WO2018210279A1 - Toll样受体激动剂与免疫效应细胞的联用 - Google Patents

Toll样受体激动剂与免疫效应细胞的联用 Download PDF

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WO2018210279A1
WO2018210279A1 PCT/CN2018/087162 CN2018087162W WO2018210279A1 WO 2018210279 A1 WO2018210279 A1 WO 2018210279A1 CN 2018087162 W CN2018087162 W CN 2018087162W WO 2018210279 A1 WO2018210279 A1 WO 2018210279A1
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cells
antigen
receptor
cell
immune effector
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PCT/CN2018/087162
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French (fr)
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李宗海
狄升蒙
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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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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
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    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • A61K39/4643—Vertebrate antigens
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    • A61K39/4644—Cancer antigens
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    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N5/10—Cells modified by introduction of foreign genetic material
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    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/49—Breast
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    • A61K2239/50—Colon
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • the invention belongs to the field of immunotherapy; relates to the combined use of immune effector cells and TOLL-like receptor agonists with targeted recognition of tumor antigens or pathogen antigens.
  • CAR-T cells chimeric antigen receptor-modified T cells
  • CAR-NK cells chimeric antigen receptor-modified NK cells
  • TCR-T TCR-modified T cells
  • immune effector cells usually do not exert anti-tumor effects in vivo.
  • the present invention aims to provide a use of a combination of a TOLL-like receptor agonist and an immune effector cell for the treatment of tumors, infectious diseases and other immune-related diseases.
  • TOLL-like receptor agonists are capable of increasing the efficacy of immune effector cells.
  • composition comprising an immune effector cell and a receptor for recognizing a tumor antigen or a pathogen antigen, the immune effector cell
  • the potentiator is a TOLL-like receptor agonist.
  • the present invention provides a composition
  • a composition comprising an immune effector cell having a receptor that targets a tumor antigen or a pathogen antigen, the immune effector cell being associated with expression of a tumor antigen or a pathogen antigen
  • an immune effector cell having a receptor that targets a tumor antigen or a pathogen antigen, the immune effector cell being associated with expression of a tumor antigen or a pathogen antigen
  • the receptor has an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain has an antigen binding domain that binds to a tumor antigen or a pathogen antigen associated with the disease;
  • the agent that increases the efficacy of immune effector cells is a TOLL-like receptor agonist.
  • the intracellular region of the receptor has a domain involved in eliciting activation of immune cells.
  • the TOLL-like receptor agonist is a Toll-like receptor 3 (TLR3) agonist.
  • TLR3 Toll-like receptor 3
  • the TLR3 agonist is selected from natural or synthetic double-stranded ribonucleic acids.
  • the TLR3 agonist is polyinosinic acid cytidine (poly(I:C)).
  • the TLR3 agonist is a mismatched double-stranded ribonucleic acid poly (I: C11-14U), exemplified by poly (I: C12U), poly (I: C13U).
  • the recognition of a tumor antigen or a pathogen antigen receptor comprises a chimeric antigen receptor (CAR), a T cell (antigen) receptor (TCR), a T cell fusion protein (TFP), or a T cell.
  • CAR chimeric antigen receptor
  • TCR T cell
  • T cell fusion protein T cell
  • TAC Antigen coupler
  • the chimeric antigen receptor comprises:
  • the TFP comprises: (a) a TCR subunit comprising a TCR partial extracellular domain, a transmembrane domain, and a TCR intracellular domain, said intracellular structure
  • the domain includes a stimulation signaling domain; (b) an antigen binding domain, the TCR subunit and the antigen binding domain are operably linked, and the extracellular, transmembrane and intracellular signal domains of the TCR subunit are derived CD3 epsilon or CD3 gamma, which integrates into the TCR expressed on T cells.
  • the TAC comprises: (a) an extracellular domain comprising: a single chain antibody having an antigen binding domain and binding to CD3; (b) a transmembrane region; c) an intracellular domain that links to protein kinase LCK.
  • the immune effector cells are selected from the group consisting of T cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, or bone marrow-derived phagocytic cells.
  • the immune cell is a T cell
  • the intracellular domain of the receptor is capable of triggering activation of T cells.
  • the immune effector cells also express other exogenous proteins.
  • the exogenous protein is selected from the group consisting of FLT3L, interferon, interleukin, interleukin receptor, PD-1 inhibition.
  • Agents such as antibodies to PD-1
  • PD-L1 inhibitors such as antibodies to PD-L1, soluble PD-1, or proteins containing soluble PD-1, or a combination thereof.
  • composition further comprises an MDSC inhibitor, and/or an immunological checkpoint inhibitor.
  • the MDSC inhibitor is selected from the group consisting of an anti-Gr1 antibody, a cyclooxygenase-2 inhibitor, a prostaglandin-type stem cell factor inhibitor, a macrophage colony-stimulating factor inhibitor, a granule mononuclear A cell colony stimulating factor inhibitor, a vascular endothelial growth factor inhibitor, or a combination thereof.
  • the immunological checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor (such as an antibody to PD-1), a PD-L1 inhibitor (such as an antibody to PD-L1), and CTLA-4.
  • a PD-1 inhibitor such as an antibody to PD-1
  • a PD-L1 inhibitor such as an antibody to PD-L1
  • CTLA-4 an inhibitor (such as a CTLA-4 antibody), or a combination thereof.
  • the immune effector cell is a TCR knockdown or PD-1 knockout T cell.
  • the expression of the TCR gene or the PD-1 gene in the immune effector cell is silenced.
  • the "expression of the gene is silenced” means that the silenced gene is not expressed or underexpressed.
  • the "low expression” refers to a ratio of the expression level G1 of the immune effector cell to the corresponding gene expression level G0 of the normal T cell, that is, G1/G0 ⁇ 0.5, preferably G1/G0. ⁇ 0.3, more preferably ⁇ 0.2, more preferably ⁇ 0.1, most preferably 0.
  • an immune effector cell and an immune effector cell synergist for the preparation of an antitumor or antipathogenic drug or a drug having low immunity, the immune effector cell having a target To a receptor that recognizes a tumor antigen or a pathogen antigen, the immune effector cell potentiator is a TOLL-like receptor agonist.
  • the present invention provides the use of a combination of a TOLL-like receptor agonist and an immune effector cell for the preparation of an anti-tumor drug or an anti-pathogenic drug or an anti-immune drug, the immune effector cell having a targeted recognition tumor antigen or A receptor for a pathogen antigen having an extracellular region, a transmembrane region, and an intracellular region, the extracellular region having an antigen binding domain that binds to the tumor antigen or the pathogen antigen.
  • the TOLL-like receptor agonist is a Toll-like receptor 3 (TLR3) agonist, preferably a natural or synthetic double-stranded ribonucleic acid.
  • TLR3 Toll-like receptor 3
  • the TLR3 agonist is polyinosinic acid cytidine (poly(I:C)).
  • the TLR3 agonist is a mismatched double-stranded ribonucleic acid, exemplified by poly (I: C12U), poly (I: C13U).
  • the amount of the mismatched double-stranded ribonucleic acid is sufficient to bind to Toll-like receptor 3 (TLR3) and reduce or eliminate proliferation of the tumor or other transformed cell in the subject.
  • TLR3 Toll-like receptor 3
  • the TOLL-like receptor agonist has a synergistic effect on the immune effector cells.
  • the TOLL-like receptor agonist and the immune effector cells are administered parenterally, preferably intravenously and intratumorally; thus the TOLL-like receptor agonist
  • the immunological effector cells can be prepared for parenteral administration, preferably intravenous and intratumoral injection.
  • the TOLL-like receptor agonist and the immune effector cells may be administered in the same or different manner.
  • the method of administration is co-administering the TOLL-like receptor agonist and the immune effector cell, or administering the immune effector cell first, and then administering the TOLL-like receptor agonist; or The TOLL-like receptor agonist is administered and the immune effector cells are administered.
  • the method of administration is to first administer the immune effector cells and then to the TOLL-like receptor agonist.
  • the tumor antigen is selected from the group consisting of EGFRvIII, GPC3, CLD18A2, CD19, CD20, CD22, CD30, WT1, CLDN6, MUC1, BCMA, IL-11Ra, IL-13Ra, FAP.
  • the TOLL-like receptor agonist can be administered in an amount from 1 mg/kg to 4 mg/kg, preferably from 2 mg/kg to 3 mg/kg, more preferably from 2.08 mg/kg to 2.77 mg/ Kg.
  • the TOLL-like receptor agonist is administered in a number of 2 to 4 times, preferably 2-3 times.
  • the immunotherapeutic cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a mast cell or a bone marrow-derived phagocytic cell, preferably a T cell.
  • the immune effector cell is a T cell
  • the intracellular domain of the receptor is capable of triggering activation of T cells
  • the recognition of a tumor antigen or a pathogen antigen receptor comprises a chimeric antigen receptor (CAR), a T cell (antigen) receptor (TCR), a T cell fusion protein (TFP), or a T cell.
  • CAR chimeric antigen receptor
  • TCR T cell
  • T cell fusion protein T cell
  • TAC Antigen coupler
  • the immune effector cells are selected from the group consisting of CAR-expressing T cells, TCR-expressing T cells, CAR-expressing NK cells, TFP-expressing T cells, TFP-expressing NK cells, TAC-expressing T cells.
  • NK cells expressing TAC are selected from the group consisting of CAR-expressing T cells, TCR-expressing T cells, CAR-expressing NK cells, TFP-expressing T cells, TFP-expressing NK cells, TAC-expressing T cells.
  • the chimeric antigen receptor (CAR) comprises:
  • the TFP comprises: (a) a TCR subunit comprising a TCR partial extracellular domain, a transmembrane domain, and a TCR intracellular domain, said intracellular structure
  • the domain includes a stimulation signaling domain; (b) an antigen binding domain, the TCR subunit and the antigen binding domain are operably linked, and the extracellular, transmembrane and intracellular signal domains of the TCR subunit are derived CD3 epsilon or CD3 gamma, which integrates into the TCR expressed on T cells.
  • the TAC comprises: (a) an extracellular domain comprising: a single chain antibody having an antigen binding domain and binding to CD3; (b) a transmembrane region; c) an intracellular domain that links to protein kinase LCK.
  • the immune effector cells are also expressed with other proteins, preferably, antibodies such as PD1, PDL1, type I interferons, and the like.
  • the medicament further comprises an MDSC inhibitor or an immunological checkpoint inhibitor.
  • the MDSC inhibitor is selected from the group consisting of an anti-Gr1 antibody, a cyclooxygenase-2 inhibitor, a prostaglandin-type stem cell factor inhibitor, a macrophage colony-stimulating factor inhibitor, a granule mononuclear A cell colony stimulating factor inhibitor, a vascular endothelial growth factor inhibitor, or a combination thereof.
  • the immunological checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor (such as an antibody to PD-1), a PD-L1 inhibitor (such as an antibody to PD-L1), and CTLA-4.
  • a PD-1 inhibitor such as an antibody to PD-1
  • a PD-L1 inhibitor such as an antibody to PD-L1
  • CTLA-4 an inhibitor (such as a CTLA-4 antibody), or a combination thereof.
  • a combination of an immune effector cell, an immune effector cell synergist and a lymphocyte scavenger for the preparation of a medicament against or against a pathogen or against immunity.
  • the lymphocyte scavenger is administered prior to administering the immune effector cells and the immune effector cell potentiator to a subject in need thereof.
  • the immune effector cell potentiator is a TOLL-like receptor agonist.
  • the present invention also provides the use of a combination of an immune effector cell, a TOLL-like receptor agonist, and a lymphocyte scavenger in the preparation of an antitumor drug or an antipathogenic drug or an anti-immune drug, the immune effector cell a receptor having a target for recognizing a tumor antigen or a pathogen antigen, the receptor having an extracellular region, a transmembrane region, and an intracellular region, the extracellular region having an antigen binding domain that binds to the tumor antigen or the pathogen antigen .
  • the lymphocyte scavenger is administered prior to administration of the TOLL-like receptor agonist and immune effector cells.
  • the lymphocyte scavenger comprises a drug that clears lymphocytes or is subjected to a radiation treatment.
  • the drug that clears lymphocytes includes, but is not limited to, fludarabine and/or cyclophosphamide.
  • administering a TOLL-like receptor agonist and an immune effector cell to a subject in need thereof comprises simultaneous or sequential administration; for example, first administering a TOLL-like receptor agonist to the immune effector cell, or The immune effector cells are administered with a TOLL-like receptor agonist; more preferably, the immune effector cells are first administered with a TOLL-like receptor agonist.
  • a method of treating a tumor or pathogen infection or immunodeficiency comprising: administering a combination of an immune effector cell and an immune effector cell synergist to a subject in need thereof;
  • a combination of an immune effector cell, an immune effector cell synergist, and a lymphocyte scavenger is administered to a subject in need thereof, wherein the lymphocyte is administered to the subject in need thereof before administration of the immune effector cell and the immune effector cell synergist Cell scavenger.
  • the invention provides a method of treating a tumor or pathogen infection or low immunity, the method comprising:
  • a combination of an immune effector cell and a TOLL-like receptor agonist having a receptor targeting a tumor antigen or a pathogen antigen having an extracellular region, a transmembrane region, and the like is administered to a subject in need thereof
  • An intracellular region having an antigen binding domain that binds to said tumor antigen or a pathogen antigen is administered to a subject in need thereof
  • An intracellular region having an antigen binding domain that binds to said tumor antigen or a pathogen antigen or
  • a combination of immune effector cells, a TOLL-like receptor agonist, and a lymphocyte scavenger is administered to a subject in need thereof.
  • the TOLL-like receptor agonist is a Toll-like receptor 3 (TLR3) agonist, preferably a natural or synthetic double-stranded ribonucleic acid.
  • TLR3 Toll-like receptor 3
  • the TLR3 agonist is polyinosinic acid cytidine (poly(I:C)).
  • the TLR3 agonist is a mismatched double-stranded ribonucleic acid, exemplified by poly (I: C12U), poly (I: C13U).
  • the amount of the mismatched double-stranded ribonucleic acid is sufficient to bind to Toll-like receptor 3 (TLR3) and reduce or eliminate proliferation of the tumor or other transformed cell in the subject.
  • TLR3 Toll-like receptor 3
  • the TOLL-like receptor agonist has a synergistic effect on the immune effector cells.
  • administering a TOLL-like receptor agonist and an immune effector cell to a subject in need thereof comprises simultaneous or sequential administration; for example, first administering a TOLL-like receptor agonist to the immune effector cell, or The immune effector cells are administered with a TOLL-like receptor agonist; more preferably, the immune effector cells are first administered with a TOLL-like receptor agonist.
  • a fifth aspect of the invention provides a composition product, comprising:
  • a first pharmaceutical composition comprising at least one immune effector cell potentiator comprising a TOLL-like receptor agonist; and a pharmaceutically acceptable carrier ;
  • a second pharmaceutical composition comprising an immune effector cell having a receptor that targets a tumor antigen or a pathogen antigen; and a pharmaceutically acceptable carrier;
  • a third pharmaceutical composition comprising an MDSC inhibitor or an immunological checkpoint inhibitor; and a pharmaceutically acceptable carrier.
  • the first pharmaceutical composition, the second pharmaceutical composition, and the third pharmaceutical composition are mixed or independently present.
  • the total content of the first pharmaceutical composition, the second pharmaceutical composition and the third pharmaceutical composition is from 70 to 100% by weight, preferably from 80 to 100% by weight, more preferably 90%. ⁇ 100% by weight, based on the total weight of the product of the composition.
  • the immune effector cells are administered in an amount of from 1 ⁇ 10 6 to 1 ⁇ 10 12 cells/kg, more preferably from 1 ⁇ 10 7 to 1 ⁇ 10 10 cells/kg.
  • the MDSC inhibitor is selected from the group consisting of an anti-Gr1 antibody, a cyclooxygenase-2 inhibitor, a prostaglandin-type stem cell factor inhibitor, a macrophage colony-stimulating factor inhibitor, a granule mononuclear A cell colony stimulating factor inhibitor, a vascular endothelial growth factor inhibitor, or a combination thereof.
  • the immunological checkpoint inhibitor is selected from the group consisting of a PD-1 inhibitor (such as an antibody to PD-1), a PD-L1 inhibitor (such as an antibody to PD-L1), and CTLA-4.
  • a PD-1 inhibitor such as an antibody to PD-1
  • a PD-L1 inhibitor such as an antibody to PD-L1
  • CTLA-4 an inhibitor (such as a CTLA-4 antibody), or a combination thereof.
  • the composition product is a pharmaceutical composition for treating a tumor or pathogen infection or low immunity.
  • the dosage form of the composition includes an injectable dosage form, a topical pharmaceutical dosage form, and an oral dosage form.
  • composition in another preferred embodiment, can be administered by subcutaneous injection, intravenous injection, intramuscular injection.
  • the oral dosage form comprises a tablet, a capsule, a film, and a granule.
  • the dosage form of the composition comprises a sustained release dosage form, and a non-slow release dosage form.
  • a sixth aspect of the invention provides the use of a composition product according to the fifth aspect of the invention for the preparation of an antitumor or antipathogenic drug or an anti-immune drug.
  • the immune effector cell synergist may be administered in an amount of 1 mg/kg to 4 mg/kg, preferably 2 mg/kg to 3 mg/kg, more preferably selected from the group consisting of 2.08 mg/kg-2.77 mg/kg.
  • the immune effector cells are administered in an amount of 1 ⁇ 10 6 - 1 ⁇ 10 12 cells/kg, more preferably, 1 ⁇ 10 7 - 1 ⁇ 10 10 Cells / kg.
  • the MDSC inhibitor is administered in an amount of from 2 mg/kg to 5 mg/kg, preferably from 2 mg/kg to 4 mg/kg, in the composition product.
  • the number of administrations of the immune effector cell synergist is 2 to 4 times, preferably 2-3 times.
  • the number of administration of the immune effector cells is 2 to 4 times, preferably 2-3 times.
  • the number of administrations of the MDSC inhibitor is 2 to 4 times, preferably 2-3 times.
  • a seventh aspect of the present invention provides a method for improving the viability of an immune effector cell, comprising the steps of:
  • Administering (i) an immune effector cell; (ii) an immune effector cell potentiator; and/or (iii) an optional MDSC inhibitor or immunological checkpoint inhibitor, wherein the immune effector cell has a target A receptor that recognizes a tumor antigen or a pathogen antigen, the immune effector cell potentiator being a TOLL-like receptor agonist.
  • the subject comprises a human or a non-human mammal.
  • the non-human mammals include rodents and primates, preferably mice, rats, rabbits, monkeys.
  • component (i), component (ii), component (iii) are applied simultaneously or sequentially.
  • immune effector cells are administered as compared to administration of immune effector cells alone.
  • the tumor inhibition rate is increased by ⁇ 20%, preferably ⁇ 30%.
  • immune effector cells are administered as compared to administration of immune effector cells alone.
  • the level of interferon release is increased by ⁇ 20%, preferably ⁇ 30%, more preferably ⁇ 50%.
  • Figure 1 is a schematic representation of the structure of the recombinant retroviral vector EGFRvIII-28Z.
  • Figure 2 is the efficiency of retrovirus-infected mouse T lymphocytes.
  • Figure 3 is a graph showing the in vitro killing function of EGFRvIII-28Z CAR-T cells.
  • Figure 4A is a graph showing the anti-tumor effect of EGFRvIII-28Z CAR-T cells and poly I:C in a mouse breast cancer model
  • Figure 4B shows EGFRvIII-28Z CAR-T cells and poly I:C in a mouse breast cancer model. Tumor weight after end of anti-tumor experiment in vivo
  • Figure 4C shows IFN ⁇ level in peripheral blood of EGFRvIII-28Z CAR-T cells and poly I:C in vivo in anti-tumor experiments in mouse breast cancer model
  • Figure 4D shows anti-tumor The end of the experiment was the number of CAR-T cell copies in the spleen and tumor of the mouse breast cancer model.
