已有报道[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.]，该方法既具有较高的转导效率又能够实现目的基因的定点整合。在本发明的一个实施方案中，实现嵌合抗原受体基因修饰的免疫效应细胞的转导方法是基于病毒如逆转录病毒或慢病毒的转导方法。该方法具有转导效率高，外源基因能够稳定表达，且可以缩短体外培养免疫效应细胞到达临床级数量的时间等优点。在该转基因免疫效应细胞表面，转导的核酸通过转录、翻译表达在其表面。通过对各种不同的培养的肿瘤细胞进行体外细胞毒实验证明，本发明的嵌合抗原修饰的免疫效应细胞具有高度特异性的肿瘤细胞杀伤效果(亦称细胞毒性)，且能够在肿瘤组织中有效存活。因此本发明的编码嵌合抗原受体的核酸，包含该核酸的质粒，包含该质粒的病毒和转导有上述核酸，质粒或病毒的转基因免疫效应细胞可以有效地用于肿瘤的免疫治疗。
本发明所述的嵌合抗原修饰的免疫效应细胞还可以表达除了上述嵌合受体以外的另一种嵌合受体，该受体不含有CD3ζ，但含有CD28的胞内信号结构域、CD137的胞内信号结构域或者这两者的组合。
本发明的嵌合抗原受体修饰的免疫效应细胞可以应用于制备药物组合物或诊断试剂。所述的组合物除了包括有效量的所述免疫细胞，还可包含药学上可接受的载体。术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。
可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如
Figure PCTCN2019087077-appb-000001
润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液和磷酸盐缓冲液等。
本发明的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式可以采用注射或其它治疗方式。
本发明的优点：
1.本文所提供的免疫效应细胞可有效地增加所述免疫效应细胞在肿瘤内的增殖、存活及功能；降低抑制性免疫检查点的表达，从而缓解T细胞的耗竭。
2.本文所提供的免疫效应细胞对实体瘤细胞具备更优异的杀伤效果以及体外扩增性能。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
本发明的示例性的抗原受体，包括CAR，以及用于工程改造和将受体导入细胞中的方法，参考例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018/018958A1中公开的那些。
实施例1.表达嵌合抗原受体的T细胞的构建
本实施例选择Claudin18.2作为CAR-T细胞的靶点，为了更准确的验证在小鼠体内的抗肿瘤效果，因此，选择的信号肽、跨膜区、胞内区等为鼠源的。制备的方法按照本领域常规的CAR-T细胞制备方法操作。
1、质粒构建
采用本领域常规分子生物学方法，本实施例采用的scFv为靶向人Claudin18.2抗体，核酸序列如SEQ ID NO:1所示，所采用的嵌合抗原受体为二代的嵌合抗原受体，具有mCD8的跨膜域、mCD28的胞内域和/或m4-1BB胞内域、及mCD3ζ。
1.以MSCV.pBABE 5(购自addgene公司)为载体，构建了表达二代嵌合抗原受体的逆转录病毒质粒MSCV-hu8E5(2I)-28Z。hu8E5(2I)-28Z的核酸序列包含了CD8α信号肽(SEQ ID NO:3)、scFv(SEQ ID NO:1)、mCD8铰链区和跨膜区(SEQ ID NO:5)和mCD28胞内信号传导结构域(SEQ ID NO:7)以及mCD3的胞内段mCD3ζ(SEQ ID NO:9)组成。hu8E5(2I)-28Z的质粒构建图如图1A所示。
在MSCV-hu8E5(2I)-m28Z质粒的基础上插入F2A-mIL7-P2A-mCCL21a或F2A- mIL7-P2A-mCCL21b的基因，构建表达CAR、IL7及CCL21的逆转录病毒质粒MSCV-hu8E5(2I)-m28Z-F2A-mIL7-P2A-mCCL21a(质粒图如图1B所示)和MSCV-hu8E5(2I)-m28Z-F2A-mIL7-P2A-mCCL21b(质粒图如图1C所示)。
F2A-mIL7-P2A-mCCL21a由F2A(SEQ ID NO:11)、mouse IL7(SEQ ID NO:13)组成、P2A(SEQ ID NO:16)、mouse CCL21a(SEQ ID NO:14)组成；F2A-mIL7-P2A-mCCL21b由F2A(SEQ ID NO:11)、mouse IL7(SEQ ID NO:13)组成、P2A(SEQ ID NO:16)、mouse CCL21b(SEQ ID NO:15)组成。
