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WO2023113545A1 - DENDRITIC CELL-MIMETIC FUNCTIONAL NANOSTRUCTURE COMPRISING αPD-1, AND PREPARATION METHOD THEREFOR - Google Patents

DENDRITIC CELL-MIMETIC FUNCTIONAL NANOSTRUCTURE COMPRISING αPD-1, AND PREPARATION METHOD THEREFOR Download PDF

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WO2023113545A1
WO2023113545A1 PCT/KR2022/020602 KR2022020602W WO2023113545A1 WO 2023113545 A1 WO2023113545 A1 WO 2023113545A1 KR 2022020602 W KR2022020602 W KR 2022020602W WO 2023113545 A1 WO2023113545 A1 WO 2023113545A1
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nanostructure
cell
mimicking
αpd
dendritic cell
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French (fr)
Korean (ko)
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하상준
문채원
김다혜
홍진기
김태현
이유진
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Fortuga Bio Inc
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Fortuga Bio Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—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
    • A61K47/50—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
    • 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
    • A61K47/54—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 an organic compound
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A61K2039/585—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation wherein the target is cancer

Definitions

  • the present invention relates to a dendritic cell mimicking functional nanostructure containing ⁇ PD-1 and a method for preparing the same.
  • Cancer is the number one cause of death in Korea.
  • Representative cancer treatment methods may include surgical operation to remove cancer, radiation treatment, and chemical drug treatment, but these treatment methods have a problem in that they have a poor prognosis and serious side effects.
  • the main cause of cancer is formation of tumors by cancer cells that have not been removed according to the immune response due to reduced immunity or immune evasion of cancer cells. Therefore, immuno-anticancer therapy that can help cancer treatment by increasing the patient's own immune response can be a fundamental cancer treatment.
  • Most of the currently developed immuno-anticancer treatments directly inject drugs that enhance immune function, but drug injection still has limitations in that the delivery efficiency is very low and additional side effects may exist. Accordingly, research is focused on the development of therapeutic agents capable of dramatically increasing cancer treatment efficiency, reducing recurrence rates and side effects, and enhancing immune function.
  • the nanoparticle-cellularization technology uses the entire cell membrane of a specific cell as a coating material to physically cloak the surface of the nanoparticle. It can maintain the complex properties of the cell membrane along with proteins, lipids, and carbohydrates, and thus the surface of the nanoparticle.
  • the characteristics of a specific cell can be implemented as it is.
  • Non-Patent Document 0001 Hu, Che-Ming J., et al. Nature, 2015, 526(7571) 118-12
  • Non-Patent Document 0002 Brian T. Luk et al., Theranostics. 2016, 6(7), 1004-1101
  • Non-Patent Document 0003 Q. Jiang et al., Biomaterials 2019, 192, 292-308
  • the present disclosure seeks to more effectively implement the cancer treatment effect according to the increase in the immune response by using cells having an antigen presenting function beyond the material limitations of the prior art. Specifically, by modifying the surface of a nanostructure that mimics dendritic cells with ⁇ PD-1, a monoclonal antibody capable of binding to PD-1, it directly activates cytotoxic T cells through suppression of immune checkpoints and selectively kills cancer cells. Induce and enhance the immune response to provide excellent immuno-anticancer treatment effect.
  • a dendritic cell-mimicking nanostructure for inhibiting immune checkpoints includes a nanoparticle core; And a shell comprising a cell membrane of lipid molecules derived from dendritic cells; wherein the shell comprises an ⁇ PD-1 monoclonal antibody.
  • the ⁇ CTLA-4 monoclonal antibody may be conjugated with a lipid molecule.
  • the nanostructure may be an antigen pulsed (pursed).
  • the antigen may be a tumor antigen-derived peptide or protein.
  • the nanostructure may have an average particle diameter of 20 nm to 50 ⁇ m, and specifically, an average particle diameter of 20 nm to 1000 nm.
  • the present disclosure may provide a pharmaceutical composition for cancer treatment comprising the dendritic cell-mimicking nanostructure for inhibiting immune checkpoints as described above.
  • the present disclosure provides a method for preparing a dendritic cell-mimicking nanostructure for suppressing immune checkpoints, which includes (S10) purifying cell membranes from dendritic cells; (S20) forming a cell membrane suspension by applying energy to the cell membrane; (S30) obtaining liposomes from the cell membrane suspension; (S40) mixing the nanoparticles and the liposomes, and then compressing the filter to obtain the nanoparticles into which the cell membrane is introduced; and (S50) introducing an ⁇ PD-1 monoclonal antibody conjugated with a lipid molecule into the nanoparticle into which the cell membrane is introduced.
  • pulsing an antigen to the nanoparticle into which the cell membrane is introduced may be further included.
  • the dendritic cell-mimicking nanostructure according to the present disclosure introduces the antigen-presenting function to the surface of the nanoparticles, thereby preserving the antigen-presenting function of dendritic cells and at the same time not killing them, and additionally imparting the targeting function and photothermal effect function of the nanoparticles to enhance It can provide a functional immuno-anticancer treatment effect.
  • the dendritic cell-mimicking nanostructure according to the present disclosure can provide an effect of increasing the immune response by staying in the body for a long period of time by mimicking dendritic cells and continuously inducing the proliferation and differentiation of antigen-specific T cells.
  • the dendritic cell-mimicking nanostructure according to the present disclosure can induce binding to PD-1 present on the surface of cytotoxic T cells by including ⁇ PD-1, a monoclonal antibody capable of binding to PD-1, on its surface. Through this binding, suppression of the immune checkpoint is induced by inhibiting the binding of PD-L1 and PD-1 present in antigen-presenting cells, and markedly activating cytotoxic T cells to induce selective death of cancer cells and to suppress immune responses. It is to provide excellent immuno-anticancer treatment effect by increasing.
  • FIG. 1 is a schematic diagram showing a dendritic cell-mimicking nanostructure ( ⁇ PD-1-conjugated Nano DC) for immune checkpoint inhibition into which ⁇ PD-1 monoclonal antibody is introduced.
  • ⁇ PD-1-conjugated Nano DC dendritic cell-mimicking nanostructure
  • Figure 2 shows a method for preparing ⁇ PD-1 conjugated phospholipids according to the present disclosure.
  • 3 is a graph showing the results of surface potential analysis showing that ⁇ PD-1-conjugated phospholipids were successfully synthesized.
  • FIG. 4 is a graph showing the results of FT-IR analysis showing that ⁇ PD-1-conjugated phospholipids were successfully synthesized.
  • FIG. 5 shows the results of in vitro experiments on the T cell proliferation and differentiation inducing effect of the dendritic cell-mimicking nanostructure for immune checkpoint inhibition into which the ⁇ PD-1 monoclonal antibody was introduced according to Experimental Example 1 of the present disclosure.
  • Figure 6 shows the in vivo test method conditions of the dendritic cell-mimicking nanostructure for immune checkpoint inhibition into which ⁇ PD-1 monoclonal antibody was introduced according to Experimental Example 2 of the present disclosure.
  • Numerical ranges include lower and upper limits and all values within that range, increments logically derived from the form and breadth of the range being defined, all values defined therein, and the upper and lower limits of the numerical range defined in different forms. includes all possible combinations of Unless otherwise specifically defined in the specification of the present invention, values outside the numerical range that may occur due to experimental errors or rounding of values are also included in the defined numerical range.
  • a dendritic cell-mimicking nanostructure for inhibiting immune checkpoints includes a nanoparticle core; And a shell comprising a cell membrane of lipid molecules derived from dendritic cells; wherein the shell comprises an ⁇ PD-1 monoclonal antibody.
  • the dendritic cell-mimicking nanostructure for inhibiting immune checkpoints induces binding to PD-1 present on the surface of cytotoxic T cells by including ⁇ PD-1, a monoclonal antibody capable of binding to PD-1, on its surface, By inhibiting the binding between B7-1/2 present in antigen-presenting cells and PD-1, suppression of the immune checkpoint is induced, thereby providing an effect of significantly activating cytotoxic T cells. This further induces selective death of cancer cells and enhances the immune response to exhibit excellent immuno-anticancer treatment effects.
  • the dendritic cell-mimicking nanostructure for inhibiting immune checkpoints includes a nanoparticle core; and a shell comprising a cell membrane of lipid molecules derived from dendritic cells, wherein the shell may contain an ⁇ PD-1 monoclonal antibody.
  • PD-1 Programmed cell death protein 1
  • PD-1 is a 55 kDa type I transmembrane protein, which forms part of the Ig superfamily gene, and is well known as a T cell co-inhibitory molecule belonging to the immunoglobulin group. In normal T cells, PD-1 is not expressed under normal conditions, but its expression increases when activated. PD-1 binds to PD-L1, a ligand present on the surface of cancer cells. The interaction between PD-1 and PD-L1 reduces tumor-invasive lymphocytes, reduces T-cell receptor-mediated proliferation, Immune evasion occurs.
  • the ⁇ PD-1 antibody inhibits binding to PD-L1 and PD-1 and blocks their signal transduction, thereby increasing the activity of T cells and thereby enhancing the immune function against tumors.
  • the ⁇ PD-1 antibody is an antibody that binds to PD-1 with high specificity and affinity, blocks the binding of PD-L1 and/or PD-L2, and inhibits the immunosuppressive effect of the PD-1 signaling pathway. It may include antigen-binding portions or fragments that bind receptors and exhibit functional properties similar to whole antibodies in inhibiting ligand binding and upregulating the immune system. Specific examples of ⁇ PD-1 antibodies known in the art include HuMAb, nivolumab, pembrolizumab, pidilizumab, and the like, but are not limited thereto.
  • the dendritic cell-mimicking nanostructure for suppressing immune checkpoints includes ⁇ PD-1 antibody in the shell, thereby blocking signal transmission from binding to PD-L1 and/or PD-L2, thereby blocking T cell-mediated immunity. It can provide a reaction enhancing effect.
  • the nanoparticle core may be a one-dimensional or two-dimensional nanostructure.
  • the one-dimensional or two-dimensional nanostructures include carbon nanoribbons, carbon nanotubes, graphene, graphene oxide, reduced graphene oxide, MXene, metal nanorods, metal nanowires, or metal nanoplatelets ( platelet), but is not limited thereto.
  • the nanoparticle core may include biocompatible organic polymer nanoparticles, metal organic framework nanoparticles, metal nanoparticles, metal oxide nanoparticles, solid lipid nanoparticles, magnetic nanoparticles, light-to-heat conversion nanoparticles, and It may be one or two or more selected from the group consisting of nucleic acid-containing nanoparticles and protein-containing nanoparticles. Specifically, it is more preferable if the nanomaterial itself has no toxicity and has photosensitivity, so that it can be applied to photothermal therapy.