  • Figure 5A is an in vivo anti-tumor effect of EGFRvIII-28Z CAR-T cells and poly I:C in a mouse colon cancer model
  • Figure 5B shows EGFRvIII-28Z CAR-T cells and poly I:C in a mouse colon cancer model Tumor weight after the end of the anti-tumor experiment
  • Figure 5C shows the IFN ⁇ level of peripheral blood after the end of the anti-tumor experiment of EGFRvIII-28Z CAR-T cells and poly I:C in the mouse colon cancer model
  • Figure 5D shows the anti-tumor experiment The CAR-T cell copy number in the spleen and tumor of the mouse colon cancer model was terminated.
  • Figure 6 is a plasmid map of MSCV-EGFRvIII-28Z-FLT3L.
  • Figure 7 shows the effect of EGFRvIII-28Z-FLT3L CAR-T+poly I:C cells in a mouse colon cancer model.
  • Figure 8A shows the growth of colon cancer xenografts after type I interferon is blocked
  • Figure 8B shows the tumor weight of colon cancer xenografts after type I interferon is blocked.
  • Figure 9A shows the growth of breast cancer xenografts after type I interferon is blocked
  • Figure 9B shows the tumor weight of breast cancer xenografts after type I interferon is blocked.
  • Figure 10 shows a comparison of the effects of MDSC inhibitors, CAR T cells, and polyI:C on tumor volume over time in a mouse tumor model (Figure 10A), tumor body weight (Figure 10B), and tumor photo comparison ( Figure 10C). ).
  • TLR agonists TLR agonists
  • chimeric antigen receptors optional MDSC inhibitors and/or immunological checkpoints.
  • MDSC inhibitors optional MDSC inhibitors and/or immunological checkpoints.
  • the combination of inhibitors in the treatment of tumors, infectious diseases and other immune-related diseases has significantly improved immunity to the body and the ability to kill tumors or pathogens.
  • the terms "the agent that increases the efficacy of immune effector cells”, “immune effector cell synergist” are used interchangeably and refer to a drug or formulation that enhances the efficacy of immune effector cells.
  • measurable values such as the amount administered, etc., including ⁇ 20% of the specified value, or in some cases ⁇ 10%, or in some cases ⁇ 5%, or in some cases ⁇ 1%, Or in some cases ⁇ 0.1% change, so such a change is suitable for carrying out the disclosed method.
  • immune effector cells refers to cells involved in an immune response, for example, to promote an immune effector response.
  • immune effector cells include T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytic cells.
  • T cells may be T cells directly derived from peripheral blood, or may be subtypes of T cells, such as CD8+ T cells, CD4+ T cells, ⁇ / ⁇ T cells, ⁇ / ⁇ T cells, and the like.
  • chimeric antigen receptor refers to a group of polypeptides that, when administered in an immune effector cell, provide said cells with specificity for a target cell, typically a cancer cell, and have Intracellular signal production.
  • CAR typically includes at least one extracellular antigen binding domain, a transmembrane domain (also known as a transmembrane domain), and a cytoplasmic signaling domain (also referred to herein as an "intracellular region”), which includes a stimulus derived from the definitions below.
  • Functional signaling domains of sex molecules and/or costimulatory molecules are contiguous with each other.
  • a polypeptide group includes a dimerization switch that can couple the polypeptides to each other in the presence of a dimerization molecule, for example, an antigen binding domain can be coupled to an intracellular signaling domain.
  • the stimulatory molecule is an ⁇ chain that binds to a T cell receptor complex.
  • the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule as defined below.
  • the costimulatory molecule is selected from a costimulatory molecule described herein, such as 4-1BB (ie, CD137), CD27, and/or CD28.
  • a CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and a functional signaling domain comprising a costimulatory molecule and a functionality derived from a stimulatory molecule The intracellular signaling domain of the signaling domain.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and two functional signaling comprising one or more costimulatory molecules.
  • signaling domain refers to a functional portion of a protein that functions by transmitting information within a cell, either by generating a second messenger or by acting in response to such a messenger effector via a determined signal
  • the conduction pathway regulates the activity of the cells.
  • TCR T cell receptor
  • the TCR molecule belongs to the immunoglobulin superfamily, and its antigen specificity exists in the V region; the V region (V ⁇ , V ⁇ ) has three hypervariable regions CDR1, CDR2, and CDR3, among which the CDR3 mutation is the largest, which directly determines the TCR antigen. Binding specificity. When the TCR recognizes the MHC-antigen peptide complex, CDR1, CDR2 recognizes and binds to the side wall of the MHC molecule antigen binding groove, while CDR3 binds directly to the antigen peptide.
  • TCR is divided into two categories: TCR1 and TCR2; TCR1 consists of two chains of ⁇ and ⁇ , and TCR2 consists of two chains of ⁇ and ⁇ . Any T cell expresses only one of TCR2 and TCR1.
  • T CELL FUSION PROTEIN P includes various polypeptide-derived recombinant polypeptides constituting a TCR, which can i) bind to a surface antigen on a target cell, and ii) and intact Other polypeptide interactions of the TCR complex are usually localized on the surface of T cells.
  • TFP consists of an antigen binding domain consisting of a TCR subunit and a human or humanized antibody domain, wherein the TCR subunit comprises at least a portion of the TCR extracellular domain, the transmembrane domain, and the TCR intracellular domain.
  • the TCR TCR subunit is operably linked to the antibody domain, wherein the extracellular, transmembrane, and intracellular signal domains of the TCR subunit are derived from CD3 epsilon or CD3 gamma, and the TFP integration TCR expressed on T cells.
  • an “effective link” is meant a functional link between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of a heterologous nucleic acid sequence.
  • the first nucleic acid sequence is located in a functional regulatory region of the second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence.
  • this promoter can be operably linked to the coding sequence.
  • Linkable DNA sequences can be contiguous with each other, for example, two protein coding regions can be placed in the same reading frame.
  • T CELL ANTIGEN COUPLER includes three functional domains: a tumor targeting domain, including a single-chain antibody, a designed ankyrin repeat protein (DARPin). Or other targeting group 2; is an extracellular domain domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor closer to other TCR receptors; the transmembrane region and the intracellular region of the CD4 co-receptor, wherein The intracellular domain is linked to the protein kinase LCK, which catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as an initial step in T cell activation.
  • ITAMs immunoreceptor tyrosine activation motifs
  • TLR Toll-like receptor
  • PAMP pathogen-associated molecular pattern
  • TLR agonist refers to a molecule that interacts with a TLR and stimulates the activity of the receptor.
  • a synthetic TLR agonist is a compound designed to interact with a TLR and stimulate the activity of the receptor. Examples of TLR agonists include TLR-7 agonists, TLR-3 agonists or TLR-9 agonists.
  • TLR3 The "Toll-like receptor 3 (TLR3) is a member of the Toll-like receptor family, which is capable of specifically recognizing the double-stranded RNA of the virus and its analog Poly(I:C).
  • the dsRNA has the ability to replicate in the virus. Two complementary strands of RNA formed during the cycle.
  • TLR3 Upon recognition, TLR3 induces activation of transcription factors such as NF- ⁇ B and interferon regulatory factor 3 (IRF3) to increase the type I signaling to other cells to increase their antiviral defense. Interferon production.
  • IRF3 interferon regulatory factor 3
  • agonists of TLR3 can also promote the maturation of dendritic cells (DC) cells, which are dendritic cell
  • poly(I:C)) in this document, has the same meaning as Poly I: C, and pIC in the drawing also refers to Poly (I: C)
  • Chinese is a polymuscular liver, an analog of synthetic dsRNA, a double-stranded RNA molecule having a MW distribution of up to, for example, 3,600,000 Daltons.
  • Poly(I:C) as an agonist of TLR3, promotes the growth and antigen presentation of B cells. Furthermore, in the absence of specific antigen stimulation, the TLR3 agonist Poly(I:C) can directly induce B cells to produce IgG1 kappa antibodies.
  • anti-tumor effect mainly depends on CD8+ T cells and NK cells, and can coordinate the induction of DC cells, macrophages/neutrophils, NK cells and T cells and other immune cells to secrete type I interferon to promote anti-resistant Tumor effect.
  • DC dendritic cell
  • progenitor cells in the bone marrow, which migrate as immature cells to peripheral tissues where they endocytose antigens and undergo complex maturation processes.
  • Antigens are endocytosed via a number of surface molecules, including complement receptors (eg, CD11c/CD18) and endocytic receptors (eg, DEC-205, DC-SIGN, and Toll-like receptors).
  • Immature DCs also receive a "danger signal" in the form of a pathogen-associated molecule such as bacterial cell wall lipopolysaccharide (LPS) during antigen acquisition, or inflammatory stimulation via cytokines such as IFN-[gamma].
  • LPS bacterial cell wall lipopolysaccharide
  • the DC then migrates to the secondary lymphoid organs and matures into competent APCs.
  • Receptors such as CD11c/CD18, DEC-205, DC-SIGN, and Toll-like receptors play key roles in Ag capture and presentation, and are predominantly expressed on DCs.
  • dendritic cell maturation agent refers to a substance that induces maturation of immature DC cells to competent APC, including a group selected from the group consisting of: a STING agonist; a TLR agonist such as heat inactivated or formalin.
  • Treated BCG preferably cell wall component of BCG, BCG-derived lip arabinose or BCG component; lipopolysaccharide (LPS) derived from E.
  • LPS lipopolysaccharide
  • coli coli
  • Picibanil OK432 or inactivated Gram-positive or Gram-negative microorganism
  • imidazoquinoline compound preferably imidazoquinolin-4-amine compound, especially 4-amino-2-ethoxymethyl-x-dimethyl-1H-imidazole [ 4,5-c]quinoline-1-ethanol or 1-(2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-amine, or a derivative thereof
  • synthetic double strand Polyribonucleotides preferably poly I:C; natural double-stranded RNA or RNA viruses or RNA fragments, or synthetic analogs, or synthetic or natural nucleic acid molecules comprising unmethylated CpG motifs;
  • Combination of cytokines preferably tumor necrosis factor alpha (TNF- ⁇ ), interferon gamma (IFN- ⁇ ), IL-1, IL-6, IL-12 or prostaglandin E6, CD40L, preferably recombinant CD40L or CD4 a
  • tumor refers to a disease characterized by pathological hyperplasia of cells or tissues, and its subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, and does not induce or inhibit normal cell proliferation.
  • a tumor can affect a variety of cells, tissues or organs including, but not limited to, selected from the group consisting of bladder, bone, brain, breast, cartilage, glial cells, esophagus, fallopian tubes, gallbladder, heart, intestine, kidney, liver, lung, lymph nodes, Nerve tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, testis, thymus, thyroid, trachea, urethra, ureter, urethra, uterus, vaginal organs, or tissue or corresponding cells.
  • Tumors include cancers such as sarcomas, carcinomas, or plasmacytomas (malignant tumors of plasma cells).
  • the tumor of the present invention may include, but is not limited to, leukemia (such as acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, acute promyelocytic leukemia, acute granulocyte-monocytic leukemia, Acute monocytic leukemia, acute leukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, polycythemia vera), lymphoma (Hodgkin's disease, non-Hodgkin's disease), primary macroglobulinemia Disease, heavy chain disease, solid tumors such as sarcoma and cancer (such as fibrosarcoma, mucinous sarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, endotheli
  • the "tumor” includes, but is not limited to, pancreatic cancer, liver cancer, lung cancer, gastric cancer, esophageal cancer, head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, Kidney cancer, leukemia, multiple myeloma, ovarian cancer, cervical cancer and glioma.
  • tumor antigen refers to a molecule (usually a protein, carbohydrate or lipid) that is expressed on the surface of a tumor cell, either in whole or in the form of a fragment (eg, MHC/peptide), and which is used for pharmacological treatment.
  • the agent preferentially targets tumor cells.
  • the tumor antigen may be a marker expressed by normal cells and cancer cells, such as a lineage marker such as CD19 on B cells; the tumor antigen may also be a cell surface molecule that is overexpressed in comparison with normal cells in cancer cells, for example, Normal cells are overexpressed by 1x overexpression, 2x overexpression, 3x or more; tumor antigens can also be cell surface molecules that are improperly synthesized in cancer cells, for example, molecules expressed on normal cells Than a molecule containing a deletion, addition or mutation; the tumor antigen can also be specifically expressed on the cell surface of the cancer cell, either in whole or in fragment form (eg, MHC/peptide), rather than on the surface of normal cells or expression.
  • a lineage marker such as CD19 on B cells
  • the tumor antigen may also be a cell surface molecule that is overexpressed in comparison with normal cells in cancer cells, for example, Normal cells are overexpressed by 1x overexpression, 2x overexpression, 3x or more
  • tumor antigens can also be cell surface molecules that
  • a CAR of the invention comprises a CAR comprising an antigen binding domain (eg, an antibody or antibody fragment) that binds to an MHC presenting peptide.
  • an antigen binding domain eg, an antibody or antibody fragment
  • peptides derived from endogenous proteins fill the pockets of class I molecules of the major histocompatibility complex (MHC) and are recognized by T cell receptors (TCRs) on CD8+ T lymphocytes.
  • MHC major histocompatibility complex
  • TCRs T cell receptors
  • the MHC class I complex is constitutively expressed by all nucleated cells.
  • virus-specific and/or tumor-specific peptide/MHC complexes represent a unique type of cell surface target for immunotherapy.
  • Tumor antigens include, but are not limited to, thyroid stimulating hormone receptor (TSHR); CD171; CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1) Ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); Tn antigen (Tn Ag); CD19; CD20; CD 22; CD 30 CD 70; CD 138; CD 33; CD 44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit alpha (IL-13Ra); 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100;
  • the tumor antigen can be, but is not limited to, EGFRvIII, GPC3, CLD18A2, CD19, CD20, CD22, CD30, WT1, CLDN6, MUC1, BCMA, IL-11Ra, IL-13Ra, FAP.
  • pathogen refers to a protozoan capable of causing a disease, including: a virus, a bacterium, a fungus or a parasite.
  • viral antigen refers to a polypeptide expressed by a virus capable of inducing an immune response.
  • antibody refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen.
  • Antibodies can be polyclonal or monoclonal, multi-stranded or single-stranded, or intact immunoglobulins, and can be derived from natural or recombinant sources.
  • the antibody can be a tetramer of immunoglobulin molecules.
  • antibody fragment refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (eg, by binding, steric hindrance, stabilization/destabilization, spatial distribution).
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, disulfide-linked Fvs (sdFv), Fd fragments consisting of VH and CH1 domains, Linear antibodies, single domain antibodies such as sdAb (VL or VH), camelid VHH domain, multispecific antibodies formed by antibody fragments (eg, bivalent fragments comprising two Fab fragments joined by a disulfide bond in the hinge region) and An isolated CDR or other epitope binding fragment of an antibody.
  • Antigen-binding fragments can also be incorporated into single domain antibodies, maximal antibodies, minibodies, Nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, for example, Hollinger and Hudson, Nature). Biotechnology 23: 1126-1136, 2005).
  • the antigen-binding fragment can also be grafted to a polypeptide-based scaffold, such as type III fibronectin (Fn3) (see U.S. Patent No. 6,703,199, which describes fibronectin polypeptide minibodies).
  • scFv refers to a fusion protein comprising at least one variable region antibody fragment comprising a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein said light and heavy chain variable regions are contiguous (for example, via a synthetic linker such as a short flexible polypeptide linker), and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • a synthetic linker such as a short flexible polypeptide linker
  • an scFv can have the VL and VH variable regions in any order (eg, relative to the N-terminus and C-terminus of the polypeptide), and the scFv can include a VL-linker-VH or A VH-linker-VL can be included.
  • binding domain refers to a protein, eg, an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence.
  • binding domain or “antibody molecule” includes antibodies and antibody fragments.
  • the antibody molecule may be a multispecific antibody molecule, for example, it comprises a plurality of immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence has binding specificity for the first epitope and a plurality of The di-immunoglobulin variable domain sequence has binding specificity for the second epitope.
  • Multispecific antibody molecules can also be bispecific antibody molecules. Bispecific antibodies have specificity for no more than two antigens.
  • a bispecific antibody molecule is characterized by having a binding specific bispecific antibody molecule to a first epitope characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope and for a second epitope A second immunoglobulin variable domain sequence having binding specificity.
  • antigen refers to a molecule that elicits an immune response.
  • the immune response can involve activation of the antibody-producing or cells with specific immunity or both.
  • any macromolecule comprising virtually all proteins or peptides can serve as an antigen.
  • the antigen can be derived from recombinant or genomic DNA.
  • any DNA comprising a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response, thus encoding an "antigen.”
  • the antigen need not be encoded only by the full length nucleotide sequence of the gene. It will be apparent that the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to encode a polypeptide that elicits a desired immune response.
  • the antigen does not need to be encoded by a "gene” at all. It will be apparent that the antigen may be produced synthetically, or may be derived from a biological sample, or may be a macromolecule other than a polypeptide. Such biological samples can include, but are not limited to, tissue samples, tumor samples, cells or liquids with other biological components.
  • anticancer effect refers to a biological effect that can be manifested by a variety of means including, but not limited to, for example, a reduction in tumor volume, a decrease in the number of cancer cells, a decrease in the number of metastases, an increase in life expectancy, a decrease in cancer cell proliferation, and a cancer cell survival rate. Amelioration of various physiological symptoms associated with or associated with cancerous conditions. "Anticancer effects” also prevent the first appearance of cancer by peptides, polynucleotides, cells and antibodies.
  • anticancer effect refers to a biological effect that can be manifested by various means including, but not limited to, for example, a reduction in tumor volume, a decrease in the number of tumor cells, a decrease in tumor cell proliferation, or a decrease in tumor cell survival.
  • derived from means the relationship between the first and second molecules. It generally refers to the structural similarity between the first molecule and the second molecule and does not imply or include limitations on the process or source of the first molecule derived from the second molecule.
  • the intracellular signaling domain maintains a sufficient CD3 ⁇ structure such that it has the desired function, i.e., the ability to generate a signal under appropriate conditions. It does not imply or include limitations on the specific process by which the intracellular signaling domain is produced. For example, it does not imply that in order to provide an intracellular signaling domain, it is necessary to start with a CD3 ⁇ sequence and to delete an unwanted sequence, or to apply Mutation to obtain an intracellular signaling domain.
  • disease associated with expression of a tumor antigen as described herein includes, but is not limited to, a disease associated with expression of a tumor antigen as described herein or a condition associated with a cell expressing a tumor antigen as described herein, Included, for example, are proliferative diseases such as cancer or malignancy or precancerous conditions, such as myelodysplasia, myelodysplastic syndrome or pre-leukemia; or non-cancer-related indications associated with cells expressing tumor antigens as described herein. .
  • the cancer associated with expression of a tumor antigen described herein is a blood cancer.
  • the cancer associated with expression of a tumor antigen described herein is a solid cancer.
  • tumor antigens described herein include, but are not limited to, for example, atypical and/or non-classical cancers, malignant tumors, precancerous conditions or hyperplasia associated with expression of a tumor antigen as described herein.
  • sexual disease Non-cancer related indications associated with expression of a tumor antigen as described herein include, but are not limited to, for example, autoimmune diseases (eg, lupus), inflammatory conditions (allergy and asthma), and transplantation.
  • Tumor antigen expression can be expressed by cells, or expressed at any time, encoding the mRNA of the tumor antigen.
  • Tumor antigen-expressing cells can produce tumor antigen proteins (e.g., wild-type or mutant), and the tumor antigen proteins can be present at normal or reduced levels. Tumor antigen-expressing cells can produce detectable levels of tumor antigen protein at one point and subsequently produce a substantially undetectable tumor antigen protein.
