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WO2021057906A1 - 表达il-15的免疫效应细胞 - Google Patents

表达il-15的免疫效应细胞 Download PDF

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WO2021057906A1
WO2021057906A1 PCT/CN2020/117759 CN2020117759W WO2021057906A1 WO 2021057906 A1 WO2021057906 A1 WO 2021057906A1 CN 2020117759 W CN2020117759 W CN 2020117759W WO 2021057906 A1 WO2021057906 A1 WO 2021057906A1
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cancer
cell
cells
seq
receptor
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PCT/CN2020/117759
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English (en)
French (fr)
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李宗海
蒋华
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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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Publication of WO2021057906A1 publication Critical patent/WO2021057906A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4261—Proteoglycans, e.g. glypican, brevican or CSPG4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterized by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the cancer treated
    • A61K2239/53—Liver

Definitions

  • the invention belongs to the field of cell therapy, and relates to immune effector cells expressing IL-15, in particular to an immune effector cell targeting GPC3 that co-expresses IL-15 and recognizes a receptor that specifically recognizes a target antigen.
  • TCR T Cell Receptor
  • scFv antibodies against tumor cell-related antigens
  • CD3 ⁇ or Fc ⁇ RI ⁇ of T lymphocyte receptors.
  • the intracellular signal activation motif is fused into a chimeric antigen receptor (CAR), which is genetically modified on the surface of T lymphocytes through methods such as lentivirus infection.
  • CAR T lymphocytes can selectively direct T lymphocytes to tumor cells and specifically kill tumors in a non-limiting manner of Major Histocompatibility Complex (MHC).
  • MHC Major Histocompatibility Complex
  • the chimeric antigen receptor includes an extracellular binding domain, a transmembrane domain and an intracellular signaling domain.
  • the extracellular domain contains scFv that can recognize tumor-associated antigens
  • the transmembrane domain uses CD8, CD28 and other molecular transmembrane regions
  • the intracellular signal domain uses immunoreceptor tyrosine activation motif (ITAM) CD3 ⁇ or Fc ⁇ RI ⁇ and costimulatory signals.
  • ITAM immunoreceptor tyrosine activation motif
  • immune effector cells have shown attractive application prospects in the treatment of hematomas, they still do not have good therapeutic effects for a variety of solid tumors, such as For some tumors, the survival rate of immune effector cells in tumor tissues is poor, and tumor killing activity is not high. Therefore, how to improve the effect of immune effector cells such as CAR T cells on solid tumors, especially some refractory solid tumors, is a huge problem faced by many researchers.
  • the present invention relates to an immune effector cell expressing IL-15, characterized in that the cell expresses IL-15 and a receptor that recognizes a specific target antigen, or the cell expresses IL-15R binding Molecules and receptors that recognize specific target antigens.
  • the IL-15 is exogenous IL-15.
  • the IL-15 is constitutively expressed or inducible expressed IL-15.
  • the IL-15 is an inducible expression of IL-15, and its expression is regulated by the receptor.
  • the expression of the IL-15 can be initiated after the receptor recognizes the target antigen.
  • the receptor induces the expression of the IL-15 through an inducible promoter.
  • the immune effector cells also express chemokines, chemokine receptors, cytokines other than IL-15, siRNA that reduces PD-1 expression, and block PD-L1 A protein that binds to PD-1, or a safety switch.
  • the chemokine is a lymphocyte chemokine
  • the lymphocyte chemokine is CCL21;
  • the CCL21 has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 26 or SEQ ID NO: 39, 40 or is the amino acid shown in SEQ ID NO: 26 or SEQ ID NO: 39, 40 A truncated fragment of the sequence; or has at least 90% identity with the amino acid sequence encoded by the nucleotides shown in SEQ ID NO: 24 or SEQ ID NO: 37, 38, or SEQ ID NO: 24 or SEQ ID A truncated fragment of the amino acid sequence encoded by the nucleotides shown in NO: 37 and 38.
  • the chemokine receptor is selected from CCR2, CCR5, CXCR2, or CXCR4.
  • the other cytokine is selected from IL-21, IL18, or type I interferon.
  • the protein that blocks the binding of PD-L1 to PD-1 is selected from the group consisting of an antibody against PD-L1, an antibody against PD-1, natural PD-1 or a short fragment of natural PD-1 Segment, a fusion peptide containing natural PD-1 or a truncated fragment of natural PD-1.
  • the safety switch is selected from iCaspase-9, Truncated EGFR, RQR8, and a protein that has a killing effect on immune effector cells.
  • the immune effector cells are selected from T cells, NK cells, NKT cells, mast cells, macrophages, dendritic cells, CIK cells, or stem cell-derived immune effector cells.
  • the inducible promoter comprises a transcription factor binding motif and a minimal promoter, and the activation of the inducible promoter depends on the activation of the receptor or on the receptor and The target antigen binds.
  • the binding motif includes NFAT, NF- ⁇ B or AP-1 binding motif, or a combination of at least two of NFAT, NF- ⁇ B, AP-1 binding motif,
  • the binding motif is an NFAT binding motif.
  • the binding motif comprises 1-12 NFAT binding motifs, 1-12 NF- ⁇ B binding motifs, 1-12 AP-1 binding motifs, or 1-12 A combination of at least two of NFAT, NF- ⁇ B, and AP-1 binding motifs;
  • the binding motif includes 1-6 NFAT binding motifs, 1-6 NF- ⁇ B binding motifs, 1-6 AP-1 binding motifs, or 1-6 NFAT, NF- ⁇ B A combination of at least two of the AP-1 binding motifs.
  • sequence of the NFAT binding motif is shown in SEQ ID NO: 23.
  • the minimal promoter is a cytokine minimal promoter.
  • the minimal promoter includes interleukin, interferon, tumor necrosis factor superfamily, colony stimulating factor, chemokine, growth factor minimal promoter;
  • IFN- ⁇ , TNF- ⁇ or IL-2 minimal promoter Preferably, it is IFN- ⁇ , TNF- ⁇ or IL-2 minimal promoter
  • it is an IL-2 minimal promoter.
  • sequence of the IL-2 minimal promoter is shown in SEQ ID NO: 25.
  • the IL-15 is natural IL-15, or a truncated fragment of natural IL-15 that has the same function as natural IL-15, or a mutant of natural IL-15;
  • the natural IL-15 is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 36, or is the amino acid shown in SEQ ID NO: 1 or SEQ ID NO: 36 A truncated fragment of the sequence; or has at least 90% identity with the amino acid sequence encoded by the nucleotide shown in SEQ ID NO: 33 or SEQ ID NO: 35, or is sequenced by SEQ ID NO: 33 or SEQ ID NO: A truncated fragment of the amino acid sequence encoded by the nucleotide shown in 35.
  • the protein or safety switch is constitutively expressed or inducible; preferably, it is constitutively expressed.
  • the target antigen is a tumor antigen.
  • the solid tumor antigen is GPC3, EGFR, EGFRvIII, mesothelin or Claudin 18.2.
  • the receptor is selected from chimeric antigen receptor (CAR), T cell receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler (TAC) or its combination;
  • CAR chimeric antigen receptor
  • TCR T cell receptor
  • TCP T cell fusion protein
  • TAC T cell antigen coupler
  • the receptor is a chimeric antigen receptor
  • the receptor is a chimeric antigen receptor, including
  • the antigen binding domain of the chimeric antigen receptor (CAR), T cell fusion protein (TFP), or T cell antigen coupler (TAC) is an antibody with SEQ ID NO: HCDR1 shown in 2, HCDR2 shown in SEQ ID NO: 3, HCDR3 shown in SEQ ID NO: 4, and LCDR1 shown in SEQ ID NO: 5, LCDR2 shown in SEQ ID NO: 6, SEQ ID NO: : LCDR3 shown in 7; or
  • the antibody has a light chain variable region shown in SEQ ID NO: 8 and a heavy chain variable region shown in SEQ ID NO: 9; or a light chain variable region shown in SEQ ID NO: 10 and SEQ ID NO: The variable region of the heavy chain shown in 11; or
  • the antibody has the amino acid sequence shown in SEQ ID NO: 12.
  • amino acid sequence of the receptor has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 13, 14, 15.
  • nucleic acid sequence of the immune cell inducible promoter inducing the expression of IL-15 is shown in SEQ ID NO: 22.
  • the receptor and IL-15 are expressed by the same nucleic acid molecule, or expressed by different nucleic acid molecules;
  • the receptor and IL-15 are expressed by the same nucleic acid molecule, and the IL-15 expression cassette and the receptor, and between the expression cassettes are directly connected or connected by a tandem fragment, and the tandem fragment is selected from F2A , P2A, T2A, and/or E2A.
  • the present invention relates to a nucleic acid molecule that expresses the aforementioned IL-15R binding molecule of the present invention or IL-15 or IL-15 and chemokines, chemokine receptors, other than IL- 7 other cytokines, siRNAs that reduce PD-1 expression, proteins that block the binding of PD-L1 to PD-1, or safety switches; the nucleic acid molecules also express the receptors of the present invention that specifically recognize the target antigen. body;
  • the nucleic acid is composed of DNA and/or RNA; or the nucleic acid is mRNA; or the nucleic acid contains nucleotide analogs.
  • the present invention relates to a vector comprising the aforementioned nucleic acid molecule
  • the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, Rous sarcoma virus (RSV) vector or retroviral vector.
  • RSV Rous sarcoma virus
  • the fourth aspect of the present invention relates to a cell comprising the above-mentioned vector of the present invention or the above-mentioned nucleic acid molecule of the present invention integrated in its genome;
  • the cells are human T cells, preferably allogeneic T cells.
  • the present invention relates to a method for preparing cells, the method comprising transducing T cells with the vector according to the present invention or the nucleic acid molecule according to the present invention.
  • RNA engineered cell population comprising introducing in vitro transcribed RNA or synthetic RNA into the cell, (i) wherein the RNA comprises the aforementioned nucleic acid of the present invention .
  • the seventh aspect of the present invention relates to a method for providing anti-tumor immunity in a mammal, the method comprising administering to the mammal an effective amount of the cell of the present invention, the nucleic acid molecule of the present invention, or the present invention.
  • the carrier of the invention comprising administering to the mammal an effective amount of the cell of the present invention, the nucleic acid molecule of the present invention, or the present invention.
  • the mammal is a human.
  • the eighth aspect of the present invention relates to a method for treating a mammal suffering from a disease related to the expression of GPC3 or claudin 18.2, the method comprising administering to the mammal an effective amount of the above-mentioned cell of the present invention, the above-mentioned The nucleic acid molecule of the present invention, or the above-mentioned vector of the present invention;
  • the disease related to the expression of GPC3 or claudin18.2 is selected from colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer , Skin or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, endocrine system cancer, thyroid Carcinoma, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, renal or ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumor, tumor angiogenesis, spine tumor, brainstem glial Tumor, pituitary adenoma, Kaposi’s sarcoma, epidermoid carcinoma, squamous
  • the above-mentioned cell of the present invention is administered in combination with an agent that increases the efficacy of the above-mentioned cell of the present invention, preferably, in combination with a chemotherapeutic drug;
  • the cells of the present invention described above are administered in combination with an agent that improves one or more side effects associated with the administration of the cells of the present invention described above;
  • the above-mentioned cells of the present invention are administered in combination with an agent for the treatment of diseases related to GPC3 or claudin 18.2, preferably in combination with a chemotherapeutic agent.
  • the ninth aspect of the present invention relates to the use of the above-mentioned cells of the present invention, the above-mentioned nucleic acid molecules of the present invention, and the above-mentioned vectors of the present invention for use as a medicine, preferably as a medicine for inhibiting tumors or inhibiting pathogens, more preferably, The use is for preparing a drug for inhibiting tumors or inhibiting pathogens.
  • the present invention relates to a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the above-mentioned cell of the present invention and a pharmaceutically acceptable carrier or excipient.
  • Figure 1 shows the schematic diagram of pRRLSIN-GPC3-BBZ, pRRLSIN-GPC3-BBZ-IL15, pRRLSIN-GPC3-BBZ-CCL21*15;
  • Figure 2 shows the in vitro killing effect of GPC3-BBZ-IL15 CAR-T cells
  • Figure 3 shows the in vivo tumor suppression results of GPC3-BBZ-IL15 CAR-T cells
  • Figure 4 shows the body weight changes of mice in the in vivo tumor suppression experiment of GPC3-BBZ-IL15 CAR-T cells
  • Figure 5 shows the in vitro killing effect of GPC3-BBZ-CCL21*IL15 CAR-T cells
  • Figure 6 shows the MSCV-GPC3-mBBZ; MSCV-GPC3-mBBZ-mNFAT6-mIL15 plasmid diagram;
  • Figure 7A shows the killing of tumor cells by MSCV-GPC3-mBBZ CAR-T, MSCV-GPC3-mBBZ-mNFAT6-mIL15 CAR-T cells in vitro;
  • Figure 7B shows MSCV-GPC3-mBBZ CAR-T, MSCV-GPC3-mBBZ -Cytokine secretion of mNFAT6-mIL15 CAR-T cells;
  • Figures 8A-C respectively show the in vivo tumor suppression results of MSCV-GPC3-mBBZ CAR-T, MSCV-GPC3-mBBZ-mNFAT6-mIL15 CAR-T cells, the effect on the weight of mice, and the comparison of tumor weight.