  • the nanoparticle core may be a gold nanoparticle.
  • the nanoparticle core may have an average particle diameter of 5 to 1000 nm or 10 to 500 nm.
  • the nanostructure including the nanoparticle core and the shell may have an average particle diameter of 20 nm to 50 ⁇ m, specifically, 20 nm to 10 ⁇ m, more specifically 20 nm to 1000 nm, and more specifically 50 nm to 500 nm. It may have an average particle diameter of
  • the ⁇ PD-1 monoclonal antibody may be conjugated with a lipid molecule.
  • a lipid molecule having biocompatibility may be used without limitation, and may specifically be a phospholipid, and more specifically, a phosphatidylethanolamine-based phospholipid.
  • phosphatidylethanolamine-based phospholipids and ⁇ PD-1 monoclonal antibody may be covalently bonded through a coupling agent.
  • the ⁇ PD-1 monoclonal antibody covalently bound to phospholipids can be easily inserted into the cell membrane-containing shell of lipid molecules using spontaneous cell membrane insertion according to the interaction between the phospholipid and the lipid bilayer present in the cell membrane. It can be stably introduced to the surface of the nanostructure.
  • Dendritic cells are powerful antigen-presenting cells, and on the surface of their cell membrane, major histocompatibility complex I/II (MHC I/II) and co-stimulatory molecules such as CD80 and CD86 and ICAM-1 are released.
  • MHC I/II major histocompatibility complex
  • Cell adhesion molecules are highly expressed, and various cytokines such as interferon, IL-12, and IL-18 related to T cell activation are secreted in large amounts, thereby performing a strong antigen presenting function.
  • the nanostructure according to the present disclosure may preferably be one in which an antigen is pulsed.
  • the antigen may be a peptide or protein derived from a tumor antigen for use in cancer treatment.
  • a tumor antigen for use in cancer treatment.
  • it may be a protein or peptide derived from ovalbumin (OVA), lymphocytic choriomeningitis mammarenavirus (LCMV) glycoprotein, or retrovirus protein. More specifically, it may be OVA 257-264 , GP 33-41 , or a cancer antigen peptide or protein of the p15E model.
  • OVA ovalbumin
  • LCMV lymphocytic choriomeningitis mammarenavirus
  • retrovirus protein More specifically, it may be OVA 257-264 , GP 33-41 , or a cancer antigen peptide or protein of the p15E model.
  • HER2/Neu tyrosinase
  • gp100 gp100
  • MART HPV E6/E7
  • EBV EBNA-1 carcinoembryonic antigen
  • GM2 GM2, GD2
  • testis antigen prostate antigen
  • CD20 tumor-specific antigens including neoantigens that can be produced by various mutations.
  • the tumor antigen-derived peptide may be GP 33-41 .
  • the shell of the nanostructure according to the present disclosure may be labeled with a physiologically active polymer, a physiologically active component, or a protein.
  • the physiologically active component may mean a cytokine for cell signaling.
  • cytokines include BMP (Bone morphogenetic protein) family, CCL (Cheomkine ligands) family, CMTM (CKLF-like MARVEL transmembrane domain containing member) family, CXCL (C-X-C motif ligand ligand) family, GDF (Growth/differentiation factor) family, growth hormone, IFN (Interferon) family, IL (Interleukin) family, TNF (Tumor necrosis factors) family, GPI (glycophosphatidylinositol), SLUPR-1 (Secreted Ly-6/uPAR-Related Protein 1), SLUPR-2 ( Secreted Ly-6/uPAR-Related Protein 2) and combinations thereof.
  • the cytokine induces or inhibits transcription factors or growth factors essential for the differentiation of T cells, and mediates the growth and differentiation of other immune cells, thereby further enhancing the effect of immunotherapeutic treatment of the dendritic cell-mimicking structure according to the present disclosure.
  • the physiologically active component is not limited as long as it is an ⁇ PD-1 monoclonal antibody, immune checkpoint inhibitors such as ⁇ CTLA-4 and ⁇ PD-L1, or anti-cancer drugs such as chemotherapy drugs using the same.
  • the present disclosure may provide a pharmaceutical composition for cancer treatment comprising the dendritic cell-mimicking nanostructure for inhibiting immune checkpoints as described above.
  • the ⁇ PD-1 monoclonal antibody-introduced dendritic cell-mimicking nanostructure for suppression of immune checkpoints is very effective in inducing proliferation and differentiation of cytotoxic T cells, and as it activates T cells, the pharmaceutical composition for cancer treatment is a strong immuno-anticancer treatment. effect can be provided.
  • Cancer is a general term for various malignant solid tumors that can expand through local and metastasis by invasion, and specific examples include B-cell lymphoma, non-small cell lung cancer, small cell lung cancer, basal cell carcinoma, squamous cell carcinoma of the skin, colorectal cancer, and melanoma.
  • B-cell lymphoma non-small cell lung cancer, small cell lung cancer, basal cell carcinoma, squamous cell carcinoma of the skin, colorectal cancer, and melanoma.
  • squamous cancer hepatocellular carcinoma
  • gastric cancer sarcoma
  • compositions for cancer treatment include formulation materials for modifying, maintaining or preserving pH, osmoticity, viscosity, sterility, transparency, color, isotonicity, odor, stability, rate of dissolution or release, adsorption or permeation. can do.
  • the pharmaceutical composition may be administered orally or parenterally.
  • parenteral administration include intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intrarectal, intracisternal, intravascular, and intradermal administration to a patient, and may be administered to a patient through a skin patch or transdermally. It can also be administered through the skin using iontophoresis, respectively.
  • the pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other anticancer agents, or may be administered sequentially or simultaneously with conventional anticancer agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art.
  • a method for treating cancer of a subject comprising administering the dendritic cell-mimicking nanostructure for suppressing the immune checkpoint or a pharmaceutical composition containing the same to a subject suffering from cancer.
  • a tumor-specific immune response can be induced in the subject or symptoms of cancer can be treated and/or alleviated in the subject.
  • the subject may be a human or a non-human mammal.
  • the present disclosure presents antigens on the surface by pulsing dendritic cell-mimicking nanostructures (anDCs), inserts phospholipids conjugated with ⁇ PD-1 into cell membranes, and finally introduces ⁇ PD-1 monoclonal antibodies into immune checkpoints. It is possible to provide a method for preparing a dendritic cell-mimicking nanostructure for inhibition.
  • the manufacturing method includes (S10) purifying cell membranes from dendritic cells; (S20) forming a cell membrane suspension by applying energy to the cell membrane; (S30) obtaining liposomes from the cell membrane suspension; (S40) mixing the nanoparticles and the liposomes, and then compressing the filter to obtain the nanoparticles into which the cell membrane is introduced; and (S50) introducing an ⁇ PD-1 monoclonal antibody conjugated with a lipid molecule into the nanoparticle into which the cell membrane is introduced.
  • the step of purifying the cell membrane from the dendritic cells may use a method known in the art without limitation, and may be performed, for example, through rapid freezing-thawing and centrifugation.
  • a cell membrane suspension may be formed by applying energy to the purified cell membrane. At this time, as an example, a cell membrane suspension of hundreds of nanometers or several micrometers may be obtained by treating with ultrasonic waves. Thereafter (S30), by filtering the cell membrane suspension through a membrane filter, cell membrane liposomes having a desired size can be obtained. Although not limited thereto, liposomes having an average particle diameter of specifically 10 to 200 nm, more specifically 20 to 150 nm can be obtained. Thereafter, a form in which the cell membrane of dendritic cells is coated on the surface of the nanoparticle can be obtained by mixing and co-extruding the cell membrane liposome and the nanoparticle.
  • the antigen may be a tumor antigen-derived peptide or protein for use in cancer treatment, and various tumor-specific antigens may be utilized as described above.
  • GP 33-41 may be used as a non-limiting example.
  • the lipid molecule may be used without limitation as long as it is a biocompatible lipid molecule, and may be specifically a phospholipid, and more specifically, a phosphatidylethanolamine-based phospholipid.
  • a specific example of the phosphatidylethanolamine-based phospholipid may be 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). 2 shows the process of obtaining ⁇ PD-1 conjugated to a lipid molecule.
  • One end of PEG may be conjugated to the hydrophilic group of DSPE, and ⁇ PD-1 may be conjugated to the other end of PEG.
  • the weight average molecular weight of PEG may be 200 to 10,000 g/mol or 500 to 5,000 g/mol.
  • ⁇ PD-1 conjugated with a phospholipid is easily incorporated according to the lipid-lipid interaction of the cell membrane of the dendritic cell nanostructure, and can effectively induce T cell activation.
  • Bone marrow cells were collected from the hind limbs of 6- to 8-week-old Naive 6 mice (Orient Bio), treated with ACK lysis buffer at room temperature for 3 minutes to remove red blood cells (RBCs), and only RBC-free BM was obtained. After counting the total number of bone marrow cells and seeding 7.5 ⁇ 10 5 cells/well in a 6 well non-tissue plate, GM-CSF (20 ng/ml) media was added to 2 ml/well at 37°C and 5% CO 2 conditions. cultured. On day 3, 2 ml of GM-CSF (20 ng/ml) media was added and maintained until day 6 to induce differentiation into dendritic cells.
  • GM-CSF 20 ng/ml
  • differentiated dendritic cells were obtained and seeded at 4 ⁇ 10 6 cells/well in media supplemented with LPS (Lipopolysaccaride) (50 ng/ml). At this time, 4 ml of LPS-added media is used in a 6 well non-tissue plate. On the 7th day, finally matured dendritic cells were obtained.
  • LPS Lipopolysaccaride
  • the matured dendritic cells were centrifuged at 2,000 rpm for 5 minutes to separate pure dendritic cells, and then treated with Protease Inhibitor Tablet-PBS buffer and dispersed at a concentration of 1 to 2 ⁇ 10 6 cells/ml. Then, only cell membranes were purified through rapid freezing at -70 ° C, thawing at room temperature, and centrifugation. Thereafter, ultrasonic waves (VC505, Sonics & materials) performed with 20% amplitude, 3 sec on/off and 3 sec 1 rotation with 2 sec cooling between cycles for a total of 60 times were processed to obtain cell membrane suspension in micro units.
  • Ultrasonic waves VC505, Sonics & materials
  • Example 1 Preparation of dendritic cell-mimicking nanostructure for immune checkpoint inhibition into which ⁇ PD-1 monoclonal antibody was introduced
  • Dendritic cell-mimicking nanostructure ⁇ PD-1-anDC
  • dendritic cell-mimicking nanostructure anDC
  • dendritic cell BMDC
  • Example 1 Dendritic cell-mimicking nanostructure prepared according to Example 1 in a 96-well U bottom plate, respectively 1 ⁇ 10 4 each 5 ⁇ 10 4 CD8 T cells isolated from P14 mice were added to the same well through 200 ⁇ l of 10% RPMI culture medium and co-cultured at 37 °C for 3 days. Restimulation was given using GP, GS, DNase1, and GP 33-41 peptide 6 hours before the end of co-culture. After 6 hours, the plate was recovered from the incubator, stained with a fluorescent antibody, and the degree of activation of surface proteins and CD8 T cells was analyzed by flow cytometry. The results are shown in FIG. 5 .
  • BMDC/GP33 and anDC/GP33 pulsed antigens induced T cell proliferation, CD44 expression and cytokines IFN ⁇ , TNF ⁇ , It can be confirmed that the expression of IL-2 is induced.