  • tumor antigen proteins e.g., wild-type or mutant
  • Tumor antigen-expressing cells can produce detectable levels of tumor antigen protein at one point and subsequently produce a substantially undetectable tumor antigen protein.
  • stimulation refers to the binding of a stimulatory molecule (eg, a TCR/CD3 complex or CAR) to its cognate ligand (or a tumor antigen in the case of a CAR), thereby mediating signal transduction events (eg, However, it is not limited to the initial response induced via signal transduction of the TCR/CD3 complex or via signal transduction of a suitable NK receptor or CAR signaling domain. Stimulation can mediate altered expression of certain molecules.
  • a stimulatory molecule eg, a TCR/CD3 complex or CAR
  • the term "irritating molecule” refers to a molecule expressed by an immune cell that provides a cytoplasmic signaling sequence that modulates the activation of immune cells for at least some aspects of the immune cell signaling pathway in an irritating manner.
  • the signal is a primary signal initiated by binding of, for example, a TCR/CD3 complex to a peptide-loaded MHC molecule, and which results in a T cell response, including, but not limited to, proliferation, activation, differentiation, and the like.
  • the primary cytoplasmic signaling sequence (also referred to as "primary signaling domain") that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAM-containing cytoplasmic signaling sequences specifically for use in the present invention include, but are not limited to, those derived from CD3 ⁇ , common FcR ⁇ (FCER1G), Fc ⁇ RIIa, FcR ⁇ (FcEpsilon R1b), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, DAP10 and DAP12.
  • the intracellular signaling domain in any one or more of the CARs of the invention comprises an intracellular signaling sequence, such as a CD3- ⁇ primary Signaling sequence.
  • the primary signaling sequence of CD3- ⁇ is an equivalent residue derived from a human or non-human species such as mouse, rodent, monkey, donkey, etc.
  • the primary signaling sequence of CD3- ⁇ is an equivalent residue from a human or non-human species such as mouse, rodent, monkey, donkey, and the like.
  • APC antigen presenting cell
  • MHC major histocompatibility complex
  • T-cells can recognize these complexes using their T-cell receptor (TCR).
  • TCR T-cell receptor
  • intracellular signaling domain refers to an intracellular portion of a molecule.
  • the intracellular signaling domain produces a signal that promotes the immune effector function of cells containing CAR, such as CART cells.
  • immune effector functions in, for example, CART cells include cell lytic activity and helper activity, including secretion of cytokines.
  • the intracellular signaling domain can comprise a first order intracellular signaling domain.
  • Exemplary primary intracellular signaling domains include those derived from molecules responsible for primary stimulation or antigen dependent stimulation.
  • the intracellular signaling domain can comprise a costimulatory intracellular domain.
  • Exemplary costimulatory intracellular signaling domains include those derived from molecules responsible for costimulatory signals or antigen independent stimuli.
  • the primary intracellular signaling domain may comprise a cytoplasmic sequence of a T cell receptor
  • the costimulatory intracellular signaling domain may comprise a cytoplasmic sequence from a co-receptor or a costimulatory molecule.
  • the first-level intracellular signaling domain can include a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.
  • ITAM immunoreceptor tyrosine-based activation motif
  • Examples of primary cytoplasmic signaling sequences containing ITAM 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.
  • ⁇ or alternatively “ ⁇ chain”, “CD3- ⁇ ” or “TCR ⁇ ” is defined as a protein as provided by GenBan Acc. No. BAG36664.1 or from a non-human species (eg mouse, rodent, Equivalent residues of monkeys, baboons, etc.
  • ⁇ stimulating domain or alternatively "CD3- ⁇ stimulating domain” or “TCR- ⁇ stimulating domain” are defined as amino acid residues from the cytoplasmic domain of the ⁇ chain A base or a functional derivative thereof sufficient to functionally deliver the initiation signal required for T cell activation.
  • the cytoplasmic domain of sputum comprises residues 52 to 164 of GenBank Acc. No. BAG36664.1 or from a non-human species (eg, mouse, rodent, monkey, ape, etc.) as a functional homolog thereof Equivalent residue of ).
  • co-stimulatory molecule refers to a homologous binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response of a T cell, such as, but not limited to, proliferation.
  • a costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that promotes an effective immune response.
  • Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) and 4- 1BB (CD137).
  • costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1 CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD22), CD19
  • the costimulatory intracellular signaling domain can be an intracellular portion of a costimulatory molecule.
  • Costimulatory molecules can be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), and NK cell receptors.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, antigen-related antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and ligands that specifically bind to CD83.
  • the intracellular signaling domain may comprise part or all of the native intracellular signaling domain, or a functional fragment or derivative thereof, of all cells within the molecule.
  • 4-1BB refers to a member of the TNFR superfamily having an amino acid sequence as provided by GenBank Acc. No. AAA62478.2, or an equivalent residue from a non-human species such as a mouse, a rodent, a monkey, a donkey, or the like.
  • 4-1BB costimulatory domain is defined as amino acid residues 214-255 of GenBank Acc. No.. AAA62478.2, or equivalents from non-human species such as mice, rodents, monkeys, baboons, etc. Residues.
  • the "4-1BB costimulatory domain” is an equivalent residue from a human or from a non-human species such as a mouse, rodent, monkey, donkey, and the like.
  • encoding refers to the intrinsic property of a particular sequence of a nucleotide in a polynucleotide, such as a gene, cDNA, or mRNA, as a template for the synthesis of other polymers and macromolecules in a biological process, the polymer and macromolecules.
  • a defined nucleotide sequence eg, rRNA, tRNA, and mRNA
  • a defined amino acid sequence and biological properties derived therefrom eg, rRNA, tRNA, and mRNA
  • a non-coding strand whose nucleotide sequence is identical to the mRNA sequence and which is usually provided in the sequence listing and a non-coding strand which is used as a template for the transcription gene or cDNA may be referred to as a protein or other encoding the gene or cDNA.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate forms of each other and that encode the same amino acid sequence.
  • the phrase nucleotide sequence encoding a protein or RNA may also include an intron to the extent that the nucleotide sequence encoding the protein may contain introns in some forms.
  • an effective amount or “therapeutically effective amount” is used interchangeably herein and refers to an amount of a compound, formulation, substance or composition effective to achieve a particular biological result as described herein.
  • expression refers to the transcription and/or translation of a particular nucleotide sequence driven by a promoter.
  • transfer vector refers to a composition of matter that includes an isolated nucleic acid and that can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids and viruses.
  • the term "transfer vector” includes autonomously replicating plasmids or viruses.
  • the term should also be interpreted to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.
  • expression vector refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed.
  • the expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, including cosmids incorporated into recombinant polynucleotides, plasmids (eg, naked or contained in liposomes), and viruses (eg, lentiviruses, retroviruses, glands) Virus and adeno-associated virus).
  • nucleobases normally present below are used.
  • A means adenosine
  • C means cytosine
  • G means guanosine
  • T means thymidine
  • U means uridine.
  • nucleic acid or “polynucleotide” refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form.
  • RNA ribonucleic acid
  • the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, and well-defined sequences.
  • degenerate codon substitutions can be made by generating sequences in which three positions of one or more selected (or all) codons are mixed bases and/or deoxyinosine residues Substituent substitution (Batzer et al, Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al, J. Biol. Chem. 260: 2605-2608 (1985); and Rossolini et al, Mol. Cell. Probes 8: 91-98 ( 1994)).
  • peptide refers to a compound consisting of amino acid residues covalently linked by a peptide bond.
  • the protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can include the sequence of the protein or peptide.
  • a polypeptide includes any peptide or protein comprising two or more amino acids that are bonded to each other by a peptide bond.
  • the term refers to short chains (which are also commonly referred to in the art as, for example, peptides, oligopeptides, and oligomers) and longer chains (which are also commonly referred to in the art as proteins, which are present in many Type).
  • Polypeptide includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, or a combination thereof.
  • the term "treat, treatment, treating” refers to slowing or ameliorating a proliferative disorder by administration of one or more therapies (eg, one or more therapeutic agents, such as the CAR of the invention).
  • therapies eg, one or more therapeutic agents, such as the CAR of the invention.
  • the progression, severity and/or duration, or amelioration of one or more symptoms of the proliferative disorder preferably one or more discernible symptoms.
  • treatment can also refer to at least one measurable physical parameter, such as tumor growth, that ameliorates a proliferative disorder, and is not necessarily discernible by the patient.
  • the term “treating” can also mean inhibiting the progression of a proliferative disorder physically, for example, by stabilizing a discernible symptom, by physiological means, for example, by stabilizing physical parameters, or both.
  • the term “treating” can also mean reducing or stabilizing tumor size or cancer cell count.
  • signal transduction pathway refers to the biochemical relationship between a variety of signal transduction molecules that function in transducing a signal from one part of a cell to another part of the cell.
  • cell surface receptor includes molecular and molecular complexes that are capable of receiving signals and transmitting signals across the cell membrane.
  • subject is meant to include living organisms (eg, mammals, humans) in which an immune response can be elicited.
  • substantially purified cells refers to cells that are substantially free of other cell types.
  • a substantially purified cell refers to a cell that has been isolated from other cell types that are normally associated with its naturally occurring state.
  • a substantially purified population of cells refers to a homologous population of cells.
  • the term refers only to cells that are isolated from cells with which they are naturally associated in their natural state.
  • the cell is cultured in vitro. In other aspects, the cells are not cultured in vitro.
  • treatment refers to treatment.
  • the therapeutic effect is obtained by alleviating, inhibiting, alleviating or eradicating the disease state.
  • prevention refers to the prophylactic or protective treatment of a disease or condition.
  • transfected or transformed or transduced refers to the process by which an exogenous nucleic acid is transferred or introduced into a host cell.
  • Transfected or transformed or transduced refers to the process by which an exogenous nucleic acid is transferred or introduced into a host cell.
  • Transfected or transformed or transduced cells A cell that has been transfected, transformed or transduced with an exogenous nucleic acid, including cells of the primary subject and progeny thereof.
  • the term "specifically binds” refers to an antibody or ligand that recognizes and binds to a binding partner (eg, tumor antigen) protein present in a sample, but the antibody or ligand does not substantially recognize or bind other molecules in the sample. .
  • a range such as 95-99% identity includes a range having 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This does not apply regardless of the width of the range.
  • FLT3L may be human FLT3L, a type IV transmembrane protein consisting of 235 amino acid residues (NCBI Reference Sequence: NP_001191431.1), and the amino acid sequence is set forth in SEQ ID NO: 8; a polypeptide having at least 80% similarity to SEQ ID NO: 8, preferably a polypeptide having at least 85% or at least 90% similarity to SEQ ID NO: 8; said “FLT3L” may also be derived from other species, including but Not limited to rats, monkeys, and pigs.
  • FLT3L may be naturally occurring, such as it may be isolated or purified from a mammal; it may also be artificially prepared, such as recombinant elements or FLT3L, according to conventional genetic engineering recombination techniques.
  • the invention may employ recombinant elements or FLT3L.
  • the present invention provides a composition comprising an immune effector cell, the composition further comprising a TOLL-like receptor agonist having a receptor targeted to recognize a tumor antigen or a pathogen antigen, the TOLL-like receptor agonist
  • the effect of the immune effector cells can be increased in the treatment of a subject, wherein: the receptor has an extracellular region, a transmembrane region, and an intracellular region, wherein the extracellular region has a tumor antigen or An antigen binding domain of a pathogen antigen that binds to a tumor antigen or a pathogen antigen associated with the disease.
  • the compositions of the invention may further comprise an MDSC inhibitor or an immunological checkpoint inhibitor.
  • the "immune effector cells” may be derived from autologous cells or from allogeneic cells.
  • the receptor for recognizing a tumor antigen or a pathogen antigen includes, but is not limited to, a chimeric antigen receptor (CAR), a T cell (antigen) receptor (TCR), a T cell fusion protein (TFP), or a T cell antigen coupler. (TAC).
  • CAR chimeric antigen receptor
  • TCR T cell
  • T cell fusion protein T cell antigen coupler.
  • TAC T cell antigen coupler.
  • the “immune effector cells” may also express other exogenous proteins, for example, the exogenous protein may be, but not limited to, FLT3L, interferon, interleukin, PD-1 or PD-L1 inhibitor.
  • the exogenous protein may be, but not limited to, FLT3L, interferon, interleukin, PD-1 or PD-L1 inhibitor.
  • the presence of these exogenous proteins enhances the ability of "immune effector cells” to fight tumors or against pathogenic microorganisms.
  • the interferon comprises, but is not limited to, a type I interferon; the interleukin comprises, but is not limited to, IL-2, IL-12, IL-21, IL18; the interleukin receptor comprises but Not limited to receptors for IL-4; inhibitors of PD-1 or PD-L1 include, but are not limited to, soluble PD-1 or fusion peptides containing soluble PD-1 (see WO2017080377), anti-PD-1 or anti-PD-1 PD-L1 antibody.
  • the TOLL-like receptor agonist can be, but is not limited to, a Toll-like receptor 3 (TLR3) agonist.
  • TLR3 agonist may be a natural or synthetic double-stranded ribonucleic acid such as polyinosinic acid cytidine (poly(I:C)).
  • the TLR3 agonist may also be a mismatched double-stranded ribonucleic acid poly (I: C11-14U), exemplified by poly (I: C12U), poly (I: C13U).
  • the recognition of a tumor antigen or a pathogen antigen receptor comprises a chimeric antigen receptor (CAR), a T cell (antigen) receptor (TCR), a T cell fusion protein (TFP), or a T cell.
  • CAR chimeric antigen receptor
  • TCR T cell
  • T cell fusion protein T cell
  • TAC Antigen coupler
  • the chimeric antigen receptor may comprise all of the conventional chimeric antigen receptor structures, and the transmembrane region may optionally be a protein or protein fragment having a transmembrane function, and the intracellular region may also be selected according to the prior art. Domains that trigger activation of immune cells, including but not limited to CD3 ⁇ , FcR ⁇ (FCER1G), Fc ⁇ RIIa, FcR ⁇ (FcEpsilon R1b), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, DAP10, DAP12, CD137, CD27, CD28, etc. Full length or active fragment (eg CN201580013987 for a description of the intracellular domain).
  • a chimeric antigen receptor can also have the following structures:
  • the TFP comprises: (a) a TCR subunit comprising a TCR partial extracellular domain, a transmembrane domain, and a TCR intracellular domain, the intracellular domain comprising a stimulation signaling domain (b) an antigen binding domain, the TCR subunit and the antigen binding domain being operably linked.
  • the extracellular, transmembrane and intracellular signal domains of the TCR subunit are derived from CD3 epsilon or CD3 gamma, which integrates into the TCR expressed on T cells.
  • the TAC comprises: (a) an extracellular domain comprising: a single chain antibody having an antigen binding domain and binding to CD3; (b) a transmembrane region; (c) an intracellular domain, The intracellular domain is linked to protein kinase LCK.
  • the immune cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a natural killer T (NKT) cell, a mast cell, or a bone marrow-derived phagocytic cell.
  • the immune cell is a T cell, and the intracellular domain of the receptor is capable of triggering activation of T cells.
  • the invention also provides a composition
  • a composition comprising an immune effector cell expressing a chimeric antigen receptor and a pharmaceutically acceptable carrier or excipient, the composition further comprising a TLR agonist and/or a dendritic cell maturation promoter .
  • the invention provides a composition comprising an immune effector cell, in addition to comprising a TOLL-like receptor agonist, an MDSC inhibitor, an anti-PD-1 antibody, or an antibody against PD-L1.
  • kits comprising an immune effector cell expressing a chimeric antigen receptor, a TLR agonist and/or a dendritic cell maturation promoter, and instructions for how to administer the cell to an individual.
  • the invention also provides a method of treating, controlling, or preventing a tumor or pathogen infection, the method comprising administering to the subject a therapeutically effective amount of an immune effector cell and a TOLL-like receptor agonist.
  • the immune effector cell has a receptor that specifically recognizes a tumor antigen or a pathogen antigen
  • the TOLL-like receptor agonist can increase the efficacy of the immune effector cell in the treatment of the subject, wherein: the receptor Having an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain has an antigen binding domain that recognizes the tumor antigen or pathogen antigen, and the antigen binding domain binds to a tumor antigen or pathogen associated with the disease antigen.
  • the immune effector cells and the TOLL-like receptor agonist may be administered simultaneously, or the immune effector cells may be administered first, then the TOLL-like receptor agonist may be administered, or the TOLL-like receptor agonist may be administered first.
  • the immune effector cells and the TOLL-like receptor agonist may be administered simultaneously, or the immune effector cells may be administered first, then the TOLL-like receptor agonist may be administered, or the TOLL-like receptor agonist may be administered first.
  • the immune effector cells and the TOLL-like receptor agonist may be administered simultaneously, or the immune effector cells may be administered first, then the TOLL-like receptor agonist may be administered, or the TOLL-like receptor agonist may be administered first.
  • the "immune effector cells” may be derived from autologous cells or from allogeneic cells.
  • the receptor for recognizing a tumor antigen or a pathogen antigen includes, but is not limited to, a chimeric antigen receptor (CAR), a T cell (antigen) receptor (TCR), a T cell fusion protein (TFP), or a T cell antigen coupler. (TAC).
  • CAR chimeric antigen receptor
  • TCR T cell
  • T cell fusion protein T cell antigen coupler.
  • TAC T cell antigen coupler.
  • the “immune effector cells” may also express other exogenous proteins, for example, the exogenous protein may be, but not limited to, FLT3L, interferon, interleukin, PD-1 or PD-L1 inhibitor.
  • the exogenous protein may be, but not limited to, FLT3L, interferon, interleukin, PD-1 or PD-L1 inhibitor.
  • the presence of these exogenous proteins enhances the ability of "immune effector cells” to fight tumors or against pathogenic microorganisms.
  • the interferon comprises, but is not limited to, a type I interferon; the interleukin comprises, but is not limited to, IL-2, IL-12, IL-21, IL18; the interleukin receptor comprises but Not limited to receptors for IL-4; inhibitors of PD-1 or PD-L1 include, but are not limited to, soluble PD-1 or fusion peptides containing soluble PD-1 (see WO2017080377), anti-PD-1 or anti-PD-1 PD-L1 antibody.
  • the TOLL-like receptor agonist can be, but is not limited to, a Toll-like receptor 3 (TLR3) agonist.
  • TLR3 agonist may be a natural or synthetic double-stranded ribonucleic acid such as polyinosinic acid cytidine (poly(I:C)).
  • the TLR3 agonist may also be a mismatched double-stranded ribonucleic acid poly (I: C 11-14 U), exemplified by poly (I: C 12 U), poly (I: C 13 U).
  • the chimeric antigen receptor may comprise all of the conventional chimeric antigen receptor structures, and the transmembrane region may optionally be a protein or protein fragment having a transmembrane function, and the intracellular region may also be selected according to the prior art. Domains that trigger activation of immune cells, including but not limited to CD3 ⁇ , FcR ⁇ (FCER1G), Fc ⁇ RIIa, FcR ⁇ (FcEpsilon R1b), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, DAP10, DAP12, CD137, CD27, CD28, etc. Full length or active fragment (eg CN201580013987 for a description of the intracellular domain).
  • a chimeric antigen receptor can also have the following structures:
  • the TFP comprises: (a) a TCR subunit comprising a TCR partial extracellular domain, a transmembrane domain, and a TCR intracellular domain, the intracellular domain comprising a stimulation signaling domain (b) an antigen binding domain, the TCR subunit and the antigen binding domain being operably linked.
  • the extracellular, transmembrane and intracellular signal domains of the TCR subunit are derived from CD3 epsilon or CD3 gamma, which integrates into the TCR expressed on T cells.
  • the TAC comprises: (a) an extracellular domain comprising: a single chain antibody having an antigen binding domain and binding to CD3; (b) a transmembrane region; (c) an intracellular domain, The intracellular domain is linked to protein kinase LCK.