  • the present invention found that immune effector cells that express IL-15 and recognize receptors that specifically recognize target antigens not only have a more excellent killing effect on tumors, but also improve the survival of immune effector cells in tumor tissues, even for refractory The solid tumors also show more excellent anti-tumor ability.
  • engineered can refer to one or more alterations of nucleic acids, such as nucleic acids within the genome of an organism.
  • engineered can refer to changes, additions, and/or deletions of genes.
  • Engineered cells can also refer to cells with added, deleted and/or changed genes.
  • genetically engineered cell refers to a cell modified by means of genetic engineering.
  • the cell is an immune effector cell.
  • the cell is a T cell.
  • the genetically engineered cell described herein refers to a cell that expresses an exogenous receptor that specifically binds to a target antigen.
  • the genetically engineered cells described herein refer to immune effector cells that express exogenous receptors that specifically bind to the target antigen and induce the expression of exogenous IL15.
  • the genetically engineered cell described herein refers to an immune effector cell that expresses an exogenous receptor that specifically binds to a target antigen, expresses exogenous CLL21, and induces expression of exogenous IL15.
  • the genetically engineered cells described herein may also be T cells that co-express chimeric antigen receptors that specifically bind to tumor antigens and induce the expression of exogenous IL15.
  • the genetically engineered cells described herein can also be T cells that co-express chimeric antigen receptors that specifically bind to tumor antigens, express exogenous CLL21, and induce the expression of exogenous IL15.
  • the genetically engineered cells described herein can also co-express chimeric antigen receptors that specifically bind to tumor antigens, CLL21, and induce the expression of IL-15R binding protein or exogenous IL-15. T cells.
  • immune effector cells refers to cells that participate in immune responses and produce immune effects, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells and bone marrow-derived phagocytes, Immune effector cells derived from macrophages, dendritic cells, CIK cells, or stem cells.
  • the immune effector cells are T cells, NK cells, NKT cells.
  • the T cell may be an autologous T cell, a heterologous T cell, or an allogeneic T cell.
  • the NK cells may be allogeneic NK cells.
  • peptide refers to a compound composed of amino acid residues covalently linked by peptide bonds.
  • 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.
  • Polypeptides include any peptide or protein containing two or more amino acids joined to each other by peptide bonds.
  • the term refers to short chains (which are also commonly referred to in the art as peptides, oligopeptides, and oligomers, for example) and longer chains (which are also commonly referred to as proteins in the art. Types).
  • Polypeptide includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides or a combination thereof.
  • IL15 Interleukin15, interleukin 15 or IL15 or IL-15
  • cytokine interleukin-15 is a member of the four alpha-helix bundle lymphokine family produced by many cells in the body.
  • IL-15 plays a key role in regulating the activity of the innate and adaptive immune system, such as maintaining the response of memory T cells to invading pathogens, inhibiting apoptosis, activating dendritic cells, and inducing the proliferation and proliferation of natural killer (NK) cells. Cytotoxic activity.
  • IL15 can interact (eg bind) with IL-15R (preferably from mammals, such as murine or human IL-15R), preferably from mammals, such as murine or human.
  • IL15 can also have anti-tumor effects through non-IL-15R pathways .
  • IL15 has one of the following characteristics: (i) is the amino acid sequence or fragments of naturally occurring mammalian IL-15, such as the amino acid sequence or fragments thereof shown in SEQ ID NO: 1 (human) or SEQ ID NO: 36 (mouse); ii) Basically have at least 85%, 90%, 95%, 96%, 97%, 98%, 99% homology with the amino acid sequence shown in SEQIDNO:1 (human) or SEQIDNO:36 (mouse) or fragments thereof, for example sexual amino acid sequence; (iii) the nucleotide sequence of naturally occurring mammalian IL-15 or a fragment thereof, such as the amino acid sequence encoded by SEQ ID NO: 35 (human) or a fragment thereof, or SEQ ID NO: 33 (mouse) Or the amino acid sequence encoded by the fragment thereof; (iv) the nucleotide sequence or fragment thereof shown in SEQ ID NO: 35 (human) or SEQ ID NO: 33 (mouse) has, for example, at
  • Exogenous includes recombinant nucleic acid molecules or polypeptides expressed in cells, such as exogenous, heterologous and overexpressed nucleic acid molecules and polypeptides.
  • exogenous IL-15 refers to IL-15 recombinant nucleic acid molecules or IL-15 expressed in cells, such as exogenous, heterologous and overexpressed nucleic acid molecules encoding IL-15 and IL-15 .
  • CTL21 Cyhemokine (CC motif) ligand 21)
  • CCL21 in the present invention has one of the following features: (i) is the amino acid sequence of naturally occurring mammalian CCL21 or a fragment thereof, such as the amino acid shown in SEQ ID NO: 26 (human), or SEQ ID NO: 39 or 40 (mouse) Sequence or fragments thereof; (ii) is the amino acid sequence shown in SEQ ID NO: 26 (human), or SEQ ID NO: 39 or 40 (mouse) or fragments thereof having, for example, at least 85%, 90%, 95%, 96%, Amino acid sequences with 97%, 98%, 99% homology; (iii) the amino acid sequence encoded by the naturally-occurring mammalian CCL21 nucleotide sequence or its fragments (such as SEQ ID NO:
  • Exogenous IL-15R binding molecule or binding protein refers to all molecules or proteins that can specifically bind to IL-15R and enhance the activity of IL-15R.
  • "Enhancement of IL-15R activity” should be understood to mean that IL-15R binding protein can enhance any one or more activities of naturally occurring IL-15R, including but not limited to stimulating the proliferation, cytotoxicity or maturation of NK cells; Proliferation or differentiation of B cells and T cells; stimulates antibody production and affinity maturation in B cells; stimulates the cytotoxicity of CD8+ T cells; stimulates interferon gamma production in T cells and NK cells; inhibits dendritic cells (DC) Activation and maturation; inhibit the release of inflammatory mediators from mast cells; enhance the phagocytosis of macrophages; inhibit the production or survival of TReg cells; and stimulate the proliferation of bone marrow progenitor cells.
  • DC dendritic cells
  • amino acid modification includes amino acid substitutions, additions, and/or deletions, and "amino acid substitution” means replacing an amino acid at a specific position in the parent polypeptide sequence with another amino acid.
  • amino acid insertion as used herein means the addition of an amino acid at a specific position in the parent polypeptide sequence.
  • amino acid deletion or “deletion” means the removal of an amino acid at a specific position in the parent polypeptide sequence.
  • conservative modification as used herein means an amino acid modification that does not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions.
  • Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are those that replace amino acid residues with amino acid residues with similar side chains.
  • a family of amino acid residues with similar side chains has been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), and uncharged acute side chains (e.g.
  • Glycine asparagine, serine, threonine, tyrosine, cysteine, tryptophan
  • non-polar side chains e.g., alanine, valine, leucine, isoleucine
  • Acid proline, phenylalanine, methionine
  • beta branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, benzene Alanine, Tryptophan, Histidine).
  • wild type has the same meaning when referring to protein and DNA.
  • mutation has the same or better biological activity as the natural protein or natural DNA, which has one or more amino acid substitutions, additions or deletions in the amino acid sequence of the natural protein ; Or one or more nucleotide substitutions, additions or deletions have occurred in the nucleic acid sequence of natural 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 97% of the amino acid sequence of the natural protein or the nucleic acid sequence of the natural DNA.
  • a "variant of IL-15” generally refers to a polypeptide having similar or better biological activity to IL-15 obtained by amino acid modification of wild-type IL-15.
  • the term "truncated fragment” refers to the non-full-length form of natural protein or natural DNA, which has consecutive or non-contiguous deletions of multiple amino acid residues or nucleotides in the natural amino acid sequence or nucleic acid sequence, and the deletion occurs in the sequence Any position, such as head, middle, tail, and combinations thereof. In the present invention, the truncated fragment of the protein still retains the same function as the natural protein from which it originated.
  • Consitutive expression also known as continuous expression, refers to the continuous expression of genes in cells under almost all physiological conditions.
  • inducible expression refers to expression under certain conditions, such as when T cells bind to an antigen.
  • an effective amount refers to the amount of a compound, preparation, substance, or composition that is effective to achieve a specific biological result, for example, including, 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 cells or therapeutic agent of the present invention can be determined by the physician It is determined in consideration of the individual's age, weight, tumor size, or degree of metastasis, and the condition of the patient (subject).
  • An effective amount of immune effector cells refers to, including but not limited to, the number of immune effector cells capable of increasing, enhancing or prolonging the anti-tumor activity of immune effector cells; including but not limited to the number of immune effector cells capable of making anti-tumor immune effector cells or activating immune effector cells.
  • the increased number of immune effector cells including but not limited to the number of immune effector cells that can promote IFN- ⁇ secretion; tumor regression, tumor shrinkage, and tumor necrosis.
  • chimeric antigen receptor refers to a set of polypeptides that, when in immune effector cells, provide said cells with specificity against target cells (usually cancer cells or tumor cells) Recognize, and have intracellular signal generation.
  • CAR usually includes at least one extracellular antigen binding domain (also called extracellular region), transmembrane domain (also called transmembrane region), and cytoplasmic signaling domain (also referred to herein as “intracellular signaling structure”).
  • Domain or “intracellular region”
  • groups of polypeptides are adjacent to each other.
  • the group of polypeptides includes a dimerization switch that can couple polypeptides to each other in the presence of a dimerization molecule, for example, an antigen binding domain can be coupled to an intracellular signal transduction domain.
  • the stimulatory molecule is the zeta chain that binds to the T cell receptor complex.
  • the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one costimulatory molecule as defined below.
  • the costimulatory molecule is selected from the costimulatory molecules described herein, such as 4-1BB (ie, CD137), CD27, CD28, or a combination thereof.
  • the CAR includes a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein, the fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and a functional signaling domain derived from a costimulatory molecule and functionality derived from a stimulatory molecule.
  • the intracellular signaling domain of the signaling domain comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and comprising two functional signaling derived from one or more costimulatory molecules.
  • the present invention contemplates modification of the amino acid sequence of the starting antibody or fragment (e.g., scFv) that produces a functionally equivalent molecule.
  • the VH or VL of the antigen-binding domain of the cancer or tumor-associated antigen described herein, such as the scFv contained in the CAR can be modified to retain the start of the antigen-binding domain of the cancer or tumor-associated antigen described herein At least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% of the VH or VL framework region (eg scFv) , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % Identity.
  • the present invention envisages the modification of the entire CAR construct, such as the modification of one or more amino acid sequences of multiple domains of the CAR construct, to produce 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% of the starting CAR construct, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • transmembrane domain (also referred to as transmembrane region) used herein may include one or more amino acid fragments adjacent to the transmembrane region, for example, one or more proteins derived from the transmembrane protein Amino acids associated with the extracellular region (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 up to 15 amino acids in the extracellular region).
  • the transmembrane domain is a domain related to one of the other domains of the chimeric receptor.
  • the transmembrane domain may be derived from a signal transduction domain, a co-stimulator The same protein from which the domain or hinge domain is derived.
  • the transmembrane domain can be selected or replaced by amino acids to prevent such a domain from binding to a transmembrane domain of the same or different surface membrane protein, for example, to bind to a receptor containing the transmembrane domain.
  • the interaction of the other members of the complex is minimized.
  • the transmembrane domain is capable of homodimerization with another chimeric receptor on the cell surface of the cell expressing the chimeric receptor.
  • the amino acid sequence of the transmembrane domain can be modified or substituted in order to minimize interaction with the binding domain of the natural binding partner present in cells expressing the same chimeric receptor.
  • the transmembrane domain can be derived from natural or recombinant sources.
  • the domain can be derived from any membrane-bound protein or transmembrane protein.
  • the transmembrane domain can transmit signals to the intracellular domain.
  • the transmembrane domain specifically used in the present invention may include at least the following transmembrane domains: for example, ⁇ , ⁇ or ⁇ chains of T-cell receptors, CD28, CD27, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 transmembrane domains.
  • the transmembrane domain may include at least the following transmembrane regions: for example, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , 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, 2
  • the transmembrane domain may be connected to the extracellular region of the CAR, such as the antigen binding domain of the CAR, via a hinge (for example, a hinge from a human protein).
  • a hinge for example, a hinge from a human protein.
  • the hinge may be a human Ig (immunoglobulin) hinge (eg, IgG4 hinge, IgD hinge), GS linker (eg, GS linker described herein), KIR2DS2 hinge, or CD8a hinge.
  • the transmembrane domain can be recombinant, in which case it will mainly contain 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 dimer provides a particularly suitable linker.
  • intracellular domain includes intracellular signaling domain.
  • the intracellular signaling domain is generally 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 a specialized function of a cell.
  • the effector function of T cells can be, for example, cytolytic activity or auxiliary activity, including secretion of cytokines. Therefore, the term “intracellular signaling domain” refers to a part of a protein that transduces effector function signals and guides cells to perform specific functions. Although the entire intracellular signaling domain can usually be used, in many cases it is not necessary to use the entire chain.