以MSCV.pBABE 5为载体，构建了表达二代嵌合抗原受体的逆转录病毒质粒MSCV-hu8E5(2I)-mBBZ。hu8E5(2I)-mBBZ序列由CD8α信号肽(SEQ ID NO:3)、scFv(SEQ ID NO:1)、mCD8hinge和跨膜区(SEQ ID NO:5)和m4-1BB胞内信号传导结构域(SEQ ID NO:24)以及mCD3的胞内段CD3ζ(SEQ ID NO:9)组成。质粒图如图1D所示。
在MSCV-hu8E5(2I)-mBBZ质粒的基础上分别插入了F2A-mIL-7-P2A-mCCL21a、F2A-mIL7-P2A-mCCL21b，构建了表达CAR、IL7及CCL21的逆转录病毒质粒MSCV-hu8E5(2I)-mBBZ-F2A-mIL-7-P2A-mCCL21a(质粒图如图1E所示)、MSCV-hu8E5(2I)-mBBZ-F2A-mIL7-P2A-mCCL21b(质粒图如图1F所示)。
2.将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-P2A-mCCL21a、MSCV-hu8E5(2I)-mBBZ-F2A-mIL7-P2A-mCCL21b分别转染293T细胞，得到逆转录病毒hu8E5(2I)-28Z、IL7-CCL21a-28Z、IL7-CCL21b-28Z、hu8E5(2I)-BBZ、IL7-CCL21a-BBZ、IL7-CCL21b-BBZ。
3.小鼠T细胞提取以及活化：剥离C57BL/6小鼠脾脏提取小鼠T细胞，经培养和活化后，将所得的逆转录病毒hu8E5(2I)-28Z、IL7-CCL21a-28Z、IL7-CCL21b-28Z、hu8E5(2I)-BBZ、IL7-CCL21a-BBZ、IL7-CCL21b-BBZ分别感染T细胞，得到m28Z CAR-T细胞、m28Z-7*21A CAR-T细胞、m28Z-7*21B CAR-T细胞、mBBZ CAR-T细胞、mBBZ-7*21A CAR-T细胞、及mBBZ-7*21B CAR-T细胞。
实施例2.体外细胞因子检测
首先利用丝裂霉素C对小鼠胰腺癌细胞PANC02(claudin18.2表达阴性，购自ATCC)与PANC02-A2(claudin18.2表达阳性)进行预处理(40μg/ml，37℃，2-3h)。
按2×10 5细胞/400ul接种于24孔板，并按照效靶比1:1分别接种Untransduced T细 胞(UTD)、mBBZ CAR-T细胞、mBBZ-7*21A CAR-T细胞、mBBZ-7*21B CAR-T细胞于24孔板。其中设置不加靶细胞的对照组，第3天后收集细胞上清，利用ELISA试剂盒检测各细胞因子IL7和CCL21的分泌情况，结果如图2所示。
其中，PANC02-A2细胞时利用pwpt-mclaudin18.2慢病毒感染PANC02细胞构建。pWPT-mclaudin18.2质粒构建过程为：体外合成鼠claudin18.2基因(GeneBank参考序列号为：NM_001194921)，通过酶切、连接的方法将其装入慢病毒表达载体pWPT，构建成pwpt-mclaudin18.2质粒。
实施例3.体外CAR-T细胞表型检测
取的UTD、mBBZ CAR-T细胞、mBBZ-7*21A CAR-T细胞、mBBZ-7*21B CAR-T细胞检测细胞表面免疫检查点：PD-1、LAG-3、TIM-3。首先分别收集不同CAR-T细胞于EP管中，每种细胞分别分为3管，用预冰浴的流式洗涤液(1％NCS加PBS配制)洗涤2次，不同检测管分别按1:50比例稀释加入BV421标记的anti-PD-1抗体、APC标记的anti-LAG-3抗体、APC标记的anti-TIM-3抗体，冰上孵育45min，洗涤3次后转至流式管检测。结果如图3A～3F所示。
图3A显示了不同组的细胞表达PD-1的情况，结果显示，mBBZ组的PD-1的分泌达到30.2％，mBBZ-7*21A组PD-1的分泌仅有11.7％，mBBZ-7*21B组PD-1的分泌仅有9.4％，图3B显示了PD-1的表达强度，从图3B看，mBBZ组PD-1的表达高于mBBZ-7*21A组和mBBZ-7*21B组。
图3C显示了不同组的细胞表达LAG-3的情况，结果显示，mBBZ组的LAG-3的分泌达到80.7％，mBBZ-7*21A组LAG-3的分泌为53.4％，mBBZ-7*21B的分泌为13.7％。图3D显示了LAG-3的表达强度，从图3D看，mBBZ组LAG-3的表达高于mBBZ-7*21A组和mBBZ-7*21B组。
图3E显示了不同组的细胞表达TIM-3的情况，结果显示，mBBZ组的TIM-3的分泌达到41.3％，mBBZ-7*21A组TIM-3的分泌为16.2％，mBBZ-7*21B的分泌为13.2％。图3F显示了TIM-3的表达强度，从图3F看，mBBZ组TIM-3的表达高于mBBZ-7*21A组和mBBZ-7*21B。