  • the immune cells include, but are not limited to, any one or a combination of T cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, or bone marrow-derived phagocytic cells.
  • the immune cell is a T cell, and the intracellular domain of the receptor is capable of triggering activation of T cells.
  • the invention provides a method of treating, managing, or preventing a tumor or a pathogen infection, the method comprising administering to the subject a therapeutically effective amount of an immune effector cell, a TOLL-like receptor agonist, and administering to the MDSC An inhibitor, an anti-PD-1 antibody, or an antibody against PD-L1.
  • the invention also provides a method for treating a tumor, an infectious disease and other immune-related diseases in combination with a TOLL-like receptor agonist and an immune effector cell, the tumor including but not limited to: pancreatic cancer, liver cancer, lung cancer, gastric cancer, Head and neck squamous cell carcinoma, prostate cancer, colon cancer, breast cancer, lymphoma, gallbladder cancer, kidney cancer, leukemia, myeloma, ovarian cancer, cervical cancer, ovarian cancer, cervical cancer or glioma;
  • Pathogens include, but are not limited to, viruses, bacteria, fungi, protozoa or parasites; preferably, the viruses include: cytomegalovirus, Epstein-Barr virus, human immunodeficiency virus or influenza virus.
  • the TOLL-like receptor agonist is a Toll-like receptor 3 (TLR3) agonist, preferably a natural or synthetic double-stranded ribonucleic acid.
  • TLR3 agonist is polyinosinic acid cytidine (poly(I:C)).
  • the TLR3 agonist may also be a mismatched double-stranded ribonucleic acid, exemplified by, for example, poly(I:C12U), poly(I:C13U).
  • the amount of the mismatched double-stranded ribonucleic acid is sufficient to bind to Toll-like receptor 3 (TLR3) and reduce or eliminate proliferation of the tumor or other transformed cell in the subject.
  • TLR3 Toll-like receptor 3
  • the TOLL-like receptor agonist has a synergistic effect on the immune effector cells.
  • the TOLL-like receptor agonist and the immune effector cells may be administered in the same or different manner.
  • the method of administration is co-administering the TOLL-like receptor agonist and the immune effector cells; or administering the immune effector cells first, and then administering the TOLL-like receptor agonist; or administering the TOLL-like sample first.
  • the receptor agonist is administered to the immune effector cell.
  • the method of administration is to first administer the immune effector cells and then to the TOLL-like receptor agonist.
  • the TOLL-like receptor agonist can be administered in an amount from 1 mg/kg to 4 mg/kg, preferably from 2 mg/kg to 3 mg/kg, more preferably from 2.08 mg/kg to 2.77 mg/ Kg. In a preferred embodiment, the TOLL-like receptor agonist is administered in a number of 2 to 4 times, preferably 2-3 times.
  • the immune cells are selected from the group consisting of T cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells or bone marrow-derived phagocytic cells, preferably T cells.
  • the immune cell is a T cell, and the intracellular domain of the receptor is capable of triggering activation of T cells.
  • the recognition of a tumor antigen or a pathogen antigen receptor comprises a chimeric antigen receptor (CAR), a T cell (antigen) receptor (TCR), a T cell fusion protein (TFP), or a T cell.
  • Antigen coupler (TAC) the immune cells may be CAR-expressing T cells, TCR-expressing T cells, CAR-expressing NK cells, TFP-expressing T cells, TFP-expressing NK cells, TAC-expressing T cells, NK cells expressing TAC.
  • the chimeric antigen receptor may be, but is not limited to, the following structures: (i) an antibody that specifically binds to the antigen, a transmembrane region of CD28, a costimulatory signal domain of CD28, and CD3 ⁇ ; or (ii) specific An antibody that binds to the antigen, a transmembrane region of CD28, a costimulatory signal domain of CD137, and CD3 ⁇ ; or (iii) an antibody that specifically binds to the antigen, a transmembrane region of CD28, a costimulatory signal domain of CD28 , the co-stimulatory signal domain of CD137 and CD3 ⁇ .
  • the TFP comprises: (a) a TCR subunit comprising a TCR partial extracellular domain, a transmembrane domain, and a TCR intracellular domain, said intracellular structure The domain includes a stimulation signaling domain; (b) an antigen binding domain, the TCR subunit and the antigen binding domain being operably linked.
  • the extracellular, transmembrane and intracellular signal domains of the TCR subunit are derived from CD3 epsilon or CD3 gamma, which integrates into the TCR expressed on T cells.
  • the TAC comprises: (a) an extracellular domain comprising: a single chain antibody having an antigen binding domain and binding to CD3; (b) a transmembrane region; c) an intracellular domain that links to protein kinase LCK.
  • Exemplary antigen receptors of the present invention including CAR, and methods for engineering and introducing a receptor into a cell, are described, for example, in Chinese Patent Application Publication No. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A. , CN105331585A, CN106397593A, CN106467573A, CN104140974A, International Patent Application Publication No. WO2017186121A1, WO2018006882A1, WO2015172339A8, WO2018018958A1.
  • the target of EGFRvIII-acting CAR-T cells was selected, and in order to more accurately verify the anti-tumor effect in mice, the selected signal peptide, transmembrane region, intracellular region and the like were mouse-derived.
  • the method of preparation is operated according to conventional CAR-T cell preparation methods in the art.
  • Mouse CD8 ⁇ signal peptide (SEQ ID NO: 1), anti-EGFRvIII monoclonal antibody (SEQ ID NO: 2), CD8 ⁇ gelatin region and transmembrane region (SEQ ID NO: 3), CD28 intracellular domain (SEQ ID) NO: 4), CD3 sputum intracellular domain (SEQ ID NO: 5) was ligated in turn, the EGFRvIII-28Z gene fragment was obtained by in vitro gene synthesis, and the retroviral vector MSCV- was replaced with Mlu I and Sal I double restriction sites. The IRES-GFP fragment in IRES-GFP obtained the recombinant vector MSCV-EGFRvIII-28Z. The order of combination of the constituent segments is as shown in FIG.
  • mice Take healthy Balb/c mice, remove the spleen after cervical dislocation, and prepare the spleen cells into a single cell suspension; add 200 ul of red blood cell lysate and use the mouse CD3+ T cell sorting kit (STEMCELL, #19851) The mouse spleen T lymphocytes were obtained by sorting.
  • Mouse CD3+ T lymphocytes were purified and added to Dynabeads Mouse T-activator CD3/CD28 in a ratio of 1:1, and cultured in RPMI 1640 complete medium for 24 hours.
  • Each well of a 48-well plate was added to 400 ⁇ l of retroNectin solution (5 ⁇ g/mL) overnight, and then retroNectn was discarded; the spleen T lymphocytes activated for 24 hours were inoculated into the plate, the number of cells per well was 1 ⁇ 106, and 1 ml of reverse transcription was added.
  • the virus was supplemented to 2 mL, cultured and subcultured after centrifugation.
  • the in vitro cytotoxicity of EGFRvIII-28Z CAR T lymphocytes to target cells was determined using a cytoTox 96 non-radioactive cytotoxicity assay kit (Promega, Madison, USA).
  • the target cells were adjusted to a concentration of CT26, CT26-EGFRvIII, E0771, E0771-EGFRvIII of 2 ⁇ 105/mL, and 50 ⁇ L was inoculated into a 96-well plate; the target ratio was 0.3:1, 1:1, and 3:1 to the 96-well plate.
  • Add CAR-T cells and control T cells 50 ul), set each control well according to the kit instructions, and set 5 replicate wells in each group; incubate for 18 hours. After the incubation, the cytotoxicity of EGFRvIII-28Z CAR T lymphocytes to target cells was determined according to the kit instructions. The results are shown in Figure 3.
  • EGFRvIII-28Z CAR-T cells showed no significant toxicity to CT26 and E0771 cells with negative expression of EGFRvIII, but showed strong killing effect on CT26-EGFRvIII and E0771-EGFRvIII cells expressing EGFR-vIII target antigen.
  • the EGFRvIII-28Z CAR-T cells killed 71% and 67.8% of the two cells, respectively; the uninfected T cells showed no significant killing activity on the target cells.
  • the average tumor volume of each group was about 150mm 3 ; after grouping, 5.0 ⁇ 10 6 EGFRvIII-28Z CAR-T cells were infused through the tail vein (day 0 of the injection day), while the UT cell group was used as a control, and the CAR-T cells were returned after the third and sixth cells. 50 ⁇ g of polyI:C was injected into the tumor, and the UT cell group was intratumorally injected with the same dose of physiological saline. The growth of the transplanted tumor was observed, and the results are shown in Fig. 4A.
  • the tumor weight of the combined group of Poly: IC was decreased as compared with the administration of CAR-T alone. 77.8%
  • mice peripheral blood was taken for measurement of IFN ⁇ levels, and as shown in Fig. 4C, spleen and tumor tissues were taken, and CAR-T cell copy number was measured, and the results are shown in Fig. 4D.
  • mice 6-week-old female Balb/c mice (purchased from Shanghai Lingchang Biotechnology Co., Ltd.) were inoculated with 3 ⁇ 105 CT26-EGFRvIII cells subcutaneously after 3Gy gamma ray irradiation (ie, lymphocyte clearance); tumor inoculation for 13 days
  • the tumor-bearing mice were divided into 4 groups, which were divided into UT cell group, UT+poly I:C group, EGFRvIII-28Z CAR-T cell group and EGFRvIII-28Z CAR-T+poly I:C group.
  • the volume average was approximately 150 mm3; after grouping, 5.0 ⁇ 106 EGFRvIII-28Z CAR-T cells were infused through the tail vein, while the U T cell group was used as a control, and the intratumoral injection was performed on the 3rd and 6th days after CAR-T cell reinfusion. 50ug polyI: C.
  • the therapeutic effect of GFRvIII-28Z CAR-T combined with poly I:C on CT26-EGFRvIII xenografts is shown in Figure 5.
  • the tumor weight of the combined group of Poly: IC was decreased as compared with the administration of CAR-T alone. 48.5%.
  • Fig. 5C peripheral blood was taken for measurement of IFN ⁇ levels, and as shown in Fig. 5C, spleens and tumor tissues were taken, and CAR-T cell copy number was measured, and the results are shown in Fig. 5D.
  • the mouse muFLT3L gene fragment (SEQ ID No: 6) was ligated with the gene sequence of EGFRvIII-28Z constructed in Example 1 by F2A (SEQ ID No: 7), ie, F2A, according to a conventional CAR-T cell preparation method.
  • F2A SEQ ID No: 7
  • the EGFRvIII-28Z-FLT3L retroviral vector MSCV-EGFRvIII-28Z-FLT3L was constructed.
  • the plasmid map is shown in Figure 6.
  • the mouse spleen T lymphocytes were infected to prepare the expression EGFRvIII- 28Z-FLT3L CAR T cells.
  • Example 4 a mouse colon cancer model was constructed, and the tumor-bearing mice were divided into EGFRvIII-28Z CAR-T cells + poly I: C group and EGFRvIII-28Z-FLT3L CAR-T + poly I: C cell group.
  • a mouse breast cancer model and a colon cancer model were constructed, respectively.
  • the antibody using the interferon receptor alpha chain (clone number MAR1-5A3, purchased from Bioxcell) was used as a blocking agent to block the release of type I interferon.
  • the mouse intestinal cancer model was divided into UT group (injected saline), EGFRvIII-28Z CAR-T cells + poly I:C group, and EGFRvIII-28Z CAR-T cells + poly I:C group + interferon receptor alpha Chain sealer.
  • 50ug polyI:C was injected intratumorally, and on the 0th day and the 2nd day after administration of poly I:C or normal saline, 50ug of interferon receptor alpha chain was blocked by intratumoral injection.
  • Agent Tumor growth was measured and the results are shown in Figure 8A.
  • the experiment was sacrificed, and after the tumors were isolated, the tumor weight was measured, and the results are shown in Fig. 8B.
  • Mouse breast cancer models were divided into UT group (injected saline), EGFRvIII-28Z CAR-T cells + poly I:C group, and EGFRvIII-28Z CAR-T cells + poly I:C group + interferon receptor alpha Chain sealer.
  • 50ug polyI:C was injected intratumorally, and on the 0th day and the 2nd day after administration of poly I:C or normal saline, intratumoral injection of interferon receptor ⁇ chain blocker was given.
  • Tumor growth was measured and the results are shown in Figure 9A.
  • the experiment was sacrificed, and after the tumors were isolated, the tumor weight was measured, and the results are shown in Fig. 9B.
  • Example 4 a mouse colon cancer model was constructed.
  • the MDSC scavenger anti-Gr1 antibody purchased from BioXell e, clone number RB6-8C5 was used to inhibit MDSC.
  • MDSC is a bone marrow-derived suppressor cell and is positive for CD11b+Gr1+.
  • the anti-Gr1 antibody alone did not have significant antitumor activity compared to the UT group.
  • the mouse intestinal cancer model was divided into UT group (injected saline), UT+anti-Gr1 group (administered anti-Gr1 antibody and normal saline), CAR-T+poly I:C group (administered to EGFRvIII-28Z-CAR- T and poly I: C), CAR-T+anti-Gr1 group (administered EGFRvIII-28Z-CAR-T and anti-Gr1 antibodies), CAR-T+poly I: C+anti-Gr1 group (administered EGFRvIII-28Z) -CAR-T, poly I: C and anti-Gr1 antibodies).
  • anti-Gr1 antibody was intraperitoneally injected into 10 mg/kg mice, followed by three injections per week for a total of two weeks.
  • 50ug polyI:C was injected into the tumor.
  • the above examples select CAR-T cells that target EGFR, it being understood that selection of CAR-T cells acting on other targets also has the same effect, such as GPC3, CLD18A2, CD19, BCMA, and the like.
  • the antibody to be used may be mouse anti-human or humanized, and the transmembrane domain and the intracellular domain may be different species depending on the purpose, such as human.
  • the T cells can also express other cytokines that enhance CAR-T cell function, such as CAR-T cells co-expressed by CAR and type I interferons. CAR-T cells co-expressed by CAR and PD1.
  • NK cells and NK-T cells may also be selected, and specific subtypes of immune cells, such as ⁇ / ⁇ T cells, may be specifically selected. Wait.
  • the above embodiment selects a CAR of a murine source, but its signal peptide, hinge region, transmembrane region, and the like may be selected from other species depending on the purpose. These include, but are not limited to, human signal peptides, hinge regions, transmembrane domains, intracellular regions. Antibodies can also be selected for murine anti- or humanized antibodies or whole human antibodies against different targets for different purposes.

