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WO2024151124A1 - Procédé de production d'une thérapie cellulaire immunitaire dérivée de lymphocytes t à mémoire présentant une excellente capacité anticancéreuse - Google Patents

Procédé de production d'une thérapie cellulaire immunitaire dérivée de lymphocytes t à mémoire présentant une excellente capacité anticancéreuse Download PDF

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WO2024151124A1
WO2024151124A1 PCT/KR2024/000602 KR2024000602W WO2024151124A1 WO 2024151124 A1 WO2024151124 A1 WO 2024151124A1 KR 2024000602 W KR2024000602 W KR 2024000602W WO 2024151124 A1 WO2024151124 A1 WO 2024151124A1
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cells
memory
antagonist
pam3csk4
cell
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Korean (ko)
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윤정호
남재국
이연택
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Immunomax Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/11—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from blood or immune system cells

Definitions

  • the present invention relates to a culture medium composition for memory T cell culture and a method for producing a memory T cell-based immune cell therapy using the same. More specifically, it relates to a method for converting uncontacted T cells containing a TLR1 antagonist or TLR2 antagonist as an active ingredient into memory T cells. It relates to a medium composition for promoting differentiation or proliferation of memory T cells and a method for manufacturing a memory T cell-based immune cell therapy with excellent anticancer activity using the same.
  • T cells or T lymphocytes are a type of lymphocyte that controls antigen-specific adaptive immunity.
  • T cells include na ⁇ ve T cells that have not yet encountered the antigen, effector T cells or TE cells that have matured after encountering the antigen (helper T cells, cytotoxic T cells, natural killer T cells), and memory T cells. It is classified as a cell (Memory T cell or TM cell).
  • Na ⁇ ve T cells are T cells that have undergone differentiation and maturation but have not yet encountered antigen in the periphery.
  • Helper T cells or Th cells are cells that promote humoral immunity by regulating the differentiation and activation of other white blood cells among effector T cells.
  • Cytotoxic T cells are cells that kill virus-infected cells or tumor cells by secreting cytotoxic substances such as granzyme or perforin.
  • Natural Killer T cells Natural Killer T cells (Natural Killer T cells or NK/T cells) are one of the effector T cells distributed in a smaller proportion than helper T cells and cytotoxic T cells.
  • Memory T cells are cells that survive for a long period of time after T cells that recognize an antigen go through differentiation and selection processes and are quickly activated when the antigen invades again, with the potential ability to function as effector T cells.
  • Antigen-specific memory T cells specific for viruses or other microbial molecules can be found in the Central Memory T cell (TCM Cell) and Effector Memory T cell (TEM cell) subsets. Memory T cells have been studied intensively recently, but most information is based on observations in the cytotoxic T cell (CD8+) subset, with similar populations existing for both helper T cells (CD4+) and cytotoxic T cells. It appears that The main function of memory T cells is to increase the immune response after the cells are reactivated when the relevant pathogen is reintroduced into the body.
  • Clones of memory T cells that express specific T cell receptors can persist in our bodies for decades. Because memory T cells have a shorter half-life than naive T cells, continuous replication and replacement of old cells are likely involved in the maintenance process (Farber DL et al., Nature Reviews. Immunology . 14:24, 2014). Currently, the mechanisms of memory T cell maintenance are not fully known, and activation through T cell receptors may play a role. Memory T cells have sometimes been shown to be able to respond to new antigens, potentially triggered by the inherent diversity and breadth of T cell receptor binding targets, and can proliferate by cross-reacting with environmental or resident antigens in our body (such as gut bacteria). You can. This will help maintain memory T cell populations. Cross-reactivity mechanisms may be important for memory T cells in mucosal tissues, as these sites have higher antigen densities.
  • T cells undergo different changes and play different roles at different stages of human life.
  • T cells present in the peripheral blood of birth and infancy are mainly na ⁇ ve T cells, and a population of memory T cells accumulates through frequent antigen exposure.
  • the number of memory T cells plateaus and is stabilized by homeostasis (Kumar BV et al., Immunity . 48:202, 2018).
  • the immune response shifts toward maintaining homeostasis as new antigens are rarely generated, and tumor surveillance is also important at this stage.
  • the immune aging stage occurs, and at this stage, impaired immune regulation, decreased T cell function, and increased susceptibility to pathogens are observed.
  • the present inventors have made diligent efforts to develop a method to promote the differentiation of memory T cells, which play an important role in the immune response of anti-cancer and infectious diseases, and to promote the growth of memory T cells.
  • naive T cells are treated with a TLR antagonist.
  • TLR antagonist When cultured, it was confirmed that differentiation into memory T cells was activated, proliferation rate increased, and anticancer activity improved, and the present invention was completed.
  • the purpose of the present invention is to provide a method for manufacturing an immune cell therapy containing memory T cells as an active ingredient.
  • Another object of the present invention is to provide an immune cell therapy containing memory T cells as an active ingredient.
  • Another object of the present invention is to provide a medium composition for promoting differentiation of naive T cells into memory T cells or for proliferation of memory T cells.
  • Another object of the present invention is to provide a method for promoting differentiation and proliferation of naive T cells into memory T cells.