综上所述，mBBZ-7*21A CAR-T细胞和mBBZ-7*21B CAR-T细胞的PD-1、LAG-3和TIM-3的表达均比mBBZ-CAR-T细胞低，说明过表达细胞因子IL7和CCL21后能够降低这些抑制性免疫检查点的表达，从而缓解T细胞的耗竭。
实施例4.体外杀伤毒性检测
采用CytoTox 96非放射性细胞毒性检测试剂盒(Promega公司)进行。具体方法参照CytoTox 96非放射性细胞毒性检测试剂盒说明书。
效应细胞：按效靶比3:1、1:1或1:3分别接种UTD细胞、m28Z CAR-T细胞、28Z-7*21A CAR-T细胞、m28Z-7*21B CAR-T、mBBZ CAR-T细胞、mBBZ-7*21A CAR-T细胞、mBBZ-7*21B CAR-T细胞于96孔板。
靶细胞：分别接种50μL 2×10 5/mL的小鼠胰腺癌细胞系PANC02-A2与PANC02细胞到相应的96孔板。
每组均设置5个复孔。将培养板置于细胞培养箱中孵育18h。
其中各实验组及各对照组设置如下：实验组：各靶细胞+不同的CAR-T细胞；对照组1：靶细胞最大释放LDH；对照组2：靶细胞自发释放LDH；对照组3：效应细胞自发释放LDH。计算公式为：％细胞毒性＝[(实验组-效应细胞自发组-靶细胞自发组)/(靶细胞最大-靶细胞自发)]*100。实验结果如图4A和4B所示。
图4A可见，m28Z CAR-T细胞、m28Z-7*21A CAR-T细胞、或m28Z-7*21B CAR-T与对照组UTD相比，对PANC02-A2在效靶比为3:1和1:1都有显著的毒性杀伤作用，对PANC02细胞没有杀伤作用。
图4B可见，mBBZ CAR-T细胞、mBBZ-7*21A CAR-T细胞、mBBZ-7*21B CAR-T与对照组UTD相比，对PANC02-A2在效靶比为3:1和1:1都有显著的毒性杀伤作用，对PANC02细胞没有杀伤作用。
实施例5.体外增殖检测
采用丝裂霉素C处理靶细胞PANC02-A2细胞(40μg/ml，37度，2-3h)，对效应细胞UTD细胞、mBBZ CAR-T细胞、mBBZ-7*21A CAR-T细胞、mBBZ-7*21B CAR-T细胞分别进行CFSE染色，然后按照效靶比(1×10 6细胞/ml)1:1共孵2天，
流式检测CAR-T细胞增殖情况，结果如图5所示，mBBZ-7*21A CAR-T细胞和mBBZ-7*21B CAR-T细胞相较于mBBZ CAR-T细胞均能够更快的增殖。
实施例6.PANC02-A2胰腺癌皮下移植瘤模型肿瘤治疗
1)实验分组：C57BL/6小鼠(购自上海西普尔-必凯实验动物有限公司)6-8周龄随机分组，每组5-6只，分别为UTD细胞、mBBZ CAR-T细胞、mBBZ-7*21A CAR-T细胞、mBBZ-7*21B CAR-T细胞治疗组。
2)皮下移植瘤的接种：胰酶消化法收集处于对数生长期且生长状态良好的PANC02-A2细胞，用PBS洗涤1次后，调整细胞密度为6×10 6/mL，在C57BL/6小鼠右侧腹部皮下注射200μL细胞悬液，即每只小鼠接种1.2×10 6的肿瘤细胞，接种日记为第0天。
3)CAR-T细胞回输：皮下接种肿瘤细胞后D11天，肿瘤平均体积约60mm 3。注射未处理T细胞或CAR-T细胞，注射剂量：2.5×10 6/只。
结果如图6所示，CAR-T注射后20天，肿瘤抑制率分别为：mBBZ CAR-T组：35.5％，mBBZ-7*21A CAR-T组：63％，mBBZ-7*21B CAR-T组：62.4％，说明mBBZ-7*21A CAR-T细胞和mBBZ-7*21B CAR-T细胞治疗组的抗肿瘤效果好于mBBZ CAR-T细胞(P<0.05)。
实施例7.表达不同趋化因子的肿瘤杀伤比较
本实施例选择表达IL7和CCL19的CAR-T细胞(mBBZ-7*19 CAR-T细胞)作为对照。mBBZ-7*19 CAR-T细胞的制备参照实施例1进行，在MSCV-hu8E5(2I)-mBBZ质粒的基础上插入F2A-mIL7-P2A-mCCL19，构建了表达CAR、IL7及CCL19的逆转录病毒质粒，质粒图如图7所示，mCCL19的核酸序列如SEQ ID NO:34所示。质粒感染小鼠的T细胞，得到mBBZ-7*19 CAR-T细胞。
1)、参照实施例6的操作制作小鼠的胰腺癌皮下移植瘤模型，在肿瘤平均体积约65mm 3时，分别注射mBBZ CAR-T细胞、mBBZ-7*19 CAR-T细胞、mBBZ-7*21B CAR-T细胞治疗组，注射剂量：2.5×10 6细胞/只，肿瘤杀伤结果如图8A所示，肿瘤抑制率分别是：mBBZ CAR-T组：22.8％，mBBZ-7*19 CAR-T组：32.7％，mBBZ-7*21B CAR-T组：76.6％，mBBZ-7*21B CAR-T细胞治疗组的抗肿瘤效果好于mBBZ CAR-T细胞和mBBZ-7*19 CAR-T细胞组。