  • intracellular signaling domain is meant to include a truncated portion of the intracellular signaling domain sufficient to transduce effector function signals.
  • T cell activation can be said to be mediated by two different kinds of cytoplasmic signaling sequences: those that trigger antigen-dependent primary activation through TCR (primary intracellular signaling domains) and those that are antigen-independent. Those that function to provide secondary or costimulatory signals (secondary cytoplasmic domains, such as costimulatory domains).
  • stimulation refers to the binding of a stimulating molecule (for example, TCR/CD3 complex or CAR) to its cognate ligand (or tumor antigen in the case of CAR), thereby mediating a signal transduction event (such as However, it is not limited to the initial response induced by signal transduction via the TCR/CD3 complex or signal transduction via a suitable NK receptor or the signal transduction domain of CAR). Stimulation can mediate the altered expression of certain molecules.
  • a stimulating molecule for example, TCR/CD3 complex or CAR
  • CAR cognate ligand
  • the term "stimulatory molecule” refers to a molecule expressed by immune cells (for example, T cells, NK cells, B cells) that provides cytoplasmic signal transduction sequences that modulate the signal transduction pathways used in immune cells in a stimulating manner. Activation of immune cells in at least some aspects.
  • the signal is a primary signal initiated by, for example, the binding of a TCR/CD3 complex to a peptide-loaded MHC molecule, and it leads to mediation of T cell responses, including, but not limited to, proliferation, activation, differentiation, and the like.
  • the primary cytoplasmic signal transduction sequence (also called “primary signal transduction domain") that acts in a stimulating manner can contain an activation motif called immunoreceptor tyrosine-based activation motif (ITAM). ) Of the signaling motif.
  • ITAM-containing cytoplasmic signaling sequences particularly used 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 CARs of the present invention includes an intracellular signaling sequence, such as a primary signaling sequence of CD3- ⁇ .
  • the primary signal transduction sequence of CD3- ⁇ is the equivalent residue from human or non-human species such as mouse, rodent, monkey, ape and the like.
  • costimulatory molecule refers to a homologous binding partner on T cells, which specifically binds to a costimulatory ligand, thereby mediating the costimulatory response of T cells, including but not limited to proliferation.
  • Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which promote an effective immune response.
  • Co-stimulatory molecules include but are not limited to MHC class I molecules, BTLA and Toll ligand receptors, as well as OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) and 4- 1BB (CD137).
  • 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 is the intracellular part of costimulatory molecules.
  • Co-stimulatory molecules are represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and NK cell receptors.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, antigen-1 (LFA-1) associated with lymphocyte function, CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3 and ligands that specifically bind to CD83, etc.
  • the intracellular signal transduction domain includes all the intracellular part or all of the natural intracellular signal transduction domain of the molecule, or a functional fragment or derivative thereof.
  • 4-1BB refers to a member of the TNFR superfamily with the amino acid sequence provided in GenBank Accession No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.;
  • 4-1BB costimulatory domain is defined as the amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, and apes.
  • the "4-1BB costimulatory domain” is an equivalent residue from humans or from non-human species such as mice, rodents, monkeys, apes, and the like.
  • T cell receptor is a characteristic mark on the surface of all T cells, which binds to CD3 by a non-covalent bond to form a TCR-CD3 complex.
  • TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules.
  • TCR is a heterodimer composed of two different peptide chains, composed of two peptide chains ⁇ and ⁇ . Each peptide chain can be divided into variable region (V region), constant region (C region), and transmembrane. Several parts such as cytoplasmic region and cytoplasmic region; its characteristic is that the cytoplasmic region is very short.
  • TCR molecules belong to the immunoglobulin superfamily, and their antigen specificity exists in the V region; V regions (V ⁇ , V ⁇ ) each have three hypervariable regions CDR1, CDR2, and CDR3, of which CDR3 has the largest variation, which directly determines the antigen of TCR Binding specificity.
  • CDR1 and CDR2 recognize and bind to the sidewall of the antigen binding groove of the MHC molecule, and CDR3 directly binds to the antigen peptide.
  • TCR is divided into two categories: TCR1 and TCR2; TCR1 is composed of two chains, ⁇ and ⁇ , and TCR2 is composed of two chains, ⁇ and ⁇ .
  • T cell fusion protein includes recombinant polypeptides derived from various polypeptides that constitute TCR, which can bind to the surface antigens of target cells and interact with other polypeptides in the complete TCR complex. The role is usually co-localized on the surface of T cells.
  • TFP consists of a TCR subunit and an antigen binding domain composed of a human or humanized antibody domain.
  • the TCR subunit includes at least part of the TCR extracellular domain, transmembrane domain, and TCR intracellular domain.
  • the stimulation domain of the internal signal domain; the TCR subunit and the antibody domain are effectively connected, wherein the extracellular, transmembrane, and intracellular signal domains of the TCR subunit are derived from CD3 ⁇ or CD3 ⁇ , and the TFP is integrated into 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 groups; 2. The extracellular domain, a single-chain antibody that binds to CD3, so that the TAC receptor and the TCR receptor are close; 3. The transmembrane region and the intracellular region of the CD4 co-receptor, Among them, the intracellular region is connected to the protein kinase LCK to catalyze the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as the initial step of T cell activation.
  • ITAMs immunoreceptor tyrosine activation motifs
  • NF-kb Nuclear factor kB
  • NF-kB nuclear transcription factor family
  • IkB inhibitory protein IkB
  • NF-kB enters the cell nucleus, binds to the specific protein on the DNA module, induces the production of specific mRNA, and finally transcribes, produces and releases various cytokines.
  • AP-1 Activator protein 1
  • AP-1 Activator protein 1
  • AP-1 Activator protein 1
  • AP-1 up-regulates the transcription of genes containing TPA DNA response elements (TRE; 5'-TGAG/CTCA-3').
  • TRE TPA DNA response elements
  • AP-1 heterodimer is formed by a leucine zipper, and the expression of the gene is initiated by the combination of a specific conserved sequence with the gene.
  • NFAT Nuclear factor of activated T cells
  • NFAT protein plays an important role in a variety of cytokines and cell surface receptors that can regulate important immune functions ( For example, interleukin-2, interleukin-4, interleukin-5, interleukin-13, interferon- ⁇ , tumor necrosis factor- ⁇ , GM-CSF, CD40L and CTLA-4) play an important role in the transcriptional regulation.
  • the NFAT proteins that have been discovered so far can be divided into five: NFAT1, NFAT2, NFAT3, NFAT4 and NFAT5. Among them, the activation of NFATc1-4 depends on the intracellular calcium signal pathway.
  • NFAT protein activation is regulated by a process including NFAT protein dephosphorylation, nuclear translocation and DNA binding.
  • the phosphorylated NFAT protein resides in the cytoplasm and has low DNA binding affinity.
  • Various stimuli that can trigger calcium movement can cause rapid dephosphorylation of NFAT protein through a process regulated by Ca2+/calmodulin-dependent protein phosphatase, that is, calcineurin.
  • the dephosphorylated NFAT protein with an exposed nuclear localization signal migrates to the nucleus, which binds to DNA with high affinity and regulates target gene transcription.
  • NFAT plays an important role in the transcriptional expression of cytokines during T cell activation.
  • IL15 is inducible expression using an inducible promoter.
  • the inducible promoter is the NFAT promoter.
  • the coding sequence of IL15 is placed under the control of a minimal promoter containing an NFAT binding motif.
  • the IL2 minimal promoter containing 6 NFAT binding motifs is a promoter composed of 6 NFAT binding sites and IL2 minimal promoter in tandem.
  • the activated TCR signal after the receptor recognizes the target antigen can activate NFAT in the cell and bind to the NFAT binding motif in the promoter to initiate IL-15 transcription.
  • promoter as used herein is defined as a DNA sequence recognized by a synthetic mechanism of the cell or an introduced synthetic mechanism required to initiate the specific transcription of a polynucleotide sequence.
  • a promoter is a DNA sequence that RNA polymerase recognizes, binds and starts transcription, and it contains conserved sequences required for RNA polymerase specific binding and transcription initiation.
  • 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 related factors (TAF) can be combined to initiate transcription.
  • polyII RNA polymerase II
  • TBP TATA binding protein
  • TAF TBP related factors
  • NFAT plays an important role in the transcriptional expression of cytokines during T cell activation. Based on this consideration, the present inventors placed the coding sequence of the cytokine under the control of the minimal promoter containing the NFAT binding motif.
  • the endogenous TCR ⁇ chain of CAR-GPC3 T cells carrying NFAT-regulated and expressing cytokine genes can also be knocked out through gene editing technology to eliminate non-tumor Target antigens (such as non-GPC3 antigens) induce the expression of cytokines through the TCR/CD3 signaling pathway, and realize that only the tumor target antigen specifically induces CAR-GPC3 T cells to express cytokines, such as IL15.
  • non-tumor Target antigens such as non-GPC3 antigens
  • the IL2 minimal promoter containing 6 NFAT binding motifs is connected in series with 6 NFAT binding sites (nucleic acid sequence shown in SEQ ID NO: 23) and IL2 minimal promoter.
  • the constitutive promoter regulates the expression of IL15 in T cells.
  • antibody refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds to an antigen.
  • Antibodies can be polyclonal or monoclonal, multi-chain or single-chain, or whole immunoglobulins, and can be derived from natural sources or recombinant sources.
  • the antibody may 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, through 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 composed of VH and CH1 domains, linear antibodies , Single domain antibodies (such as sdAb), multispecific antibodies formed by antibody fragments (such as bivalent fragments including two Fab fragments connected by disulfide bonds in the hinge region) and the isolated CDR or other epitope binding of the antibody Fragment.
  • scFv refers to a fusion protein comprising at least one variable region antibody fragment including a light chain and at least one antibody fragment including a heavy chain variable region, wherein the light chain 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.
  • the scFv may have the VL and VH variable regions in any order (for example, relative to the N-terminus and C-terminus of the polypeptide), and the scFv may include VL-linker-VH or May include VH-Linker-VL.
  • antibody heavy chain refers to the larger of the two polypeptide chains present in the antibody molecule in its naturally occurring configuration and usually determines the type of antibody to which it belongs.
  • antibody light chain refers to the smaller of the two polypeptide chains present in the antibody molecule in its naturally occurring configuration. Kappa(k) and lambda(l) light chains refer to the two main isotypes of antibody light chains.
  • recombinant antibody refers to an antibody produced using recombinant DNA technology, such as, for example, an antibody expressed by a phage or yeast expression system.
  • the term should also be interpreted as referring to antibodies that have been produced by synthesizing a DNA molecule encoding the antibody (and wherein the DNA molecule expresses the antibody protein) or the amino acid sequence of the specified antibody, wherein the DNA or amino acid sequence has been recombinant DNA or is available in the art And the well-known amino acid sequence technology is obtained.
  • antigen refers to a molecule that causes an immune response.
  • the immune response may involve the production of antibodies or the activation of cells with specific immunity, or both.
  • any macromolecule including virtually all proteins or peptides can serve as an antigen.
  • the antigen can be derived from recombinant or genomic DNA.
  • a protein or peptide encoded by any DNA that includes a nucleotide sequence or a partial nucleotide sequence of a protein that causes an immune response is included.
  • the antigen need not be encoded only by the full-length nucleotide sequence of the gene.
  • the present 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 polypeptides that elicit a desired immune response.
  • the antigen may not need to be encoded by a "gene”. It is obvious that the antigen can be produced synthetically, or it can be derived from a biological sample, or it can be a macromolecule other than a polypeptide.
  • biological samples may include, but are not limited to tissue samples, tumor samples, cells or fluids with other biological components.
  • tumor antigen refers to an antigen that is newly emerged or overexpressed during the occurrence and development of hyperproliferative diseases.
  • the hyperproliferative disorder of the present invention refers to cancer.
  • the tumor antigens of the present invention can be solid tumor antigens or hematoma antigens.
  • the tumor antigens of the present invention include but are not limited to: Thyroid Stimulating Hormone Receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; Ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3(CD276), B7H6; KIT(CD117); Interleukin 13 receptor subunit ⁇ (IL-13R ⁇ ); Interleukin 11 Receptor ⁇ (IL-11R ⁇ ); Prostate Stem Cell Antigen (PSCA); Prostate Specific Membrane Antigen (PSMA); Carcinoembryonic Antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; Mesothelin; EpCAM; Protease Serine 21 (PRSS21); Vascular Endothelial Growth Factor Receptor, Vascular Endo
  • the pathogen antigen is selected from: virus, bacteria, fungus, protozoa, or parasite antigen; virus antigen is selected from: cytomegalovirus antigen, Epstein-Barr virus antigen, human immunodeficiency virus antigen, or influenza virus antigen.
  • tumor refers to a broad category of disorders in which hyperproliferative cell growth occurs in vitro (e.g., transformed cells) or in vivo.
  • Conditions that can be treated or prevented by the method of the present invention include, for example, various neoplasms, including benign or malignant tumors, various hyperplasias, and the like.