  • Another object of the present invention is to provide a method for producing memory T cells.
  • the present invention provides a method for producing an immune cell therapy containing memory T cells comprising the following steps:
  • the present invention also provides an immune cell therapy agent containing memory T cells produced by the above method as an active ingredient.
  • the present invention also provides a medium composition for promoting differentiation of naive T cells into memory T cells or for proliferation of memory T cells, containing a TLR1 antagonist or TLR2 antagonist as an active ingredient.
  • the present invention also provides a method for promoting differentiation of memory T cells comprising the following steps:
  • PBMC peripheral blood mononuclear cells
  • the present invention also provides a method for proliferating memory T cells comprising the following steps:
  • the present invention also provides a method for producing memory T cells comprising the following steps:
  • the present invention also provides a method for preventing or treating cancer using memory T cells produced by the above method.
  • the present invention also provides the use of memory T cells produced by the above method for preventing or treating cancer.
  • the present invention also provides the use of memory T cells produced by the above method for producing an immune cell therapy for preventing or treating cancer.
  • Figure 1 shows an experimental method for analyzing the effect of enhancing memory T cells by TLR antagonist treatment.
  • Figure 2 shows the results of analyzing the proliferation ability and survival rate of human peripheral blood mononuclear cells by treatment with a TLR antagonist.
  • FIG 3 shows the results of analyzing changes in T cell phenotype in CD3+ T cells (T E : effector T cells, T EM : effector memory T cells, TCM: central memory T cells, T SCM / Naive : Stem cell-like memory T cells/naive T cells).
  • Figure 4 shows the results of analyzing changes in T cell phenotype in CD3+ CD4+ T cells.
  • Figure 5 shows the results of analyzing changes in T cell phenotype in CD3+ CD8+ T cells.
  • Figure 6 shows the results of analyzing the absolute cell number of the group treated with Pam3CSK4, a TLR antagonist.
  • Figure 7 shows the results of analyzing changes in cytokine secretion from human peripheral blood mononuclear cells caused by TLR antagonists.
  • Figure 8 shows changes in absolute cell number and cytokine secretion of human peripheral blood mononuclear cells in the TLR antagonist-treated group.
  • Figure 9 shows the results of analyzing the effect of enhancing memory T cells according to high concentration Pam3CSK4 treatment.
  • Figure 10 shows the results of analyzing memory T cell phenotypic changes according to Pam3CSK4 treatment.
  • FIG 11 shows the results of comparative analysis of the proliferation ability of human peripheral blood mononuclear cells by TLR antagonists (Pam3CSK4, Pam2CSK4).
  • Figure 12 shows the results of a comparative analysis of the memory T cell enhancing ability by TLR antagonists (Pam3CSK4, Pam2CSK4).
  • Figure 13 shows the results of analyzing the absolute cell number of central memory T cells increased by TLR antagonists (Pam3CSK4, Pam2CSK4).
  • Figure 14 shows the results of analyzing the memory T cell enhancement effect by Pam3CSK4, a TLR antagonist.
  • Figure 15 shows the results of analyzing the in vitro cancer cell killing ability of memory T cells against human chronic leukemia cell line (K562) and liver cancer cell line (HepG2) by treatment with Pam3CSK4, a TLR antagonist.
  • Figure 16 shows the results of analyzing the in vivo efficacy of treatment with Pam3CSK4, a TLR antagonist.
  • Figure 17 shows the results of analyzing the in vivo anti-tumor effect of spleen cells of OT-1 mice treated with Pam3CSK4, a TLR antagonist, on the E.G7 cell line.
  • Figure 18 shows the results of analyzing immune cells after treating spleen cells of OT-1 mice with Pam3CSK4, a TLR antagonist.
  • Figure 19 shows the results of analyzing tumor infiltration immune cells (Tumor Infiltration Leukocyte, TIL) after administering spleen cells from OT-1 mice treated with Pam3CSK4, a TLR antagonist, to mice engrafted with the E.G7 cancer cell line.
  • TIL tumor infiltration immune cells
  • Figure 20 shows the analysis of the correlation between immune cells present within tumor-infiltrating immune cells and tumor size after administering spleen cells from OT-1 mice treated with Pam3CSK4, a TLR antagonist, to mice engrafted with E.G7 cancer cell line. will be.
  • Figure 21 shows the results of analyzing the in vivo efficacy of memory T cells cultured by treatment with Pam3CSK4, a TLR antagonist, for tumor cell killing ability in the Xenograft animal model of HepG2, a human liver cancer cell line.
  • Figure 22 shows the results of analyzing changes in the memory T cell phenotype of CD3+ TCR V ⁇ 2+ T cells according to treatment at different Pam3CSK4 concentrations.
  • Figure 23 shows the results of analyzing changes in the memory T cell phenotype of CD3+ TCR V ⁇ 2+ T cells according to Pam3CSK4 and IL-21 treatment.
  • peripheral blood mononuclear cells peripheral blood mononuclear cells
  • TLR antagonist Toll-like receptor agonist
  • T cell refers to a type of lymphocyte that hosts antigen-specific adaptive immunity.
  • T cells include na ⁇ ve T cells that have not yet encountered an antigen, mature T cells that have encountered an antigen, and memory T cells.