同时，检测到各组小鼠体重变化(如图8B所示)，结果显示各组小鼠之间体重没有显著性差异(ns)，提示细胞因子的分泌对小鼠没有毒性作用。
2)、在肿瘤接种后的第31天，将小鼠实施安乐死，剥离小鼠肿瘤并称量瘤重，具体统计结果如图8C所示。结果显示mBBZ-7*21B CAR-T治疗组肿瘤重量明显小于mBBZ组(P<0.05)，说明嵌合抗原受体修饰的T细胞共表达IL7、CCL21能显著增加T细胞对体内肿瘤的抑制。
3)、回输CAR-T细胞后，在d31天结束实验的时候剥离肿瘤组织，检测肿瘤组织CAR-T拷贝数。
取1mg肿瘤组织块进行机械研磨，然后利用
Figure PCTCN2019087077-appb-000002
genomic DNA kits提取肿瘤中的DNA，分别测量每个样品浓度。利用实时定量PCR(qPCR)检测CAR拷贝数。根据模板质粒进行标准曲线制作，最后算出每个样品中CAR的拷贝数。
结果如图8D所示，显示mBBZ-7*21B CAR-T的CAR-T拷贝数较高
4)、免疫组化检测CD8 +细胞的肿瘤浸润
取步骤2)安乐死后小鼠的肿瘤组织，制备石蜡组织切片，常规脱蜡后，对标本进行水化；水化完成后将切片置于摇床上PBS清洗3次，将盛有柠檬酸缓冲液煮沸后将组织切片放入柠檬酸缓冲液中，进行抗原热修复；修复完成后采用1％BSA封闭。
将封闭好的切片加入相应的CD8a的抗体(anti-mouse CD8αantibody，购自：Cell Signaling)或空白对照试剂，4℃孵育过夜；用0.5％PBST缓冲液洗涤；然后用PBS缓冲液洗涤。
将洗涤好的切片加入二抗羊抗兔-HRP，37℃孵育1h。用0.5％PBST缓冲液洗涤2次后，用PBS缓冲液洗涤1次，DAB(Dako REAL TMEnVision TMDetection System,Peroxidase/DAB+,1:50稀释)显色。
苏木紫复染至细胞核染至深红色，将复染后的组织切片置于1％盐酸乙醇分化液分化3～5秒钟；自来水冲洗20min后脱水透明；90％乙醇浸泡1min；100％乙醇I浸泡1min；100％乙醇II浸泡1min；二甲苯浸泡3min；中性树胶封片，风干。
显微镜下观察，结果如图8E所示，虽然mBBZ-7*19组和mBBZ-7*21B组肿瘤组织中都有明显的CD8 +T细胞的浸润，但其中mBBZ-7*21B CAR-T组CD8 +T细胞浸润较多。
实施例8.小鼠的乳腺癌原位移植瘤模型
制作小鼠的乳腺癌皮下移植瘤模型，胰酶消化法收集处于对数生长期且生长状态良好的E0771-A2细胞(制备方法：利用pwpt-mclaudin18.2质粒包装慢病毒感染E0771细胞)，用PBS洗涤1次后，调整细胞密度为2×10 7/mL，在C57BL/6小鼠右侧腹部第四对乳房皮下注射50μL细胞悬液，即每只小鼠接种1×10 6的E0771-A2细胞，接种日记为第0天。
CAR-T细胞回输：皮下接种肿瘤细胞后D12天，肿瘤平均体积约150mm 3。注射未处理T细胞或CAR-T细胞，注射剂量：2.5×10 6/只。
每隔3-4天测量E0771-A2移植瘤体积的大小，记录每组小鼠瘤体积变化，结果如图9A所示，相比mBBZ-7*19 CAR-T组，mBBZ-7*21B CAR-T治疗组的肿瘤杀伤能力显 著增强。
在肿瘤接种后的第31天，将小鼠实施安乐死，剥离小鼠肿瘤并称量瘤重，具体统计结果如图9B所示。结果显示mBBZ-7*21B CAR-T治疗组肿瘤重量明显小于mBBZ-7*19 CAR-T组(P<0.05)和mBBZ CAR-T组(P<0.001)，说明嵌合抗原受体修饰的T细胞共表达IL7、CCL21能显著增加T细胞对体内肿瘤的抑制。
参照实施例7步骤3)的操作，检测乳腺癌皮下移植瘤模型的CAR-T的细胞拷贝，结果如图9C所示，mBBZ-7*19 CAR-T和mBBZ-7*21B CAR-T组的CAR-T拷贝数高于UTD和BBZ组。
参照实施例7步骤4)的操作，检测CD8 +细胞的肿瘤浸润情况，结果如图9D所示，mBBZ-7*19 CAR-T组和mBBZ-7*21B CAR-T组肿瘤组织中有明显的CD8T细胞的浸润，其中mBBZ-7*21B CAR-T组CD8 +T细胞浸润较多。
实施例9.小鼠的肝癌皮下移植瘤模型
制作小鼠的肝癌移植瘤模型，胰酶消化法收集处于对数生长期且生长状态良好的Hepa1-6-A2细胞(利用pwpt-mclaudin18.2质粒包装慢病毒感染Hepa1-6细胞)，用PBS洗涤1次后，调整细胞密度为5×10 7/mL，在C57BL/6小鼠右侧腹部皮下注射200μL细胞悬液，即每只小鼠接种1×10 7的Hepal1-6-A2肝癌细胞，接种日记为第0天。