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Abstract

本发明提供了TOLL样受体激动剂和免疫效应细胞联合在制备抗肿瘤药物或抗病原体药物或抗免疫低下的药物中的用途，TOLL样受体激动剂可以增强免疫效应细胞的效应，本发明还提供了TOLL样受体激动剂和免疫效应细胞的组合物。

Description

TOLL样受体激动剂与免疫效应细胞的联用
本申请要求CN201710344558.5(申请日：2017.5.16)以及CN201710561825.4(申请日：2017.7.11)两个专利的优先权，通过引用的形式将其全部并入本文。
技术领域
本发明属于免疫治疗领域；涉及具有靶向识别肿瘤抗原或病原体抗原的免疫效应细胞和TOLL样受体激动剂的联合应用。
背景技术
近年来，基因工程化的免疫细胞越来越多的应用于肿瘤治疗领域或者感染性疾病治疗领域，如嵌合抗原受体修饰的T细胞(CAR-T细胞)、嵌合抗原受体修饰的NK细胞(CAR-NK细胞)、TCR修饰的T细胞(TCR-T)等。
但由于肿瘤微环境的复杂性，在肿瘤治疗特别是实体瘤治疗中，免疫效应细胞在体内通常不能较好的发挥抗肿瘤作用。
发明内容
本发明目在于提供一种TOLL样受体激动剂和免疫效应细胞联合应用在肿瘤、传染病及其他免疫相关性疾病的治疗中的用途。TOLL样受体激动剂能够增加免疫效应细胞的功效。
在本发明的第一方面，提供了包含免疫效应细胞和所述免疫效应细胞增效剂的组合物，所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗原的受体，所述免疫效应细胞增效剂为TOLL样受体激动剂。
具体地，本发明提供了包含免疫效应细胞的组合物，所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗原的受体，所述免疫效应细胞与在具有与肿瘤抗原或病原体抗原的表达相关的疾病的受试者的治疗中增加所述免疫效应细胞的功效的作用剂组合使用，其中：
(i)所述受体具有胞外区、跨膜区和胞内区，其中，胞外区具有抗原结合结构域，所述抗原结合结构域结合至与该疾病相关的肿瘤抗原或病原体抗原；
(ii)增加免疫效应细胞功效的所述作用剂为TOLL样受体激动剂。
在优选的实施方案中，所述受体的胞内区具有参与引发免疫细胞活化的结 构域。
在优选的实施方案中，所述TOLL样受体激动剂为Toll样受体3(TLR3)激动剂。
在优选的实施方案中，所述的TLR3激动剂选自天然或合成的双链核糖核酸。
在一优选例中，所述的TLR3激动剂为聚肌苷酸胞苷酸(poly(I:C))。
在一优选例中，所述的TLR3激动剂为错配的双链核糖核酸poly(I:C11-14U)，示例性的，如poly(I:C12U)、poly(I:C13U)。
在优选的实施方案中，所述的识别肿瘤抗原或病原体抗原受体包括嵌合抗原受体(CAR)、T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、或T细胞抗原耦合器(TAC)。
在一优选例中，所述嵌合抗原受体包括：
(i)特异性结合所述抗原的抗体、CD28的跨膜区、CD28的共刺激信号结构域和CD3ζ胞浆信号传导结构域；或
(ii)特异性结合所述抗原的抗体、CD28的跨膜区、CD137的共刺激信号结构域和CD3ζ胞浆信号传导结构域；或
(iii)特异性结合所述抗原的抗体、CD28的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ胞浆信号传导结构域。
在优选的实施方案中，所述的TFP包括:(a)TCR亚基，所述TCR亚基包括TCR部分胞外结构域，跨膜结构域，和TCR胞内结构域，所述胞内结构域包括刺激信号传导结构域；(b)抗原结合结构域，所述TCR亚基和所述抗原结合结构域有效连接，所述TCR亚基的胞外、跨膜和胞内信号结构域来源于CD3ε或CD3γ，所述TFP整合进T细胞上表达的TCR。
在优选的实施方案中，所述的TAC包括：(a)胞外结构域：所述胞外结构域包括具有抗原结合结构域和与CD3结合的单链抗体；(b)跨膜区；(c)胞内结构域，所述胞内结构域连接蛋白激酶LCK。
在优选的实施方案中，所述的免疫效应细胞选自T细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞或骨髓源性吞噬细胞。
在一优选例中，所述的免疫细胞为T细胞，所述的受体的胞内域能引发T细胞的活化。
在另一优选例中，所述的免疫效应细胞还表达其他外源性蛋白，优选的，所述外源性蛋白选自下组：FLT3L、干扰素、白介素、白介素受体、PD-1抑制 剂(如PD-1的抗体)、PD-L1抑制剂(如PD-L1的抗体、可溶性的PD-1、或者含有可溶性的PD-1的蛋白)、或其组合。
在另一优选例中，所述组合物还包括MDSC抑制剂、和/或免疫检查点抑制剂。
在另一优选例中，所述MDSC抑制剂选自下组：抗Gr1抗体、环氧化酶-2抑制剂、前列腺素类干细胞因子抑制剂，巨噬细胞集落刺激因子抑制剂、粒单核细胞集落刺激因子抑制剂、血管内皮细胞生长因子抑制剂、或其组合。
在另一优选例中，所述免疫检查点抑制剂选自下组：PD-1抑制剂(如PD-1的抗体)、PD-L1抑制剂(如PD-L1的抗体)、CTLA-4抑制剂(如CTLA-4抗体)、或其组合。
在另一优选例中，所述的免疫效应细胞是TCR敲除或者PD-1敲除的T细胞。
在另一优选例中，所述的免疫效应细胞中的TCR基因或PD-1基因的表达是被沉默的。
在另一优选例中，所述“基因的表达是被沉默的”指被沉默基因不表达或低表达。
在另一优选例中，所述“低表达”指所述免疫效应细胞被沉默基因表达量G1与正常T细胞相应基因表达量G0的比值，即G1/G0≤0.5，较佳地G1/G0≤0.3，更佳地≤0.2，更佳地≤0.1，最佳地为0。
在本发明的第二方面，还提供了免疫效应细胞和免疫效应细胞增效剂的组合在制备抗肿瘤药物或抗病原体药物或抗免疫力低下的药物中的用途，所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗原的受体，所述免疫效应细胞增效剂是TOLL样受体激动剂。
具体地，本发明提供TOLL样受体激动剂和免疫效应细胞的组合在制备抗肿瘤药物或抗病原体药物或抗免疫力低下的药物中的用途，所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗原的受体，该受体具有胞外区、跨膜区和胞内区，所述的胞外区具有结合所述的肿瘤抗原或病原体抗原的抗原结合结构域。
在优选的实施方案中，所述TOLL样受体激动剂为Toll样受体3(TLR3)激动剂，所述的TLR3激动剂优选自天然或合成的双链核糖核酸。
在一优选例中，所述的TLR3激动剂为聚肌苷酸胞苷酸(poly(I:C))。
在一优选例中，所述的TLR3激动剂为错配双链核糖核酸，示例性的，如 poly(I:C12U)、poly(I:C13U)。
在一优选例中，所述错配双链核糖核酸的量足以与Toll样受体3(TLR3)结合并且减少或消除所述受试对象中的所述肿瘤或其它转化细胞的增殖。
在优选的实施方案中，所述TOLL样受体激动剂对所述免疫效应细胞具有协同作用。
在优选的实施方案中，所述TOLL样受体激动剂和所述免疫效应细胞的给药方式为非胃肠道给药，优选静脉注射和瘤内注射；从而所述TOLL样受体激动剂和所述免疫效应细胞可制备成非胃肠道给药，优选静脉注射和瘤内注射给药的药物。
在优选的实施方案中，所述TOLL样受体激动剂和所述免疫效应细胞的给药方式可以相同，也可以不同。
在优选的实施方案中，给药方法为共同给予所述TOLL样受体激动剂和所述免疫效应细胞，或者先给予所述免疫效应细胞，再给予所述TOLL样受体激动剂；或者先给予所述TOLL样受体激动剂，再给予所述免疫效应细胞。
在一优选例中，所述的给药方法为先给予所述免疫效应细胞，再给予所述TOLL样受体激动剂。
在优选的实施方案中，所述的肿瘤抗原选自EGFRvIII、GPC3、CLD18A2、CD19、CD20、CD22、CD30、WT1、CLDN6、MUC1、BCMA、IL-11Ra、IL-13Ra、FAP。
在优选的实施方案中，所述TOLL样受体激动剂给药量可以是1mg/kg～4mg/kg，优选2mg/kg～3mg/kg，更优的选自2.08mg/kg-2.77mg/kg。
在优选的实施方案中，所述TOLL样受体激动剂的给药次数为2～4次，优选2-3次。
在优选的实施方案中，所述的免效应疫细胞选自T细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞或骨髓源性吞噬细胞，优选T细胞。
在一优选例中，所述的免疫效应细胞为T细胞，所述的受体的胞内域能引发T细胞的活化。
在优选的实施方案中，所述的识别肿瘤抗原或病原体抗原受体包括嵌合抗原受体(CAR)、T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、或T细胞抗原耦合器(TAC)。
在优选的实施方案中，所述的免疫效应细胞选自表达CAR的T细胞、表达TCR的T细胞、表达CAR的NK细胞，表达TFP的T细胞、表达TFP的NK细胞、表 达TAC的T细胞、表达TAC的NK细胞。
在一优选例中，所述嵌合抗原受体(CAR)包括：
(i)特异性结合所述抗原的抗体、CD28的跨膜区、CD28的共刺激信号结构域和CD3ζ胞浆信号传导结构域；或
(ii)特异性结合所述抗原的抗体、CD28的跨膜区、CD137的共刺激信号结构域和CD3ζ胞浆信号传导结构域；或
(iii)特异性结合所述抗原的抗体、CD28的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ胞浆信号传导结构域。
在优选的实施方案中，所述的TFP包括：(a)TCR亚基，所述TCR亚基包括TCR部分胞外结构域，跨膜结构域，和TCR胞内结构域，所述胞内结构域包括刺激信号传导结构域；(b)抗原结合结构域，所述TCR亚基和所述抗原结合结构域有效连接，所述TCR亚基的胞外、跨膜和胞内信号结构域来源于CD3ε或CD3γ，所述TFP整合进T细胞上表达的TCR。
在优选的实施方案中，所述的TAC包括：(a)胞外结构域：所述胞外结构域包括具有抗原结合结构域和与CD3结合的单链抗体；(b)跨膜区；(c)胞内结构域，所述胞内结构域连接蛋白激酶LCK。
在优选的实施方案中，所述的免疫效应细胞还表达有其他蛋白，优选的，如PD1、PDL1的抗体、I型干扰素等。
在另一优选例中，所述药物还包括MDSC抑制剂或免疫检查点抑制剂。
在另一优选例中，所述MDSC抑制剂选自下组：抗Gr1抗体、环氧化酶-2抑制剂、前列腺素类干细胞因子抑制剂，巨噬细胞集落刺激因子抑制剂、粒单核细胞集落刺激因子抑制剂、血管内皮细胞生长因子抑制剂、或其组合。
在另一优选例中，所述免疫检查点抑制剂选自下组：PD-1抑制剂(如PD-1的抗体)、PD-L1抑制剂(如PD-L1的抗体)、CTLA-4抑制剂(如CTLA-4抗体)、或其组合。
在本发明的第三方面，还提供了免疫效应细胞、免疫效应细胞增效剂和淋巴细胞清除剂的组合在制备抗肿瘤或抗病原体或抗免疫力低下的药物中的用途。
在优选的实施方式中，在将免疫效应细胞和免疫效应细胞增效剂给予有此需要的对象之前，先给予淋巴细胞清除剂。
在优选的实施方式中，所述免疫效应细胞增效剂是TOLL样受体激动剂。
具体地，本发明还提供了免疫效应细胞、TOLL样受体激动剂和淋巴细胞清除剂的组合在制备抗肿瘤药物或抗病原体药物或抗免疫力低下的药物中的用途，所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗原的受体，该受体具有胞外区、跨膜区和胞内区，所述的胞外区具有结合所述的肿瘤抗原或病原体抗原的抗原结合结构域。
在优选的实施方式中，在给予TOLL样受体激动剂和免疫效应细胞之前，先给予淋巴细胞清除剂。
在优选的实施方式中，所述淋巴细胞清除剂包括清除淋巴细胞的药物或者进行辐射处理。在一优选例中，清除淋巴细胞的药物包括但不限于氟达拉滨和/或环磷酰胺。
在优选的实施方式中，将TOLL样受体激动剂和免疫效应细胞给予有此需要的对象包括同时给予或顺次给予；例如，先给予TOLL样受体激动剂再给予免疫效应细胞，或者先给予免疫效应细胞再给予TOLL样受体激动剂；更优选地，先给予免疫效应细胞再给予TOLL样受体激动剂。
在本发明的第四方面，还提供了治疗肿瘤或病原体感染或免疫力低下的方法，所述方法包括：将免疫效应细胞和免疫效应细胞增效剂的组合给予有此需要的对象；或者
将免疫效应细胞、免疫效应细胞增效剂和淋巴细胞清除剂的组合给予有此需要的对象，其中，在将免疫效应细胞和免疫效应细胞增效剂给予有此需要的对象之前，先给予淋巴细胞清除剂。
具体地，本发明提供治疗肿瘤或病原体感染或免疫力低下的方法，所述方法包括：
将免疫效应细胞和TOLL样受体激动剂的组合给予有此需要的对象，所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗原的受体，该受体具有胞外区、跨膜区和胞内区，所述的胞外区具有结合所述的肿瘤抗原或病原体抗原的抗原结合结构域；或者
将免疫效应细胞、TOLL样受体激动剂和淋巴细胞清除剂的组合给予有此需要的对象。
在优选的实施方案中，所述TOLL样受体激动剂为Toll样受体3(TLR3)激动剂，所述的TLR3激动剂优选自天然或合成的双链核糖核酸。
在一优选例中，所述的TLR3激动剂为聚肌苷酸胞苷酸(poly(I:C))。
在一优选例中，所述的TLR3激动剂为错配双链核糖核酸，示例性的，如poly(I:C12U)、poly(I:C13U)。
在一优选例中，所述错配双链核糖核酸的量足以与Toll样受体3(TLR3)结合并且减少或消除所述受试对象中的所述肿瘤或其它转化细胞的增殖。
在优选的实施方案中，所述TOLL样受体激动剂对所述免疫效应细胞具有协同作用。
在优选的实施方式中，将TOLL样受体激动剂和免疫效应细胞给予有此需要的对象包括同时给予或顺次给予；例如，先给予TOLL样受体激动剂再给予免疫效应细胞，或者先给予免疫效应细胞再给予TOLL样受体激动剂；更优选地，先给予免疫效应细胞再给予TOLL样受体激动剂。
本发明第五方面提供了一种组合物产品，其特征在于，包括：
(i)第一药物组合物，所述第一药物组合物包括至少一种免疫效应细胞增效剂，所述免疫效应细胞增效剂包括TOLL样受体激动剂；和药学上可接受的载体；
(ii)第二药物组合物，所述第二药物组合物包括免疫效应细胞，所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗原的受体；和药学上可接受的载体；和
(iii)第三药物组合物，所述第三药物组合物包括MDSC抑制剂或免疫检查点抑制剂；和药学上可接受的载体。
在另一优选例中，所述第一药物组合物、第二药物组合物、第三药物组合物是混合或独立存在的。
在另一优选例中，所述的第一药物组合物、第二药物组合物和第三药物组合物的总含量为70～100wt％，较佳地为80～100wt％，更佳地为90～100wt％，按所述组合物产品的总重量计。
在另一优选例中，所述免疫效应细胞的给药量为1×10 6－1×10 12细胞/kg，更佳地，1×10 7－1×10 10细胞/kg。
在另一优选例中，所述MDSC抑制剂选自下组：抗Gr1抗体、环氧化酶-2抑制剂、前列腺素类干细胞因子抑制剂，巨噬细胞集落刺激因子抑制剂、粒单核细胞集落刺激因子抑制剂、血管内皮细胞生长因子抑制剂、或其组合。
在另一优选例中，所述免疫检查点抑制剂选自下组：PD-1抑制剂(如PD-1 的抗体)、PD-L1抑制剂(如PD-L1的抗体)、CTLA-4抑制剂(如CTLA-4抗体)、或其组合。
在另一优选例中，所述组合物产品是用于治疗肿瘤或病原体感染或免疫力低下的药物组合物。
在另一优选例中，所述的组合物的剂型包括注射剂型、外用药物剂型和口服剂型。
在另一优选例中，所述组合物可以通过皮下注射、静脉注射、肌内注射的方式给药。
在另一优选例中，所述口服剂型包括片剂、胶囊剂、膜剂、和颗粒剂。
在另一优选例中，所述组合物的剂型包括缓释型剂型、和非缓释型剂型。
本发明第六方面提供了一种本发明第五方面所述的组合物产品的用途，用于制备抗肿瘤药物或抗病原体药物或抗免疫低下的药物。
在另一优选例中，所述组合物产品中，所述免疫效应细胞增效剂的给药量可以是1mg/kg～4mg/kg，优选2mg/kg～3mg/kg，更优的选自2.08mg/kg-2.77mg/kg。
在另一优选例中，所述组合物产品中，所述免疫效应细胞的给药量为1×10 6－1×10 12细胞/kg，更佳地，1×10 7－1×10 10细胞/kg。
在另一优选例中，所述组合物产品中，所述MDSC抑制剂的给药量为2mg/kg-5mg/kg，较佳地，2mg/kg-4mg/kg。
在另一优选例中，所述组合物产品中，所述免疫效应细胞增效剂的给药次数为2～4次，优选2-3次。
在另一优选例中，所述组合物产品中，所述免疫效应细胞的给药次数为2～4次，优选2-3次。
在另一优选例中，所述组合物产品中，所述MDSC抑制剂的给药次数为为2～4次，优选2-3次。
本发明第七方面提供了一种提高免疫效应细胞活力的方法，其特征在于，包括步骤：
给需要的对象施用(i)免疫效应细胞；(ii)免疫效应细胞增效剂；和/或(iii)任选的MDSC抑制剂或免疫检查点抑制剂，其中所述免疫效应细胞具有靶向识别肿 瘤抗原或病原体抗原的受体，所述免疫效应细胞增效剂为TOLL样受体激动剂。
在另一优选例中，所述对象包括人或非人哺乳动物。
在另一优选例中，所述非人哺乳动物包括啮齿动物和灵长目动物，优选小鼠、大鼠、兔、猴。
在另一优选例中，所述组分(i)、组分(ii)、组分(iii)同时或先后施用。
在另一优选例中，与单独施用免疫效应细胞相比，施用(i)免疫效应细胞；(ii)免疫效应细胞增效剂；和(iii)任选的MDSC抑制剂或免疫检查点抑制剂的抑瘤率提高了≥20％，较佳地，≥30％。
在另一优选例中，与单独施用免疫效应细胞相比，施用(i)免疫效应细胞；(ii)免疫效应细胞增效剂；和(iii)任选的MDSC抑制剂或免疫检查点抑制剂的干扰素释放水平提高了≥20％，较佳地，≥30％，更佳地，≥50％。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1是重组逆转录病毒载体EGFRvIII-28Z结构示意图。
图2是逆转录病毒感染小鼠T淋巴细胞效率。
图3是EGFRvIII-28Z CAR-T细胞体外杀伤功能检测。
图4A是显示了小鼠乳腺癌模型中EGFRvIII-28Z CAR-T细胞与poly I:C体内抗肿瘤作用；图4B显示了小鼠乳腺癌模型中EGFRvIII-28Z CAR-T细胞与poly I:C体内抗肿瘤实验结束后的肿瘤重量；图4C显示了小鼠乳腺癌模型中EGFRvIII-28Z CAR-T细胞与poly I:C体内抗肿瘤实验结束后外周血的IFNγ水平；图4D显示了抗肿瘤实验结束小鼠乳腺癌模型的脾脏和肿瘤中的CAR-T细胞拷贝数。
图5A是小鼠结肠癌模型中EGFRvIII-28Z CAR-T细胞与poly I:C的体内抗肿瘤作用；图5B显示了小鼠结肠癌模型中EGFRvIII-28Z CAR-T细胞与poly I:C体内抗肿瘤实验结束后的肿瘤重量；图5C显示了小鼠结肠癌模型中EGFRvIII-28Z CAR-T细胞与poly I:C体内抗肿瘤实验结束后外周血的IFNγ水平；图5D显示了抗肿瘤实验结束小鼠结肠癌模型的脾脏和肿瘤中的CAR-T细胞拷贝数。
图6是MSCV-EGFRvIII-28Z-FLT3L的质粒图。
图7显示了EGFRvIII-28Z-FLT3L CAR-T+poly I:C细胞在小鼠结肠癌模型中的作用。
图8A显示了I型干扰素被封闭后肠癌移植瘤的生长情况；图8B显示了I型干扰素被封闭后肠癌移植瘤的肿瘤重量。
图9A显示了I型干扰素被封闭后乳腺癌移植瘤的生长情况；图9B显示了I型干扰素被封闭后乳腺癌移植瘤的肿瘤重量。
图10显示了使用MDSC抑制剂、CAR T细胞和polyI:C在小鼠肿瘤模型中随时间对肿瘤体积影响的对比图(图10A)、肿瘤体重(图10B)以及肿瘤照片对比图(图10C)。
具体实施方式
为了克服现有技术中的缺陷，本发明进行了深入的研究，提供了(a)TLR激动剂和(b)嵌合抗原受体以及(c)任选的MDSC抑制剂和/或免疫检查点抑制剂联合应用肿瘤、传染病及其他免疫相关性疾病的治疗方法，具有显著优异的提高机体免疫力，杀伤肿瘤或病原体的能力。
术语
如本文所用，术语“增加免疫效应细胞功效的所述作用剂”、“免疫效应细胞增效剂”可互换使用，均指增强免疫效应细胞功效的药物或制剂。
本文采用的科技术语具有与本领域技术人员常规理解的相同或相似的含义。为便于理解本发明，一些术语定义如下。
当涉及可测量值比如给药量等时，包括指定值的±20％，或在某些情况下±10％，或在某些情况下±5％，或在某些情况下±1％，或在某些情况下±0.1％的变化，因此这样的变化适于进行所公开的方法。