  • the mature T cells include cytotoxic T cells, ⁇ T cells, and ⁇ T cells, and “memory T cells (Central memory T cells, TCM)” include T cells with TCR ⁇ and TCR ⁇ .
  • ⁇ T cells used in the present invention exist in a very small proportion of about 0.5 to 5% in human peripheral blood, and functionally involve two types of immune responses: adaptive immunity and innate immunity. It is known that they are all involved in.
  • Human ⁇ T cells have two main subtypes of TCR chains: the V ⁇ 1 chain or the V ⁇ 2 chain. Most ⁇ T cells in the blood have the V ⁇ 2 chain, which exists in pairs with the V ⁇ 9 chain.
  • TCR ⁇ T cells develop before TCR ⁇ T cells in the thymus, and have limited TCR diversity against several antigens, including phosphoantigens, but initiate an immune response more rapidly than TCR ⁇ T cells, inducing an innate immune response. do.
  • the present invention relates to a method for producing an immune cell therapy containing memory T cells comprising the following steps:
  • the TLR antagonist is an agent that binds to a toll-like receptor and promotes the activity of TLR, including LPS, CpG motif, dsRNA, poly (I:C), Pam3Cys, Pam3CSK4, lipid A, heat shock protein, plasma Gelin, lipotichoic acid, MPLAs, Resiquimod, Gariquimod, etc. are known.
  • the TLR1 antagonist or TLR2 antagonist includes Pam3Cys, Pam3CSK4, CU-T12-9, Diprovocim, EBV-encoded dUTPase Glycoprotein B, Glucuronoxylomannan, Glycosylphosphatidylinositol (GPI) anchors, Heat-labile enterotoxins (b subunit), Hepatitis B Capsid, Lipomannan/lipoarabinomannan, Lipophosphoglycan, Lipopeptidophosphoglycan, Lipoprotein, Lipoteichoic acid (LTA), Peptidoglycan (PG), Lysophosphatidylserine, Zymosan, Porins, Phospholipomannan, Triacyl lipopeptides, Triacylated lipoproteins, Yeast phase-specific protein (Yps3p), etc. can be used. , preferably Pam3Cys or Pam3CSK4, but is not limited thereto.
  • Pam3CSK4 or Pam2CSK4 contained in the cell culture medium composition of the present invention binds to Toll-like receptor 2 and Toll-like receptor 1 or forms a heterodimer with toll-like receptor 6.
  • the agonist binds to Toll-like receptor 2 and Toll-like receptor 1 or to Toll-like receptor 2 and Toll-like receptor 6, MyD88 is activated, and NF-kB and AP-1 are produced, causing an innate immune response. do.
  • Pam3CSK4 or Pam2CSK4 (Toll-like receptor 2 agonist) of the present invention has the characteristic of inducing differentiation and activation of naive T cells into memory T cells.
  • the present invention relates to a medium composition for promoting differentiation of naive T cells into memory T cells or for proliferation of memory T cells, containing a TLR1 antagonist or a TLR2 antagonist as an active ingredient.
  • the TLR1 antagonist or TLR2 antagonist includes Pam3Cys, Pam3CSK4, CU-T12-9, Diprovocim, EBV-encoded dUTPase Glycoprotein B, Glucuronoxylomannan, Glycosylphosphatidylinositol (GPI) anchors, Heat-labile enterotoxins (b subunit), Hepatitis B Capsid, Lipomannan/lipoarabinomannan, Lipophosphoglycan, Lipopeptidophosphoglycan, Lipoprotein, Lipoteichoic acid (LTA), Peptidoglycan (PG), Lysophosphatidylserine, Zymosan, Porins, Phospholipomannan, Triacyl lipopeptides, Triacylated lipoproteins, Yeast phase-specific protein (Yps3p), etc. can be used. , preferably Pam3Cys or Pam3CSK4, but is not limited thereto.
  • the medium composition may further include a reducing agent, and the reducing agent is selected from the group consisting of ⁇ -mercaptoethanol, DTT (dithiothreitol), and TCEP (tris-(2-carboxyl)phosphine). Preferred, more preferably ⁇ -mercaptoethanol, but not limited thereto.
  • the base medium used in the medium composition is preferably selected from the group consisting of DMEM, IMDM, MEM, RPMI-1640 and EMEM, more preferably RPMI-1640, but is not limited thereto. .
  • the medium composition is a basic medium containing 8.0 to 10% by weight of fetal bovine serum (FBS) relative to the total weight, RPMI-1640, and 1.0 to 20 mM HEPES (4-(2) -hydroxyethyl)-1-piperazineethanesulfonic acid), 0.1 to 5 mM L-glutamine, 30 to 70 ⁇ M 2-mercaptoethanol, 0.1 to 3.0 mM Sodium private, 1 wt % penicillin/streptomycin, and 1.0 to 5.0 ⁇ M.
  • FBS fetal bovine serum
  • HEPES 4-(2) -hydroxyethyl)-1-piperazineethanesulfonic acid
  • 0.1 to 5 mM L-glutamine 30 to 70 ⁇ M 2-mercaptoethanol
  • 0.1 to 3.0 mM Sodium private 1 wt % penicillin/streptomycin
  • Those containing the daurinol compound can be used.