CAR-T细胞回输：皮下接种肿瘤细胞后D7天，肿瘤平均体积约300mm 3。注射未处理T细胞或CAR-T细胞，注射剂量：1×10 6/只。
每隔3-4天测量Hepal1-6-A2移植瘤体积的大小，记录每组小鼠瘤体积变化，结果如图10A所示，相比mBBZ-7*19 CAR-T组，mBBZ-7*21B CAR-T治疗组的肿瘤杀伤能力显著增强。
在肿瘤接种后的第31天，将小鼠实施安乐死，剥离小鼠肿瘤并称量瘤重，具体统计结果如图10B所示。结果显示mBBZ-7*21B CAR-T治疗组肿瘤重量明显小于mBBZ-7*19 CAR-T组(p<0.01)和8E5-2I-mBBZ CAR-T组(p<0.05)，说明嵌合抗原受体修饰的T细胞共表达IL7、CCL21能显著增加T细胞对体内肿瘤的抑制。
参照实施例7步骤3)的操作，检测CAR-T细胞拷贝数，结果如图10C所示，mBBZ-7*21B CAR-T组的CAR-T拷贝数较高。
参照实施例7步骤4)的操作，检测CD8 +细胞的肿瘤浸润情况，结果如图10D所示，mBBZ-7*19 CAR-T组和mBBZ-7*21B CAR-T组肿瘤组织中有明显的CD8 +T细胞的浸润，其中mBBZ-7*21B CAR-T组CD8 +T细胞浸润较多。
实施例10.体外IFN-γ检测
将UTD、8E5-2I-mBBZ-CAR、mBBZ-7*21B CAR-T、及mBBZ-7*19 CAR-T分别与靶细胞PANC02-A2，按照1:1共孵24h后收上清后ELISA检测上清中IFN-γ的分泌水平。采用的ELISA试剂盒为联科生物mouse IFN-γ检测试剂盒。
结果如图11所示，mBBZ-7*21B CAR-T细胞与claudin18.2阳性的肿瘤细胞共孵育后有较多的IFN-γ分泌。
实施例11.小鼠PANC02-A2胰腺癌皮下瘤清淋模型的治疗
参照实施例6的操作，制备C57BL/6小鼠的PANC02-A2皮下移植瘤模型，接种日记为第0天，肿瘤接种后第14天，肿瘤平均体积约60mm 3，按照100mg/kg，尾静脉注射环磷酰胺，肿瘤接种后第15天，注射未处理T细胞或CAR-T细胞，注射剂量：2.5×10 6/只。
每隔3-4天测量PANC02-A2移植瘤体积的大小，记录每组小鼠瘤体积变化，结果如图12A所示，在肿瘤接种后的第38天，将小鼠实施安乐死，剥离小鼠肿瘤并称量瘤重，具体统计结果如图12B所示。
参照实施例7步骤3)的操作，检测CAR-T细胞拷贝数，结果如图12C所示，mBBZ-7*19 CAR-T和mBBZ-7*21B CAR-T组的CAR-T拷贝数高于UTD和BBZ组。
参照实施例7步骤4)的操作，检测CD8 +细胞的肿瘤浸润情况，结果如图12D所示。mBBZ-7*19 CAR-T组和mBBZ-7*21B CAR-T组肿瘤组织中有明显的CD8T细胞的浸润，其中mBBZ-7*21B CAR-T组CD8 T细胞浸润较多。
实施例12.小鼠PANC02-A2胰腺癌皮下瘤模型的CAR-T细胞检测分析
参照实施例6的操作，制备小鼠胰腺癌皮下移植瘤模型，在C57BL/6小鼠右侧腹部皮下注射2×10 6的PANC02-A2胰腺癌细胞，皮下接种肿瘤细胞后D14天，肿瘤平均体积约60mm 3，分别注射未处理T细胞或CAR-T细胞(mBBZ CAR-T细胞、mBBZ-7*21A CAR-T细胞、及mBBZ-7*19 CAR-T细胞)，注射剂量：4×10 6/只。
1、分别提取CAR-T细胞治疗后第10天(d10)、第20天(d20)小鼠的脾脏、骨髓检测Tcm(中心记忆性T细胞)的含量(各治疗组取2只小鼠)。实验方法如下：
1)脾脏细胞提取：通过颈椎脱臼的方法处死小鼠，取脾脏，置于干净的2mL EP 管内，用PBS清洗掉血污。利用40um滤膜进行研磨取脾细胞。将脾细胞混合液400g5min离心，去掉上清，加入400μL小鼠红细胞裂解液(1×)，静置5min，加入1.5mL PBS中和反应，离心，加入PBS重悬，分管进行抗体孵育，抗体标记为CD8(PerCP)、CD44(BV510)、CD62L(APC)。
2)骨髓细胞提取：通过颈椎脱臼的方法处死小鼠，取小鼠股骨和胫骨，剔除肌肉，置于干净的2mL EP管内，用PBS清洗掉血污。取2mL注射器，抽吸2mL的PBS，将针头沿着股骨或胫骨一端穿刺进去，用小镊子固定住，挤压活塞，冲洗取骨髓细胞。将细胞混合液400g 5min离心，去掉上清，加入400μL小鼠红细胞裂解液(1×)，静置5min，加入400μL小鼠红细胞裂解液(1×)，静置5min，加入1.5mL PBS中和反应，离心，加入PBS重悬，分管进行抗体孵育，抗体标记为CD8(PerCP)、CD44(BV510)、CD62L(APC)。
