WO2018210279A1 - Utilisation d'un agoniste du récepteur de type toll combiné à une cellule effectrice immunitaire - Google Patents

Utilisation d'un agoniste du récepteur de type toll combiné à une cellule effectrice immunitaire 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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Chinese (zh)
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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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Publication of WO2018210279A1 publication Critical patent/WO2018210279A1/fr

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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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    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
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    • A61K39/46—Cellular immunotherapy
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    • A61K39/4644—Cancer antigens
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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
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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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    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/31—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by the route of administration
    • A—HUMAN NECESSITIES
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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
    • C07—ORGANIC CHEMISTRY
    • 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

La présente invention concerne l'utilisation d'un agoniste du récepteur de type TOLL combiné à une cellule effectrice immunitaire dans la préparation d'un médicament antitumoral, d'un médicament anti-pathogène, ou d'un médicament immunosuppresseur. L'agoniste du récepteur de type TOLL peut améliorer l'effet de la cellule effectrice immunitaire. La présente invention concerne également une composition de l'agoniste du récepteur de type TOLL et de la cellule effectrice immunitaire.
PCT/CN2018/087162 2017-05-16 2018-05-16 Utilisation d'un agoniste du récepteur de type toll combiné à une cellule effectrice immunitaire WO2018210279A1 (fr)

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WO2022028623A1 (fr) 2020-08-07 2022-02-10 佧珐药业有限公司 Cellules modifiées et procédé de modification de cellules
WO2022214089A1 (fr) 2021-04-08 2022-10-13 克莱格医学有限公司 Utilisation d'immunothérapie cellulaire
WO2023274303A1 (fr) 2021-06-29 2023-01-05 科济生物医药（上海）有限公司 Polypeptide chimérique pour la régulation de l'activité physiologique cellulaire

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CN116333141A (zh) * 2019-01-15 2023-06-27 浙江道尔生物科技有限公司 抗cld18a2纳米抗体及其应用
CN111875708B (zh) * 2020-05-28 2021-05-04 浙江康佰裕生物科技有限公司 一种嵌合抗原受体t淋巴细胞及其在制备治疗实体肿瘤的产品中的应用
JP2024500254A (ja) * 2019-08-29 2024-01-09 クレージュ メディカル カンパニー，リミテッド 移植拒絶反応に抵抗する細胞及び方法
CN112062859B (zh) * 2020-07-24 2022-08-09 沣潮医药科技（上海）有限公司 用于病原体清除的嵌合抗原受体及其应用
CN113501885B (zh) * 2021-07-20 2023-02-24 中国农业大学 嵌合抗病基因及其相关生物材料与应用

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WO2022028623A1 (fr) 2020-08-07 2022-02-10 佧珐药业有限公司 Cellules modifiées et procédé de modification de cellules
WO2022214089A1 (fr) 2021-04-08 2022-10-13 克莱格医学有限公司 Utilisation d'immunothérapie cellulaire
WO2023274303A1 (fr) 2021-06-29 2023-01-05 科济生物医药（上海）有限公司 Polypeptide chimérique pour la régulation de l'activité physiologique cellulaire

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