  • Tumors include but are not limited to: breast cancer, prostate cancer, leukemia, lymphoma, nasopharyngeal cancer, glioma, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small bowel cancer, esophageal cancer, melanin Tumor, 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, vagina cancer, thyroid cancer, parathyroid cancer, adrenal cancer, Soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumor, hemangioma, spinal tumor, glioma, astrocytoma, pituitary adenoma Describe the metastatic lesions of cancer.
  • CNS central nervous system
  • transfected or “transformed” or “transduced” refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell.
  • a “transfected” or “transformed” or “transduced” cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid.
  • the cells include primary subject cells and their progeny.
  • the term "specifically binds” means that the antibody or ligand binds to a binding partner (e.g., tumor antigen) present in the sample, but does not substantially recognize or bind to other molecules in the sample.
  • a binding partner e.g., tumor antigen
  • Refractory refers to a disease, such as a tumor, which does not respond to treatment.
  • the refractory tumor may be resistant to the treatment before or at the beginning of the treatment.
  • a refractory tumor may be a tumor that is resistant to the treatment during treatment.
  • refractory tumors include but are not limited to tumors that are not sensitive to radiotherapy, relapse after radiotherapy, are not sensitive to chemotherapy, relapse after chemotherapy, are not sensitive to CAR-T treatment, or relapse after treatment.
  • Refractory or recurrent malignant tumors can use the treatment regimens described herein.
  • relapsed refers to a period of improvement, for example, after an effective tumor treatment, the patient reappears with the signs and symptoms before the effective treatment.
  • an enhanced response refers to allowing a subject or tumor cell to improve its ability to respond to the treatment disclosed herein.
  • an enhanced response may include 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% of the responsiveness. %, 75%, 80%, 85%, 90%, 95% or 98% or more.
  • "enhancing" can also refer to increasing the number of subjects responding to treatment, such as immune effector cell therapy.
  • an enhanced response can refer to the total percentage of subjects responding to treatment, where the percentages are 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 based on clinical results; through the increase, enhancement or extension of the anti-tumor activity of T cells; compared with the number before treatment, the increase in the number of anti-tumor T cells or activated T cells promotes the secretion of IFN- ⁇ , or Combination to decide.
  • 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 genetic markers indicative of T cell inflammation, promotion of IFN- ⁇ secretion, or a combination thereof.
  • the immune effector cells as disclosed herein can be administered to individuals by various routes, including, for example, orally or parenterally, such as intravenous, intramuscular, subcutaneous, intraorbital, intrasaccular, intraperitoneal, intrarectal, intracisternal, intratumoral Passive or promoted absorption through the skin using, for example, skin patches or transdermal iontophoresis, intranasally, intradermally, or respectively.
  • routes including, for example, orally or parenterally, such as intravenous, intramuscular, subcutaneous, intraorbital, intrasaccular, intraperitoneal, intrarectal, intracisternal, intratumoral Passive or promoted absorption through the skin using, for example, skin patches or transdermal iontophoresis, intranasally, intradermally, or respectively.
  • the total amount of agent to be administered in practicing the method of the present invention can be administered to the subject as a single dose as a bolus or by infusion over a relatively short period of time, or can be administered using a graded treatment regimen, wherein Multiple doses are administered in segments.
  • a graded treatment regimen wherein Multiple doses are administered in segments.
  • the amount of the composition to treat a pathological condition in a subject depends on many factors, including the age and general health of the subject, as well as the route of administration and the number of treatments to be administered. Taking these factors into account, the technician will adjust the specific dosage as needed. In general, initially, phase I and phase II clinical trials are used to determine the formulation of the composition and the route and frequency of administration.
  • Range Throughout this disclosure, various aspects of the present invention may be presented in a range format. It should be understood that the description in range format is only for convenience and brevity, and should not be regarded as an unchangeable limitation on the scope of the present invention. Therefore, the description of a range should be considered as specifically disclosing all possible subranges and individual values within that range. For example, a description of a range such as from 1 to 6 should be considered to specifically disclose subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and within the range Individual values for, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6.
  • a range such as 95-99% identity includes a range with 95%, 96%, 97%, 98%, or 99% identity, and includes sub-ranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98% and 98-99% identity. This applies regardless of the width of the range.
  • GPC3 or “Glypican 3” is a member of the Glypican family, with gene registration numbers: NM_016697.3, NP_057906.2, which play an important role in regulating cell growth and differentiation.
  • Abnormal expression of GPC3 is closely related to the occurrence and development of a variety of tumors, such as liver cancer, lung cancer, breast cancer, ovarian cancer, kidney cancer, thyroid cancer, gastric cancer, colorectal cancer, and so on.
  • GPC3 includes any post-translational modified variants, isoforms, and interspecies homologs of GPC3 that are naturally expressed by cells or expressed by cells transfected with the GPC3 gene.
  • GPC3 variant shall include (i) GPC3 splice variants, (ii) GPC3 post-translational modification variants, especially including variants with different N glycosylation states, (iii) GPC3 conformational variants, (iv) GPC3 and homotypic/allo-associated variants located on the cell surface, (v) GPC3 cancer-related variants and GPC3 non-cancer-related variants.
  • CAR-T co-expressing IL-15 targets GPC3-positive tumors.
  • the tumor includes, but is not limited to, liver cancer, stomach cancer, lung cancer, esophageal cancer, head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, kidney cancer, pancreatic cancer, cervical cancer, liposarcoma, melanoma, Adrenal gland cancer, schwannoma, malignant fibrous histiocytoma, esophageal cancer.
  • the GPC3-positive tumors or GPC-positive tumors described herein are liver cancer, gastric cancer, lung cancer, and esophageal cancer.
  • the corresponding species can be selected.
  • murine-derived IL-15 is used to construct CAR elements such as transmembrane domain
  • the intracellular domain can also be of murine origin.
  • human-derived IL-15 and human-derived CAR elements are preferred.
  • the sequence of the CAR used may be as shown in SEQ ID NO: 13, 14, or 15.
  • the cells of the present invention can be used in combination with chemotherapeutics when used for tumor treatment.
  • chimeric antigen receptor polypeptides of the present invention can be selected from sequentially linked as follows:
  • 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 present invention also includes nucleic acid encoding the chimeric antigen receptor.
  • the present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides having the same amino acid sequence as the present invention, or polypeptide fragments, analogs and derivatives.
  • the present invention also provides a vector containing the nucleic acid of the chimeric antigen receptor.
  • the present invention also includes viruses containing the above-mentioned vectors.
  • the virus of the present invention includes a packaged virus that is infectious, and also includes a virus to be packaged that contains the necessary components for packaging as a virus that is infectious.
  • Other viruses known in the art that can be used to transfer 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, which is transduced with a nucleic acid encoding the chimeric antigen receptor or is transduced with the aforementioned recombinant plasmid containing the nucleic acid, or contains the plasmid Virus.
  • Conventional nucleic acid transduction methods in the art 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 instrument developed by Amaxa recently can directly introduce foreign genes into the nucleus to obtain efficient transduction of target genes.
  • transduction efficiency of transposon systems based on Sleeping Beauty system or PiggyBac transposon is much higher than that of ordinary electroporation, and combined application of nucleofector transfection instrument and Sleeping Beauty transposon system It has been reported [Davies JK., et al. Combining CD19 redirection and alloanergization to generate tumor-specific human cells for alllogeneic cell therapy of B-cell malignancies. Cancer Res, 2010, 70(10): OF1-10.], This method not only has high transduction efficiency but also can realize the targeted integration of the target gene.
  • the method for transduction of immune effector cells modified by chimeric antigen receptor genes is based on the method for transduction of viruses such as retroviruses or lentiviruses.
  • the method has the advantages of high transduction efficiency, stable expression of foreign genes, and shortening the time for culturing immune effector cells in vitro to reach clinical level.
  • the transduced nucleic acid is expressed on the surface through transcription and translation.
  • the immune effector cells modified by the chimeric antigen of the present invention have a highly specific tumor cell killing effect (also known as cytotoxicity), and can be found 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 containing the plasmid, and the transgenic immune effector cells transduced with the nucleic acid, plasmid or virus of the present invention can be effectively used for tumor immunotherapy.
  • the chimeric antigen-modified immune effector cells of the present invention can also express another chimeric receptor besides the above-mentioned chimeric receptor, which does not contain CD3 ⁇ , but contains the intracellular signal domain of CD28, CD137 The intracellular signal domain or a combination of the two.
  • the immune effector cells modified by the chimeric antigen receptor of the present invention can be applied to the preparation of pharmaceutical compositions or diagnostic reagents.
  • the composition may also include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable means that when the molecular entities and compositions are properly administered to animals or humans, they will not produce adverse, allergic or other adverse reactions.
  • sugars such as lactose, glucose, and sucrose
  • starches such as corn starch and potato starch
  • cellulose and its derivatives such as carboxymethyl fiber Sodium, ethyl cellulose and methyl cellulose
  • tragacanth powder malt
  • gelatin talc
  • solid lubricants such as stearic acid and magnesium stearate
  • calcium sulfate vegetable oils, such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil and cocoa butter
  • polyols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol
  • coloring agents such as sodium lauryl sulfate
  • flavoring agents such as sodium lauryl sulfate
  • coloring agents such as sodium lauryl sulfate
  • flavoring agents such as sodium lauryl
  • composition of the present invention can be made into various dosage forms according to needs, and the doctor can determine the beneficial dosage for the patient according to factors such as the patient's type, age, weight, general disease condition, and administration method.
  • the mode of administration can be injection or other treatment methods.
  • the immune effector cells provided herein have a more excellent killing effect on solid tumor cells.
  • Exemplary antigen receptors of the present invention including CARs, and methods for engineering and introducing receptors into cells, refer to, for example, Chinese Patent Application Publication Nos. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A , CN105331585A, CN106397593A, CN106467573A, CN104140974A, CN 108884459 A, CN107893052A, CN108866003A, CN108853144A, CN109385403A, CN109385400A, CN109468279A, CN109503715A, CN 109908176 A, CN109880837A, CN 11055275A, CN 110082 A public patent Nos.
  • WO2017186121A1 WO2018006882A1, WO2015172339A8, WO2018/018958A1, WO2014180306A1, WO2015197016A1, WO2016008405A1, WO2016086813A1, WO2016150400A1, WO2017032293A1, WO2017080377A1, WO2017186121A1, WO2018045811A1, WO2018/219279/WO/2019 114762, WO2019/141270, WO2019/149279, WO2019/170147A1, WO 2019/210863, WO2019/219029.
  • the scFv used in this example is a targeting GPC3 antibody.
  • the amino acid sequence is shown in SEQ ID NO: 12, and the nucleic acid sequence is shown in SEQ ID NO: 16.
  • the body is a second-generation chimeric antigen receptor.
  • the nucleic acid sequence of GPC3-BBZ includes the CD8 ⁇ signal peptide (SEQ ID NO: 20), scFv (SEQ ID NO: 16), the hinge region of CD8 (SEQ ID NO: 21), and the transmembrane domain of CD8 (the nucleic acid sequence is as SEQ ID NO: 17), CD137 intracellular signaling domain (nucleic acid sequence is shown in SEQ ID NO: 18), and CD3 intracellular segment CD3 ⁇ signal domain (nucleic acid sequence is shown in SEQ ID NO: 19).
  • NFAT-IL15 Insert the NFAT-IL15 gene on the basis of pRRLSIN-GPC3-BBZ plasmid to construct the lentiviral plasmid pRRLSIN-GPC3-BBZ-IL15 expressing CAR and NFAT-regulated IL15 (plasmid map is shown in Figure 1).
  • the gene of NFAT-IL15 includes NFAT6 binding motif (see SEQ ID NO: 23 for nucleic acid sequence), IL2 minimal promoter (see SEQ ID NO: 25 for nucleic acid sequence), and human IL15 (see SEQ ID NO: 35 for nucleic acid sequence) .
  • CCL21-NFAT-IL15 Insert the CCL21-NFAT-IL15 gene on the basis of pRRLSIN-GPC3-BBZ plasmid to construct a lentiviral plasmid pRRLSIN-GPC3-BBZ-CCL21*15 expressing CAR, IL15 and CCL21 (see Figure 1).
  • the genes of CCL21-NFAT-IL15 include CCL21 (see SEQ ID NO: 24 for nucleic acid sequence), NFAT6 binding motif (see SEQ ID NO: 23 for nucleic acid sequence), and IL2 minimal promoter (see SEQ ID NO: 25 for nucleic acid sequence) And human IL15 (see SEQ ID NO: 35 for the nucleic acid sequence).
  • pRRLSIN-GPC3-BBZ Isolate human peripheral blood PBMC cells, after culture and activation, the resulting lentiviruses pRRLSIN-GPC3-BBZ, pRRLSIN-GPC3-BBZ-IL15, and pRRLSIN-GPC3-BBZ-CCL21*15 were respectively infected with T cells to obtain GPC3 -BBZ cells, GPC3-BBZ-IL15 cells, and GPC3-BBZ-CCL21*15 cells. After flow cytometry, GPC3-BBZ cells, GPC3-BBZ-IL15 cells, and GPC3-BBZ-CCL21*15 positive cells were screened out.