d10天脾脏中Tcm的检测情况如图13A所示，d20天脾脏中Tcm的检测情况如图13B所示，相较于常规的CAR-T，表达IL7和CCL21的mBBZ-CAR-T细胞组Tcm的含量显著增多。
d10天骨髓中Tcm的含量情况如图14A所示，d20天脾脏中Tcm的检测情况如图14B所示，相较于常规的CAR-T，mBBZ-7*21BCAR-T治疗后，骨髓中Tcm的含量显著增多。
2、DC浸润检测
CAR-T细胞治疗后第10天(d10)小鼠的肿瘤组织进行冰冻切片制作检测DC的浸润，结果如图15所示，相较于mBBZ细胞组，mBBZ-7*21B细胞组小鼠肿瘤组织中有更多的DC细胞浸润。
3、MDSC(骨髓来源的抑制性细胞)的含量检测
分别提取CAR-T细胞治疗后第10天(d10)的UTD组、mBBZ组、mBBZ-7*21B组、和mBBZ-7*19 CAR-T组的小鼠的肿瘤组织，去除肿瘤表面脂肪、血管、包膜及内部坏死组织，PBS冲洗，转入5mL离心管加入2mL培养液，将肿瘤剪碎至1×1mm左右，补培养液至4.7mL按比例加入消化酶(组织分离所使用的消化酶：collagenase type I(0.05mg/ml)、collagenase type IV(0.05mg/ml、hyaluronidase(0.025mg/ml)、DNase I(0.01mg/ml)37℃摇床30min左右(中间取出观察消化情况)；消化后悬液过70um细胞筛至50mL管(冰上操作)，用注射器推棒轻撵未消化完的组织，用大量(至20mL)培养液冲洗筛网并收集，同时终止消化，400g离心8min，4℃，去上清PBS洗涤后分管进行抗体孵育，检测CD45 +、CD11b +(FITC)和Gr-1 +(PE)的细胞的含量，即MDSC 的含量。实验结果如图16所示：相较于mBBZ组，mBBZ-7*21B CAR-T治疗后，肿瘤组织中MDSC的含量较少。
作为示例性的，上述实施例选择靶向CLD18A2的CAR-T细胞，应理解，选择作用于其他靶点的CAR-T细胞也具有相同的效果，如GPC3、EGFR、EGFRvIII、CD19、BCMA等。所采用的抗体可以是鼠抗也可以是人源化的，采用的跨膜域、胞内域也可以根据目的不同采用不同种属的，如采用人的。
作为示例性的，上述实施例虽然采用的是CAR-T细胞，但该T细胞还可以表达有其他增强CAR-T细胞功能的细胞因子，如CAR和I型干扰素共表达的CAR-T细胞、CAR和PD-1共表达的CAR-T细胞等。作为示例性的，上述实施例虽然采用的是CAR-T细胞，但还可以选择其他免疫细胞，如NK细胞、NK-T细胞，还可以具体选择免疫细胞的特定亚型，如γ/δT细胞等。
本发明所用的序列总结于下表：
Figure PCTCN2019087077-appb-000003
Figure PCTCN2019087077-appb-000004
Figure PCTCN2019087077-appb-000005
Figure PCTCN2019087077-appb-000006
Figure PCTCN2019087077-appb-000007
Figure PCTCN2019087077-appb-000008
本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (40)

  1. 一种基因工程化的细胞，其特征在于，所述细胞表达特异性结合靶抗原的外源性受体和外源性CCL21；优选地，还表达促进所述细胞增殖的蛋白；更优选地，所述促进细胞增殖的蛋白为IL-7R结合蛋白或外源性IL-7。
  2. 如权利要求1所述的的细胞，其特征在于，所述IL-7R结合蛋白为外源性IL-7R结合蛋白，所述外源性IL-7R结合蛋白能特异性结合IL-7R且增强IL-7R活性；
    优选地，所述外源性IL-7R的氨基酸序列如SEQ ID NO:19所示。
  3. 如权利要求1或2所述的细胞，其特征在于，所述外源性CCL21为天然CCL21、或与天然CCL21具有相同功能的天然CCL21的截短片段或天然CCL21的突变体；
    优选地，所述天然CCL21与SEQ ID NO:21所示的氨基酸序列具有至少90％同一性、或是SEQ ID NO:21所示的氨基酸序列的截短片段；或者与由SEQ ID NO:14或15所示的核苷酸编码的氨基酸序列具有至少90％同一性，或是由SEQ ID NO:14或15所示的核苷酸编码的氨基酸序列的截短片段。
  4. 如权利要求1-3中任一项所述的细胞，其特征在于，所述外源性CCL21是组成型表达。
  5. 如权利要求1-3中任一项所述的细胞，其特征在于，所述外源性CCL21是诱导型表达；较佳地，所述诱导型表达通过免疫细胞诱导型启动子启动；更佳地，所述的免疫细胞诱导型启动子是NFAT启动子。