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Abstract

The present invention relates to a dendritic cell-mimetic nanostructure for inhibiting immune checkpoints, and a preparation method therefor, the nanostructure comprising a nanoparticle core and a shell, which comprises the cell membrane of a lipid molecule derived from dendritic cells, wherein the shell comprises αPD-1 monoclonal antibody, and the strong activation of cytotoxic T cells, caused by immune checkpoint inhibitory activity, increases an immune response, and thus an excellent cancer immunotherapy effect can be provided.

Description

αPD-1를 포함하는 수지상 세포 모방 기능성 나노구조체 및 이의 제조방법Dendritic cell mimicking functional nanostructure containing αPD-1 and method for preparing the same

본 발명은 αPD-1를 포함하는 수지상 세포 모방 기능성 나노구조체 및 이의 제조방법에 관한 것이다.The present invention relates to a dendritic cell mimicking functional nanostructure containing αPD-1 and a method for preparing the same.

암은 우리나라 국민 사망원인 중 1위를 차지하고 있다. 대표적인 암치료방법은 암을 절제하는 외과적 수술, 방사선 치료, 화학약물 치료가 있을 수 있는데, 이들 치료방법은 예후가 불량하고 심각한 부작용이 뒤따른다는 문제점이 있다.Cancer is the number one cause of death in Korea. Representative cancer treatment methods may include surgical operation to remove cancer, radiation treatment, and chemical drug treatment, but these treatment methods have a problem in that they have a poor prognosis and serious side effects.

암 발생은 면역력 감소 또는 암세포의 면역회피 작용으로 인해 면역반응에 따라 제거되지 못한 암세포의 종양 형성을 주요 원인으로 들 수 있다. 따라서 환자 본인의 면역 반응을 증대시켜 암 치료를 도울 수 있는 면역항암치료제는 근본적인 암 치료법이 될 수 있다. 현재 개발된 면역항암치료제는 면역기능을 높이는 약물을 직접 주입하는 것이 대부분인데, 약물 주입술은 전달 효율이 매우 낮고, 추가 부작용이 존재할 수 있다는 점에서 여전히 한계가 존재한다. 이에 따라 암 치료 효율을 획기적으로 높이고, 재발율 및 부작용을 낮추면서 면역기능을 증대시킬 수 있는 치료제의 개발에 연구가 집중되고 있다.The main cause of cancer is formation of tumors by cancer cells that have not been removed according to the immune response due to reduced immunity or immune evasion of cancer cells. Therefore, immuno-anticancer therapy that can help cancer treatment by increasing the patient's own immune response can be a fundamental cancer treatment. Most of the currently developed immuno-anticancer treatments directly inject drugs that enhance immune function, but drug injection still has limitations in that the delivery efficiency is very low and additional side effects may exist. Accordingly, research is focused on the development of therapeutic agents capable of dramatically increasing cancer treatment efficiency, reducing recurrence rates and side effects, and enhancing immune function.

한편, 나노기술을 바이오 의료 분야에 접목한 나노-바이오 융합기술에 대한 연구가 지속되어 오고 있다. 하지만 대부분의 나노재료는 합성을 통해 얻어지며, 나노재료를 이용한 표면 개질 시, 합성물이 첨가되는 과정이 수반되므로 체내 독성, 원치 않는 면역반응 유도, 암 유발 등의 부작용이 생길 수 있다. 그러므로 생체를 직접 모사하는 방식의 표면 개질 방법을 사용하여 부작용을 최소화하고, 생체특성에 따라 다양한 기능을 구현할 수 있도록 함으로써 기존 나노기술이 가진 한계점을 극복할 수 있다.On the other hand, research on nano-bio convergence technology that grafts nanotechnology to the biomedical field has been continued. However, most nanomaterials are obtained through synthesis, and surface modification using nanomaterials involves adding a compound, which can cause side effects such as toxicity in the body, induction of unwanted immune responses, and induction of cancer. Therefore, it is possible to overcome the limitations of existing nanotechnology by minimizing side effects by using a surface modification method that directly simulates a living body and implementing various functions according to the characteristics of a living body.

특히 나노입자-세포화 기술은 특정 세포의 전체 세포막을 코팅 재료로 활용하여 나노입자 표면을 물리적으로 클로킹(Cloaking)하는 기술로 단백질, 지질 및 탄수화물과 함께 세포막의 복합적 성질을 유지할 수 있어 나노입자 표면에 특정 세포의 특성을 그대로 구현할 수 있다.In particular, the nanoparticle-cellularization technology uses the entire cell membrane of a specific cell as a coating material to physically cloak the surface of the nanoparticle. It can maintain the complex properties of the cell membrane along with proteins, lipids, and carbohydrates, and thus the surface of the nanoparticle. The characteristics of a specific cell can be implemented as it is.

2015년 University of California 의 Liangfang Zhang연구팀에서 처음으로 혈소판을 PLGA(poly(lactic-co-glycolic) acid) 입자 표면에 도입한 기술을 바탕으로, 현재까지 다양한 세포에 적용되고 있다.In 2015, the research team of Liangfang Zhang at the University of California first introduced platelets to the surface of PLGA (poly(lactic-co-glycolic) acid) particles, and it has been applied to various cells to date.

PLGA 코어에 독소루비신(doxorubicin)을 담지한 후, 적혈구 막으로 코팅하여 면역적합성 나노캐리어를 제조해 고형 종양을 제거하거나, 적혈구 막과 암세포의 세포막을 멜라닌 나노입자에 동시에 코팅하여 하이브리드 세포막을 지닌 나노입자를 제조해 체내 순환시간을 증가시키고 종양 표적능을 증대시킨 연구가 보고되었다.After supporting doxorubicin on the PLGA core, coat it with a red blood cell membrane to prepare an immunocompatible nanocarrier to remove solid tumors, or coat the red blood cell membrane and cancer cell membrane on melanin nanoparticles at the same time to nanoparticles with hybrid cell membranes. was manufactured to increase circulation time in the body and increase tumor targeting ability.

하지만 종래의 세포막 코팅 기술은 대부분 암세포, 혈액세포 위주로 진행되었고, 그 응용 역시 혈액 내 안정적 전달 등을 목표로 하는 연구들이 주를 이루었다. 또한 암세포막을 항원으로 직접 도입하여 체내에 전달하는 경우, 항원 내성으로 인한 면역반응 저하, 암세포 유래 물질에 대한 거부감 등의 단점이 존재한다. 따라서 직접적인 항원제시 면역세포의 기능을 가질 수 있는 면역치료제를 개발하여 중간 과정 없이 T 세포의 분화 및 증식을 유도하는 것이 매우 필요하다.However, most of the conventional cell membrane coating technologies have been conducted mainly on cancer cells and blood cells, and their applications have also mainly focused on studies aimed at stable delivery in the blood. In addition, when cancer cell membranes are directly introduced as antigens and delivered into the body, there are disadvantages such as a decrease in immune response due to antigen resistance and a feeling of rejection against cancer cell-derived substances. Therefore, it is very necessary to develop an immunotherapeutic agent capable of directly functioning as an antigen-presenting immune cell and induce differentiation and proliferation of T cells without an intermediate process.

[선행기술문헌][Prior art literature]

[비특허문헌][Non-Patent Literature]

(비특허문헌 0001) Hu, Che-Ming J., et al. Nature, 2015, 526(7571) 118-12(Non-Patent Document 0001) Hu, Che-Ming J., et al. Nature, 2015, 526(7571) 118-12

(비특허문헌 0002) Brian T. Luk et al., Theranostics. 2016, 6(7), 1004-1101(Non-Patent Document 0002) Brian T. Luk et al., Theranostics. 2016, 6(7), 1004-1101

(비특허문헌 0003) Q. Jiang et al., Biomaterials 2019, 192, 292-308(Non-Patent Document 0003) Q. Jiang et al., Biomaterials 2019, 192, 292-308

본 개시는 종래기술의 재료적인 한계에서 벗어나 항원제시 기능을 가지는 세포를 사용하여 면역반응 증대에 따른 암치료 효과를 보다 효과적으로 구현하고자 한다. 구체적으로, 수지상세포를 모방하는 나노구조체에 PD-1와 결합할 수 있는 단일클론 항체 αPD-1를 표면에 수식함으로써, 면역관문의 억제를 통해 세포독성 T 세포를 직접 활성화하여 암세포의 선택적 사멸을 유도하고, 면역 반응을 증대시켜 우수한 면역항암치료 효과를 제공하는 것이다.The present disclosure seeks to more effectively implement the cancer treatment effect according to the increase in the immune response by using cells having an antigen presenting function beyond the material limitations of the prior art. Specifically, by modifying the surface of a nanostructure that mimics dendritic cells with αPD-1, a monoclonal antibody capable of binding to PD-1, it directly activates cytotoxic T cells through suppression of immune checkpoints and selectively kills cancer cells. Induce and enhance the immune response to provide excellent immuno-anticancer treatment effect.

상기 목적을 달성하기 위하여, 본 개시에 따른 면역관문 억제용 수지상세포 모방 나노구조체는 나노입자 코어; 및 수지상 세포로부터 유래된 지질 분자의 세포막을 포함하는 쉘;을 포함하는 나노구조체로서, 상기 쉘은 αPD-1 단일클론 항체를 포함하는 것을 특징으로 한다.In order to achieve the above object, a dendritic cell-mimicking nanostructure for inhibiting immune checkpoints according to the present disclosure includes a nanoparticle core; And a shell comprising a cell membrane of lipid molecules derived from dendritic cells; wherein the shell comprises an αPD-1 monoclonal antibody.

본 개시의 일 구현예에 있어서, 상기 αCTLA-4 단일클론 항체는 지질 분자와 컨쥬게이션된 것일 수 있다.In one embodiment of the present disclosure, the αCTLA-4 monoclonal antibody may be conjugated with a lipid molecule.

본 개시의 일 구현예에 있어서, 상기 나노구조체는 항원이 펄싱(pursing)된 것일 수 있다.In one embodiment of the present disclosure, the nanostructure may be an antigen pulsed (pursed).

본 개시의 일 구현예에 있어서, 상기 항원은 종양 항원 유래 펩타이드 또는 단백질일 수 있다.In one embodiment of the present disclosure, the antigen may be a tumor antigen-derived peptide or protein.

본 개시의 일 구현예에 있어서, 상기 나노구조체는 20 ㎚ 내지 50 ㎛의 평균입경을 가질 수 있고, 구체적으로, 20 ㎚ 내지 1000 ㎚의 평균입경을 가질 수 있다.In one embodiment of the present disclosure, the nanostructure may have an average particle diameter of 20 nm to 50 μm, and specifically, an average particle diameter of 20 nm to 1000 nm.