  • the Pam3CSK4 or the Pam2CSK4 may be contained at a concentration of 4.0 to 32 ⁇ g/mL, preferably 4.0 to 16 ⁇ g/mL, and more preferably 4.0 to 8.0. It may be contained at a concentration of ⁇ g/mL.
  • the present invention relates to a method for promoting differentiation of memory T cells comprising the following steps:
  • the TLR1 antagonist or TLR2 antagonist includes Pam3Cys, Pam3CSK4, CU-T12-9, Diprovocim, EBV-encoded dUTPase Glycoprotein B, Glucuronoxylomannan, Glycosylphosphatidylinositol (GPI) anchors, Heat-labile enterotoxins (b subunit), Hepatitis B Capsid, Lipomannan/lipoarabinomannan, Lipophosphoglycan, Lipopeptidophosphoglycan, Lipoprotein, Lipoteichoic acid (LTA), Peptidoglycan (PG), Lysophosphatidylserine, Zymosan, Porins, Phospholipomannan, Triacyl lipopeptides, Triacylated lipoproteins, Yeast phase-specific protein (Yps3p), etc. can be used. , preferably Pam3Cys or Pam3CSK4, but is not limited thereto.
  • the Pam3CSK4 or the Pam2CSK4 may be contained at a concentration of 4.0 to 32 ⁇ g/mL, preferably 4.0 to 16 ⁇ g/mL, and more preferably 4.0 to 8.0. It may be contained at a concentration of ⁇ g/mL.
  • memory T cells can be differentiated and activated while peripheral blood mononuclear cells are cultured in the above medium at 35-37°C for 7 days.
  • the present invention relates to a method for proliferating memory T cells comprising the following steps:
  • the TLR1 antagonist or TLR2 antagonist includes Pam3Cys, Pam3CSK4, CU-T12-9, Diprovocim, EBV-encoded dUTPase Glycoprotein B, Glucuronoxylomannan, Glycosylphosphatidylinositol (GPI) anchors, Heat-labile enterotoxins (b subunit), Hepatitis B Capsid, Lipomannan/lipoarabinomannan, Lipophosphoglycan, Lipopeptidophosphoglycan, Lipoprotein, Lipoteichoic acid (LTA), Peptidoglycan (PG), Lysophosphatidylserine, Zymosan, Porins, Phospholipomannan, Triacyl lipopeptides, Triacylated lipoproteins, Yeast phase-specific protein (Yps3p), etc. can be used. , preferably Pam3Cys or Pam3CSK4, but is not limited thereto.
  • the Pam3CSK4 or the Pam2CSK4 may be contained at a concentration of 4.0 to 32 ⁇ g/mL, preferably at a concentration of 4.0 to 16 ⁇ g/mL, and more preferably 4.0 to 16 ⁇ g/mL. It may be contained at a concentration of 8.0 ⁇ g/mL.
  • memory T cells can be differentiated and activated while peripheral blood mononuclear cells are cultured in the above medium at 35 to 37 ° C. for 7 days.
  • the present invention relates to a method for producing memory T cells comprising the following steps:
  • the TLR1 antagonist or TLR2 antagonist includes Pam3Cys, Pam3CSK4, CU-T12-9, Diprovocim, EBV-encoded dUTPase Glycoprotein B, Glucuronoxylomannan, Glycosylphosphatidylinositol (GPI) anchors, Heat-labile enterotoxins (b subunit), Hepatitis B Capsid, Lipomannan/lipoarabinomannan, Lipophosphoglycan, Lipopeptidophosphoglycan, Lipoprotein, Lipoteichoic acid (LTA), Peptidoglycan (PG), Lysophosphatidylserine, Zymosan, Porins, Phospholipomannan, Triacyl lipopeptides, Triacylated lipoproteins, Yeast phase-specific protein (Yps3p), etc. can be used. , preferably Pam3Cys or Pam3CSK4, but is not limited thereto.
  • the Pam3CSK4 or the Pam2CSK4 may be contained at a concentration of 4.0 to 32 ⁇ g/mL, preferably at a concentration of 4.0 to 16 ⁇ g/mL, and more preferably 4.0 to 16 ⁇ g/mL. It may be contained at a concentration of 8.0 ⁇ g/mL.
  • the present invention also relates to an immune cell therapy agent containing memory T cells produced by the above method as an active ingredient.
  • the immune cell therapy agent is preferably for treating cancer, more preferably adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, sarcoma, and leukemia.
  • cancer more preferably adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, sarcoma, and leukemia.
  • leukemia lymphoma, multiple myeloma, melanoma, brain tumor, spine bone tumor, germ cell tumor, neuroendocrine tumor
  • a disease selected from the group consisting of tumor
  • cancer metastasis cancer disease.
  • the memory T cell-based immune cell therapy using TLR antagonists (Pam3CSK4 and Pam2CSK4) was confirmed to have cancer cell killing ability in human chronic leukemia cell lines and liver cancer cell lines in vitro (FIG. 15).