  • CytoTox 96 non-radioactive cytotoxicity detection kit (Promega) was used. For specific methods, refer to the CytoTox 96 non-radioactive cytotoxicity detection kit instructions.
  • Effector cells Inoculate UTD cells (untransduced T cells), GPC3-BBZ cells, and GPC3-BBZ-IL15 cells in a 96-well plate according to the effective target ratio of 3:1, 1:1 or 1:3.
  • Target cells Inoculate 50 ⁇ L of 1 ⁇ 10 5 /mL Huh-7 cells (RIKEN cell bank in Japan), PLC/PRF/5 cells (ATCC in the United States), and SK-HEP-1 cells (ATCC in the United States) into the corresponding 96 wells. board.
  • Each group is equipped with 5 multiple wells, and the culture plate is placed in a cell culture incubator and incubated for 18 hours.
  • Each experimental group and each control group are set as follows: experimental group: each target cell + different CAR-T cells; control group 1: target cell maximum release of LDH; control group 2: target cell spontaneous release of LDH; control group 3: effect Cells release LDH spontaneously.
  • Fig. 2 The experimental results are shown in Fig. 2.
  • the effective target ratio of Huh7 cells with positive GPC3 expression is 1:1 or 1:3, GPC3-BBZ-IL15 cells show better killing than GPC3-BBZ cells; in GPC3
  • the positive target ratio of PLC/PRF/5 cells is 3:1, 1:1 or 1:3, GPC3-BBZ-IL15 cells show better killing than GPC3-BBZ cells; GPC3-BBZ And GPC3-BBZ-IL15 cells were not detected to kill SK-HEP-1, which was negative for GPC3 expression.
  • Tumor inoculation 3 ⁇ 10 6 liver cancer cells PLC/PRF/5 were respectively inoculated into the right axilla of NPG mice. Divided into 3 groups with 6 animals in each group. The vaccination diary is D0.
  • CAR T injection D13 days after subcutaneous inoculation of tumor tissue, the average tumor volume is about 300mm 3 .
  • the CAR T cells prepared in Example 1 were injected, and the injection dose was 2.0 ⁇ 10 6 /head.
  • the tumor suppression results are shown in Figure 3. 19 days after CAR T injection (D32), compared with the UTD group, the tumor suppression rates in the GPC3-BBZ and GPC3-BBZ-IL15 groups were 29.22% and 71.26%, respectively (P ⁇ 0.05) , GPC3-BBZ-IL15 cells showed better killing than GPC3-BBZ cells.
  • mice The weight change of the mice was weighed, and the results are shown in Fig. 4, which showed that GPC3-BBZ-IL15 cells had no significant effect on the weight of the mice.
  • Example 2 Refer to the operation of Example 2 to detect the cell killing of PLC/PRF/5 cells and SK-HEP-1 cells by GPC3-BBZ-IL15 cells and GPC3-BBZ-CCL21*15 cells.
  • the results are shown in Figure 5, showing GPC3 -BBZ-CCL21*15 showed more excellent cell killing.
  • GPC3-BBZ-IL15 and GPC3-BBZ-CCL21*15 cells were not detected to kill SK-HEP-1, which was negative for GPC3 expression.
  • mouse CD8 ⁇ signal peptide (SEQ ID NO: 27), anti-GPC3 monoclonal antibody (SEQ ID NO: 16), mouse CD8 ⁇ gel chain region and transmembrane region (SEQ ID NO: 28)
  • SEQ ID NO: 29 mouse CD8 ⁇ signal peptide
  • SEQ ID NO: 30 mouse CD8 ⁇ gel chain region and transmembrane region
  • the mNFAT6-mIL15 sequence was inserted on the basis of the MSCV-GPC3-mBBZ CAR-T plasmid, and a plasmid expressing the second-generation chimeric antigen receptor of GPC3 and mIL15, MSCV-GPC3-mBBZ-mNFAT6-mIL15 was constructed.
  • the mNFAT6-mIL15 sequence is composed of mNFAT6 binding motif (SEQ ID NO: 31), mIL2 minimal promoter (SEQ ID NO: 32), mIL15 (SEQ ID NO: 33), PA2 (SEQ ID NO: 34) ( Figure 6) Shown).
  • Example 6 In vitro killing toxicity test and in vitro cytokine secretion test
  • CytoTox 96 non-radioactive cytotoxicity detection kit (Promega) was used. For specific methods, refer to the CytoTox 96 non-radioactive cytotoxicity detection kit instructions.
  • human-derived mouse GPC3 epitopes were transferred into mouse liver cancer cells Hepa 1-6 (Cell Collection Center, Chinese Academy of Sciences (Shanghai)) to construct Hepa 1-6 GPC3 overexpressing GPC3 protein.
  • mice hepatocarcinoma cells Hepa 1-6 and Hepa 1-6 GPC3 cells in 96-well plates, according to the effective target ratio of 3:1, 1:1, 1:3, and co-culture with effector cells for 18h detection.
  • the effector cells are UTD respectively.
  • the ELISA method detected the secretion of IFN- ⁇ , IL2 and TNF- ⁇ of effector cells after stimulation of target cells.
  • GPC3-mBBZ CAR T cells and GPC3-mBBZ-mNFAT6-mIL15 CAR T cells can effectively secrete tumor-killing cytokines IFN- ⁇ , IL2 and TNF when stimulated by Hepa1-6-GPC3 cells - ⁇ , and compared with GPC3-mBBZ CAR T cells, GPC3-mBBZ-mNFAT6-mIL15 CAR T cells have stronger IL2 and TNF- ⁇ secretion ability (P ⁇ 0.001), and the secretion level of IFN- ⁇ is not statistically significant Difference (P>0.05).
  • Example 7 The therapeutic effect of GPC3-mBBZ-mNFAT6-mIL15 CAR T on Hepa 1-6 GPC3 subcutaneous transplantation tumor
  • the average tumor volume was about 300mm 3 .
  • 1.5 ⁇ 10 6 CAR T cells were injected into the tail vein.
  • the GPC3-mBBZ CAR-T and GPC3-mBBZ-mNFAT6-mIL15 CAR T treatment groups can effectively inhibit tumor growth, and the GPC3-mBBZ-mNFAT6-mIL15 CAR T cell resistance
  • the tumor effect is stronger than that of GPC3-Mbbz CAR-T cells (P ⁇ 0.001).
  • the body weight change curve of mice is shown in Figure 8B. The body weight of each group of mice did not decrease significantly, indicating that GPC3-mBBZ-mNFAT6-mIL15 CAR T treatment has no serious toxic and side effects.
  • the tumor weight in the CAR T treatment group was significantly lower than that of the GPC3- mBBZ CAR T treatment group (P ⁇ 0.05).
  • the tumor inhibition rates of GPC3-mBBZ-mNFAT6-mIL15 CAR T group and GPC3-mBBZ CAR T group were 98.1% and 70.7%, respectively.
  • the T cells can also express other cytokines that enhance the function of CAR-T cells, such as CAR-T cells co-expressed by CAR and type I interferon , CAR-T cells co-expressed with CAR and PD-1, etc.
  • CAR-T cells are used in the above embodiments, other immune cells, such as NK cells, NK-T cells, and specific subtypes of immune cells, such as ⁇ / ⁇ T cells, can also be selected. Wait.

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Abstract

表达IL-15的免疫效应细胞，所述细胞表达IL-15及识别特异性靶抗原的受体、或所述细胞表达IL-15R结合分子及识别特异性靶抗原的受体。核酸分子，所述核酸分子表达的IL-15R结合分子或IL-15或IL-15与趋化因子、趋化因子受体、除IL-7之外的其他细胞因子、降低PD-1表达的siRNA、阻断PD-L1与PD-1结合的蛋白、或安全开关。

Description

表达IL-15的免疫效应细胞 技术领域
本发明属于细胞治疗领域，涉及表达IL-15的免疫效应细胞，具体涉及一种靶向GPC3的共表达IL-15和识别特异性识别靶抗原的受体的免疫效应细胞。
背景技术
近年来，根据CTL对靶细胞的识别特异性依赖于T淋巴细胞受体(T Cell Receptor，TCR)的发现，将针对肿瘤细胞相关抗原的抗体的scFv与T淋巴细胞受体的CD3ζ或FcεRIγ等胞内信号激活基序融合成嵌合抗原受体(Chimeric antigen receptor，CAR)，并将其通过如慢病毒感染等方式基因修饰在T淋巴细胞表面。这种CAR T淋巴细胞能够以主要组织相容性复合物(Major Histocompatibility Complex，MHC)非限制性方式选择性地将T淋巴细胞定向到肿瘤细胞并特异性地杀伤肿瘤。
嵌合抗原受体包括胞外结合域，跨膜域和胞内信号域。如胞外域包含能够识别肿瘤相关抗原的scFv，跨膜域采用CD8，CD28等分子的跨膜区，胞内信号域采用免疫受体酪氨酸活化基序(ITAM)CD3ζ或FcεRIγ及共刺激信号分子CD28、CD27、CD137、CD134等的胞内信号域。
然而，由于生物体，特别是实体瘤的微环境的复杂性，尽管免疫效应细胞在血液瘤治疗中展现出诱人的应用前景，但对于多种实体瘤肿瘤仍然没有较好的治疗效果，如对于一些肿瘤，免疫效应细胞在肿瘤组织内的存活率较差、肿瘤杀伤活性不高等。因此，如何提升免疫效应细胞如CAR T细胞对实体瘤特别是一些难治性实体瘤治疗的有效应，是众多研究人员面临的巨大难题。
发明内容
在本发明的第一方面，涉及一种表达IL-15的免疫效应细胞，其特征在于，所述细胞表达IL-15及识别特异性靶抗原的受体、或所述细胞表达IL-15R结合分子 及识别特异性靶抗原的受体。
在一个具体的实施方式中，所述IL-15为外源性IL-15。
在另一个具体的实施方式中，所述的IL-15为组成型表达或诱导型表达的IL-15。