본 개시는 전술한 바와 같은 면역관문 억제용 수지상세포 모방 나노구조체를 포함하는 암 치료용 약학적 조성물을 제공할 수 있다.The present disclosure may provide a pharmaceutical composition for cancer treatment comprising the dendritic cell-mimicking nanostructure for inhibiting immune checkpoints as described above.

본 개시는 면역관문 억제용 수지상세포 모방 나노구조체의 제조방법을 제공하며, 상기 제조방법은 (S10) 수지상 세포로부터 세포막을 정제하는 단계; (S20) 상기 세포막에 에너지를 가하여 세포막 현탁액을 형성하는 단계; (S30) 상기 세포막 현탁액으로부터 리포좀을 수득하는 단계; (S40) 나노입자 및 상기 리포좀을 혼합한 후, 필터 압축하여 상기 세포막이 도입된 나노입자를 수득하는 단계; 및 (S50) 지질 분자와 컨쥬게이션된 αPD-1 단일클론 항체를 상기 세포막이 도입된 나노입자에 도입하는 단계;를 포함하는 것을 특징으로 할 수 있다.The present disclosure provides a method for preparing a dendritic cell-mimicking nanostructure for suppressing immune checkpoints, which includes (S10) purifying cell membranes from dendritic cells; (S20) forming a cell membrane suspension by applying energy to the cell membrane; (S30) obtaining liposomes from the cell membrane suspension; (S40) mixing the nanoparticles and the liposomes, and then compressing the filter to obtain the nanoparticles into which the cell membrane is introduced; and (S50) introducing an αPD-1 monoclonal antibody conjugated with a lipid molecule into the nanoparticle into which the cell membrane is introduced.

본 개시의 일 구현예에 있어서, 상기 (S40) 단계 이후, 상기 세포막이 도입된 나노입자에 항원을 펄싱하는 단계;를 더 포함할 수 있다.In one embodiment of the present disclosure, after the step (S40), pulsing an antigen to the nanoparticle into which the cell membrane is introduced; may be further included.

본 개시에 따른 수지상 세포 모방 나노구조체는 항원제시 기능을 나노입자 표면에 도입하여, 수지상세포의 항원제시 기능은 보존함과 동시에 사멸하지 않고 나노입자의 표적화 기능 및 광열효과 기능을 추가적으로 부여하여 강화된 기능의 면역항암치료 효과를 제공할 수 있다.The dendritic cell-mimicking nanostructure according to the present disclosure introduces the antigen-presenting function to the surface of the nanoparticles, thereby preserving the antigen-presenting function of dendritic cells and at the same time not killing them, and additionally imparting the targeting function and photothermal effect function of the nanoparticles to enhance It can provide a functional immuno-anticancer treatment effect.

본 개시에 따른 수지상 세포 모방 나노구조체는 수지상 세포를 모방함으로써 장기간 체내에서 체류하며 항원 특이적 T 세포의 증식 및 분화를 지속적으로 유도함으로써 면역반응을 증대시키는 효과를 제공할 수 있다. The dendritic cell-mimicking nanostructure according to the present disclosure can provide an effect of increasing the immune response by staying in the body for a long period of time by mimicking dendritic cells and continuously inducing the proliferation and differentiation of antigen-specific T cells.

본 개시에 따른 수지상세포 모방 나노구조체는 PD-1와 결합할 수 있는 단일클론 항체 αPD-1를 표면에 포함함으로써 세포독성 T 세포 표면에 존재하는 PD-1와 결합을 유도할 수 있다. 이러한 결합을 통해 항원제시 세포에 존재하는 PD-L1와 PD-1의 결합을 억제함으로써 면역관문의 억제를 유도하고, 세포독성 T 세포를 현저하게 활성화하여 암세포의 선택적 사멸을 유도하고, 면역 반응을 증대시켜 우수한 면역항암치료 효과를 제공하는 것이다.The dendritic cell-mimicking nanostructure according to the present disclosure can induce binding to PD-1 present on the surface of cytotoxic T cells by including αPD-1, a monoclonal antibody capable of binding to PD-1, on its surface. Through this binding, suppression of the immune checkpoint is induced by inhibiting the binding of PD-L1 and PD-1 present in antigen-presenting cells, and markedly activating cytotoxic T cells to induce selective death of cancer cells and to suppress immune responses. It is to provide excellent immuno-anticancer treatment effect by increasing.

도 1은 αPD-1 단일클론 항체가 도입된 면역관문 억제용 수지상세포 모방 나노구조체 (αPD-1-conjugated Nano DC)를 나타낸 모식도이다. 세포독성 T 세포의 PD-1와 나노구조체의 αPD-1가 결합됨에 따라 PD-L1가 PD-1의 결합이 억제됨으로써 세포독성 T 세포가 활성화될 수 있음을 도시하고 있다.1 is a schematic diagram showing a dendritic cell-mimicking nanostructure (αPD-1-conjugated Nano DC) for immune checkpoint inhibition into which αPD-1 monoclonal antibody is introduced. As PD-1 of the cytotoxic T cell binds to αPD-1 of the nanostructure, the binding of PD-L1 to PD-1 is inhibited, and thus the cytotoxic T cell can be activated.

도 2는 본 개시에 따른 αPD-1가 컨쥬게이션된 인지질의 제조방법을 도시한 것이다.Figure 2 shows a method for preparing αPD-1 conjugated phospholipids according to the present disclosure.

도 3은 αPD-1가 컨쥬게이션된 인지질이 성공적으로 합성되었음을 도시하는 표면전위 분석 결과를 나타낸 그래프이다.3 is a graph showing the results of surface potential analysis showing that αPD-1-conjugated phospholipids were successfully synthesized.

도 4는 αPD-1가 컨쥬게이션된 인지질이 성공적으로 합성되었음을 도시하는 FT-IR 분석 결과를 나타낸 그래프이다.4 is a graph showing the results of FT-IR analysis showing that αPD-1-conjugated phospholipids were successfully synthesized.

도 5는 본 개시의 실험예 1에 따른 αPD-1 단일클론 항체가 도입된 면역관문 억제용 수지상세포 모방 나노구조체의 T 세포 증식 및 분화 유도 효과에 대한 in vitro 실험결과를 도시한 것이다.FIG. 5 shows the results of in vitro experiments on the T cell proliferation and differentiation inducing effect of the dendritic cell-mimicking nanostructure for immune checkpoint inhibition into which the αPD-1 monoclonal antibody was introduced according to Experimental Example 1 of the present disclosure.

도 6은 본 개시 실험예 2에 따른 αPD-1 단일클론 항체가 도입된 면역관문 억제용 수지상세포 모방 나노구조체의 in vivo 실험방법 조건을 나타낸 것이다.Figure 6 shows the in vivo test method conditions of the dendritic cell-mimicking nanostructure for immune checkpoint inhibition into which αPD-1 monoclonal antibody was introduced according to Experimental Example 2 of the present disclosure.

도 7 및 도 8은 본 개시 실험예 2에 따른 αPD-1 단일클론 항체가 도입된 면역관문 억제용 수지상세포 모방 나노구조체의 T 세포 증식 및 분화 유도 효과에 대한 in vivo 실험결과를 도시한 것이다. 7 and 8 show in vivo experimental results for the T cell proliferation and differentiation inducing effect of the dendritic cell-mimicking nanostructure for immune checkpoint inhibition into which the αPD-1 monoclonal antibody was introduced according to Experimental Example 2 of the present disclosure.

이하, 첨부된 도면 및 실시예들을 참조하여 본 발명에 따른 αPD-1를 포함하는 수지상 세포 모방 기능성 나노구조체 및 이의 제조방법에 대하여 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있도록 상세히 설명한다. 다만, 본 발명은 여러 가지 상이한 형태로 구현될 수 있으며, 여기에서 설명하는 구현예에 한정되지 않는다. 또한, 청구범위에 의하여 한정되는 보호범위를 제한하고자 하는 것도 아니다.Hereinafter, with reference to the accompanying drawings and examples, those skilled in the art can easily practice the dendritic cell mimicking functional nanostructure containing αPD-1 and the manufacturing method thereof according to the present invention. Please explain in detail so that However, the present invention may be implemented in many different forms, and is not limited to the embodiments described herein. Nor is it intended to limit the scope of protection defined by the claims.

본 발명의 설명에 사용되는 기술 용어 및 과학 용어에 있어서 다른 정의가 없다면, 이 발명이 속하는 기술 분야에서 통상의 지식을 가진 자가 통상적으로 이해하고 있는 의미를 가지며, 하기의 설명에서 본 발명의 요지를 불필요하게 흐릴 수 있는 공지 기능 및 구성에 대한 설명은 생략한다.Unless otherwise defined, the technical and scientific terms used in the description of the present invention have meanings commonly understood by those of ordinary skill in the art to which this invention belongs, and the gist of the present invention in the following description Descriptions of well-known functions and configurations that may be unnecessarily obscure are omitted.

본 명세서에서 사용되는 수치 범위는 하한치와 상한치와 그 범위 내에서의 모든 값, 정의되는 범위의 형태와 폭에서 논리적으로 유도되는 증분, 이중 한정된 모든 값 및 서로 다른 형태로 한정된 수치 범위의 상한 및 하한의 모든 가능한 조합을 포함한다. 본 발명의 명세서에서 특별한 정의가 없는 한 실험 오차 또는 값의 반올림으로 인해 발생할 가능성이 있는 수치범위 외의 값 역시 정의된 수치범위에 포함된다.Numerical ranges, as used herein, include lower and upper limits and all values within that range, increments logically derived from the form and breadth of the range being defined, all values defined therein, and the upper and lower limits of the numerical range defined in different forms. includes all possible combinations of Unless otherwise specifically defined in the specification of the present invention, values outside the numerical range that may occur due to experimental errors or rounding of values are also included in the defined numerical range.

본 명세서에서 사용되는 단수 형태는 문맥에서 특별한 지시가 없는 한 복수 형태도 포함하는 것으로 의도할 수 있다.The singular form used herein may be intended to include the plural form as well, unless the context dictates otherwise.

본 명세서의 용어, "포함한다"는 "구비한다", "함유한다", "가진다" 또는 "특징으로 한다" 등의 표현과 등가의 의미를 가지는 개방형 기재이며, 추가로 열거되어 있지 않은 요소, 재료 또는 공정을 배제하지 않는다. The term "comprises" in the present specification is an open description having the same meaning as expressions such as "comprises", "includes", "has" or "characterized by", elements not additionally listed, No materials or processes are excluded.

이하, 본 발명에 대해 구체적으로 설명한다.Hereinafter, the present invention will be specifically described.

본 개시에 따른 면역관문 억제용 수지상세포 모방 나노구조체는 나노입자 코어; 및 수지상 세포로부터 유래된 지질 분자의 세포막을 포함하는 쉘;을 포함하는 나노구조체로서, 상기 쉘은 αPD-1 단일클론 항체를 포함하는 것을 특징으로 한다.A dendritic cell-mimicking nanostructure for inhibiting immune checkpoints according to the present disclosure includes a nanoparticle core; And a shell comprising a cell membrane of lipid molecules derived from dendritic cells; wherein the shell comprises an αPD-1 monoclonal antibody.