术语，所述的“免疫效应细胞(在本文中也称免疫细胞)”，是指参与免疫应答，例如，促进免疫效应子应答的细胞。免疫效应细胞的实例包括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。例如，T细胞可以是直接来自外周血的T细胞、也可以是T细胞的亚型，如CD8+的T细胞，CD4+的T细胞，α/β的T细胞、γ/δT细胞等。
本文所用的“嵌合抗原受体”或“CAR”是指一组多肽，当其在免疫效应细胞中时，给所述的细胞提供针对靶细胞(通常是癌细胞)的特异性，并且具有细胞内信号产生。CAR通常包括至少一个细胞外抗原结合结构域、跨膜结构域(也称跨膜区)和细胞质信号传导结构域(本文中也称为“胞内区”)，其包括来源于如下定义的刺激性分子和/或共刺激性分子的功能信号传导结构域。在某些方面，多肽组彼此邻接。多肽组包括在存在二聚化分子时可以使多肽彼此偶联的二聚化开关，例如，可以使抗原结合结构域偶联至胞内信号传导结构域。在一个方面，刺激性分子为与T细胞受体复合体结合的ζ链。在一个方面，细胞质信号传导结构域进一步包括一种或多种来源于至少一个如下定义的共刺激性分子的功能性信号传导结构域。在一个方面，共刺激性分子选自本文所述共刺激性分子，例如4-1BB(即，CD137)、CD27和/或CD28。在一个方面，CAR包括嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于刺激性分子的功能性信号传导结构域的胞内信号传导结构域。在一个方面，CAR包含嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于共刺激性分子的功能性信号传导结构域和来源于刺激性分子的功能性信号传导结构域的胞内信号传导结构域。在一个方面中，CAR包含嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于一个或更多个共刺激性分子的两个功能性信号传导。
术语，所述的“信号传导结构域”是指通过在细胞内传递信息而起作用的蛋白质的功能性部分，用来通过产生第二信使或通过响应这样的信使起效应物作用经由确定的信号传导途径调节细胞的活性。
所述的“T细胞(抗原)受体(T cell receptor,TCR)”，为所有T细胞表面的特征性标志，以非共价键与CD3结合，形成TCR—CD3复合物。TCR的作用是识别抗原。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。任一T细胞只表达TCR2和TCR1之一。
所述的“T细胞融合蛋白(T CELL FUSION PROTEIN，TFP)”，P包括构成TCR的各种多肽衍生的重组多肽，其能够i)结合到靶细胞上的表面抗原,和ii)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 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。
所述的“有效链接”指的是一个调控序列和异源核酸序列之间的功能联系，其能导致异源核酸序列的表达。例如，当第一核酸序列位于第二核酸序列功能调控区时，第一核酸序列可操作地与第二核酸序列连接。例如，如果需要启动子影响编码序列的转录和表达，那么这个启动子可有效地与该编码序列连接。可链接的DNA序列可以互相邻接的，例如，可以将两个蛋白编码区放在同一个阅读框里。
所述的“T细胞抗原耦合器(T CELL ANTIGEN COUPLER,TAC)”，包括三个功能结构域：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；为胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与其他TCR受体靠近；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。
所述的“Toll样受体”(TLR)是指结合至病原体相关分子模式(PAMP)并且促进哺乳动物中的免疫应答的受体家族的成员。已知10种哺乳动物TLR，例如TLR1-10。术语“toll样受体激动剂”(TLR激动剂)是指与TLR相互作用并刺激所述受体活性的分子。合成的TLR激动剂是设计为与TLR相互作用并刺激所述受体活性的化合物。TLR激动剂的实例包括TLR-7激动剂、TLR-3激动剂或TLR-9激动剂。
所述的“Toll样受体3(TLR3)是Toll样受体家族成员之一，它能够特异性识别病毒的双链RNA及其类似物Poly(I:C)。dsRNA为具有可在病毒复制循环期间形成的两个互补链的RNA。在识别时，TLR3诱发转录因子如NF-κB和干扰素调节因子3(IRF3)的活化以提高向其他细胞发出信号以增加其抗病毒防御的I型干扰素的产生。已有研究表明，体内多种免疫细胞包括NK细胞、巨噬细胞、树突状细胞以及T细胞中均可表达TLR3。而Poly(I:C)作为TLR3的激动剂，能够促进B细胞的生长增殖及抗原呈递功能。另外，TLR3的激动剂也能够促进树突细胞(DC)细胞成熟，是树突细胞成熟剂。
所述的“聚肌苷酸聚胞苷酸(Poly(I:C))”(在本申请文件中，与Poly I:C具有同等含义，在附图中的pIC也代指Poly(I:C))，中文为聚肌肝，是合成的dsRNA的类似物，为具有高达例如3,600,000道尔顿的MW分布的双链RNA分子。Poly(I:C)作为TLR3的激动剂，能够促进B细胞的生长增殖及抗原呈递功能。并且，在无特异性抗原刺激的情况下，TLR3激动剂Poly(I:C)能够直接诱导B细胞产生IgG1κ抗体。其抗肿瘤作用主要依赖于CD8+T细胞和NK细胞，并能协调诱导DC细胞、巨噬细胞/中性粒细胞、NK细胞和T细胞等多种免疫细胞分泌I型干扰素，共同促进抗肿瘤效应。
术语“树突细胞(Dendritic cell，DC)”源自骨髓中的祖细胞，作为未成熟细胞迁移到外周组织，在那里它们内吞抗原并进行复杂的成熟过程。抗原经由很多表面分子被内吞，包括补体受体(例如，CD11c/CD18)和内吞受体(例如，DEC-205、DC-SIGN和Toll样受体)。在抗原获得过程中，未成熟DC也接受例如细菌细胞壁脂多糖(LPS)的病原体相关分子形式的“危险信号”，或者经由例如IFN-γ的细胞因子的炎性刺激。然后DC迁移到次级淋巴器官，成熟成为感受态APC。受体例如CD11c/CD18、DEC-205、DC-SIGN和Toll样受体在Ag捕获和递呈过程中扮演关键角色，并且主要表达在DC上。
术语“树突细胞成熟剂”是指诱导未成熟DC细胞成熟为感受态APC的物质，包括选自下组或其组合：STING激动剂；TLR激动剂诸如热灭活的或经福尔马林处理的卡介苗(BCG)，优选BCG的细胞壁成分、BCG衍生的脂阿拉伯甘露聚糖或BCG成分；自大肠杆菌衍生的脂多糖(LPS)；溶链菌制剂(Picibanil)(OK432)或灭活的革兰氏阳性或革兰氏阴性微生物；咪唑喹啉化合物， 优选咪唑喹啉-4-胺化合物，特别是4-氨基-2-乙氧基甲基-x-二甲基-1H-咪唑[4，5-c]喹啉-1-乙醇或1-(2-甲基丙基)-1H-咪唑[4，5-c]喹啉-4-胺，或其衍生物；合成的双链多核糖核苷酸，优选多聚I:C；天然的双链RNA或RNA病毒或RNA片段、或合成的类似物、或包含未甲基化的CpG基序的合成的或天然的核酸分子；细胞因子组合，优选肿瘤坏死因子α(TNF-α)、干扰素γ(IFN-γ)、、IL-1、IL-6、IL-12或前列腺素E6、CD40L，优选重组CD40L或包含CD40L域的融合蛋白；或遗传工程化改造成表达CD40L的原代细胞或细胞系，或在生理学上上调CD40L表达的活化T淋巴细胞诸如T淋巴细胞；2-Hydroxypropyl-β-cyclodextrin(HP-β-CD)[参考文献：Front Immunol.2016 Oct 20；7:435.eCollection 2016]、galectin(Gal)-9[参考文献J Immunol.2005 Sep 1；175(5):2974-81]。
术语，所述的“肿瘤”指的是一种以细胞或组织的病理性增生为特征的疾病，及其随后的迁移或侵袭其他组织或器官。肿瘤生长通常是不受控制的和进行性的，不诱导或抑制正常细胞增殖。肿瘤可影响多种细胞、组织或器官，包括但不限于选自膀胱、骨、脑、乳腺、软骨、神经胶质细胞、食管、输卵管、胆囊、心脏、肠、肾、肝、肺、淋巴结、神经组织、卵巢、胰腺、前列腺、骨骼肌、皮肤、脊髓、脾、胃、睾丸、胸腺、甲状腺、气管、尿道、输尿管、尿道、子宫、阴道器官，或组织或相应的细胞。肿瘤包括癌症，如肉瘤，癌，或浆细胞瘤(浆细胞的恶性肿瘤)。本发明所述的肿瘤，可包括，但不限于白血病(如急性白血病、急性淋巴细胞白血病、急性髓细胞性白血病，急性粒细胞白血病，急性早幼粒细胞白血病、急性粒-单核细胞白血病、急性单核细胞白血病、急性白血病、慢性白血病、慢性粒细胞白血病、慢性淋巴细胞白血病、真性红细胞增多症)，淋巴瘤(霍奇金病、非霍奇金病)、原发性巨球蛋白血症，重链病，实体瘤如肉瘤和癌症(如纤维肉瘤、粘液肉瘤、脂肪肉瘤、软骨肉瘤、骨肉瘤、脊索瘤、内皮肉瘤、淋巴管肉瘤、血管肉瘤、淋巴管内皮肉瘤，滑膜vioma，间皮瘤，尤文氏瘤、平滑肌肉瘤、横纹肌肉瘤、结肠癌、胰腺癌、乳腺癌、卵巢癌、前列腺癌、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、癌、支气管癌、髓样癌、肾细胞癌、肝癌，尼罗河管癌，绒癌、精原细胞瘤、胚胎癌、肾母细胞瘤、宫颈癌、子宫癌、睾丸癌、肺癌、小细胞 肺癌、膀胱癌、上皮癌、胶质瘤、星形细胞瘤、髓母细胞瘤，颅咽管瘤、室管膜瘤、松果体瘤、血管母细胞瘤，听神经瘤，少突胶质瘤、神经鞘瘤、脑膜瘤、黑色素瘤、神经母细胞瘤、视网膜母细胞瘤)、食管癌、胆囊癌、肾癌、多发性骨髓瘤。较佳地，所述的“肿瘤”包括但不限于：胰腺癌、肝癌、肺癌、胃癌、食管癌、头颈部鳞状细胞癌、前列腺癌、结肠癌、乳腺癌、淋巴瘤、胆囊癌、肾癌、白血病、多发性骨髓瘤、卵巢癌、宫颈癌和胶质瘤。
术语，所述的“肿瘤抗原”是指全部地或以片段形式(例如MHC/肽)在肿瘤细胞表面上被表达的分子(通常为蛋白质、碳水化合物或脂质)，并且其用于将药理学作用剂优先靶向肿瘤细胞。肿瘤抗原可以为由正常细胞和癌细胞表达的标记物，例如谱系标记物例如在B细胞上的CD19；肿瘤抗原也可以为在癌细胞中与正常细胞相比过表达的细胞表面分子，例如与正常细胞相比1倍过表达、2倍过表达、3倍或以上过表达；肿瘤抗原也可以为癌细胞中被不适当地合成的细胞表面分子，例如，与正常细胞上被表达的分子相比含有缺失、添加或突变的分子；肿瘤抗原也可以全部或以片段形式(例如，MHC/肽)被专一性地表达在癌细胞的细胞表面上，而不是在正常细胞的表面上合成或表达。在某些实施方式中，本发明的CAR包括包含结合MHC呈递肽的抗原结合结构域(例如，抗体或抗体片段)的CAR。通常，来源于内源蛋白质的肽填充主要组织相容性复合体(MHC)的I类分子的口袋，并且被CD8+T淋巴细胞上的T细胞受体(TCRs)识别。MHC I类复合体由全部有核细胞组成型表达。在肿瘤中，病毒特异性和/或肿瘤特异性肽/MHC复合体代表免疫疗法的独特类型的细胞表面靶点。
肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1(CD2子集1,CRACC,SLAMF7,CD319,和19A24)；C型凝集素样分子-1(CLL-1)；神经节苷脂GD3(aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer)；Tn抗原(Tn Ag)；CD19；CD20；CD 22；CD 30；CD 70；CD 123；CD 138；CD 33；CD 44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Ra)；白介素11受体α(IL-11Ra)；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,EGFRvII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；蛋白酶体(Prosome，Macropain)亚基，β型，9(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(aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer；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)；claudin 6(CLDN6)，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；MAGE A1,MAGE3；KDR；Lambda；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组，成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物，基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6，位于染色体12p(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族，成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶，丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白 sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤，X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。在具体的实施方案中，肿瘤抗原可以但不限于为EGFRvIII、GPC3、CLD18A2、CD19、CD20、CD22、CD30、WT1、CLDN6、MUC1、BCMA、IL-11Ra、IL-13Ra、FAP。
术语，所述的“病原体”是指能够引起疾病的原生动物，包括：病毒、细菌、真菌或寄生虫。所述的“病毒抗原”是指病毒表达的，能够诱导免疫反应的多肽。
正如本文中使用的那样，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抗体片段、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、单域抗体比如sdAb(VL或VH)、camelid VHH结构域、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。抗原结合片段也可以被掺入单域抗体、最大抗体、微小抗体、纳米抗体、胞内抗体、双抗体、三抗体、四抗体、v-NAR和双-scFv(参见，例如Hollinger和Hudson，Nature Biotechnology 23:1126-1136,2005)。抗原结合片段也可以接枝到基于多肽的支架，比如III型纤连蛋白(Fn3)(参见美国专利号:6,703,199，其描述了纤连蛋白多肽微小抗体)。
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。
在本文中，术语“结合结构域”或“抗体分子”指的是蛋白质，例如，免疫球蛋白链或其片段，包含至少一个免疫球蛋白可变结构域序列。术语“结合结构域”或“抗体分子”包括抗体和抗体片段。抗体分子可以是多特异性抗体分子，例如，它包含多个免疫球蛋白可变结构域序列，其中第一免疫球蛋白可变结构域序列对第一表位具有结合特异性并且多个的第二免疫球蛋白可变结构域序列具有针对第二表位的结合特异性。多特异性抗体分子也可以为双特异性抗体分子。双特异性抗体具有对不超过两个抗原的特异性。双特异性抗体分子的特征在于对第一表位具有结合特异双特异性抗体分子的特征在于对第一表位具有结合特异性的第一免疫球蛋白可变结构域序列和对第二表位具有结合特异性的第二免疫球蛋白可变结构域序列。
术语“抗原”或“Ag”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，因此编码“抗原”。此外，本领域技术人员应当理解抗原无需仅通过基因的全长核苷酸序列编码。显而易见的是，本发明包括但不限于使用超过一个基因的部分核苷酸序列，并且这些核苷酸序列以不同组合排列以编码引发期望免疫应答的多肽。而且，本领域技术人员应当理解抗原根本无需由“基因”编码。显而易见的是，抗原可以合成产生，或者可以来源于生物样品，或者可以是除了多肽之外的大分子。这样的生物样品可以包括，但不限于组织样品、肿瘤样品、具有其它生物组分的细胞或液体。
术语“抗癌作用”是指可以通过多种手段表现的生物效应，包括但不限于例如肿瘤体积减小、癌细胞数量减少、转移数量减少、预期寿命增加、癌细胞增殖减少、癌细胞存活率降低或与癌性病症有关的各种生理学症状的改善。“抗 癌作用”也可以通过肽、多核苷酸、细胞和抗体预防癌症首次出现的能力。术语“抗癌作用”是指可以通过各种手段表现的生物学效应，包括但不限于例如肿瘤体积减小、肿瘤细胞数量减少、肿瘤细胞增殖减少或肿瘤细胞存活率降低。
如该术语在本文中使用的，“来源于”表示第一和第二分子之间的关系。它一般是指在第一分子和第二分子之间的结构相似性，并且不暗示或包括对来源于第二分子的第一分子的过程或来源的限制。例如，在来源于CD3ζ分子的胞内信号传导结构域的情况下，胞内信号传导结构域保持足够CD3ζ结构使得其具有所需的功能，即，在适当的条件下产生信号的能力。它不暗示或包括对产生胞内信号传导结构域的具体过程的限制，例如，它并不意味着，为了提供胞内信号传导结构域，必须从CD3ζ序列开始并缺失不需要的序列，或施加突变，以得到胞内信号传导结构域。
短语“与本文所述的肿瘤抗原的表达相关的疾病”包括，但并不限定于与如本文所述肿瘤抗原的表达相关的疾病或与表达如本文所述的肿瘤抗原的细胞相关的状况，包括例如，增生性疾病如癌症或恶性肿瘤或癌前状态，如脊髓发育不良，骨髓增生异常综合征或白血病前期；或与表达如本文所述的肿瘤抗原的细胞相关的非癌症相关的适应证。在一个方面中，与本文所述的肿瘤抗原的表达相关的癌症是血液癌症。在一个方面中，与本文所述的肿瘤抗原的表达相关的癌症是实体癌。与本文所述的肿瘤抗原的表达相关的其它疾病包括，但不限于，例如，非典型和/或非经典癌症，恶性肿瘤，癌前状态或与如本文所述的肿瘤抗原的表达相关的增生性疾病。与本文所述的肿瘤抗原的表达相关的非癌症相关适应证包括，但不限于，例如，自身免疫性疾病(例如，狼疮)，炎性病症(过敏和哮喘)以及移植。肿瘤抗原表达可以细胞表达，或在任何时间表达了，编码所述肿瘤抗原的mRNA。肿瘤抗原表达细胞可以产生肿瘤抗原蛋白(例如，野生型或突变体)，并且所述肿瘤抗原蛋白可以在正常水平或降低的水平存在。肿瘤抗原表达细胞可以在一个点产生可检测水平的肿瘤抗原蛋白，并随后产生基本上不可检测的肿瘤抗原蛋白。
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或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。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者非人类种类例如小鼠、啮齿类动物、猴、猿等的等同残基。
术语“抗原呈递细胞”或“APC”是指其表面上呈递与主要组织相容性复合体(MHC的)复合的外来抗原的免疫系统细胞，比如辅助细胞(例如，B-细胞、树突细胞等)。T-细胞可以使用其T-细胞受体(TCR)识别这些复合体。APC加工抗原且将其呈递给T-细胞。
术语“胞内信号传导结构域”是指分子的细胞内部分。胞内信号传导结构域产生促进含有CAR的细胞例如CART细胞的免疫效应子功能的信号。在例如CART细胞中免疫效应子功能的实例包括细胞裂解活性和辅助活性，包括细胞因子的分泌。在一种实施方式中，胞内信号传导结构域可以包括一级胞内信号传导结构域。示例性的一级胞内信号传导结构域包括来源于负责初次刺激或抗原依赖性刺激的分子的那些。在一种实施方式中，胞内信号传导结构域可以包括共刺激细胞内结构域。示例性的共刺激胞内信号传导结构域包括来源于负责共刺激信号或抗原独立的刺激的分子的那些。例如，在CART的情况下，一级 胞内信号传导结构域可以包含T细胞受体的细胞质序列，并且共刺激胞内信号传导结构域可以包含来自共同受体或共刺激性分子的细胞质序列。
一级胞内信号传导结构域可以包括被称为基于免疫受体酪氨酸的活化基序或ITAM的信号传导基序。含有ITAM的一级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FcRγ(FCER1G)、FcγRIIa、FcRβ(FcEpsilon R1b)、CD3γ、CD3δ、CD3ε、CD79a、CD79b、DAP10和DAP12。