在另一个具体的实施方式中，所述IL-15是诱导型表达IL-15，其表达受所述受体调控。
在另一个具体的实施方式中，当所述受体识别所述靶抗原后能够启动所述IL-15的表达。
在另一个具体的实施方式中，所述受体通过诱导型启动子诱导所述IL-15的表达。
在另一个具体的实施方式中，所述免疫效应细胞还表达趋化因子、趋化因子受体、除IL-15之外的其他细胞因子、降低PD-1表达的siRNA、阻断PD-L1与PD-1结合的蛋白、或安全开关。
在另一个具体的实施方式中，所述趋化因子为淋巴细胞趋化因子；
优选的，所述的淋巴细胞趋化因子为CCL21；
优选地，所述CCL21与SEQ ID NO:26或SEQ ID NO:39、40所示的氨基酸序列具有至少90％同一性或是SEQ ID NO:26或SEQ ID NO:39、40所示的氨基酸序列的截短片段；或者与由SEQ ID NO:24或SEQ ID NO:37、38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:24或SEQ ID NO:37、38所示的核苷酸编码的氨基酸序列的截短片段。
在另一个具体的实施方式中，所述趋化因子受体选自CCR2、CCR5、CXCR2、或CXCR4。
在另一个具体的实施方式中，所述其他细胞因子选自IL-21、IL18、或I型干扰素。
在另一个具体的实施方式中，所述阻断PD-L1与PD-1结合的蛋白选自PD-L1的抗体、PD-1的抗体、天然的PD-1或者天然PD-1的截短片段、含有天然的PD-1或者天然PD-1的截短片段的融合肽。
在另一个具体的实施方式中，所述的安全开关选自iCaspase-9、Truancated  EGFR、RQR8、对免疫效应细胞有杀伤作用的蛋白。
在另一个具体的实施方式中，所述的免疫效应细胞选自T细胞、NK细胞、NKT细胞、肥大细胞、巨噬细胞、树突细胞、CIK细胞、或干细胞衍生的免疫效应细胞。
在另一个具体的实施方式中，所述诱导型启动子包含转录因子的结合基序和最小启动子，所述诱导型启动子的活化依赖所述受体的活化或依赖于所述受体与所述靶抗原结合。
在另一个具体的实施方式中，所述结合基序包括NFAT、NF-κB或AP-1结合基序，或者NFAT、NF-κB、AP-1结合基序的至少两种的组合，
优选的，所述结合基序为NFAT结合基序。
在另一个具体的实施方式中，所述结合基序包含1-12个NFAT结合基序、1-12个NF-κB结合基序、1-12个AP-1结合基序，或1-12个NFAT、NF-κB、AP-1结合基序中至少两种的组合；
优选的，所述结合基序包含1-6个NFAT结合基序、1-6个NF-κB结合基序、1-6个AP-1结合基序，或1-6个NFAT、NF-κB、AP-1结合基序中至少两种的组合。
在另一个具体的实施方式中，所述NFAT结合基序的序列如SEQ ID NO：23所示。
在另一个具体的实施方式中，所述最小启动子为细胞因子最小启动子。
在另一个具体的实施方式中，所述最小启动子包括白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子、生长因子最小启动子；
优选的，为IFN-γ、TNF-α或IL-2最小启动子；
更优选，为IL-2最小启动子。
在另一个具体的实施方式中，所述IL-2最小启动子的序列如SEQ ID NO：25所示。
在另一个具体的实施方式中，所述IL-15为天然IL-15、或与天然IL-15具有相同功能的天然IL-15的截短片段或天然IL-15的突变体；
优选地，所述天然IL-15与SEQ ID NO:1或SEQ ID NO:36所示的氨基酸 序列具有至少90％同一性、或是SEQ ID NO:1或SEQ ID NO:36所示的氨基酸序列的截短片段；或者与由SEQ ID NO:33或SEQ ID NO:35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:33或SEQ ID NO:35所示的核苷酸编码的氨基酸序列的截短片段。
在另一个具体的实施方式中，所述趋化因子、趋化因子受体、除IL-15之外的其他细胞因子、降低PD-1表达的siRNA、阻断PD-L1与PD-1结合的蛋白、或安全开关是组成型表达或诱导型表达；优选的，为组成型表达。
在另一个具体的实施方式中，所述靶抗原为肿瘤抗原为肿瘤抗原。
优选地，所述实体瘤抗原为GPC3、EGFR、EGFRvIII、间皮素或Claudin18.2。
在另一个具体的实施方式中，所述受体选自嵌合抗原受体(CAR)、T细胞受体(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，所述受体为嵌合抗原受体；
更优选地，所述受体为嵌合抗原受体，包括
(i)抗原结合结构域、CD28或CD8的跨膜域、CD28的共刺激信号结构域和CD3ζ的胞内信号域；或
(ii)抗原结合结构域、CD28或CD8的跨膜域、4-1BB的共刺激信号结构域和CD3ζ的胞内信号域；或
(iii)抗原结合结构域、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的胞内信号域。
在另一个具体的实施方式中，所述嵌合抗原受体(CAR)、T细胞融合蛋白(TFP)、或T细胞抗原耦合器(TAC)的抗原结合结构域为抗体，具有SEQ ID NO：2所示的HCDR1，SEQ ID NO：3所示的HCDR2，SEQ ID NO：4所示的HCDR3，和SEQ ID NO：5所示的LCDR1，SEQ ID NO：6所示的LCDR2，SEQ ID NO：7所示的LCDR3；或
所述抗体具有SEQ ID NO:8所示轻链可变区和SEQ ID NO:9所示的重链可变区；或具有SEQ ID NO:10所示轻链可变区和SEQ ID NO:11所示的重链可变区；或
所述抗体具有SEQ ID NO：12所示的氨基酸序列。
在另一个具体的实施方式中，所述受体的氨基酸序列与SEQ ID NO:13、14、15所示的氨基酸序列具有至少90％的同一性。
在另一个具体的实施方式中，所述的免疫细胞诱导型启动子诱导表达IL-15的核酸序列如SEQ ID NO：22所示。
在另一个具体的实施方式中，所述受体与IL-15由同一核酸分子表达，或由不同核酸分子表达；
[根据细则91更正 25.02.2021]　
优选，所述受体与IL-15由同一核酸分子表达，所述IL-15的表达盒与受体之间、以及表达盒之间直接连接或由串联片段连接，所述串联片段选自F2A、P2A、T2A、和/或E2A。
在本发明的第二方面，涉及一种核酸分子，所述核酸分子表达上述本发明的IL-15R结合分子或IL-15或IL-15与趋化因子、趋化因子受体、除IL-7之外的其他细胞因子、降低PD-1表达的siRNA、阻断PD-L1与PD-1结合的蛋白、或安全开关；所述核酸分子还表达上述本发明的特异性识别靶抗原的受体；
优选所述核酸由DNA和/或RNA组成；或所述核酸是mRNA；或所述核酸包含核苷酸类似物。
在本发明的第三方面，涉及一种载体，包含上述核酸分子；
优选所述载体选自由以下组成的组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、劳氏肉瘤病毒(RSV)载体或逆转录病毒载体。
在本发明的第四方面，涉及一种细胞，其包含上述本发明的载体或其基因组上整合有上述本发明的核酸分子；
优选所述细胞是人T细胞，优选地，为同种异体T细胞。
在本发明的第五方面，涉及一种制备细胞的方法，所述方法包括用本发明所述的载体或本发明所述的核酸分子转导T细胞。
在本发明的第六方面，涉及一种产生RNA工程改造的细胞群的方法，所述方法包括将体外转录的RNA或合成RNA引入细胞中，(i)其中所述RNA包括上述本发明的核酸。
在本发明的第七方面，涉及一种在哺乳动物体内提供抗肿瘤免疫性的方法，所述方法包括向所述哺乳动物施用有效量的上述本发明的细胞、本发明的核酸分子、或本发明的载体；
优选所述哺乳动物是人。
在本发明的第八方面，涉及一种治疗患有与GPC3或者claudin18.2表达有关的疾病的哺乳动物的方法，所述方法包括向所述哺乳动物施用有效量的上述本发明的细胞、上述本发明的核酸分子、或上述本发明的载体；
优选所述与GPC3或者claudin18.2表达有关的疾病选自结肠癌，直肠癌，肾细胞癌，肝癌，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，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，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、与GPC3或者claudin18.2表达有关的非癌症相关适应症；优选的，选自肝癌、肺癌、乳腺癌、卵巢癌、肾癌、甲状腺癌、胃癌、结直肠癌、胰腺癌、食道癌；
优选上述本发明的细胞和增加上述本发明的细胞的功效的药剂组合施用，优选地，与化疗药物联用；
优选上述本发明的细胞与改善与施用上述本发明的细胞相关的一种或多种副作用的药剂联合施用；
优选上述本发明的细胞和治疗所述与GPC3或者claudin18.2相关的疾病的药剂组合施用，优选地，与化疗药物联用。
在本发明的第九方面，涉及上述本发明的细胞、上述本发明的核酸分子、上述本发明的载体的用途，用作药物，优选地，用作抑制肿瘤或抑制病原体的药物，进一步优选，所述用途为用于制备用于抑制肿瘤或抑制病原体的药物的用途。
在本发明的第十方面，涉及一种药物组合物，其特征在于，所述的药物组合物包括上述本发明的细胞和药学上可接受的载体或赋形剂。
附图说明
图1显示了pRRLSIN-GPC3-BBZ、pRRLSIN-GPC3-BBZ-IL15、pRRLSIN-GPC3-BBZ-CCL21*15质粒简图；
图2显示了GPC3-BBZ-IL15 CAR-T细胞的体外杀伤效果；
图3显示了GPC3-BBZ-IL15 CAR-T细胞的体内肿瘤抑制结果；
图4显示了GPC3-BBZ-IL15 CAR-T细胞的体内肿瘤抑制实验的小鼠体重变化情况；
图5显示了GPC3-BBZ-CCL21*IL15 CAR-T细胞的体外杀伤效果；
图6显示了MSCV-GPC3-mBBZ；MSCV-GPC3-mBBZ-mNFAT6-mIL15质粒简图；
图7A显示了MSCV-GPC3-mBBZ CAR-T、MSCV-GPC3-mBBZ-mNFAT6-mIL15 CAR-T细胞对肿瘤细胞的体外杀伤；图7B显示MSCV-GPC3-mBBZ CAR-T、MSCV-GPC3-mBBZ-mNFAT6-mIL15 CAR-T细胞的细胞因子分泌情况；
图8A-C分别显示了MSCV-GPC3-mBBZ CAR-T、MSCV-GPC3-mBBZ-mNFAT6-mIL15 CAR-T细胞的体内肿瘤抑制结果、对小鼠体重的影响、肿瘤重量比较。
具体实施方式
本发明发现，表达IL-15及识别特异性识别靶抗原的受体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”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诱导表达外源性的IL15的免疫效应细胞。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、诱导表达外源性的IL15的免疫效应细胞。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、诱导表达外源性的IL15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、表达外源性的CLL21、诱导表达外源性的IL15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、及诱导表达IL-15R结合蛋白或外源性的IL-15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、巨噬细胞、树突细胞、CIK细胞、或干细胞衍生的免疫效应细胞。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。
IL15(Interleukin15，白细胞介素15或IL15或IL-15)(细胞因子白介素-15)是由体内许多细胞产生的四α-螺旋束淋巴因子家族的成员。IL-15在调节先天和适应性免疫系统的活性方面发挥关键作用，例如维持记忆T细胞对入侵病原体的应答、抑制细胞凋亡、活化树突状细胞以及诱导自然杀伤(NK)细胞的增殖和细胞毒活性。IL15可以与IL-15R(优选来自哺乳动物，例如鼠或人IL-15R，相互作用(例如结合)，优选来自哺乳动物，例如鼠或人。IL15也可以通过非IL-15R通路起抗肿瘤作用。
IL15具有以下特征之一：(i)为天然存在的哺乳动物IL-15的氨基酸序列或其片段，例如SEQIDNO：1(人)或SEQIDNO：36(鼠)所示的氨基酸序列或其片段；(ii)基本上与SEQIDNO：1(人)或SEQIDNO：36(鼠)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-15核苷酸序列或其片段，例如SEQ ID NO:35(人)或其片段所编码的氨基酸序列、或SEQIDNO：33(鼠)或其片段所编码的氨基酸序列；(iv)由与SEQ ID NO:35(人)或SEQIDNO：33(鼠)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SEQIDNO：35(人)或其片段、SEQIDNO：33(鼠)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”包括在细胞内表达的重组核酸分子或多肽，如外源性、异源性和过表达的核酸分子和多肽。在本文中，外源性IL-15是在细胞内表达的IL-15重组核酸分子或IL-15，如外源性、异源性和过表达的编码IL-15的核酸分子和IL-15。“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:26(人)、或SEQIDNO：39或40(鼠)所示的氨基酸序列或其片段；(ii)为与 SEQ ID NO:26(人)、或SEQIDNO：39或40(鼠)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:24(人)或其片段所编码的氨基酸序列、SEQ ID NO:37或38(鼠)或其片段所编码的氨基酸序列；(iv)由与SEQ ID NO:24(人)、SEQ ID NO:37或38(鼠)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:24(人)或其片段、SEQ ID NO:37或38(鼠)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。
“外源性IL-15R结合分子或结合蛋白”是指能特异性结合IL-15R且增强IL-15R活性的所有分子或蛋白质。“增强IL-15R活性”应理解为意指IL-15R结合蛋白能够增强天然存在的IL-15R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。
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”意指不显著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-15的变体”通常指将野生型的IL-15进行氨基酸修饰后得到的与IL-15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、尾部及其组合。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。“诱导型表达或诱导性表达(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，例如包括，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，包括但不限于能使免疫效应细胞抗肿瘤活性增加、增强或延长的免疫效应细胞的数量；包括但不限于能使抗肿瘤免疫效应细胞或活化免疫效应细胞数目 的增加的免疫效应细胞的数量；包括但不限于能促进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(通常是癌细胞或肿瘤细胞)的特异性识别，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、CD28或其组合。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，本文所述的癌症或肿瘤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或肿瘤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氨基酸片段，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)。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，例如使与含所述跨膜结构域的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所述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跨膜结构域。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跨膜区域。