본 개시에 따른 면역관문 억제용 수지상세포 모방 나노구조체는 PD-1 와 결합할 수 있는 단일클론 항체 αPD-1를 표면에 포함함으로써 세포독성 T 세포 표면에 존재하는 PD-1와 결합을 유도하고, 항원제시 세포에 존재하는 B7-1/2와 PD-1의 결합을 억제함에 따라 면역관문의 억제를 유도함으로써, 세포독성 T 세포를 현저하게 활성화시키는 효과를 제공할 수 있다. 이는 나아가 암세포의 선택적 사멸을 유도하고, 면역 반응을 증대시켜 우수한 면역항암치료 효과를 발휘한다.The dendritic cell-mimicking nanostructure for inhibiting immune checkpoints according to the present disclosure induces binding to PD-1 present on the surface of cytotoxic T cells by including αPD-1, a monoclonal antibody capable of binding to PD-1, on its surface, By inhibiting the binding between B7-1/2 present in antigen-presenting cells and PD-1, suppression of the immune checkpoint is induced, thereby providing an effect of significantly activating cytotoxic T cells. This further induces selective death of cancer cells and enhances the immune response to exhibit excellent immuno-anticancer treatment effects.

이를 위해 본 개시에 따른 면역관문 억제용 수지상세포 모방 나노구조체는 나노입자 코어; 및 수지상 세포로부터 유래된 지질 분자의 세포막을 포함하는 쉘;을 포함하고, 이때 상기 쉘은 αPD-1 단일클론 항체를 포함하는 것을 특징으로 할 수 있다.To this end, the dendritic cell-mimicking nanostructure for inhibiting immune checkpoints according to the present disclosure includes a nanoparticle core; and a shell comprising a cell membrane of lipid molecules derived from dendritic cells, wherein the shell may contain an αPD-1 monoclonal antibody.

PD-1 (Programmed cell death protein1)은 55 kDa의 제I형 경막 단백질로서, Ig 상과 유전자의 일부를 이루고 있으며, 면역글로불린 분자군에 속하는 T 세포의 보조저해분자로 잘 알려져 있다. 일반적인 T 세포에서는 정상 상태일 때 PD-1이 발현되지 않다가 활성화되면 발현이 증가한다. PD-1은 암세포 표면에 존재하는 리간드인 PD-L1과 결합하는데, PD-1과 PD-L1 사이의 상호 작용으로 인하여 종양 침습성 림프구가 줄어들고, T 세포 수용체 매개 증식이 감소하며, 암 세포에 의한 면역 회피 현상이 발생하게 된다.PD-1 (Programmed cell death protein 1) is a 55 kDa type I transmembrane protein, which forms part of the Ig superfamily gene, and is well known as a T cell co-inhibitory molecule belonging to the immunoglobulin group. In normal T cells, PD-1 is not expressed under normal conditions, but its expression increases when activated. PD-1 binds to PD-L1, a ligand present on the surface of cancer cells. The interaction between PD-1 and PD-L1 reduces tumor-invasive lymphocytes, reduces T-cell receptor-mediated proliferation, Immune evasion occurs.

αPD-1 항체는 PD-L1 및 PD-1과의 결합을 저해하여, 이들의 신호 전달을 차단함으로써, T 세포의 활성을 증가시킬 수 있고 이를 통해 종양에 대한 면역기능을 강화시킬 수 있는 효과를 제공할 수 있다. The αPD-1 antibody inhibits binding to PD-L1 and PD-1 and blocks their signal transduction, thereby increasing the activity of T cells and thereby enhancing the immune function against tumors. can provide

αPD-1 항체는 높은 특이성 및 친화도로 PD-1에 결합하고, PD-L1 및 또는 PD-L2의 결합을 차단하고, PD-1 신호전달 경로의 면역억제 효과를 억제하는 항체로서, PD-1 수용체에 결합하고, 리간드 결합 억제 및 면역계 상향조절에서 전체 항체와 유사한 기능적 특성을 나타내는 항원-결합 부분 또는 단편을 포함할 수 있다. 당업계에 알려진 αPD-1 항체로서, 구체적인 예를 들면, HuMAb, 니볼루맙, 펨브롤리주맙, 피딜리주맙 등이 있으나, 이에 제한되지 않는다.The αPD-1 antibody is an antibody that binds to PD-1 with high specificity and affinity, blocks the binding of PD-L1 and/or PD-L2, and inhibits the immunosuppressive effect of the PD-1 signaling pathway. It may include antigen-binding portions or fragments that bind receptors and exhibit functional properties similar to whole antibodies in inhibiting ligand binding and upregulating the immune system. Specific examples of αPD-1 antibodies known in the art include HuMAb, nivolumab, pembrolizumab, pidilizumab, and the like, but are not limited thereto.

본 개시에 따른 면역관문 억제용 수지상세포 모방 나노구조체는 상기 쉘에 αPD-1 항체를 포함함으로써, 전술한 PD-L1 및 또는 PD-L2과의 결합으로부터의 신호 전달을 차단하여 T 세포 매개의 면역반응을 강화시키는 효과를 제공할 수 있다.The dendritic cell-mimicking nanostructure for suppressing immune checkpoints according to the present disclosure includes αPD-1 antibody in the shell, thereby blocking signal transmission from binding to PD-L1 and/or PD-L2, thereby blocking T cell-mediated immunity. It can provide a reaction enhancing effect.

본 개시의 일 예로서, 나노입자 코어는 1차원 또는 2차원 나노구조체일 수 있다. 상기 1차원 또는 2차원 나노구조체의 구체적인 예를 들면, 탄소 나노리본, 탄소 나노튜브, 그래핀, 산화그래핀, 환원된 산화그래핀, 맥신, 금속 나노로드, 금속 나노와이어 또는 금속 나노 판상체(platelet)일 수 있으나, 이에 제한되지 않는다.As an example of the present disclosure, the nanoparticle core may be a one-dimensional or two-dimensional nanostructure. Specific examples of the one-dimensional or two-dimensional nanostructures include carbon nanoribbons, carbon nanotubes, graphene, graphene oxide, reduced graphene oxide, MXene, metal nanorods, metal nanowires, or metal nanoplatelets ( platelet), but is not limited thereto.

또한 상기 나노입자 코어는 생체적합성을 가지는 유기 고분자 나노입자, 금속 유기골격체 나노입자, 금속 나노입자, 금속산화물 나노입자, 고체상 지질 나노입자(solid lipid nanoparticle), 자성 나노입자, 광열 변환 나노입자 및 핵산 함유 나노입자, 단백질 함유 나노입자로 이루어진 군에서 선택되는 하나 또는 둘 이상일 수 있다. 구체적으로 나노물질 자체의 독성이 없으면서 광 감응성을 가지고 있어 광열치료에 적용이 가능한 것이면 더욱 바람직하다. In addition, the nanoparticle core may include biocompatible organic polymer nanoparticles, metal organic framework nanoparticles, metal nanoparticles, metal oxide nanoparticles, solid lipid nanoparticles, magnetic nanoparticles, light-to-heat conversion nanoparticles, and It may be one or two or more selected from the group consisting of nucleic acid-containing nanoparticles and protein-containing nanoparticles. Specifically, it is more preferable if the nanomaterial itself has no toxicity and has photosensitivity, so that it can be applied to photothermal therapy.

비한정적인 일 예에 있어서, 나노입자 코어는 금 나노입자일 수 있다.In one non-limiting example, the nanoparticle core may be a gold nanoparticle.

이때 나노입자 코어는 5 내지 1000 ㎚, 또는 10 내지 500 ㎚의 평균 입경을 가질 수 있다. 나노입자 코어 및 쉘을 포함하는 나노구조체는 20 ㎚ 내지 50 ㎛의 평균 입경을 가질 수 있고, 구체적으로, 20 ㎚ 내지 10 ㎛, 보다 구체적으로 20 ㎚ 내지 1000 ㎚, 더욱 구체적으로 50 ㎚ 내지 500 ㎚의 평균 입경을 가질 수 있다.In this case, the nanoparticle core may have an average particle diameter of 5 to 1000 nm or 10 to 500 nm. The nanostructure including the nanoparticle core and the shell may have an average particle diameter of 20 nm to 50 μm, specifically, 20 nm to 10 μm, more specifically 20 nm to 1000 nm, and more specifically 50 nm to 500 nm. It may have an average particle diameter of

본 개시의 일 예에 있어서, αPD-1 단일클론 항체는 지질 분자와 컨쥬게이션된 것일 수 있다. 지질 분자는 생체적합성을 가지는 지질 분자라면 제한없이 사용될 수 있으며, 구체적으로 인지질일 수 있고, 보다 구체적으로 포스파티딜에탄올아민계 인지질일 수 있다.In one example of the present disclosure, the αPD-1 monoclonal antibody may be conjugated with a lipid molecule. Any lipid molecule having biocompatibility may be used without limitation, and may specifically be a phospholipid, and more specifically, a phosphatidylethanolamine-based phospholipid.

일 예시에 따르면 포스파티딜에탄올아민계 인지질과 αPD-1 단일클론 항체는 커플링제를 통해 공유결합될 수 있다. 인지질과 공유결합된 αPD-1 단일클론 항체는 인지질 및 세포막에 존재하는 지질 이중층과의 상호작용에 따라 자발적 세포막 삽입 현상을 이용하여 지질 분자의 세포막을 포함하는 쉘에 쉽게 삽입될 수 있으며, 이에 따라 안정적으로 나노구조체의 표면에 도입될 수 있다.According to one example, phosphatidylethanolamine-based phospholipids and αPD-1 monoclonal antibody may be covalently bonded through a coupling agent. The αPD-1 monoclonal antibody covalently bound to phospholipids can be easily inserted into the cell membrane-containing shell of lipid molecules using spontaneous cell membrane insertion according to the interaction between the phospholipid and the lipid bilayer present in the cell membrane. It can be stably introduced to the surface of the nanostructure.

수지상세포는 강력한 항원 제시 세포로서, 세포막 표면에 주조직 적합성 복합체(major histocompatibility complex I/II: MHC I/II)와 CD80 및 CD86과 같은 보조자극인자(co-stimulatory molecules) 및 ICAM-1과 같은 세포부착 분자(cell adhesion molecules)가 고도로 발현되어 있고, T 세포 활성화와 관련된 인터페론, IL-12, IL-18 등의 다양한 사이토카인을 다량 분비함으로써, 강력한 항원제시 기능을 수행할 수 있다. 이에 따라 본 개시에 따른 나노구조체는 바람직하게 항원이 펄싱(pursing)된 것일 수 있다.Dendritic cells are powerful antigen-presenting cells, and on the surface of their cell membrane, major histocompatibility complex I/II (MHC I/II) and co-stimulatory molecules such as CD80 and CD86 and ICAM-1 are released. Cell adhesion molecules are highly expressed, and various cytokines such as interferon, IL-12, and IL-18 related to T cell activation are secreted in large amounts, thereby performing a strong antigen presenting function. Accordingly, the nanostructure according to the present disclosure may preferably be one in which an antigen is pulsed.