术语“ζ”或备选地“ζ链”、“CD3-ζ”或“TCRζ”定义为如GenBan Acc.No.BAG36664.1提供的蛋白质或来自非人类种类(例如小鼠、啮齿类动物、猴子、猿等)的等同残基，且“ζ刺激结构域”或备选地“CD3-ζ刺激结构域”或“TCR-ζ刺激结构域”定义为来自ζ链的细胞质结构域的氨基酸残基或其功能性衍生物，其足以在功能上传递T细胞活化所需的起始信号。在一个方面，ζ的细胞质结构域包括GenBank Acc.No.BAG36664.1的残基52至164或作为其功能性同源物的来自非人类种类(例如小鼠、啮齿类动物、猴子、猿等)的等同残基。
术语“共刺激性分子”是指T细胞上的同源结合配偶体，其特异性地结合共刺激配体，从而介导T细胞的共刺激反应，比如但不限于增殖。共刺激性分子为除了抗原受体或其配体之外的细胞表面分子，其促进有效的免疫应答。共刺激性分子包括但不限于MHC I类分子，BTLA和Toll配体受体，以及OX40、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)和4-1BB(CD137)。这样的共刺激性分子的进一步实例包括CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a，以及特异性地结合CD83的配体。
共刺激性胞内信号传导结构域可以为共刺激性分子的细胞内部分。共刺激性分子可以以下述蛋白质家族代表:TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、信号传导淋巴细胞性活化分子(SLAM蛋白质)、和NK细胞受体。这样的分子的实例包括CD27、CD28、4-1BB(CD137)、OX40、GITR、CD30、CD40、ICOS、BAFFR、HVEM、ICAM-1、与淋巴细胞功能相关的抗原-1(LFA-1)、CD2、CDS、CD7、CD287、LIGHT、NKG2C、NKG2D、SLAMF7、NKp80、NKp30、NKp44、NKp46、CD160、B7-H3、以及特异性地结合CD83的配体等。
胞内信号传导结构域可以包括分子的全部细胞内部分或全部天然胞内信号传导结构域、或其功能片段或衍生物。
术语“4-1BB”是指具有如GenBank Acc.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 Acc.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“编码”是指多核苷酸比如基因、cDNA、或mRNA中的核苷酸的特异性序列在生物学过程中作为合成其它聚合物和大分子的模板的固有特性，该聚合物以及大分子具有确定的核苷酸序列(例如，rRNA、tRNA和mRNA)或确定的氨基酸序列及由其得到的生物学特性。因此，如果与所述基因对应的mRNA的转录和翻译在细胞或其它生物系统中产生蛋白质，则基因、cDNA或RNA编码蛋白质。其核苷酸序列与mRNA序列相同且通常被提供在序列表中的编码链和用作转录基因或cDNA的模板的非编码链这两者都可以被称为编码该基因或cDNA的蛋白或其他产物
除非另有说明，否则“编码氨基酸序列的核苷酸序列”包括作为彼此的简并形式且编码相同氨基酸序列的所有核苷酸序列。短语编码蛋白质或RNA的核苷酸序列也可包括内含子，其达到编码蛋白质的核苷酸序列可以在一些形式中含有内含子的程度。
术语“有效量”或“治疗有效量”在本文中可互换地使用，并且是指如本文 中所述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。
术语“表达”是指由启动子驱动的特定核苷酸序列的转录和/或翻译。
术语“转移载体”是指包括分离的核酸且可用于将所分离的核酸递送至细胞内部的物质组合物。大量载体是本领域已知的，包括，但不限于线性多核苷酸、与离子或两亲性化合物有关的多核苷酸、质粒和病毒。因此，术语“转移载体”包括自主复制的质粒或病毒。该术语也应当被解释为进一步包括非质粒和促进核酸转移到细胞中的非病毒化合物，比如例如聚赖氨酸化合物、脂质体等。病毒转移载体的实例包括，但不限于腺病毒载体、腺伴随病毒载体、逆转录病毒载体、慢病毒(lentiviral)载体等
术语"表达载体”是指包含重组多核苷酸的载体，其包含有效连接要表达的核苷酸序列的表达控制序列。表达载体包含足够的用于表达的顺式作用元件；用于表达的其它元件可以由宿主细胞提供或在体外表达系统中。表达载体包括本领域已知的所有那些，包括被掺入重组多核苷酸的粘粒、质粒(例如，裸的或包含在脂质体中)和病毒(例如，慢病毒、逆转录病毒、腺病毒和腺伴随病毒)。
在本发明的上下文中，使用下述通常存在的核酸碱基的缩写。“A”是指腺苷、“C”是指胞嘧啶、“G”是指鸟苷、“T”是指胸苷、和“U”是指尿苷。
术语免疫原性的组合物的“肠胃外”施用包括例如皮下(s.c.)、静脉内(i.v.)、肌肉内(i.m.)或胸骨内注射、瘤内或输注技术。术语“核酸”或“多核苷酸”是指单链或双链形式的脱氧核糖核酸(DNA)或核糖核酸(RNA)及其聚合物。除非明确地限定，否则该术语包括含有天然核苷酸的已知类似物的核酸，所述已知类似物具有与参考核酸类似的结合性质，并且以与天然存在核苷酸类似的方式代谢。除非另有说明，否则特定的核酸序列也隐含地包括其保守修饰的变体(例如，简并密码子取代)、等位基因、直向同源物、SNP和互补序列以及明确指定的序列。特别地，简并密码子取代可以通过产生序列而实现，所述序列中一个或更多个所选择的(或所有的)密码子的三个位置被混合碱基和/或去氧肌苷残基取代(Batzer等,Nucleic Acid Res.19:5081(1991)；Ohtsuka等,J.Biol.Chem.260:2605-2608(1985)；和Rossolini等,Mol.Cell.Probes 8:91-98(1994))。
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。
正如本文中使用的那样，术语“治疗(treat、treatment、treating)”是指由于施用一种或多种疗法(例如一种或多种治疗剂比如本发明的CAR)减慢或改善增生性病症的进展、严重程度和/或持续时间，或改善增生性病症的一种或多种症状(优选地，一种或多种可辨别的症状)。术语“治疗”是也可以指改善增生性病症的至少一种可测量的物理参数比如肿瘤生长，不必是患者可辨别的。术语“治疗”也可以是指通过例如稳定可辨别的症状以物理方式、通过例如稳定物理参数以生理学方式或两者抑制增生性病症的进展。术语“治疗”也可以是指减小或稳定肿瘤尺寸或癌细胞计数。
术语“信号转导途径”是指在将信号从细胞的一个部分转导到细胞的另一个部分中起作用的多种信号转导分子之间的生化关系。短语“细胞表面受体”包括能够跨越细胞膜接受信号和传递信号的分子和分子复合体。
术语“受试者”是指包括其中可以引出免疫应答的活生物体(例如，哺乳动物、人类)。
术语“基本上纯化的”细胞是指基本上不含其它细胞类型的细胞。基本上纯化的细胞是指其已经与其天然存在状态中通常有关的其它细胞类型分离的细胞。在某些情况下，基本上纯化的细胞群是指细胞的同源群体。在其它的情况下，该术语仅指在其天然状态下与其天然有关的细胞分离的细胞。在某些方面中，所述细胞是体外培养的。在其它方面中，所述细胞不是体外培养的。
正如本文中使用的那样，术语“治疗”是指治疗。治疗效果是通过减轻、抑制、缓解或根除疾病状态获得的。
正如本文中使用的那样，术语“预防”是指预防性或保护性治疗疾病或疾病状态。
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”是指识别并且结合存在于样品中的结合配偶体(例如肿瘤抗原)蛋白质的抗体或配体，但是该抗体或配体基本上不会识别或结合样品中的其它分子。
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。
术语，所述的“FLT3L”可以人的FLT3L，由235个氨基酸残基组成的IV型跨膜蛋白(NCBI Reference Sequence:NP_001191431.1)，氨基酸序列如SEQ ID NO：8所示；也可以是与SEQ ID NO：8具有至少80％相似性的多肽，优选与SEQ ID NO：8具有至少85％或至少90％相似性的多肽；所述的“FLT3L”还可以来自其他种属，包括但不限于鼠、猴、猪。FLT3L可以是天然存在的，比如其可被分离或纯化自哺乳动物；也可以是人工制备的，比如可以根据常规的基因工程重组技术来生产重组各元件或FLT3L。优选的，本发明可采用重组的各元件或FLT3L。
组合物
本发明提供了包含免疫效应细胞的组合物，该组合物还包含TOLL样受体激动剂，所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗原的受体，所述TOLL样受体激动剂在对受试者的治疗中能增加所述免疫效应细胞的功效，其中：所述受体具有胞外区、跨膜区和胞内区，其中，胞外区具有识别所述的肿 瘤抗原或病原体抗原的抗原结合结构域，所述抗原结合结构域结合至与疾病相关的肿瘤抗原或病原体抗原。在一优选实施方式中，本发明的组合物还可包括MDSC抑制剂或免疫检查点抑制剂。
所述的“免疫效应细胞”可以来自自体细胞，也可以来自同种异体细胞。
所述的识别肿瘤抗原或病原体抗原的受体包括但不限于嵌合抗原受体(CAR)、T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、或T细胞抗原耦合器(TAC)。
所述的“免疫效应细胞”还可以表达其他外源性蛋白，例如，所述外源性蛋白可以是但不限于FLT3L、干扰素、白介素、PD-1或PD-L1抑制剂。这些外源性蛋白的存在可以增强“免疫效应细胞”的抗肿瘤或抗病原微生物的能力。
在具体实施方式中，所述的干扰素包括但不限于I型干扰素；所述的白介素包括但不限于IL-2、IL-12、IL-21、IL18；所述的白介素受体包括但不限于IL-4的受体；PD-1或PD-L1的抑制剂包括但不限于可溶性的PD-1或者含有可溶性的PD-1的融合肽(可参见WO2017080377)、抗PD-1或者抗PD-L1的抗体。
所述TOLL样受体激动剂可以但不限于为Toll样受体3(TLR3)激动剂。所述的TLR3激动剂可以为天然或合成的双链核糖核酸如聚肌苷酸胞苷酸(poly(I:C))。所述的TLR3激动剂还可以为错配的双链核糖核酸poly(I:C11-14U)，示例性的，如poly(I:C12U)、poly(I:C13U)。
在优选的实施方案中，所述的识别肿瘤抗原或病原体抗原受体包括嵌合抗原受体(CAR)、T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、或T细胞抗原耦合器(TAC)。
所述嵌合抗原受体可以包括常规的所有嵌合抗原受体结构，跨膜区通常可以任选已知的具有跨膜功能的蛋白或蛋白片段，胞内区也可以根据现有技术选择常规的具有引发免疫细胞活化的结构域，包括但不限于CD3ζ、FcRγ(FCER1G)、FcγRIIa、FcRβ(FcEpsilon R1b)、CD3γ、CD3δ、CD3ε、CD79a、CD79b、DAP10、DAP12、CD137、CD27、CD28等的全长或活性片段(例如CN201580013987关于胞内域的描述)。例如，嵌合抗原受体的还可以具有以下几种结构：
(i)特异性结合所述抗原的抗体、CD28的跨膜区、CD28的共刺激信号结构域和CD3ζ胞浆信号传导结构域；或
(ii)特异性结合所述抗原的抗体、CD28的跨膜区、CD137的共刺激信号结构域和CD3ζ胞浆信号传导结构域；或
(iii)特异性结合所述抗原的抗体、CD28的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ胞浆信号传导结构域。
所述的TFP包括：(a)TCR亚基，所述TCR亚基包括TCR部分胞外结构域，跨膜结构域，和TCR胞内结构域，所述胞内结构域包括刺激信号传导结构域；(b)抗原结合结构域，所述TCR亚基和所述抗原结合结构域有效连接。在一具体实施例中，所述TCR亚基的胞外、跨膜和胞内信号结构域来源于CD3ε或CD3γ，所述TFP整合进T细胞上表达的TCR。
所述的TAC包括：(a)胞外结构域：所述胞外结构域包括具有抗原结合结构域和与CD3结合的单链抗体；(b)跨膜区；(c)胞内结构域，所述胞内结构域连接蛋白激酶LCK。
在本发明中，所述的免疫细胞选自T细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞或骨髓源性吞噬细胞。在一优选例中，所述的免疫细胞为T细胞，所述的受体的胞内域能引发T细胞的活化。
本发明还提供了包含表达有嵌合抗原受体的免疫效应细胞及医药学上可接受的载体或赋形剂的组合物，该组合物还包含TLR激动剂和/或树突细胞成熟促进剂。
在另一方面，本发明提供了包含免疫效应细胞的组合物，除了包含TOLL样受体激动剂，还可以包含MDSC抑制剂、抗PD-1抗体、或者抗PD-L1的抗体。
本发明还提供了包含表达有嵌合抗原受体的免疫效应细胞、TLR激动剂和/或树突细胞成熟促进剂及指导如何向个体给予所述细胞的说明书的试剂盒。
给药方法
本发明还提供了治疗、控制、或者预防肿瘤或者病原体感染的方法，该方法包括给予受试者治疗有效量的免疫效应细胞和TOLL样受体激动剂。所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗原的受体，所述TOLL样受体激动剂在对受试者的治疗中能增加所述免疫效应细胞的功效，其中：所述受体具有胞外区、跨膜区和胞内区，其中，胞外区具有识别所述的肿瘤抗原或病原体抗原的抗原结合结构域，所述抗原结合结构域结合至与疾病相关的肿瘤抗原或 病原体抗原。
在具体实施方式中，免疫效应细胞和TOLL样受体激动剂可以同时给药，也可以先给予免疫效应细胞，再给予TOLL样受体激动剂，也可以先给予TOLL样受体激动剂，再给予免疫效应细胞。
所述的“免疫效应细胞”可以来自自体细胞，也可以来自同种异体细胞。
所述的识别肿瘤抗原或病原体抗原的受体包括但不限于嵌合抗原受体(CAR)、T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、或T细胞抗原耦合器(TAC)。
所述的“免疫效应细胞”还可以表达其他外源性蛋白，例如，所述外源性蛋白可以是但不限于FLT3L、干扰素、白介素、PD-1或PD-L1抑制剂。这些外源性蛋白的存在可以增强“免疫效应细胞”的抗肿瘤或抗病原微生物的能力。
在具体实施方式中，所述的干扰素包括但不限于I型干扰素；所述的白介素包括但不限于IL-2、IL-12、IL-21、IL18；所述的白介素受体包括但不限于IL-4的受体；PD-1或PD-L1的抑制剂包括但不限于可溶性的PD-1或者含有可溶性的PD-1的融合肽(可参见WO2017080377)、抗PD-1或者抗PD-L1的抗体。
所述TOLL样受体激动剂可以但不限于为Toll样受体3(TLR3)激动剂。所述的TLR3激动剂可以为天然或合成的双链核糖核酸如聚肌苷酸胞苷酸(poly(I:C))。所述的TLR3激动剂还可以为错配的双链核糖核酸poly(I:C 11-14U)，示例性的，如poly(I:C 12U)、poly(I:C 13U)。
所述嵌合抗原受体可以包括常规的所有嵌合抗原受体结构，跨膜区通常可以任选已知的具有跨膜功能的蛋白或蛋白片段，胞内区也可以根据现有技术选择常规的具有引发免疫细胞活化的结构域，包括但不限于CD3ζ、FcRγ(FCER1G)、FcγRIIa、FcRβ(FcEpsilon R1b)、CD3γ、CD3δ、CD3ε、CD79a、CD79b、DAP10、DAP12、CD137、CD27、CD28等的全长或活性片段(例如CN201580013987关于胞内域的描述)。例如，嵌合抗原受体的还可以具有以下几种结构：
(i)特异性结合所述抗原的抗体、CD28的跨膜区、CD28的共刺激信号结构域和CD3ζ胞浆信号传导结构域；或
(ii)特异性结合所述抗原的抗体、CD28的跨膜区、CD137的共刺激信号结构域和CD3ζ胞浆信号传导结构域；或
(iii)特异性结合所述抗原的抗体、CD28的跨膜区、CD28的共刺激信号 结构域、CD137的共刺激信号结构域和CD3ζ胞浆信号传导结构域。
所述的TFP包括:(a)TCR亚基，所述TCR亚基包括TCR部分胞外结构域，跨膜结构域，和TCR胞内结构域，所述胞内结构域包括刺激信号传导结构域；(b)抗原结合结构域，所述TCR亚基和所述抗原结合结构域有效连接。在一具体实施例中，所述TCR亚基的胞外、跨膜和胞内信号结构域来源于CD3ε或CD3γ，所述TFP整合进T细胞上表达的TCR。
所述的TAC包括：(a)胞外结构域：所述胞外结构域包括具有抗原结合结构域和与CD3结合的单链抗体；(b)跨膜区；(c)胞内结构域，所述胞内结构域连接蛋白激酶LCK。
在本发明中，所述的免疫细胞包括但不限于T细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞或骨髓源性吞噬细胞中的任一一种或其组合。在一优选例中，所述的免疫细胞为T细胞，所述的受体的胞内域能引发T细胞的活化。
在另一方面，本发明还提供了治疗、控制、或者预防肿瘤或者病原体感染的方法，该方法包括除了给予受试者治疗有效量的免疫效应细胞、TOLL样受体激动剂，还给与MDSC抑制剂、抗PD-1抗体、或者抗PD-L1的抗体。
制备治疗/预防/抑制肿瘤或者病原体感染、或调节免疫耐受能力的药物中的用途
本发明还提供了TOLL样受体激动剂和免疫效应细胞联合应用肿瘤、传染病及其他免疫相关性疾病的治疗方法,所述的肿瘤包括但并不限于：胰腺癌、肝癌、肺癌、胃癌、头颈部鳞状细胞癌、前列腺癌、结肠癌、乳腺癌、淋巴瘤、胆囊癌、肾癌、白血病、骨髓瘤、卵巢癌、宫颈癌、卵巢癌、宫颈癌或胶质瘤；所述的病原体包括但不限于：病毒、细菌、真菌、原生动物或寄生虫；较佳地，所述的病毒包括：巨细胞病毒、爱泼斯坦-巴尔病毒、人类免疫缺陷病毒或流感病毒。
在优选的实施方案中，所述TOLL样受体激动剂为Toll样受体3(TLR3)激动剂，所述的TLR3激动剂优选自天然或合成的双链核糖核酸。在一优选例中，所述的TLR3激动剂为聚肌苷酸胞苷酸(poly(I:C))。所述的TLR3激动剂还可以为错配双链核糖核酸，示例性的，如poly(I:C12U)、poly(I:C13U)。
在一优选例中，所述错配双链核糖核酸的量足以与Toll样受体3(TLR3)结合并且减少或消除所述受试对象中的所述肿瘤或其它转化细胞的增殖。
在优选的实施方案中，所述TOLL样受体激动剂对所述免疫效应细胞具有协同作用。
本领域技术人员可以凭借专业知识和现有技术，根据患者或对象的年龄、体重、性别、待治疗疾病的严重程度等因素决定所述TOLL样受体激动剂和所述免疫效应细胞的给药方式及用量。在具体的实施方案中，所述TOLL样受体激动剂和所述免疫效应细胞的给药方式可以相同，也可以不同。例如，给药方法为共同给予所述TOLL样受体激动剂和所述免疫效应细胞；或者先给予所述免疫效应细胞，再给予所述TOLL样受体激动剂；或者先给予所述TOLL样受体激动剂，再给予所述免疫效应细胞。在优选的实施方式中，所述的给药方法为先给予所述免疫效应细胞，再给予所述TOLL样受体激动剂。
在优选的实施方案中，所述TOLL样受体激动剂给药量可以是1mg/kg～4mg/kg，优选2mg/kg～3mg/kg，更优的选自2.08mg/kg-2.77mg/kg。在优选的实施方案中，所述TOLL样受体激动剂的给药次数为2～4次，优选2-3次。
在优选的实施方案中，所述的免疫细胞选自T细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞或骨髓源性吞噬细胞，优选T细胞。在一优选例中，所述的免疫细胞为T细胞，所述的受体的胞内域能引发T细胞的活化。
在优选的实施方案中，所述的识别肿瘤抗原或病原体抗原受体包括嵌合抗原受体(CAR)、T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、或T细胞抗原耦合器(TAC)。在优选的实施方案中，所述的免疫细胞可以为表达CAR的T细胞、表达TCR的T细胞、表达CAR的NK细胞，表达TFP的T细胞、表达TFP的NK细胞、表达TAC的T细胞、表达TAC的NK细胞。
所述嵌合抗原受体(CAR)可以但不限于下列结构：(i)特异性结合所述抗原的抗体、CD28的跨膜区、CD28的共刺激信号结构域和CD3ζ；或(ii)特异性结合所述抗原的抗体、CD28的跨膜区、CD137的共刺激信号结构域和CD3ζ；或(iii)特异性结合所述抗原的抗体、CD28的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ。
在优选的实施方案中，所述的TFP包括：(a)TCR亚基，所述TCR亚基包括 TCR部分胞外结构域，跨膜结构域，和TCR胞内结构域，所述胞内结构域包括刺激信号传导结构域；(b)抗原结合结构域，所述TCR亚基和所述抗原结合结构域有效连接。在一优选例中，所述TCR亚基的胞外、跨膜和胞内信号结构域来源于CD3ε或CD3γ，所述TFP整合进T细胞上表达的TCR。
在优选的实施方案中，所述的TAC包括：(a)胞外结构域：所述胞外结构域包括具有抗原结合结构域和与CD3结合的单链抗体；(b)跨膜区；(c)胞内结构域，所述胞内结构域连接蛋白激酶LCK。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