  6. 如权利要求1-5中任一项所述的细胞，其特征在于，所述外源性IL-7为天然IL-7、或与天然IL-7具有相同功能的天然IL-7的截短片段或天然IL-7的突变体；
    优选地，所述天然IL-7与SEQ ID NO:18所示的氨基酸序列具有至少90％同一性、或是SEQ ID NO:18所示的氨基酸序列的截短片段；或者与由SEQ ID NO:13所示的核苷酸编码的氨基酸序列具有至少90％同一性、或是由SEQ ID NO:13所示的核苷酸编码的氨基酸序列的截短片段。
  7. 如权利要求1-5中任一项所述的细胞，其特征在于，所述外源性IL-7R结合蛋白或外源性IL-7是组成型表达。
  8. 如权利要求1-5中任一项所述的细胞，其特征在于，所述外源性IL-7R结合蛋白或外源性IL-7是诱导型表达；较佳地，所述诱导型表达通过免疫细胞诱导型启动子启动；更佳地，所述的免疫细胞诱导型启动子是NFAT启动子。
  9. 如权利要求1-8中任一项所述的细胞，其特征在于，所述的细胞为免疫效应细胞；
    优选地，所述免疫效应细胞为T细胞、NK细胞或者NKT细胞；
    更优选地，所述免疫效应细胞为T细胞。
  10. 如权利要求1所述的细胞，其特征在于，所述靶抗原包括肿瘤抗原和/或病原体抗原；优选地，为肿瘤抗原。
  11. 如权利要求10所述的细胞，其特征在于，所述靶抗原为实体瘤抗原；
    优选地，所述实体瘤抗原为GPC3、EGFR或Claudin18.2，更优选地，所述实体瘤抗原为Claudin18.2。
  12. 如权利要求1-11中任一项所述的细胞，其特征在于，所述外源性受体为嵌合受体，其包含特异性结合靶抗原的抗原结合结构域、跨膜结构域和细胞内结构域。
  13. 如权利要求12所述的细胞，其特征在于，所述外源性受体选自嵌合抗原受体(CAR)、修饰的T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)或其组合；
    优选地，所述外源性受体为嵌合抗原受体。
  14. 如权利要求13所述的细胞，其特征在于，所述的嵌合抗原受体包括：
    (i)特异性结合靶抗原的抗体或其片段、CD28或CD8的跨膜域、CD28的共刺激信号结构域和CD3ζ；或
    (ii)特异性结合靶抗原的抗体或其片段、CD28或CD8的跨膜域、4-1BB的共刺激信号 结构域和CD3ζ；或
    (iii)特异性结合靶抗原的抗体或其片段、CD28或CD8的跨膜域、CD28的共刺激信号结构域、4-1BB的共刺激信号结构域和CD3ζ。
  15. 如权利要求12所述的细胞，其特征在于，所述外源性受体的抗原结合结构域的氨基酸序列与SEQ ID NO:2所示的氨基酸序列具有至少90％的同一性。
  16. 如权利要求15所述的细胞，其特征在于，所述外源性受体的氨基酸序列与SEQ ID NO:26、27或35所示的氨基酸序列具有至少90％的同一性。
  17. 如权利要求1-16中任一项所述的细胞，其特征在于，所述外源性受体、和/或外源性IL-7R结合蛋白或外源性IL-7、和/或外源性CCL21利用病毒载体表达；
    较佳地，所述的病毒载体包括：慢病毒载体，逆转录病毒载体或腺病毒载体。
  18. 一种表达构建物，其特征在于，该表达构建物包括顺序连接的：特异性结合靶抗原的外源性受体的表达盒1，外源性IL-7R结合蛋白或外源性IL-7的表达盒2，及外源性CCL21的表达盒3；优选地，所述表达盒之间任选地由选自F2A、P2A、T2A、和/或E2A的串联片段连接。
  19. 如权利要求18所述的表达构建物，其特征在于，所述表达盒2含有如SEQ ID NO:17所示的核酸序列。
  20. 如权利要求18所述的表达构建物，其特征在于，所述表达盒3含有如SEQ ID NO:20所示的核酸序列。
  21. 一种表达载体，其包含权利要求18-20中任一项所述的表达构建物。
  22. 一种病毒，其特征在于，所述的病毒包含权利要求21所述的表达载体。
  23. 一种提高表达嵌合受体的免疫效应细胞在个体中的活力的方法，其特征在于在所述免疫应答细胞中共表达外源性CCL21、外源性IL-7R结合蛋白或外源性IL- 7；优选地，所述嵌合受体是嵌合抗原受体。
  24. 如权利要求23所述的方法，其特征在于，所述外源性IL-7R结合蛋白能特异性结合IL-7R且增强IL-7R活性；
    优选地，所述外源性IL-7R的氨基酸序列如SEQ ID NO:19所示。
  25. 如权利要求23或24所述的方法，其特征在于，所述的外源性CCL21为天然CCL21、或与天然CCL21具有相同功能的天然CCL21的截短片段或天然CCL21的突变体；