상기 항원은 암 치료의 용도를 위해 종양 항원 유래 펩타이드 또는 단백질일 수 있다. 구체적으로 예를 들면, 생쥐 암 모델에 있어서, 오브알부민(ovalbumin, OVA), LCMV(Lymphocytic choriomeningitis mammarenavirus) glycoprotein, retrovirus protein 유래의 단백질 또는 펩타이드일 수 있다. 보다 구체적으로 OVA257-264, GP33-41, p15E 모델의 암 항원 펩타이드 또는 단백질일 수 있다. The antigen may be a peptide or protein derived from a tumor antigen for use in cancer treatment. Specifically, for example, in a mouse cancer model, it may be a protein or peptide derived from ovalbumin (OVA), lymphocytic choriomeningitis mammarenavirus (LCMV) glycoprotein, or retrovirus protein. More specifically, it may be OVA 257-264 , GP 33-41 , or a cancer antigen peptide or protein of the p15E model.

또한 인간 암 항원에 있어서, (1) HER2/Neu, tyrosinase, gp100, MART, HPV E6/E7, EBV EBNA-1, carcinoembryonic antigen, a-fetoprotein, GM2, GD2, testis antigen, prostate antigen, 및 CD20을 포함하는 종양 연관 항원 및 (2) 다양한 돌연변이에 의해 생성될 수 있는 신생항원(neoantigen)을 포함하는 종양 특이 항원으로부터 유래된 펩타이드 또는 단백질일 수 있다.In addition, in human cancer antigens, (1) HER2/Neu, tyrosinase, gp100, MART, HPV E6/E7, EBV EBNA-1, carcinoembryonic antigen, a-fetoprotein, GM2, GD2, testis antigen, prostate antigen, and CD20 and (2) tumor-specific antigens including neoantigens that can be produced by various mutations.

비제한적인 일 예로서, 종양 항원 유래 펩타이드로는 GP33-41일 수 있다.As a non-limiting example, the tumor antigen-derived peptide may be GP 33-41 .

본 개시에 따른 나노구조체의 쉘은 αPD-1 단일클론 항체 외에도 생리활성 고분자, 생리활성 성분 또는 단백질이 표지된 것일 수 있다. In addition to the αPD-1 monoclonal antibody, the shell of the nanostructure according to the present disclosure may be labeled with a physiologically active polymer, a physiologically active component, or a protein.

구체적으로 상기 생리활성 성분은 세포 신호전달을 위한 사이토카인을 의미할 수 있다. 비제한적인 예로서, 케모카인, 인터페론, 인터루킨, 림포카인, 종양 괴사 인자, 모노카인 및 콜로니 자극 인자들을 포함할 수 있다. 보다 구체적으로 사이토카인은 BMP (Bone morphogenetic protein) 패밀리, CCL (Cheomkine ligands) 패밀리, CMTM (CKLF-like MARVEL transmembrane domain containing member) 패밀리, CXCL (C-X-C motif ligand ligand) 패밀리, GDF (Growth/differentiation factor) 패밀리, 성장 호르몬, IFN (Interferon) 패밀리, IL (Interleukin) 패밀리, TNF (Tumor necrosis factors) 패밀리, GPI(glycophosphatidylinositol), SLUPR-1(Secreted Ly-6/uPAR-Related Protein 1), SLUPR-2(Secreted Ly-6/uPAR-Related Protein 2) 및 이들의 조합으로 구성된 군으로부터 선택되는 어느 하나일 수 있다. Specifically, the physiologically active component may mean a cytokine for cell signaling. Non-limiting examples may include chemokines, interferons, interleukins, lymphokines, tumor necrosis factors, monokines and colony stimulating factors. More specifically, cytokines include BMP (Bone morphogenetic protein) family, CCL (Cheomkine ligands) family, CMTM (CKLF-like MARVEL transmembrane domain containing member) family, CXCL (C-X-C motif ligand ligand) family, GDF (Growth/differentiation factor) family, growth hormone, IFN (Interferon) family, IL (Interleukin) family, TNF (Tumor necrosis factors) family, GPI (glycophosphatidylinositol), SLUPR-1 (Secreted Ly-6/uPAR-Related Protein 1), SLUPR-2 ( Secreted Ly-6/uPAR-Related Protein 2) and combinations thereof.

상기 사이토카인은 T 세포의 분화에 필수적인 전사인자 또는 성장인자를 유도 또는 억제하며, 다른 면역세포의 성장 및 분화를 매개함으로써, 본 개시에 따른 수지상세포 모방 구조체의 면역항암 치료의 효과를 보다 강화할 수 있다. The cytokine induces or inhibits transcription factors or growth factors essential for the differentiation of T cells, and mediates the growth and differentiation of other immune cells, thereby further enhancing the effect of immunotherapeutic treatment of the dendritic cell-mimicking structure according to the present disclosure. there is.

구체적으로 상기 생리활성 성분은 αPD-1 단일클론 항체 외 αCTLA-4, αPD-L1와 같은 면역관문 억제제, 또는 이를 이용한 화학 요법 약물 등의 항암 약물이라면 제한되지 않는다.Specifically, the physiologically active component is not limited as long as it is an αPD-1 monoclonal antibody, immune checkpoint inhibitors such as αCTLA-4 and αPD-L1, or anti-cancer drugs such as chemotherapy drugs using the same.

본 개시는 전술한 바와 같은 면역관문 억제용 수지상세포 모방 나노구조체를 포함하는 암 치료용 약학적 조성물을 제공할 수 있다. The present disclosure may provide a pharmaceutical composition for cancer treatment comprising the dendritic cell-mimicking nanostructure for inhibiting immune checkpoints as described above.

αPD-1 단일클론 항체가 도입된 면역관문 억제용 수지상세포 모방 나노구조체는 세포독성 T 세포의 증식 및 분화 유도 효과가 매우 뛰어나 T 세포를 활성화시킴에 따라 이를 암 치료용 약학 조성물은 강력한 면역항암 치료 효과를 제공할 수 있다.The αPD-1 monoclonal antibody-introduced dendritic cell-mimicking nanostructure for suppression of immune checkpoints is very effective in inducing proliferation and differentiation of cytotoxic T cells, and as it activates T cells, the pharmaceutical composition for cancer treatment is a strong immuno-anticancer treatment. effect can be provided.

암은 침윤에 의해 국부적 및 전이를 통해 확장가능한 각종 악성 고형 종양 등을 총징하는 것으로서, 구체적인 예로는 B-세포 림프종, 비소세포 폐암, 소세포 폐암, 기저세포 암종, 피부 편평세포암종, 직장결장암, 흑색종, 두경부 편평암, 간세포암, 위암, 육종, 위식도암, 신세포 암종, 교모세포종, 췌장암, 방광암, 전립선암, 유방암, 피부 T-세포 림프종, 머켈세포 암종 또는 다발성 골수종일 수 있으나 이에 제한되지 않는다.Cancer is a general term for various malignant solid tumors that can expand through local and metastasis by invasion, and specific examples include B-cell lymphoma, non-small cell lung cancer, small cell lung cancer, basal cell carcinoma, squamous cell carcinoma of the skin, colorectal cancer, and melanoma. may be, but is not limited to, cancer of the head and neck, squamous cancer, hepatocellular carcinoma, gastric cancer, sarcoma, gastroesophageal cancer, renal cell carcinoma, glioblastoma, pancreatic cancer, bladder cancer, prostate cancer, breast cancer, cutaneous T-cell lymphoma, Merkel cell carcinoma, or multiple myeloma. don't

암 치료를 위한 약학적 조성물은 pH, 삼투도, 점도, 무균성, 투명도, 색상, 등장성, 냄새, 안정성, 용해 속도 또는 방출 속도, 흡착 또는 침투를 변형, 유지 또는 보존하기 위한 제형 물질을 포함할 수 있다. 상기 약학적 조성물은 경구 또는 비경구로 투여될 수 있다. 비경구의 예로는, 정맥내, 근육내, 피하, 안와내, 피막내, 복강내, 직장내, 수조내, 혈관내, 진피내를 포함하는 다양한 경로로 환자에게 투여될 수 있으며, 피부 패치 또는 경피 이온도입법을 각각 이용하여 피부를 통해서도 투여될 수 있다.Pharmaceutical compositions for cancer treatment include formulation materials for modifying, maintaining or preserving pH, osmoticity, viscosity, sterility, transparency, color, isotonicity, odor, stability, rate of dissolution or release, adsorption or permeation. can do. The pharmaceutical composition may be administered orally or parenterally. Examples of parenteral administration include intravenous, intramuscular, subcutaneous, intraorbital, intracapsular, intraperitoneal, intrarectal, intracisternal, intravascular, and intradermal administration to a patient, and may be administered to a patient through a skin patch or transdermally. It can also be administered through the skin using iontophoresis, respectively.

상기 약학적 조성물은 개별 치료제로 투여하거나 다른 항암제와 병용하여 투여될 수 있고, 종래의 항암제와 순차적 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other anticancer agents, or may be administered sequentially or simultaneously with conventional anticancer agents. And it can be single or multiple administrations. It is important to administer the amount that can obtain the maximum effect with the minimum amount without side effects in consideration of all the above factors, and can be easily determined by those skilled in the art.

아울러 본 개시에 따르면 상기 면역관문 억제용 수지상세포 모방 나노구조체 또는 이를 포함하는 약학적 조성물을 암에 걸린 개체에 투여하는 단계를 포함하는 개체의 암 치료방법을 제공할 수 있다. 상기 약학적 조성물을 개체에 투여함으로써 개체에서 종양 특이적 면역 반응을 유도하거나 개체에서 암의 증상을 치료 및/또는 완화시킬 수 있다. 이때 상기 개체는 인간 또는 비인간 포유동물일 수 있다.In addition, according to the present disclosure, it is possible to provide a method for treating cancer of a subject comprising administering the dendritic cell-mimicking nanostructure for suppressing the immune checkpoint or a pharmaceutical composition containing the same to a subject suffering from cancer. By administering the pharmaceutical composition to a subject, a tumor-specific immune response can be induced in the subject or symptoms of cancer can be treated and/or alleviated in the subject. In this case, the subject may be a human or a non-human mammal.

또한 본 개시는 수지상세포 모방 나노구조체 (anDC)에 항원을 펄싱하여 항원을 표면에 제시하고, αPD-1이 컨쥬게이션된 인지질을 세포막에 삽입하여 최종적으로 αPD-1 단일클론 항체가 도입된 면역관문 억제용 수지상세포 모방 나노구조체를 제조하는 방법을 제공할 수 있다.In addition, the present disclosure presents antigens on the surface by pulsing dendritic cell-mimicking nanostructures (anDCs), inserts phospholipids conjugated with αPD-1 into cell membranes, and finally introduces αPD-1 monoclonal antibodies into immune checkpoints. It is possible to provide a method for preparing a dendritic cell-mimicking nanostructure for inhibition.