  • memory T cell-based immune cell therapy using TLR antagonists (Pam3CSK4 and Pam2CSK4) was injected intravenously at 1 , As shown in Figure 21, after administering memory T cells co-treated with an antigen-specific material and the TLR antagonist Pam3CSK4, tumor growth was confirmed to be reduced compared to the control group or the group treated with the non-antigen-specific material.
  • the present invention relates to a method for preventing or treating cancer, including administering the immune cell therapy agent to a cancer patient.
  • a pharmaceutically acceptable carrier is a substance that can be added to an active ingredient to help formulate or stabilize the preparation and does not cause significant detrimental toxic effects in the patient.
  • the carrier refers to a carrier or diluent that does not stimulate the patient and does not inhibit the biological activity and characteristics of memory T cells according to the present invention.
  • Acceptable pharmaceutical carriers in compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed.
  • injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.
  • injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.
  • injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.
  • Other carriers are described, for example, in Remington's Pharmaceutical Sciences (E. W. Martin).
  • compositions include sterile aqueous solutions or dispersions and sterile powders for preparing sterile injectable solutions or dispersions for extemporaneous administration.
  • the use of such media and agents for pharmaceutically active substances is known in the art.
  • the composition is preferably formulated for parenteral injection.
  • Compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations.
  • the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof.
  • isotonic agents such as sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, may be included in the composition.
  • Sterile injectable solutions can be prepared by incorporating the required amount of cytotoxic T cells in an appropriate solvent with one or a combination of ingredients described above, as required, followed by sterile microfiltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle containing the basic dispersion medium and the other necessary ingredients from those described above.
  • some preparation methods include vacuum drying and freeze-drying (lyophilization), which produce powders of the active ingredient and any additional desired ingredient from a previously sterile-filtered solution thereof. )am.
  • composition according to the present invention may be administered orally or parenterally at a dosage and frequency that may vary depending on the severity of the patient's suffering.
  • the composition may be administered to the patient as a bolus or by continuous infusion as needed.
  • the present invention relates to a method for preventing or treating cancer using memory T cells produced by the above method.
  • the present invention relates to the use of memory T cells produced by the above method for the prevention or treatment of cancer.
  • the present invention relates to the use of memory T cells produced by the above method for producing an immune cell therapy agent for preventing or treating cancer.
  • Example 1 Isolation of Human Peripheral Blood Mononuclear Cell (hPBMC)
  • 100 mL of blood from a healthy donor was added to four 50 mL tubes, along with 25 mL of 1X DPBS and 25 mL of blood using a 25 mL surgical pipette.
  • the blood was separated into four layers and the white buffy coat layer between the Lymphoprep and plasma layers was collected and transferred to 50 mL tubes (3 to 4).
  • the tube containing the layer was centrifuged at 1,500 rpm (500 The cells collected in the tube were centrifuged at 1,250 rpm (350 1.0 mL of the cell suspension was suspended in 1X DPBS and dispensed into a microtube as a Day 0 sample, and the next experiment was performed.
  • Example 2 Induction of uncontacted T cells into memory T cells
  • Example 1 Human peripheral blood mononuclear cells isolated in Example 1 were induced into memory T cells.
  • CD3+, CD4+ T cells were tested to determine whether they were differentiated from uncontacted T cells to memory T cells.
  • Cells and CD8+ T cells were analyzed for the type of cultured cells using specific cell surface antibodies and flow cytometry.
  • Alexa-647-labeled anti-CCR7 (the antibodies were purchased from BD Biosciences).
  • Antibody kit used to measure human T h 1/T h 2 cytokine secretion
  • BDTM Cytometric Bead Array Human T h 1/T h 2 Cytokine Kit II (the above antibody kit was purchased from BD Biosciences).
  • TLR antagonist has a similar effect as memory T cell enhancement complex (IM3 (30%)) (see Korean Patent No. 10-2130599).
  • IM3 memory T cell enhancement complex
  • the memory T cell enhancement effect was confirmed when treating human peripheral blood mononuclear cells with a memory T cell enhancement complex, and the test method for the effectiveness of the memory T cell enhancement complex using existing human peripheral blood mononuclear cells was conducted in the same manner.
  • Monocytes were isolated from human peripheral blood. 1x10 6 cells were inoculated into each well of a 24 well plate, and cultured in a culture medium containing anti-CD3, anti-CD28, and human IL-2 at 37°C in a 5% CO 2 incubator for 7 days. The experimental and control groups were divided into a group without memory enhancement complex (NT) and a group with memory T cell enhancement complex. After 7 days of culture, cells were recovered and reacted with fluorescent anti-CD3, anti-CD8, anti-CD45RO, and anti-CCR7 antibodies. After removing the antibodies that did not attach to the cells through a washing process, the cell phenotype was confirmed using flow cytometry (FACS), BD LSR FortessaII, and the results were analyzed using FlowJo software.
  • FACS flow cytometry
  • BD LSR FortessaII BD LSR FortessaII
  • TLR antagonists Pam3CSK4 (TLR1/2), MPLAS (TLR4, invitrogen, USA), Poly(I:C) ( TLR-3, invitrogen, USA), Resiquimod (TLR7/8, Sigma-Aldrich, USA), and Gariquimod (TLR7/8, Sigma-Aldrich, USA) were evaluated using the 2-fold serial dilution method. .