本发明的示例性的抗原受体，包括CAR，以及用于工程改造和将受体导入细胞中的方法，参考例如中国专利申请公开号CN107058354A、CN107460201A、CN105194661A、CN105315375A、CN105713881A、CN106146666A、CN106519037A、CN106554414A、CN105331585A、CN106397593A、CN106467573A、CN104140974A、国际专利申请公开号WO2017186121A1、WO2018006882A1、WO2015172339A8、WO2018018958A1中公开的那些。
实施例1.CAR-T细胞的制备
本实施例选择EGFRvIII作用CAR-T细胞的靶点，为了更准确的验证在小鼠体内的抗肿瘤效果，因此，选择的信号肽、跨膜区、胞内区等为鼠源的。制备的方法按照本领域常规的CAR-T细胞制备方法操作。
1、逆转录病毒载体构建
将小鼠CD8α信号肽(SEQ ID NO：1)、抗EGFRvIII单抗抗体(SEQ ID NO：2)、CD8α胶链区及跨膜区(SEQ ID NO：3)、CD28胞内域(SEQ ID NO：4)、CD3ζ胞内域(SEQ ID NO：5)依次连接，通过体外基因合成的方法获得EGFRvIII-28Z基因片段，并用Mlu Ⅰ和Sal Ⅰ双酶切位点置换逆转录病毒载体MSCV-IRES-GFP 中的IRES-GFP片段，获得重组载体MSCV-EGFRvIII-28Z。各组成片段组合顺序如图1所示。
2、逆转录病毒制备
操作如下：
(1)以4.5×106的密度接种293T细胞于培养皿中，CO2培养过夜，培养基为添加胎牛血清的DMEM，；
(2)将9μg MSCV-EGFRvIII-28Z与9μg pCL-Eco溶入800μL无血清DMEM培养液得到质粒混合液；将54μg PEI(1μg/μl)溶解于800μl的无血清DMEM培养液中，得到PEI混合液；将质粒混合液加入PEI混合液中室温孵育，得到转染复合物。
(4)将转染复合物1.6ml滴加入含步骤(1)的培养皿中培养，收集上清，得到。
3、逆转录病毒感染小鼠脾脏T淋巴细胞
取健康Balb/c小鼠，脱颈处死后取出脾脏，并将脾脏细胞制备成单细胞悬液；加200ul红细胞裂解液裂红后采用小鼠CD3+T细胞分选试剂盒(STEMCELL，#19851)分选获得小鼠脾脏T淋巴细胞。
将小鼠CD3+T淋巴细胞纯化后按1:1的比例加入Dynabeads Mouse T-activator CD3/CD28，在RPMI 1640完全培养基中培养24小时。
将48孔板中每孔加入400μl retroNectin溶液(5μg/mL)孵育过夜后弃去retroNectn；将活化24h的小鼠脾脏T淋巴细胞接种于板中，每孔细胞数目1×106，加入1ml逆转录病毒，补充培养基至2mL，离心后培养并传代。
4、嵌合抗原受体在T淋巴细胞的表达
感染3天后，取4×105的T细胞，重悬细胞，加入Biotin标记的EGFRvIII蛋白至终浓度为25ug/ml冰上孵育后清洗；加入50μL PBS+2％FCS重悬，加入1μLPE标记的streptavidin，冰上孵育清洗后，加入400μL PBS+2％FCS重悬细胞，流式检测，结果如图2所示，EGFRvIII-28Z CART细胞感染效率为76.0％。
实施例2.EGFRvIII-28Z CAR T淋巴细胞体外毒性
将小鼠结肠癌细胞CT26(购自美国模式培养物保藏所(ATCC))和小鼠胰腺癌细胞E0771(获自贝勒医学院)，采用本领域常规的技术手段，制备得到 过表达EGFRvIII的CT26-EGFRvIII和E0771-EGFRvIII。
使用cytoTox 96非放射活性的细胞毒性测定试剂盒(Promega，Madison，USA)测定EGFRvIII-28Z CAR T淋巴细胞对靶细胞的体外细胞毒性。调整靶细胞CT26、CT26-EGFRvIII、E0771、E0771-EGFRvIII浓度为2×105/mL，取50μL接种到96孔板；按效靶比0.3:1、1:1和3:1向96孔板中加入CAR-T细胞及对照T细胞(50ul)，按试剂盒说明书设置各对照孔，各组均设5个复孔；共孵育18小时。孵育结束后按照试剂盒说明书进行操作测定EGFRvIII-28Z CAR T淋巴细胞对靶细胞的体外细胞毒性，结果如图3所示。
结果显示，EGFRvIII-28Z CAR-T细胞对EGFRvIII表达阴性的CT26和E0771细胞无明显毒性，而对表达EGFR-vIII靶抗原的CT26-EGFRvIII、E0771-EGFRvIII细胞表现出较强的杀伤作用。在效靶比3:1的时候，EGFRvIII-28Z CAR-T细胞对两种细胞的杀伤分别为71％和67.8％；未感染T细胞则对靶细胞均无明显的杀伤活性。且EGFRvIII-28Z CAR T与未感染T细胞对EGFR-vIII靶抗原阳性细胞的杀伤效果存在显著性差异。这些结果表明我们构建的EGFRvIII-28Z CAR T淋巴细胞在体外可以特异性杀伤EGFRvIII表达阳性的肿瘤细胞。
实施例3.EGFRvIII-28Z CAR-T联合poly I:C在小鼠乳腺癌模型中的抗肿瘤作用
接种1×106 E0771-EGFRvIII细胞于6周龄的雌性C57小鼠(体重18g-24g，购自上海灵畅生物科技有限公司)的第四对乳腺脂肪垫；肿瘤接种13天，将荷瘤小鼠分为4组，分为UT细胞组、UT+poly I:C组、EGFRvIII-28Z CAR-T细胞组和EGFRvIII-28Z CAR-T+poly I:C组，每组肿瘤体积平均值约为150mm 3；分组后，通过尾静脉输注5.0×10 6EGFRvIII-28Z CAR-T细胞(注射日为第0天)，同时以U T细胞组作为对照，CAR-T细胞回输后第3、6天瘤内注射50ug polyI:C，UT细胞组为瘤内注射相同剂量的生理盐水。观察移植瘤的生长，结果如图4A所示。
结果显示，回输5×106 EGFRvIII-28Z虽然能够抑制E0771-EGFRvIII移植瘤的生长；而联合poly I:C后，对E0771-EGFRvIII移植瘤生长的抑制效果则更加显 著，表明poly I:C进一步增强了CAR-T细胞的抗肿瘤能力。相对于UT组，在第16天时EGFRvIII-28Z CAR-T细胞组抑瘤率为36.1％，而联用poly I:C组则达到69.6％，差异具有显著性(P<0.05)，抑瘤率提高超过30％。
待对照组小鼠肿瘤大小达到2000mm 3时，即将实验处死，分离肿瘤后，测定肿瘤重量，结果如图4B所示，与单独给予CAR-T相比，联合给予Poly:IC组的肿瘤重量降低77.8％
处死后的小鼠，取外周血测定IFNγ水平，结果如图4C所示，取脾脏和肿瘤组织，测定CAR-T细胞拷贝数，结果如图4D所示。
实施例4.EGFRvIII-28Z CAR-T联合poly I:C在小鼠结肠癌模型中的抗肿瘤作用
6周龄雌性Balb/c小鼠(购自上海灵畅生物科技有限公司)采用3Gy gamma射线辐照(即进行淋巴细胞清除)后，当天皮下接种3×105 CT26-EGFRvIII细胞；肿瘤接种13天，将荷瘤小鼠分为4组，分为UT细胞组、UT+poly I:C组、EGFRvIII-28Z CAR-T细胞组和EGFRvIII-28Z CAR-T+poly I:C组，每组肿瘤体积平均值约为150mm3；分组后，通过尾静脉输注5.0×106 EGFRvIII-28Z CAR-T细胞，同时以U T细胞组作为对照，CAR-T细胞回输后第3、6天瘤内注射50ug polyI:C。GFRvIII-28Z CAR-T联合poly I:C对CT26-EGFRvIII移植瘤的治疗效果如图5所示。
结果显示单独回输5×106 CT26-EGFRvIII可以明显抑制CT26-EGFRvIII移植瘤的生长；而联合poly I:C后，对CT26-EGFRvIII移植瘤生长的抑制效果则更加显著，表明poly I:C进一步增强了CAR-T细胞的抗肿瘤能力。相对于UT组，在第16天时EGFRvIII-28Z CAR-T细胞组抑瘤率为36.1％，而联用poly I:C组则达到74.3％，差异具有显著性(P<0.05，P<0.01)，抑瘤率提高超过30％。
待对照组小鼠肿瘤大小达到2000mm 3时，即将实验处死，分离肿瘤后，测定肿瘤重量，结果如图5B所示，与单独给予CAR-T相比，联合给予Poly:IC组的肿瘤重量降低了48.5％。
处死后的小鼠，取外周血测定IFNγ水平，结果如图5C所示，取脾脏和肿瘤组织，测定CAR-T细胞拷贝数，结果如图5D所示。
实施例5 表达FLT3L CAR-T细胞与Poly I:C的联用
参照常规的CAR-T细胞制备方法，将小鼠muFLT3L基因片段(SEQ ID No：6)与实施例1中构建的EGFRvIII-28Z的基因序列通过通过F2A(SEQ ID No：7)连接，即将F2A-FLT3L基因插入EGFR-28Z基因中CD3ζ片段后，构建EGFRvIII-28Z-FLT3L逆转录病毒载体MSCV-EGFRvIII-28Z-FLT3L，质粒图见图6，感染小鼠脾脏T淋巴细胞，制备得到表达EGFRvIII-28Z-FLT3L CAR T细胞。
参照实施例4的操作，构建小鼠结肠癌模型，将荷瘤小鼠分为EGFRvIII-28Z CAR-T细胞+poly I:C组和EGFRvIII-28Z-FLT3L CAR-T+poly I:C细胞组
结果如图7所示，在Balb/c小鼠CT26-EGFRvIII移植瘤模型中，EGFRvIII-28Z-FLT3L CAR-T+poly I:C细胞组的肿瘤抑制效果明显优于EGFRvIII-28Z CAR-T细胞+poly I:C组，EGFRvIII-28Z-FLT3L CAR-T+poly I:C联合治疗组的抑瘤率在CAR T细胞输注后第7、10、14天分别达到47.8％、50.1％和54.0％。
实施例6 干扰素抑制剂对CAR-T+poly I:C的抗肿瘤作用的影响
参照实施例3和4，分别构建小鼠乳腺癌模型和结肠癌模型。采用干扰素受体α链的抗体(克隆号MAR1-5A3,购自Bioxcell)作为封闭剂封闭I型干扰素的释放。
取小鼠肠癌模型分为UT组(注射生理盐水)、EGFRvIII-28Z CAR-T细胞+poly I:C组、以及EGFRvIII-28Z CAR-T细胞+poly I:C组+干扰素受体α链封闭剂。CAR-T细胞回输后第3、6天瘤内注射50ug polyI:C，在给予poly I:C或生理盐水后第0天和第2天，瘤内注射给予50ug干扰素受体α链封闭剂。测量肿瘤的生长情况，结果如图8A所示。待对照组小鼠肿瘤大小达到2000mm 3时，即将实验处死，分离肿瘤后，测定肿瘤重量，结果如图8B所示。
取小鼠乳腺癌模型分为UT组(注射生理盐水)、EGFRvIII-28Z CAR-T细胞+poly I:C组、以及EGFRvIII-28Z CAR-T细胞+poly I:C组+干扰素受体α链封闭剂。CAR-T细胞回输后第3、6天瘤内注射50ug polyI:C，在给予poly I:C或生理盐水后第0天和第2天，瘤内注射给予干扰素受体α链封闭剂。测量肿瘤的生长情况，结果如图9A所示。待对照组小鼠肿瘤大小达到2000mm 3时，即将实验处死，分离肿瘤后，测定肿瘤重量，结果如图9B所示。
以上结果显示，封闭I型干扰素后，给予CAR-T细胞和poly I:C，引起抗肿瘤活性降低，即I型干扰素的存在将有利于CAR-T细胞和poly I:C的抗肿瘤作用。
实施例7 MDSC抑制剂对CAR-T+poly I:C的抗肿瘤作用的影响
参照实施例4，构建小鼠结肠癌模型。采用MDSC清除剂anti-Gr1抗体(购自BioXell e，克隆号RB6-8C5)，抑制MDSC。
MDSC为骨髓来源的抑制性细胞，呈CD11b+Gr1+阳性。单独给予anti-Gr1抗体与UT组相比，没有明显的抗肿瘤活性。
取小鼠肠癌模型分为UT组(注射生理盐水)、UT+anti-Gr1组(给予anti-Gr1抗体和生理盐水)、CAR-T+poly I:C组(给予EGFRvIII-28Z-CAR-T和poly I:C)、CAR-T+anti-Gr1组(给予EGFRvIII-28Z-CAR-T和anti-Gr1抗体)、CAR-T+poly I:C+anti-Gr1组(给予EGFRvIII-28Z-CAR-T、poly I:C和anti-Gr1抗体)。
CAR-T细胞回输后第2天，按照10mg/kg小鼠腹腔注射anti-Gr1抗体，接下来每周3次注射，共计两周。CAR-T细胞回输后第3、6天瘤内分别注射50ug polyI:C。
测量肿瘤的生长情况，结果如图10A所示。待对照组小鼠肿瘤大小达到2000mm3时，即将实验处死，分离肿瘤后，测定肿瘤重量，结果如图10B所示。分离肿瘤后进行拍照，结果如图10C所示。
作为示例性的，上述实施例选择靶向EGFR的CAR-T细胞，应理解，选择作用于其他靶点的CAR-T细胞也具有相同的效果，如GPC3、CLD18A2、CD19、BCMA等。所采用的抗体可以是鼠抗也可以是人源化的，采用的跨膜域、胞内域也可以根据目的不同采用不同种属的，如采用人的。
作为示例性的，上述实施例虽然采用的是CAR-T细胞，但该T细胞还可以表达有其他增强CAR-T细胞功能的细胞因子，如CAR和I型干扰素共表达的CAR-T细胞、CAR和PD1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。
作为示例性的，上述实施例选择鼠源的CAR，但其信号肽、铰链区、跨膜 区等可以根据目的的不同选用其他种属。包括但不限于人的信号肽、铰链区、跨膜域、胞内区。抗体也可以根据不同的目的，选择针对不同靶点的鼠抗或者人源化的抗体或者全人的抗体。
在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 (24)

  1. 包含免疫效应细胞的组合物，所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗原的受体，所述免疫效应细胞与在具有与肿瘤抗原或病原体抗原的表达相关的疾病的受试者的治疗中增加所述免疫效应细胞的功效的作用剂组合使用，其中：
    (i)所述受体具有胞外区、跨膜区和胞内区，其中，胞外区具有识别所述的肿瘤抗原或病原体抗原的抗原结合结构域，
    (ii)增加免疫细胞功效的所述作用剂为TOLL样受体激动剂。
  2. 如权利要求1所述的组合物，所述TOLL样受体激动剂为Toll样受体3(TLR3)激动剂。
  3. 如权利要求2所述的组合物，所述的TLR3激动剂选自天然或合成的双链核糖核酸，优选聚肌苷酸胞苷酸(poly(I:C))。
  4. 如权利要求1-3任一所述的组合物，所述的识别肿瘤抗原或病原体抗原的受体包括嵌合抗原受体(CAR)、T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、或T细胞抗原耦合器(TAC)。
  5. 如权利要求4所述的组合物，其特征在于，所述嵌合抗原受体包括：
    (i)特异性结合所述抗原的抗体、CD28的跨膜区、CD28的共刺激信号结构域和CD3ζ胞浆信号传导结构域；或
    (ii)特异性结合所述抗原的抗体、CD28的跨膜区、CD137的共刺激信号结构域和CD3ζ胞浆信号传导结构域；或
    (iii)特异性结合所述抗原的抗体、CD28的跨膜区、CD28的共刺激信号结构域、CD137的共刺激信号结构域和CD3ζ胞浆信号传导结构域。
  6. 如权利要求4所述的组合物，所述的TFP包括:
    (a)TCR亚基，所述TCR亚基包括：
    TCR部分胞外结构域，跨膜结构域，和TCR胞内结构域，所述胞内结构域包括刺激信号传导结构域；
    (b)抗原结合结构域，所述TCR亚基和所述抗原结合结构域有效连接，
    其中，所述TCR亚基的胞外、跨膜和胞内信号结构域来源于CD3ε或CD3γ，所 述TFP整合进T细胞上表达的TCR。
  7. 如权利要求4所述的组合物，所述的TAC包括：
    (a)胞外结构域：所述胞外结构域包括具有抗原结合结构域和与CD3结合的单链抗体；
    (b)跨膜区；
    (c)胞内结构域，所述胞内结构域连接蛋白激酶LCK。
  8. 如权利要求1-7任一所述的组合物，其特征在于，所述的免疫细胞选自T细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞或骨髓源性吞噬细胞。
  9. TOLL样受体激动剂和免疫效应细胞的组合在制备抗肿瘤药物或抗病原体药物或抗免疫低下的药物中的用途，所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗原的受体，该受体具有胞外区、跨膜区和胞内区，所述的胞外区具有结合所述的肿瘤抗原或病原体抗原的抗原结合结构域；
    所述的受体优选嵌合抗原受体(CAR)、T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、或T细胞抗原耦合器(TAC)。
  10. 如权利要求9所述的用途，其特征在于，所述TOLL样受体激动剂和所述免疫效应细胞制备成非胃肠道给药，优选静脉注射和瘤内注射给药的药物。
  11. 如权利要求9所述的用途，其特征在于，所述的免疫细胞选自T细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞或骨髓源性吞噬细胞。
  12. 如权利要求9-11任一所述的用途，其特征在于，所述的免疫细胞选择表达CAR的T细胞、表达CAR的NK细胞、或表达TCR的T细胞。
  13. 如权利要求9-11任一所述的用途，所述TOLL样受体激动剂为Toll样受体3(TLR3)激动剂，所述的TLR3激动剂优选自天然或合成的双链核糖核酸，更优的为聚肌苷酸胞苷酸(poly(I:C))。
  14. 如权利要求1所述的组合物，其特征在于，所述的免疫效应细胞还表达其他外源性蛋白，优选的，所述外源性蛋白选自下组：FLT3L、干扰素、白介素、白介素受体、PD-1抑制剂、PD-L1抑制剂、或其组合。
  15. 如权利要求1所述的组合物，其特征在于，所述组合物还包括MDSC抑制剂、和/或免疫检查点抑制剂。
  16. 如权利要求15所述的组合物，其特征在于，所述MDSC抑制剂选自下组：抗Gr1抗体、环氧化酶-2抑制剂、前列腺素类干细胞因子抑制剂，巨噬细胞 集落刺激因子抑制剂、粒单核细胞集落刺激因子抑制剂、血管内皮细胞生长因子抑制剂、或其组合。
  17. 如权利要求15所述的组合物，其特征在于，所述免疫检查点抑制剂选自下组：PD-1抑制剂、PD-L1抑制剂、CTLA-4抑制剂、或其组合。
  18. 一种组合物产品，其特征在于，包括：
    (i)第一药物组合物，所述第一药物组合物包括至少一种免疫效应细胞增效剂，所述免疫效应细胞增效剂包括TOLL样受体激动剂；和药学上可接受的载体；
    (ii)第二药物组合物，所述第二药物组合物包括免疫效应细胞，所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗原的受体；和药学上可接受的载体；和
    (iii)第三药物组合物，所述第三药物组合物包括MDSC抑制剂或免疫检查点抑制剂；和药学上可接受的载体。
  19. 一种权利要求18所述的组合物产品的用途，其特征在于，用于制备抗肿瘤药物或抗病原体药物或抗免疫低下的药物。
  20. 一种提高免疫效应细胞活力的方法，其特征在于，包括步骤：
    给需要的对象施用(i)免疫效应细胞；(ii)免疫效应细胞增效剂；和/或(iii)任选的MDSC抑制剂或免疫检查点抑制剂，其中所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗原的受体，所述免疫效应细胞增效剂为TOLL样受体激动剂。
  21. 如权利要求20所述的方法，其特征在于，与单独施用免疫效应细胞相比，施用(i)免疫效应细胞；(ii)免疫效应细胞增效剂；和(iii)任选的MDSC抑制剂或免疫检查点抑制剂的抑瘤率提高了≥20％，较佳地，≥30％。
  22. 如权利要求20所述的方法，其特征在于，与单独施用免疫效应细胞相比，施用(i)免疫效应细胞；(ii)免疫效应细胞增效剂；和(iii)任选的MDSC抑制剂或免疫检查点抑制剂的干扰素释放水平提高了≥20％，较佳地，30％，更佳地，≥50％。
  23. 免疫效应细胞、免疫效应细胞增效剂和淋巴细胞清除剂的组合在制备抗肿瘤或抗病原体或抗免疫力低下的药物中的用途。
  24. 一种治疗肿瘤或病原体感染或免疫力低下的方法，其特征在于，所述方法包括：将免疫效应细胞、免疫效应细胞增效剂和淋巴细胞清除剂的组合给予有此需要的对象，其中所述免疫效应细胞具有靶向识别肿瘤抗原或病原体抗 原的受体，所述免疫效应细胞增效剂为TOLL样受体激动剂。
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