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，包括但不限于增殖。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是共刺激性分子的细胞内部分。共刺激性分子是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“NF-kb(Nuclear factor kB)”是转录因子家族中的成员，是细胞内最重要的核转录因子，在许多细胞刺激介导的细胞信息的转录调控中起核心作用，参与多种基因的表达和调控，是细胞激活的标志。NF-κB一般以同源或异源二聚体形式存在。在静息细胞中，NF-kB二聚体通过非共价键的形式与其抑制蛋白IkB结合而分散在细胞质内，包括内质网应激在内的许多因素可激活NF-kB，激活后的NF-kB进入细胞核，与DNA模块上的特异蛋白结合，诱导特异mRNA的产生，最后转录、产生和释放各种细胞因子。
术语“AP-1(Activator protein 1)”是细胞内的一个转录激活因子，是由c-Fos和c-Jun组成的异二聚体。它通过调节基因的表达来应对多种刺激，包括细胞因子、生长因子、压力、细菌和病毒感染；因此AP-1控制了许多的细胞进程，包括分化、增殖和凋亡。AP-1上调含有TPA DNA应答元件(TRE；5'-TGAG/CTCA-3')的基因的转录。AP-1异二聚体通过亮氨酸拉链形成，并通过特定的保守序列与基因结合来启动基因的表达。
术语“活化T细胞核因子”(Nuclear factor of activated T cells，NFAT)在细胞因 子基因的转录调节中起着重要的作用，NFAT蛋白质在多种可调节重要免疫功能的细胞因子及细胞表面受体(例如，白介素-2、白介素-4、白介素-5、白介素-13、干扰素-γ、肿瘤坏死因子-α、GM-CSF、CD40L及CTLA-4)的转录调节中均起到重要作用。迄今已发现的NFAT蛋白可分为5个：NFAT1、NFAT2、NFAT3、NFAT4和NFAT5，其中，NFATc1-4的激活均依赖于胞内钙信号途径。
NFAT蛋白质活化是通过一包括NFAT蛋白质去磷酸化、核移位及DNA结合的过程来调节的。在静止细胞中，磷酸化的NFAT蛋白质驻留于细胞质中且具有较低的DNA结合亲和性。能够触发钙移动的各种刺激可通过一由Ca2+/钙调素依赖性蛋白磷酸酶，即钙调磷酸酶调节的过程而引起NFAT蛋白质的快速去磷酸化。具有一暴露的核定位信号的去磷酸化NFAT蛋白质移位于核中，其以高亲和性与DNA结合并调节靶基因转录。在一些实施方案中，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，IL15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IL15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。在本发明的实施方案中，当所述受体识别所述靶抗原之后活化的TCR信号能激活细胞内的NFAT，并与启动子中的NFAT结合基序结合从而启动IL-15的转录。
本文使用的术语“启动子”定义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。启动子是RNA聚合酶识别、结合和开始转录的一段DNA序列，它含有RNA聚合酶特异性结合和转录起始所需的保守序列。
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。通过将最小启 动子与不同的顺式调控元件组合而得到的嵌合启动子的构建描述在例如美国专利6555673中。
在一些实施方案中，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。基于这样考虑，本发明人将细胞因子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。另外，为了进一步提高表达细胞因子的CAR-T细胞的特异性，还可以通过基因编辑技术将携带NFAT调控表达细胞因子基因的CAR-GPC3 T细胞的内源性TCRα链敲除，以消除非肿瘤靶抗原(如非GPC3的抗原)通过TCR/CD3信号通路诱导的细胞因子的表达，实现了只有肿瘤靶抗原特异性诱导CAR-GPC3 T细胞表达细胞因子，如IL15。
实施例中，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(如SEQ ID NO：23所示的核酸序列)与IL2的最小启动子(minimal promoter)串联在一起组成的启动子来调节IL15在T细胞中的表达。
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可以无需由“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。
术语“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-1Gag；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；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。肿瘤包括但不限于：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，难治性肿瘤可以是治疗期间肿瘤对所述治疗出现抗性的。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，或其组合来决定。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。
本文所用的“GPC3”或“磷脂酰肌醇蛋白聚糖3”是Glypican家族的一员，基因登记号：NM_016697.3，NP_057906.2，其在调控细胞生长和分化方面起重要作用。GPC3异常表达与多种肿瘤的发生、发展关系密切，如在肝癌、肺癌、乳腺癌、卵巢癌、肾癌、甲状腺癌、胃癌、结直肠癌、等中呈现异常表达。
“GPC3”包括细胞天然表达的或转染了GPC3基因的细胞所表达的任何GPC3的翻译后修饰变体、同工型和种间同源物。
术语“GPC3变体”应包括(i)GPC3剪接变体，(ii)GPC3翻译后修饰变体，特别是包括N糖基化状态不同的变体，(iii)GPC3构象变体，(iv)位于细胞表面的GPC3和同型/异型相关联变体，(v)GPC3癌症相关变体和GPC3非癌症相关变体。
在本发明中，共表达IL-15的CAR-T靶向GPC3阳性表达的肿瘤。在具体的实施方式中，所述肿瘤包括但不限于肝癌、胃癌、肺癌、食道癌、头颈癌、膀胱癌、卵巢癌、宫颈癌、肾癌、胰腺癌、宫颈癌、脂肪肉瘤、黑色素瘤、肾上腺癌、神经鞘瘤、恶性纤维组织细胞瘤、食道癌。本领域技术人员知晓，有些肿瘤细胞，例如肝癌细胞对很多药物不敏感，因此，即便在体外有效的药物，有时候在体内的效果也不佳，甚至没有效果。因此，在优选的实施方式中，本文所述的GPC3阳性表达的肿瘤或GPC阳性肿瘤是肝癌、胃癌、肺癌、食道癌。
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。
当采用的共表达IL-15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IL-15，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IL-15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：13、14、或15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。
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：
胞外抗原结合域-CD8跨膜区-4-1BB-CD3ζ，
胞外抗原结合域-CD8跨膜区-CD28b-CD3ζ，
胞外抗原结合域-CD28a-CD28b-CD3ζ，
胞外抗原结合域-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 PCTCN2020117759-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：
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。
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、CN 108884459 A、CN107893052A、CN108866003A、CN108853144A、CN109385403A、CN109385400A、CN109468279A、CN109503715A、CN 109908176 A、CN109880803A、CN 110055275 A、CN110123837A、CN 110438082 A、CN 110468105 A国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018/018958A1、WO2014180306A1、WO2015197016A1、WO2016008405A1、WO2016086813A1、WO2016150400A1、WO2017032293A1、WO2017080377A1、WO2017186121A1、WO2018045811A1、WO2018108106A1、WO 2018/219299、WO2018/210279、WO2019/024933、WO2019/114751、WO2019/114762、WO2019/141270、WO2019/149279、WO2019/170147A1、WO 2019/210863、WO2019/219029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
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GPC3抗体，氨基酸序列如SEQ ID NO：12所示，核酸序列如SEQ ID NO:16所示，所采用的嵌合抗原受体为二代的嵌合抗原受体。
以pRRLSIN-cPPT.EF-1α为载体，构建了表达二代嵌合抗原受体的慢病毒质粒pRRLSIN-GPC3-BBZ。GPC3-BBZ的核酸序列包含了CD8α信号肽(SEQ ID NO:20)、scFv(SEQ ID NO:16)、CD8的铰链区(SEQ ID NO:21)、CD8的跨膜域(核酸序列如SEQ ID NO:17所示)、CD137胞内信号传导结构域(核酸序列如SEQ ID NO:18所示)，及CD3的胞内段CD3ζ信号域(核酸序列如SEQ ID NO:19所示)。
在pRRLSIN-GPC3-BBZ质粒的基础上插入NFAT-IL15的基因，构建表达CAR和NFAT调控的IL15的慢病毒质粒pRRLSIN-GPC3-BBZ-IL15(质粒图如图1所示)。NFAT-IL15的基因包括NFAT6结合基序(核酸序列见SEQ ID NO：23)、IL2最小启动子(核酸序列见SEQ ID NO：25)、和人的IL15(核酸序列见SEQ ID NO：35)。
在pRRLSIN-GPC3-BBZ质粒的基础上插入CCL21-NFAT-IL15的基因，构建表达CAR、IL15及CCL21的慢病毒质粒pRRLSIN-GPC3-BBZ-CCL21*15(见图1)。CCL21-NFAT-IL15的基因包括CCL21(核酸序列见SEQ ID NO：24)、NFAT6结合基序(核酸序列见SEQ ID NO：23)、和IL2最小启动子(核酸序列见SEQ ID NO：25)和人的IL15(核酸序列见SEQ ID NO：35)。
2、CAR T细胞的构建
分离人的外周血PBMC细胞，经培养和活化后，将所得的慢病毒pRRLSIN-GPC3-BBZ、pRRLSIN-GPC3-BBZ-IL15、和pRRLSIN-GPC3-BBZ-CCL21*15分别感染T细胞，得到GPC3-BBZ细胞、GPC3-BBZ-IL15细胞、和GPC3-BBZ-CCL21*15细胞。经流式检测，分别筛选出GPC3-BBZ细胞、GPC3-BBZ-IL15细胞、和GPC3-BBZ-CCL21*15阳性细胞。
实施例2.体外杀伤实验
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(未转导T细胞)、GPC3-BBZ细胞、GPC3-BBZ-IL15细胞于96孔板。
靶细胞：分别接种50μL 1×10 5/mL的Huh-7细胞(日本RIKEN细胞库)、PLC/PRF/5细胞(美国ATCC)、SK-HEP-1细胞(美国ATCC)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2所示，GPC3表达阳性的Huh7细胞在效靶比为1:1或1:3时，相对GPC3-BBZ细胞，GPC3-BBZ-IL15细胞显示出更为优异的杀伤；在GPC3表达阳性的PLC/PRF/5细胞中效靶比为3:1、1:1或1:3时，相对GPC3-BBZ细胞，GPC3-BBZ-IL15细胞显示出更为优异的杀伤；GPC3-BBZ和GPC3-BBZ-IL15细胞对GPC3表达阴性的SK-HEP-1均没检测到杀伤。
实施例3.NPG小鼠皮下移植瘤模型的体内杀伤
接种瘤块：分别接种3×10 6的肝癌细胞PLC/PRF/5于NPG小鼠右侧腋部皮下。分为3组，每组6只。接种日记为D0。
注射CAR T：皮下接种瘤组织后D13天，肿瘤平均体积约300mm 3。注射实施例1制备的CAR T细胞，注射剂量：2.0×10 6/只。
肿瘤抑制结果如图3所示，CAR T注射后19天(D32)，与UTD组相比，GPC3-BBZ、GPC3-BBZ-IL15组抑瘤率分别为29.22％和71.26％(P<0.05)，GPC3-BBZ-IL15细胞显示出比GPC3-BBZ细胞更为优异的杀伤。
称量小鼠体重变化，结果如图4所示，显示GPC3-BBZ-IL15细胞对小鼠的体重无显著影响。
实施例4.GPC3-BBZ-CCL21*15细胞的体外杀伤
参照实施例2的操作，检测GPC3-BBZ-IL15细胞和GPC3-BBZ-CCL21*15细胞对PLC/PRF/5细胞和SK-HEP-1细胞的细胞杀伤，结果如图5所示，显示GPC3-BBZ-CCL21*15显示出更优异的细胞杀伤。在GPC3表达阳性的PLC/PRF/5 细胞中当效靶比为3:1、1:1或1:3时，相对UTD,GPC3-BBZ-IL15、GPC3-BBZ-CCL21*15细胞均显示显著增加的肿瘤细胞杀伤力，且GPC3-BBZ-CCL21*15显示出更优异的肿瘤细胞杀伤。GPC3-BBZ-IL15、GPC3-BBZ-CCL21*15细胞对GPC3表达阴性的SK-HEP-1均没检测到杀伤。
实施例5、小鼠GPC3-mBBZ CAR T细胞和GPC3-mBBZ-mNFAT6-mIL15 CAR T细胞的制备
1、质粒构建