  • Cell culture continued until Day 7, and on Day 3, 0.5 mL was removed and 1.5 mL of new culture medium was added.
  • the effect of memory T cell enhancement by TLR antagonists was analyzed using each specific cell surface antibody and flow cytometry, and the number of cells was measured to analyze proliferation ability and survival rate (Figure 1).
  • NK cells Natural Killer Cells
  • CD4+ T cells CD4+ T cells
  • CD8+ T cells were cultured using specific cell surface antibodies and flow cytometry. The types were analyzed.
  • the group treated with Pam3CSK4 and Poly(I:C) showed similar proliferation and survival rates as the group added with the memory T cell enhancement complex.
  • MPLA, R848, and Grmd appear to maintain proliferation and survival rates only at the lowest concentrations, and MPLA and Grmd appear to maintain low proliferation and survival rates in human peripheral blood mononuclear cells at high concentrations.
  • the rate of phenotypic change of T cells in CD3+ T cells was evaluated.
  • T E effector T cells
  • T EM effector memory T cells
  • TCM central memory T cells
  • TS CM /Naive stem cell-like memory T cells/naive T cells
  • the control group the (+) control group with memory T cell enhancement complex added, and the TLR antagonists Pam3CSK4, MPLAS, Poly(I:C), Resiquimod, and Gariquimod were treated.
  • the cells were grown and cultured until Day 7, and changes in the ratio of CD3+ CD4+ T cells were compared and analyzed.
  • CD3+ and CD4+ T cells increased in central memory T cells, but the increase was not significant compared to the untreated group.
  • the increase rate of central memory T cells in CD8+ T cells treated with Pam3CSK4 was found to be the largest compared to other effector memory T cells or effector T cells. Only the group treated with Pam3CSK4 showed a similar effect as the memory T cell enhancement complex, and the memory T cell enhancement effect of the memory T cell enhancement complex was confirmed to be able to promote central memory CD8 T cells due to signaling of TLR2. .
  • Th1 cytokines IFN- ⁇ , TNF, IL-2
  • the untreated group, (+) control group, memory T cell enhancement complex, 4.0 ⁇ g/ml, 8.0 ⁇ g/ml, 16 ⁇ g/ml, and 32 ⁇ g/ml of Pam3CSK4 were treated and analyzed.
  • memory T cells increased in a concentration-dependent manner from 4.0 to 16 ⁇ g/ml Pam3CSK4 concentration, but decreased at a concentration of 32 ⁇ g/ml.
  • the memory T cell enhancement effect by Pam3CSK4 appears to be lower than that of IM3, a conventional memory T cell enhancement complex, but in the group treated with 4.0 to 16 ⁇ g/ml Pam3CSK4, the memory T cell enhancement effect was observed in a concentration-dependent manner.
  • Example 4 Comparative analysis of in vitro effect of enhancing memory T cells in human peripheral blood mononuclear cells using TLR antagonists (Pam3CSK4 and Pam2CSK4)
  • Example 1 1.0 x 10 6 cells/mL of human peripheral blood mononuclear cells obtained by the method of Example 1 were added to each lane.
  • the control group, the (+) control group with memory T cell enhancement complex (IM3) addition group, and the TLR antagonists Pam3CSK4 and Pam2CSK4 were evaluated using a 2-fold serial dilution method.
  • Cell culture continued until Day 7, and on Day 3, 0.5 mL was removed and 1.5 mL of new culture medium was added.
  • the effect of memory T cell enhancement by TLR antagonists was analyzed using each specific cell surface antibody and flow cytometry, and the number of cells was measured to analyze proliferation ability and survival rate.
  • the proliferative ability of human peripheral blood mononuclear cells decreased in a concentration-dependent manner for both types of TLR antagonists (Pam3CSK4, Pam2CSK4).
  • the control group untreated group
  • the control group showed an 11-fold increase in proliferation ability, but when treated with Pam3CKS4, it increased 6.5-fold, but the survival rate (Viability) remained above 90%.
  • Pam3CSK4 tends to reduce the proliferative ability of human peripheral blood mononuclear cells, but the proliferative ability was also reduced when treated with Pam2CSK4.
  • the absolute cell numbers of central memory T cells were analyzed by two types of TLR antagonists, Pam3CSK4 and Pam2CSK4.
  • the proliferative ability of human peripheral blood mononuclear cells decreased as the concentration increased, and in the 16 ⁇ g/ml concentration treatment group, it decreased to a level similar to that of the (+) control group, memory T cell enhancement complex.
  • the effect of enhancing memory T cells by Pam3CSK4 was analyzed. As the concentration increased, the proportion of memory T cells in human peripheral blood mononuclear cells increased, but it actually decreased when treated at a high concentration of 32 ⁇ g/ml, and when treated with 8.0 ⁇ g/ml and 16 ⁇ g/ml, the enhancing effect of central memory T cells was observed in memory T cells. It was similar to the enhancement complex.
  • the results of analyzing the change in the absolute number of memory T cells by Pam3CSK4 showed that as the concentration of Pam3CSK4 increases, the proportion of memory T cells in human peripheral blood mononuclear cells increases, but as the fold value decreases, the number of memory T cells decreases. There is a possibility. The number of memory T cells was found to be the highest when treated with 8.0 ⁇ g/ml.