구체적으로 상기 제조방법은 (S10) 수지상 세포로부터 세포막을 정제하는 단계; (S20) 상기 세포막에 에너지를 가하여 세포막 현탁액을 형성하는 단계; (S30) 상기 세포막 현탁액으로부터 리포좀을 수득하는 단계; (S40) 나노입자 및 상기 리포좀을 혼합한 후, 필터 압축하여 상기 세포막이 도입된 나노입자를 수득하는 단계; 및 (S50) 지질 분자와 컨쥬게이션된 αPD-1 단일클론 항체를 상기 세포막이 도입된 나노입자에 도입하는 단계;를 포함하는 것을 특징으로 할 수 있다.Specifically, the manufacturing method includes (S10) purifying cell membranes from dendritic cells; (S20) forming a cell membrane suspension by applying energy to the cell membrane; (S30) obtaining liposomes from the cell membrane suspension; (S40) mixing the nanoparticles and the liposomes, and then compressing the filter to obtain the nanoparticles into which the cell membrane is introduced; and (S50) introducing an αPD-1 monoclonal antibody conjugated with a lipid molecule into the nanoparticle into which the cell membrane is introduced.

(S10) 수지상세포로부터 세포막을 정제하는 단계는 당업계에 알려진 방법을 제한없이 이용할 수 있고, 일 예로서, 급속 냉동-해동 및 원심분리를 통하여 진행될 수 있다. (S20) 정제된 세포막에 에너지를 가하여 세포막 현탁액을 형성할 수 있다. 이때, 일 예로서, 초음파를 처리하여 수백 나노 또는 수 마이크로 단위의 세포막 현탁액을 얻을 수 있다. 이후 (S30) 상기 세포막 현탁액을 멤브레인 필터를 통해 여과하면 원하는 크기의 세포막 리포좀을 수득할 수 있다. 이에 제한되는 것은 아니지만, 구체적으로 10 내지 200 ㎚, 보다 구체적으로 20 내지 150 ㎚ 의 평균 입경을 가지는 리포좀을 수득할 수 있다. 이후, 세포막 리포좀 및 나노입자를 혼합하고, 공압출함으로써 수지상세포의 세포막이 나노입자 표면에 코팅된 형태가 얻어질 수 있다.(S10) The step of purifying the cell membrane from the dendritic cells may use a method known in the art without limitation, and may be performed, for example, through rapid freezing-thawing and centrifugation. (S20) A cell membrane suspension may be formed by applying energy to the purified cell membrane. At this time, as an example, a cell membrane suspension of hundreds of nanometers or several micrometers may be obtained by treating with ultrasonic waves. Thereafter (S30), by filtering the cell membrane suspension through a membrane filter, cell membrane liposomes having a desired size can be obtained. Although not limited thereto, liposomes having an average particle diameter of specifically 10 to 200 nm, more specifically 20 to 150 nm can be obtained. Thereafter, a form in which the cell membrane of dendritic cells is coated on the surface of the nanoparticle can be obtained by mixing and co-extruding the cell membrane liposome and the nanoparticle.

상기 (S40) 단계 이후, 상기 세포막이 도입된 나노입자에 항원을 펄싱하는 단계;를 더 포함할 수 있다. 상기 항원은 암 치료의 용도를 위해 종양 항원 유래 펩타이드 또는 단백질일 수 있고, 전술한 바에 따라 다양한 종양 특이 항원을 활용할 수 있다. 본 개시에 있어서, 비제한적인 일 예로 GP33-41를 이용할 수 있다.After the step (S40), pulsing an antigen to the nanoparticle into which the cell membrane is introduced; may be further included. The antigen may be a tumor antigen-derived peptide or protein for use in cancer treatment, and various tumor-specific antigens may be utilized as described above. In the present disclosure, GP 33-41 may be used as a non-limiting example.

(S50) 단계에 있어서, 지질 분자는 생체적합성을 가지는 지질 분자라면 제한없이 사용될 수 있으며, 구체적으로 인지질일 수 있고, 보다 구체적으로 포스파티딜에탄올아민계 인지질일 수 있다. 포스파티딜에탄올아민계 인지질의 구체적인 예를 들면, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)일 수 있다. 도 2에 지질 분자에 컨쥬게이션된 αPD-1를 수득하는 과정이 도시되어 있다. DSPE의 친수성 기에 PEG의 일 말단이 컨쥬게이션되고, PEG의 타 말단은 αPD-1이 컨쥬게이션될 수 있다. PEG의 중량평균분자량은 200 내지 10,000 g/mol 또는 500 내지 5,000 g/mol일 수 있다.In step (S50), the lipid molecule may be used without limitation as long as it is a biocompatible lipid molecule, and may be specifically a phospholipid, and more specifically, a phosphatidylethanolamine-based phospholipid. A specific example of the phosphatidylethanolamine-based phospholipid may be 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). 2 shows the process of obtaining αPD-1 conjugated to a lipid molecule. One end of PEG may be conjugated to the hydrophilic group of DSPE, and αPD-1 may be conjugated to the other end of PEG. The weight average molecular weight of PEG may be 200 to 10,000 g/mol or 500 to 5,000 g/mol.

인지질과 컨쥬게이션된 αPD-1는 수지상세포 나노구조체의 세포막의 지질-지질 상호작용에 따라 용이하게 삽입되어, 효과적으로 T 세포의 활성화를 유도하는 역할을 할 수 있다. αPD-1 conjugated with a phospholipid is easily incorporated according to the lipid-lipid interaction of the cell membrane of the dendritic cell nanostructure, and can effectively induce T cell activation.

이상과 같이 본 발명에서는 특정된 사항들과 한정된 실시예에 의해 설명되었으나 이는 본 발명의 보다 전반적인 이해를 돕기 위해서 제공된 것일 뿐, 본 발명은 상기의 실시예에 한정되는 것은 아니며, 본 발명이 속하는 분야에서 통상의 지식을 가진 자라면 이러한 기재로부터 다양한 수정 및 변형이 가능하다. As described above, the present invention has been described with specific details and limited examples, but this is only provided to help a more general understanding of the present invention, the present invention is not limited to the above examples, and the field to which the present invention belongs Those skilled in the art can make various modifications and variations from these descriptions.

따라서, 본 발명의 사상은 설명된 실시예에 국한되어 정해져서는 아니되며, 후술하는 특허청구범위뿐 아니라 이 특허청구범위와 균등하거나 등가적 변형이 있는 모든 것들은 본 발명 사상의 범주에 속한다고 할 것이다.Therefore, the spirit of the present invention should not be limited to the described embodiments, and it will be said that not only the claims to be described later, but also all modifications equivalent or equivalent to these claims belong to the scope of the present invention. .

제조예 1. 수지상세포 모방 나노구조체 제조Preparation Example 1. Preparation of dendritic cell-mimicking nanostructures

6주~8주령 Naive 6 마우스(Orient Bio)의 뒷다리에서 골수세포를 채취하고, ACK lysis buffer를 실온에서 3분간 처리하여 적혈구 세포(RBC)를 제거한 뒤 RBC가 제거된 BM만을 수득하였다. 총 골수세포 수를 세어 6 well non-tissue plate에 7.5×105 cells/well씩 시딩한 후 GM-CSF (20 ng/㎖) media 2 ㎖/well와 함께 37°C, 5% CO2 조건에서 배양하였다. 3일차에 2 ㎖의 GM-CSF (20 ng/㎖) media를 추가해주고, 6일차까지 유지하여 수지상세포로 분화를 유도하였다. 6일차에 분화가 완료된 수지상세포를 수득하여 LPS (Lipopolysaccaride) (50 ng/㎖)가 첨가된 media에 4×106 cells/well로 시딩하였다. 이 때 LPS 첨가된 media는 6 well non-tissue plate에 4 ㎖씩 사용된다. 7일차에 최종적으로 성숙이 완료된 수지상세포를 수득하였다.Bone marrow cells were collected from the hind limbs of 6- to 8-week-old Naive 6 mice (Orient Bio), treated with ACK lysis buffer at room temperature for 3 minutes to remove red blood cells (RBCs), and only RBC-free BM was obtained. After counting the total number of bone marrow cells and seeding 7.5×10 5 cells/well in a 6 well non-tissue plate, GM-CSF (20 ng/ml) media was added to 2 ml/well at 37°C and 5% CO 2 conditions. cultured. On day 3, 2 ml of GM-CSF (20 ng/ml) media was added and maintained until day 6 to induce differentiation into dendritic cells. On day 6, differentiated dendritic cells were obtained and seeded at 4×10 6 cells/well in media supplemented with LPS (Lipopolysaccaride) (50 ng/ml). At this time, 4 ml of LPS-added media is used in a 6 well non-tissue plate. On the 7th day, finally matured dendritic cells were obtained.

성숙이 완료된 수지상세포를 2,000 rpm에서 5분간 원심분리하여 순수한 수지상세포만을 분리한 후, Protease Inhibitor Tablet-PBS buffer를 처리하여 1~2Х106 cells/㎖ 농도로 분산시켰다. 그리고 -70 ℃에서 급속 냉동, 상온에서의 해동 과정 및 원심분리를 거쳐 세포막만을 정제하였다. 이후 20%의 진폭으로, 3초 켜기/끄기 및 사이클간 2초의 냉각기를 갖는 3초 1회전을 총 60회 수행하는 초음파(VC505, Sonics & materials)를 처리하여, 마이크로 단위의 세포막 현탁액을 확보하였으며, 이를 나노 사이즈의 필터를 갖는 폴리카보네이트 막(기공크기 1㎛, 400 ㎚, 100 ㎚)으로 걸러내 각 기공 크기의 직경을 갖는 나노 리포좀을 생성하였다. 나노입자 표면 상에 코팅되는 세포막의 표면적을 고려하여, 나노입자 및 리포좀이 최적화된 혼합비율을 가지는 경우를 XDM=1로 전제하였다. The matured dendritic cells were centrifuged at 2,000 rpm for 5 minutes to separate pure dendritic cells, and then treated with Protease Inhibitor Tablet-PBS buffer and dispersed at a concentration of 1 to 2Х10 6 cells/㎖. Then, only cell membranes were purified through rapid freezing at -70 ° C, thawing at room temperature, and centrifugation. Thereafter, ultrasonic waves (VC505, Sonics & materials) performed with 20% amplitude, 3 sec on/off and 3 sec 1 rotation with 2 sec cooling between cycles for a total of 60 times were processed to obtain cell membrane suspension in micro units. , This was filtered with a polycarbonate membrane (pore size of 1 μm, 400 nm, and 100 nm) having a nano-sized filter to produce nano-liposomes having a diameter of each pore size. Considering the surface area of the cell membrane coated on the surface of the nanoparticle, the case where the nanoparticle and the liposome have an optimized mixing ratio was assumed to be X DM = 1.