    优选地，所述天然CCL21与SEQ ID NO:21所示的氨基酸序列具有至少90％同一性、或是SEQ ID NO:21所示的氨基酸序列的截短片段；或者与由SEQ ID NO:14或15所示的核苷酸编码的氨基酸序列具有至少90％同一性、或是由SEQ ID NO:14或15所示的核苷酸编码的氨基酸序列的截短片段。
  26. 如权利要求23-25中任一项所述的方法，其特征在于，所述外源性CCL21是组成型表达。
  27. 如权利要求23-25中任一项所述的方法，其特征在于，所述外源性CCL21是诱导型表达；较佳地，所述诱导型表达通过免疫细胞诱导型启动子启动；更佳地，所述的免疫细胞诱导型启动子是NFAT启动子。
  28. 如权利要求23-27中任一项所述的方法，其特征在于，所述外源性IL-7为天然IL-7、或与天然IL-7具有相同功能的天然IL-7的截短片段或天然IL-7的突变体；
    优选地，所述IL-7与SEQ ID NO:18所示的氨基酸序列具有至少90％同一性、或是SEQ ID NO:18所示的氨基酸序列的截短片段；或者与由SEQ ID NO:13所示的核苷酸编码的氨基酸序列具有至少90％同一性、或是由SEQ ID NO:13所示的核苷酸编码的氨基酸序列的截短片段。
  29. 如权利要求23-27中任一项所述的方法，其特征在于，所述外源性IL-7R结合蛋白或外源性IL-7是组成型表达。
  30. 如权利要求23-27中任一项所述的方法，其特征在于，所述外源性IL-7R结合蛋白或外源性IL-7是诱导型表达；较佳地，所述诱导型表达通过免疫细胞诱导型启动子启动；更佳地，所述的免疫细胞诱导型启动子是NFAT启动子。
  31. 如权利要求23-30中任一项所述的方法，其特征在于，所述的免疫效应细胞是T细胞、NK细胞或NKT细胞。
  32. 如权利要求1-17中任一项所述的细胞，或权利要求18-20中任一项所述的表达构建物，或权利要求21所述的表达载体，或权利要求22所述的病毒的用途，用于制备抑制肿瘤或抑制病原体的药物；优选地，用于制备抑制肿瘤的药物。
  33. 如权利要求32所述的用途，其特征在于，所述制备抑制肿瘤的药物与化疗药物联用。
  34. 一种药物组合物，其特征在于，所述的药物组合物包括权利要求1-17任一所述的细胞和药学上可接受的载体或赋形剂。
  35. 一种药盒，其包括药盒A和药盒B，所述药盒A包括基因工程化的细胞，所述细胞表达特异性结合靶抗原的外源性受体；所述药盒B包括CCL21，和/或，促进所述细胞增殖的蛋白；优选地，所述促进细胞增殖的蛋白为IL-7R结合蛋白或IL-7；更优选地，所述药盒A和药盒B的施用不分先后。
  36. 如权利要求35所述的药盒，其特征在于，所述IL-7R结合蛋白能特异性结合IL-7R且增强IL-7R活性；优选地，所述外源性IL-7R的氨基酸序列如SEQ ID NO:19所示。
  37. 如权利要求35或36所述的药盒，其特征在于，所述CCL21为天然CCL21、或与天然CCL21具有相同功能的天然CCL21的截短片段或天然CCL21的突变体；
    优选地，所述天然CCL21与SEQ ID NO:21所示的氨基酸序列具有至少90％同一性、或是SEQ ID NO:21所示的氨基酸序列的截短片段；或者与由SEQ ID NO:14或15所示的核苷酸编码的氨基酸序列具有至少90％同一性，或是由SEQ ID NO:14或15 所示的核苷酸编码的氨基酸序列的截短片段。
  38. 如权利要求35-37中任一项所述的药盒，其特征在于，所述IL-7为天然IL-7、或与天然IL-7具有相同功能的天然IL-7的截短片段或天然IL-7的突变体；
    优选地，所述天然IL-7与SEQ ID NO:18所示的氨基酸序列具有至少90％同一性、或是SEQ ID NO:18所示的氨基酸序列的截短片段；或者与由SEQ ID NO:13所示的核苷酸编码的氨基酸序列具有至少90％同一性、或是由SEQ ID NO:13所示的核苷酸编码的氨基酸序列的截短片段。
  39. 如权利要求35所述的药盒，其特征在于，所述药盒A包括嵌合受体修饰的免疫效应细胞；
    优选地，所述嵌合受体为嵌合抗原受体。
  40. 如权利要求35所述的药盒，其特征在于，所述免疫效应细胞为T细胞、NK细胞或NKT细胞。
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WO2022028623A1 (zh) 2020-08-07 2022-02-10 佧珐药业有限公司 工程化改造的细胞以及工程化改造细胞的方法
CN114805596A (zh) * 2021-01-22 2022-07-29 华东师范大学 一种以磷脂酰肌醇聚糖3为靶点的嵌合抗原受体及其应用
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