  • Example 5 Comparative analysis of the in vitro effect of enhancing anticancer activity of memory T cell-based immune cell therapy using TLR antagonists (Pam3CSK4 and Pam2CSK4)
  • T cells non-specific to cancer antigens were treated with ⁇ -CD3/CD28 and cultured.
  • the cancer cell killing ability for chronic leukemia cell line (K562: ATCC, USA) showed cancer cell-specific killing ability of about 75-80% or more at an E:T ratio of 20:1, and also for liver cancer cell line (HepG2: ATCC, USA) also showed a specific killing ability against about 60% of cancer cells when treated with a TLR antagonist (Pam3CSK4) at an E:T ratio of 20:1.
  • Example 6 In vivo evaluation of antitumor activity of memory T cell-based Splenocytes treated with TLR antagonists (Pam3CSK4 and Pam2CSK4)
  • the E.G7 cancer cell line (ATCC, USA) expressing OVA as an antigen was subcutaneously injected into C57BL/6 to evaluate the effect of enhancing memory T cells when the tumor size reached 100-150 mm3.
  • Control group (+ )
  • memory T cell enhancement complex addition group mouse T cells cultured with the TLR antagonist Pam3CSK4 (invitrogen, USA) were injected into the tail vein of the mouse, and the change in tumor size was measured by 2% for about 30 days. -Observed and analyzed once every three days.
  • Mouse T cells were isolated from the spleens of enucleated OT-1 mice and prepared by in vitro culture under the same culture conditions as the experimental and control groups described above.
  • mice We analyzed whether T cells treated with memory T cell enhancement complex or TLR antagonist showed antitumor activity in mice.
  • IM3 memory T cell enhancement complex
  • Pam3CSK4 TLR antagonist
  • the mouse group injected with OT-1 spleen cells (SPL) treated with IM3 or Pam3CSK4 showed anti-tumor activity, and the IM3-treated group showed statistical improvement compared to the group treated with OT-1 spleen cells alone.
  • the Pam3CSK treatment group had similar results to the IM3 treatment group, but was not statistically significant compared to the OT-1 spleen cell treatment group alone.
  • FIG. 18A-Top when T cells treated with Pam3CSK4, a TLR antagonist, were administered, approximately 10-20% more CD3+ T cells were comprised in the blood compared to the control group.
  • Figure 18A-Middle shows that within CD3+ T cells, when T cells treated with memory T cell enhancement complex or Pam3CSK4 were administered, there were about 15% more CD8+ T cells in the blood and about 15% fewer CD4+ T cells than in the control group. It was composed.
  • Figure 18A-Bottle shows that when T cells treated with Pam3CSK4, a TLR antagonist, were administered, more than 30% more antigen-specific CD8+ T cells were formed in the blood compared to the rest of the groups.
  • TIL tumor infiltration leukocytes
  • Isolated OT-1 mouse spleen cells were treated with Pam3CSK4, a TLR antagonist, cultured, and then administered to mice with E.G7 cancer cell line engraftment, and tumor infiltration immune cells (Tumor Infiltration Leukocyte, TIL) were analyzed.
  • TIL Tumor Infiltration Leukocyte
  • CD3+ T cells when memory T cells were administered, the distribution ratio of CD3+ T cells within TILs increased by about 20% or more compared to the PBS-administered group, and when T cells treated with memory T cell enhancement complex or Pam3CSK4 were administered.
  • CD3+ T cells When compared to the control group, CD3+ T cells were distributed at a rate of approximately 10% higher.
  • antigen-specific T cells in TIL constitute about 30% of CD8+ T cells when cultured with Pam3CSK4 and then administered, and are distributed at a higher rate of about 15% or more compared to other groups.
  • the upper graph of Figure 19B shows that effector T cells were distributed at a rate more than 15% lower than that of other groups when T cells treated with memory T cell enhancement complex or Pam3CSK4 were administered, and memory T cells were distributed within TIL. was analyzed as less than 1%.
  • the middle graph in Figure 19B shows that in the case of the immune checkpoint factors PD-1 and Tim3, both total CD8 T cells and antigen-specific CD8+ T cells increased by more than 50% when T cells treated with memory T cell enhancement complex or Pam3CSK4 were administered. A decreasing trend appeared.
  • the lower graph of Figure 19 shows that the expression rate of PD-1 and Tim3, which are immune checkpoint factors, in antigen-specific CD8+ T cells treated with memory T cell enhancement complex or Pam3CSK4 in TILs decreased to about 50% or more lower than that in the control group. .
  • Isolated OT-1 mouse spleen cells were treated with Pam3CSK4, a TLR antagonist, cultured, and then administered to mice engrafted with E.G7 cancer cell line. The correlation between immune cells present within tumor-infiltrating immune cells and tumor size was analyzed.
  • the tumor size tended to decrease as the number of CD3+ T cells and antigen-specific CD8+ T cells among tumor-infiltrating immune cells (TIL) present in the blood increased, and PD-, an immune checkpoint factor, In case 1, the size of the tumor tended to decrease as the expression of antigen-specific CD8+ T cells decreased.