以pMSCV(购自addgene)为载体，将鼠CD8α信号肽(SEQ ID NO：27)、抗GPC3单抗抗体(SEQ ID NO：16)、鼠CD8α胶链区及跨膜区(SEQ ID NO：28)、鼠CD137胞内域(SEQ ID NO：29)、鼠CD3ζ胞内域(SEQ ID NO：30)的基因序列依次连接，通过体外基因合成的方法获得重组质粒MSCV-GPC3-mBBZ。在MSCV-GPC3-mBBZ CAR-T质粒的基础上插入了mNFAT6-mIL15序列，构建了表达GPC3的二代嵌合抗原受体以及mIL15的质粒，MSCV-GPC3-mBBZ-mNFAT6-mIL15。mNFAT6-mIL15序列是由mNFAT6 binding motif(SEQ ID NO：31)、mIL2最小启动子(SEQ ID NO：32)、mIL15(SEQ ID NO：33)、PA2(SEQ ID NO：34)组成(图6所示)。
2、小鼠CAR-T淋巴细胞的制备
取健康Balb/c小鼠，采用Cell isolation kit(STEMCELL Technologies)，按照说明书所述，分离得到小鼠脾脏T淋巴细胞，经培养和活化后，将所得的逆转录病毒MSCV-GPC3-mBBZ、MSCV-GPC3-mBBZ-mNFAT6-mIL15分别感染小鼠T细胞，得到GPC3-mBBZ CAR-T细胞、GPC3-mBBZ-mNFAT6-mIL15 CAR-T细胞。经流式检测，分别筛选出GPC3-mBBZ CAR-T细胞、GPC3-mBBZ-mNFAT6-mIL15 CAR-T阳性细胞。
实施例6、体外杀伤毒性检测及体外细胞因子分泌检测
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。利用常规分子生物学技术分别将表位人源的鼠源GPC3转入鼠肝癌细胞Hepa 1-6(中国科学院细胞典藏中心(上海)构建成过表达GPC3蛋白的Hepa 1-6 GPC3。
接种小鼠肝癌细胞Hepa 1-6以及Hepa 1-6 GPC3细胞于96孔板，按效靶比3:1、1:1、1:3，与效应细胞共培养18h检测，效应细胞分别为UTD,GPC3-mBBZ CAR-T细胞及GPC3-mBBZ-mNFAT6-mIL15 CAR-T细胞。
实验结果如图7A所示，GPC3-mBBZ CAR T细胞和GPC3-mBBZ-mNFAT6-mIL15 CAR T细胞均能特异性杀伤Hepa1-6-GPC3肿瘤细胞，而对Hepa1-6肿瘤细胞几乎没有杀伤能力。
ELISA法检测了靶细胞刺激后的效应细胞IFN-γ、IL2和TNF-α的分泌情况。如图7B所示，GPC3-mBBZ CAR T细胞和GPC3-mBBZ-mNFAT6-mIL15 CAR T细胞在Hepa1-6-GPC3细胞刺激下，可有效分泌杀伤肿瘤效应相关的细胞因子IFN-γ、IL2和TNF-α，且相比于GPC3-mBBZ CAR T细胞，GPC3-mBBZ-mNFAT6-mIL15 CAR T细胞具有更强的IL2和TNF-α分泌能力(P<0.001)，IFN-γ的分泌水平无统计学差异(P>0.05)。而上述CAR T细胞与Hepa1-6细胞共培养后，几乎均没有细胞因子的分泌。以上结果表明，激活后的GPC3-mBBZ-mNFAT6-mIL15 CAR T细胞可特异性杀伤GPC3表达阳性的肿瘤细胞并且可提高CAR T细胞释放细胞因子的能力。
实施例7、GPC3-mBBZ-mNFAT6-mIL15 CAR T对Hepa 1-6 GPC3皮下移植瘤的治疗效果
分别接种1×10 7的肝癌细胞Hepa 1-6 GPC3于雌性C57BL/6小鼠(上海西普尔－必凯实验动物有限公司)右侧腋部皮下，分3组，每组6只，接种日记为D0。
皮下接种瘤组织后D6天，肿瘤平均体积约300mm 3。尾静脉注射1.5×10 6的CAR T细胞。每隔3-4天测量Hepa 1-6 GPC3移植瘤体积的大小，记录每组小鼠瘤体积的变化，瘤体积计算公式为：(长×宽 2)/2。
如图8A所示，与UTD组相比，GPC3-mBBZ CAR-T和GPC3-mBBZ-mNFAT6-mIL15 CAR T治疗组均能有效抑制肿瘤生长，而且 GPC3-mBBZ-mNFAT6-mIL15 CAR T细胞的抗肿瘤作用比GPC3-Mbbz CAR-T细胞更强(P<0.001)。小鼠体重变化曲线，如图8B所示，每组小鼠的体重均未出现明显下降，表明GPC3-mBBZ-mNFAT6-mIL15 CAR T治疗无严重毒副作用。CAR T细胞注射后20天，将小鼠实施安乐死，剥取残余皮下肿瘤并称取肿瘤重量，结果如图8C所示，GPC3-mBBZ-mNFAT6-mIL15 CAR T治疗组瘤显著重低于GPC3-mBBZ CAR T治疗组(P<0.05)。GPC3-mBBZ-mNFAT6-mIL15 CAR T组和GPC3-mBBZ CAR T组的抑瘤率分别为98.1％和70.7％。实验终止时，GPC3-mBBZ-mNFAT6-mIL15 CAR T组有4只小鼠肿瘤完全消退。
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 PCTCN2020117759-appb-000002
Figure PCTCN2020117759-appb-000003
Figure PCTCN2020117759-appb-000004
Figure PCTCN2020117759-appb-000005
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 (37)

  1. 表达IL-15的免疫效应细胞，其特征在于，所述细胞表达IL-15及识别特异性靶抗原的受体、或所述细胞表达IL-15R结合分子及识别特异性靶抗原的受体。
  2. 如权力要求1所述的细胞，其特征在于，所述IL-15为外源性IL-15。
  3. 如权利要求1所述的细胞，其特征在于，所述的IL-15为组成型表达或诱导型表达。
  4. 如权利要求1所述的细胞，其特征在于，所述IL-15是诱导型表达，其表达受所述受体调控。
  5. 如权利要求4所述的免疫效应细胞，其特征在于，当所述受体识别所述靶抗原后能够启动所述IL-15的表达。
  6. 如权利要求5所述的免疫效应细胞，其特征在于，所述受体通过诱导型启动子诱导所述IL-15的表达。
  7. 如权利要求1-6任一所述的免疫效应细胞，其特征在于，所述免疫效应细胞还表达趋化因子、趋化因子受体、除IL-15之外的其他细胞因子、降低PD-1表达的siRNA、阻断PD-L1与PD-1结合的蛋白、或安全开关。
  8. 如权利要求7所述的免疫效应细胞，其特征在于，所述趋化因子为淋巴细胞趋化因子；
    优选的，所述的淋巴细胞趋化因子为CCL21；
    优选地，所述CCL21与SEQ ID NO:26、SEQ ID NO:39或40所示的氨基酸序列具有至少90％同一性或是SEQ ID NO:26、SEQ ID NO:39或40所示的氨基酸序列的截短片段；或者与由SEQ ID NO:24、SEQ ID NO:37或38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:24、SEQ ID NO:37或38所示的核苷酸编码的氨基酸序列的截短片段。
  9. 如权利要求7所述的免疫效应细胞，其特征在于，所述趋化因子受体选自CCR2、CCR5、CXCR2、或CXCR4。
  10. 如权利要求7所述的免疫效应细胞，其特征在于，所述其他细胞因子选自IL-21、IL18、或I型干扰素。
  11. 如权利要求7所述的免疫效应细胞，其特征在于，所述阻断PD-L1与PD-1结合的蛋白选自PD-L1的抗体、PD-1的抗体、天然的PD-1或者天然PD-1的截短片段、含有天然的PD-1或者天然PD-1的截短片段的融合肽。
  12. 如权利要求7所述的免疫效应细胞，其特征在于，所述的安全开关选自iCaspase-9、Truancated EGFR、RQR8、对免疫效应细胞有杀伤作用的蛋白。
  13. 如权利要求1-12所述的免疫效应细胞，其特征在于，所述的免疫效应细胞选自T细胞、NK细胞、NKT细胞、肥大细胞、巨噬细胞、树突细胞、CIK细胞、或干细胞衍生的免疫效应细胞。
  14. 如权利要求6所述的免疫效应细胞，其特征在于，所述诱导型启动子包含转录因子的结合基序和最小启动子，所述诱导型启动子的活化依赖所述受体的活化或依赖于所述受体与所述靶抗原结合。
  15. 如权利要求14所述的免疫效应细胞，其特征在于，所述结合基序包括NFAT、NF-κB或AP-1结合基序，或者NFAT、NF-κB、AP-1结合基序的至少两种的组合，
    优选的，所述结合基序为NFAT结合基序。
  16. 如权利要求15所述的免疫效应细胞，其特征在于，所述结合基序包含1-12个NFAT结合基序、1-12个NF-κB结合基序、1-12个AP-1结合基序，或1-12个NFAT、NF-κB、AP-1结合基序中至少两种的组合；
    优选的，所述结合基序包含1-6个NFAT结合基序、1-6个NF-κB结合基序、1-6个AP-1结合基序，或1-6个NFAT、NF-κB、AP-1结合基序中至少两种的组合。
  17. 如权利要求15所述的免疫效应细胞，其特征在于，所述NFAT结合基序的序列如SEQ ID NO：23所示。
  18. 如权利要求14所述的免疫效应细胞，其特征在于，所述最小启动子为细胞因子最小启动子。
  19. 如权利要求18所述的免疫效应细胞，其特征在于，所述最小启动子包括白细胞介素、干扰素、肿瘤坏死因子超家族、集落刺激因子、趋化因子、生长因子最小启动子；
    优选的，为IFN-γ、TNF-α或IL-2最小启动子；
    更优选，为IL-2最小启动子。
  20. 如权利要求19所述的免疫效应细胞，其特征在于，所述IL-2最小启动子的序列如SEQ ID NO：25所示。
  21. 如权利要求1所述的细胞，其特征在于，所述IL-15为天然IL-15、或与天然IL-15具有相同功能的天然IL-15的截短片段或天然IL-15的突变体；
    优选地，所述天然IL-15与SEQ ID NO:1或SEQ ID NO:36所示的氨基酸序列具有至少90％同一性、或是SEQ ID NO:1或SEQ ID NO:36所示的氨基酸序列的截短片段；或者与由SEQ ID NO:33或SEQ ID NO:35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:33或SEQ ID NO:35所示的核苷酸编码的氨基酸序列的截短片段。
  22. 如权利要求7所述的细胞，其特征在于，所述趋化因子、趋化因子受体、除IL-15之外的其他细胞因子、降低PD-1表达的siRNA、阻断PD-L1与PD-1结合的蛋白、或安全开关是组成型表达或诱导型表达；优选的，为组成型表达。
  23. 如权利要求1所述的细胞，其特征在于，所述靶抗原为肿瘤抗原为肿瘤抗原；
    优选地，所述实体瘤抗原为GPC3、EGFR、EGFRvIII、间皮素或Claudin18.2。
  24. 如权利要求23所述的细胞，其特征在于，所述受体选自嵌合抗原受体(CAR)、T细胞受体(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，所述受体为嵌合抗原受体；
    更优选地，所述受体为嵌合抗原受体，包括
    (i)抗原结合结构域、CD28或CD8的跨膜域、CD28的共刺激信号结构域和CD3ζ的胞内信号域；或
    (ii)抗原结合结构域、CD28或CD8的跨膜域、4-1BB的共刺激信号结构域和CD3ζ的胞内信号域；或
    (iii)抗原结合结构域、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的胞内信号域。
  25. 如权利要求26所述的细胞，其特征在于，所述嵌合抗原受体(CAR)、T细胞融合蛋白(TFP)、或T细胞抗原耦合器(TAC)的抗原结合结构域为抗体，具有SEQ ID NO：2所示的HCDR1，SEQ ID NO：3所示的HCDR2，SEQ ID NO：4所示的HCDR3，和SEQ ID NO：5所示的LCDR1，SEQ ID NO：6所示的LCDR2，SEQ ID NO：7所示的LCDR3；或
    所述抗体具有SEQ ID NO:8所示轻链可变区和SEQ ID NO:9所示的重链可变区；或具有SEQ ID NO:10所示轻链可变区和SEQ ID NO:11所示的重链可变区；或
    所述抗体具有SEQ ID NO：12所示的氨基酸序列。
  26. 如权利要求25所述的细胞，其特征在于，所述受体的氨基酸序列与SEQ ID NO:13、14、15所示的氨基酸序列具有至少90％的同一性。
  27. 如权利要求25所述细胞，其特征在于，所述的免疫细胞诱导型启动子诱导表达IL-15的核酸序列如SEQ ID NO：22所示。
  28. [根据细则91更正 25.02.2021]
    如权利要求1-27任一所述的细胞，其特征在于，所述受体与IL-15由同一核酸分子表达，或由不同核酸分子表达；
    优选，所述受体与IL-15由同一核酸分子表达，所述IL-15的表达盒与受体之间、以及表达盒之间直接连接或由串联片段连接，所述串联片段选自F2A、P2A、T2A、和/或E2A。
  29. 一种核酸分子，所述核酸分子表达权利要求1-28中任一项所述的IL-15R结合分子或IL-15或IL-15与趋化因子、趋化因子受体、除IL-7之外的其他细胞因子、降低PD-1表达的siRNA、阻断PD-L1与PD-1结合的蛋白、或安全开关；所述核酸分子还表达权利要求1-28中任一项所述的特异性识别靶抗原的受体；
    优选所述核酸由DNA和/或RNA组成；或所述核酸是mRNA；或所述核酸包含核苷酸类似物。
  30. 一种载体，包含权利要求29所述的核酸分子；
    优选所述载体选自由以下组成的组：DNA、RNA、质粒、慢病毒载体、腺病毒载体、劳氏肉瘤病毒(RSV)载体或逆转录病毒载体。
  31. 一种细胞，其包含权利要求30所述的载体或其基因组上整合有权利要求29所述的核酸分子；
    优选所述细胞是人T细胞，优选地，为同种异体T细胞。
  32. 一种制备细胞的方法，所述方法包括用权利要求30所述的载体或权利要求29所述的核酸分子转导T细胞。
  33. 一种产生RNA工程改造的细胞群的方法，所述方法包括将体外转录的RNA或合成RNA引入细胞中，
    (i)其中所述RNA包括权利要求15所述的核酸。
  34. 一种在哺乳动物体内提供抗肿瘤免疫性的方法，所述方法包括向所述哺乳动物施用有效量的权利要求1-28、31中任一项所述细胞、权利要求29所述的核酸分子、或权利要求30所述的载体；
    优选所述哺乳动物是人。
  35. 一种治疗患有与GPC3或者claudin18.2表达有关的疾病的哺乳动物的方法，所述方法包括向所述哺乳动物施用有效量的权利要求1-28、31中任一项所述细胞、权利要求29所述的核酸分子、或权利要求30所述的载体；
    优选所述与GPC3或者claudin18.2表达有关的疾病选自结肠癌，直肠癌，肾细胞癌，肝癌，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，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，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、与GPC3或者claudin18.2表达有关的非癌症相关适应症；优选的，选自肝癌、肺癌、乳腺癌、卵巢癌、肾癌、甲状腺癌、胃癌、结直肠癌、胰腺癌、食道癌；
    优选权利要求1-28、31中任一项所述细胞和增加权利要求1-28中任一项细胞的功效的药剂组合施用，优选地，与化疗药物联用；
    优选权利要求1-28、31中任一项所述细胞与改善与施用权利要求1-28中任一项所述细胞相关的一种或多种副作用的药剂联合施用；
    优选权利要求1-28、31中任一项所述细胞和治疗所述与GPC3或者claudin18.2相关的疾病的药剂组合施用，优选地，与化疗药物联用。
  36. 如权利要求1-28、31中任一项所述的细胞、权利要求29所述的核酸分子、权利要求30所述的载体的用途，用作药物，优选地，用作抑制肿瘤或抑制病原体的药物，进一步优选，所述用途为用于制备用于抑制肿瘤或抑制病原体的药物的用途。
  37. 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1-28、31任一所述的细胞和药学上可接受的载体或赋形剂。
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