  • TIL tumor-infiltrating immune cells
  • Example 7 Comparative analysis of the in vivo effect of enhancing anticancer activity of memory T cell-based cell therapy using TLR antagonists (Pam3CSK4 and Pam2CSK4)
  • TLR antagonists Pam3CSK4 and Pam2CSK4
  • the cancer cell killing ability was evaluated in human melanoma cell lines and lung cancer cell lines, and T cells non-specific to cancer antigens.
  • Control group was treated with ⁇ -CD3/CD28 and cultured.
  • the human liver cancer cell line HepG2 (ATCC, USA) Xenograft animal model was created by subcutaneously injecting 5 15 days after tumor cell administration, a single 1
  • Example 8 Induction of na ⁇ ve ⁇ T cells into memory T cells
  • T cells were proliferated from human peripheral blood mononuclear cells isolated in Example 1 and then induced into memory T cells.
  • the ⁇ T cells of this example can be used by proliferation and culture by the method described in Korean Patent Publication No. 2023-0105166, but are not limited thereto.
  • Peripheral blood mononuclear cells were stimulated with 1 ⁇ M Zoledronic acid (USV) in CTS TM OpTmizerTCellExpansionSFM (Gibco, USA) culture medium containing 10% heat-inactivated fetal bovine serum to induce ⁇ T cells, and 1.0 ⁇ g/mL After treatment with Pam3CSK4, the cells were cultured for 7 days and differentiated into memory T cells.
  • USV Zoledronic acid
  • CTS TM OpTmizerTCellExpansionSFM Gibco, USA
  • Example 9 Analysis of differentiated T cells following treatment with Pam3CSK4 and cytokine IL-21
  • Memory T cells cultured for 7 days were differentiated from uncontacted T cells into memory T cells by using cell surface antibodies specific for CD3+ T cells and ⁇ T cells and flow cytometry to determine the type of cultured cells. analyzed.
  • BV605-labeled anti-CD27 (The above antibodies were purchased from Biolegend and BD biosciences)
  • the proportion of central memory T cells increased in CD3+ TCR V ⁇ 2+ cells cultured for 7 days after IL-21 or mixed treatment including Pam3CSK4 and IL-21, compared to the IL-21 treated group.
  • the proportion of central memory T cells increased more significantly in the mixed treatment group containing both Pam3CSK4 and IL-21.
  • the memory T cell-based immune cell therapy using TLR antagonists (Pam3CSK4 and Pam2CSK4) according to the present invention has excellent cancer cell killing ability and exhibits a tumor suppressive effect
  • the cell culture medium composition according to the present invention is a memory T cell Cultivating cells for the treatment of immune diseases using memory T cells, which have the potential ability to function as effector T cells by promoting differentiation into cells, activating the proliferation of memory T cells, and quickly being activated when antigens invade again. It is suitable for use as a culture medium for:
  • cells for the treatment of immune diseases can be maintained for a long period of time, making it useful for cultivating cell therapy products with excellent therapeutic efficacy.

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Abstract

La présente invention se rapporte à une composition de milieu pour la culture de lymphocytes T à mémoire et un procédé de production d'une thérapie cellulaire immunitaire anticancéreuse à base de lymphocytes T à mémoire utilisant cette dernière. Plus spécifiquement, la présente invention se rapporte à une composition de milieu comprenant un antagoniste de TLR1 ou un antagoniste de TLR2 comme principe actif pour favoriser la différenciation de lymphocytes T à mémoire à partir de lymphocytes T sans contact ou la prolifération de lymphocytes T à mémoire et un procédé de production d'une thérapie cellulaire immunitaire à base de lymphocytes T à mémoire présentant une excellente capacité anticancéreuse utilisant cette dernière. La thérapie cellulaire immunitaire selon la présente invention présente une excellente capacité de destruction de cellules cancéreuses et présente des effets de suppression de tumeur.
PCT/KR2024/000602 2023-01-12 2024-01-12 Procédé de production d'une thérapie cellulaire immunitaire dérivée de lymphocytes t à mémoire présentant une excellente capacité anticancéreuse Ceased WO2024151124A1 (fr)

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Citations (4)

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JP2011016811A (ja) * 2002-12-30 2011-01-27 Three M Innovative Properties Co 組み合わせ免疫賦活薬
KR20190017702A (ko) * 2017-08-10 2019-02-20 주식회사 굳티셀 암 치료를 위한 t 세포의 활성화 방법
KR20220044242A (ko) * 2019-05-08 2022-04-07 바이오엔테크 유에스 인크. T 세포 제조 조성물 및 방법
KR20220098351A (ko) * 2019-10-07 2022-07-12 노쓰웨스트 바이오써라퓨틱스, 인크. 수지상 세포 및 T 세포의 활성화를 증진시키고 Th-1 면역 반응을 유도하기 위한 시험관내 방법 및 조성물

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JP2011016811A (ja) * 2002-12-30 2011-01-27 Three M Innovative Properties Co 組み合わせ免疫賦活薬
KR20190017702A (ko) * 2017-08-10 2019-02-20 주식회사 굳티셀 암 치료를 위한 t 세포의 활성화 방법
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