[식 1][Equation 1]

Mole fraction (XDM )=(세포막의 표면적)/(나노입자의 표면적)Mole fraction (X DM )=(cell membrane surface area)/(nanoparticle surface area)

상기 XDM = 1이 되도록 60 ㎚ 금 나노입자 5 ㎕ 및 리포좀 1 ㎖을 혼합한 후, 함께 필터압축 (filter extrusion)하여, 리포좀이 금 나노입자 표면에 코팅된 수지상세포 모방 나노구조체를 제조하였다. 상기 제조된 수지상세포 모방 나노구조체에 0.2 ㎍/㎖의 GP33-41 항원을 약 30분간 37 ℃에서 처리함으로써 MHC class I, II 표면에 항원을 제시할 수 있도록 유도하여, 항원이 펄싱된 수지상세포 모방 나노구조체를 제조하였다.After mixing 5 μl of 60 nm gold nanoparticles and 1 ml of liposomes so that X DM = 1, filter extrusion was performed together to prepare a dendritic cell-mimicking nanostructure coated with liposomes on the surface of the gold nanoparticles. By treating the prepared dendritic cell-mimicking nanostructure with 0.2 μg/ml of GP 33-41 antigen at 37° C. for about 30 minutes, the antigen is induced to present on the surface of MHC class I and II, and the antigen is pulsed dendritic cells Mimic nanostructures were prepared.

실시예 1. αPD-1 단일클론 항체가 도입된 면역관문 억제용 수지상세포 모방 나노구조체 제조Example 1. Preparation of dendritic cell-mimicking nanostructure for immune checkpoint inhibition into which αPD-1 monoclonal antibody was introduced

도 2에 도시된 방법에 따라, 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) 의 친수성 기에 PEG 말단이 컨쥬게이션 된 분자에 PEG의 타 말단에 αPD-1 단일클론 항체를 컨쥬게이션 시켜 준비하고, 상기 제조예 1에 따라 제조된 수지상세포 모방 나노구조체의 세포막에 인지질이 결합된 αPD-1 단일클론 항체를 자발적 세포막 삽입 현상을 이용하여 도입함으로써, 면역관문 억제용 수지상세포 모방 나노구조체를 제조하였다.Prepared by conjugating αPD-1 monoclonal antibody to the other end of PEG to a molecule in which the PEG end was conjugated to the hydrophilic group of 2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) according to the method shown in FIG. and introducing αPD-1 monoclonal antibody bound to a phospholipid into the cell membrane of the dendritic cell-mimicking nanostructure prepared in Preparation Example 1 using spontaneous cell membrane insertion, thereby preparing a dendritic cell-mimicking nanostructure for immune checkpoint inhibition. did

실험예 1. 면역관문 억제용 수지상세포 모방 나노구조체의 T 세포 증식 및 분화유도 효과 평가Experimental Example 1. Evaluation of T cell proliferation and differentiation inducing effect of dendritic cell-mimicking nanostructure for immune checkpoint inhibition

96 well U bottom plate에 실시예 1에 따라 제조된 면역관문 억제용 수지상세포 모방 나노구조체(αPD-1-anDC), 수지상세포 모방 나노구조체(anDC), 수지상세포(BMDC) 각각 1×104개에 대하여 P14 mouse로부터 분리한 CD8 T 세포 5×104개를 같은 well에 배양액 10% RPMI 200 ㎕을 통해 넣고, 37 ℃에서 3일간 공배양하였다. 공배양이 끝나기 6시간 전에 GP, GS, DNase1, GP33-41 peptide를 이용하여 재자극을 주었다. 6시간 후, 해당 플레이트를 인큐베이터에서 회수하여 형광이 달린 항체를 통해 염색을 진행하고 유세포 분석기를 통하여 표면 단백질과 CD8 T 세포의 활성화 정도를 분석하였다. 그 결과는 도 5에 도시되었다.Dendritic cell-mimicking nanostructure (αPD-1-anDC), dendritic cell-mimicking nanostructure (anDC), and dendritic cell (BMDC) prepared according to Example 1 in a 96-well U bottom plate, respectively 1 × 10 4 each 5 × 10 4 CD8 T cells isolated from P14 mice were added to the same well through 200 μl of 10% RPMI culture medium and co-cultured at 37 °C for 3 days. Restimulation was given using GP, GS, DNase1, and GP 33-41 peptide 6 hours before the end of co-culture. After 6 hours, the plate was recovered from the incubator, stained with a fluorescent antibody, and the degree of activation of surface proteins and CD8 T cells was analyzed by flow cytometry. The results are shown in FIG. 5 .

T 세포만 배양한 그룹과 달리 T 세포를 anDC와 함께 배양한 경우 BMDC와 마찬가지로 T 세포의 증식을 유도하였고, CD44의 발현과 사이토카인인 IFNγ, TNFα, IL-2의 발현을 유도함을 확인할 수 있다. 이러한 결과를 통해 anDC가 항원제시세포로서 기능하고 있음을 알 수 있다.Unlike the group in which only T cells were cultured, when T cells were cultured with anDCs, the proliferation of T cells was induced as in BMDCs, and the expression of CD44 and cytokines IFNγ, TNFα, and IL-2 were induced. . These results suggest that anDCs function as antigen-presenting cells.

또한 anDC의 경우 BMDC와 비교하였을 때 T 세포가 발현하는 CD44의 MFI값이 낮고 사이토카인을 발현하는 비율이 낮기 때문에 상대적으로 T 세포를 활성화 시키는 능력이 떨어지는 것을 알 수 있다. 반면, anDC에 αPD-1을 컨쥬게이션한 경우 anDC에 비해 CD44의 MFI값이 증가하고 사이토카인을 발현하는 비율이 증가하였으며, 사이토카인을 발현하는 비율의 경우 BMDC와 비슷하였고, IL-2의 경우 더 높게 나타났다. CTV+ frequency를 통해 BMDC, anDC, aPD-1을 컨쥬게이션한 anDC 세 가지 그룹의 경우 90% 이상의 T 세포들이 잘 증식한 것을 확인할 수 있다. In addition, in the case of anDC, compared to BMDC, it can be seen that the ability to activate T cells is relatively low because the MFI value of CD44 expressed by T cells is low and the ratio of expressing cytokines is low. On the other hand, when αPD-1 was conjugated to anDC, the MFI value of CD44 increased and the ratio of cytokine expression increased compared to anDC. The ratio of cytokine expression was similar to that of BMDC, and IL-2 appeared higher. In the case of the three groups of anDC conjugated with BMDC, anDC, and aPD-1 through CTV+ frequency, it was confirmed that more than 90% of T cells proliferated well.

실험예 2. 면역관문 억제용 수지상세포 모방 나노구조체의 T 세포 증식 및 분화유도 효과 평가 (in vivo)Experimental Example 2. Evaluation of T cell proliferation and differentiation inducing effects of dendritic cell mimicking nanostructures for immune checkpoint inhibition (in vivo)

congenic marker(Ly5.1)가 달린 P14 mouse에서 CD8 T 세포를 분리한 후 naive recipient mouse에 양자면역세포이입(adoptive transfer)를 해주고, 24시간 후 수지상세포 (BMDC), 수지상세포 모방 나노구조체 (anDC) 및 면역관문 억제용 수지상세포 모방 나노구조체 (αCTLA4 conjugated anDC, αPD-1 conjugated anDC) 를 각 그룹마다 다른 조건으로 양자면역세포이입을 진행하였다. 구체적인 조건은 도 6에 도시되었다. 그 후 48시간이 경과한 다음, 마우스의 비장에서 면역세포를 분리하여, 형광이 달린 항체를 통해 염색을 진행하고 유세포 분석기를 통하여 CD8 T 세포의 활성화 정도를 분석하였다. 그 결과는 도 7 및 도 8에 도시되었다.After isolating CD8 T cells from P14 mice with congenic markers (Ly5.1), adoptive transfer was performed on naive recipient mice, and after 24 hours, dendritic cells (BMDC) and dendritic cell-mimicking nanostructures (anDC ) and dendritic cell-mimicking nanostructures for immune checkpoint inhibition (αCTLA4 conjugated anDC, αPD-1 conjugated anDC) were subjected to adoptive immunocytotransduction under different conditions for each group. Specific conditions are shown in FIG. 6 . After 48 hours had elapsed, immune cells were isolated from the spleen of the mouse, stained with a fluorescent antibody, and the degree of activation of CD8 T cells was analyzed by flow cytometry. The results are shown in FIGS. 7 and 8 .

대조군인 T 세포 특이적인 항원을 탑재하지 않은 BMDC 및 anDC와 달리 항원을 펄싱한 BMDC/GP33, anDC/GP33을 넣어준 경우 T 세포의 증식을 유도하였고, CD44의 발현과 사이토카인인 IFNγ, TNFα, IL-2의 발현을 유도함을 확인할 수 있다. 이러한 결과를 통해 anDC가 항원제시세포로서 기능하고 있음을 알 수 있다.Unlike BMDCs and anDCs that did not load T cell-specific antigens as controls, BMDC/GP33 and anDC/GP33 pulsed antigens induced T cell proliferation, CD44 expression and cytokines IFNγ, TNFα, It can be confirmed that the expression of IL-2 is induced. These results suggest that anDCs function as antigen-presenting cells.

anDC의 경우 BMDC와 비교하였을 때 증식한 T 세포의 비율이 낮았으며, 또한 anDC의 경우 BMDC와 비교하였을 때 T 세포가 발현하는 CD44의 MFI값이 낮고 사이토카인을 발현하는 비율이 낮기 때문에 상대적으로 T 세포를 활성화 시키는 능력이 떨어지는 것을 알 수 있다. 반면, anDC에 αPD-1을 컨쥬게이션한 경우 anDC에 비해 CD44의 MFI값이 증가하고 사이토카인을 발현하는 비율이 증가하였다. 즉, 항체를 결합한 경우 T 세포 활성화 능력이 현저히 향상됨을 확인하였는데, 이러한 결과는 본 개시에 따른 면역관문 억제용 수지상세포 모방 나노구조체를 통한 항암 활성이 매우 우수할 것임을 시사한다. In the case of anDC, the ratio of proliferating T cells was lower than that of BMDC, and in the case of anDC, compared to BMDC, the MFI value of CD44 expressed by T cells was low and the ratio of cytokine expression was low. It can be seen that the ability to activate cells is reduced. On the other hand, when αPD-1 was conjugated to anDC, the MFI value of CD44 increased and the ratio of cytokine expression increased compared to anDC. That is, it was confirmed that the T cell activation ability was remarkably improved when the antibody was combined. These results suggest that the anticancer activity through the dendritic cell-mimicking nanostructure for suppressing the immune checkpoint according to the present disclosure will be very excellent.

Claims (10)

나노입자 코어; 및 수지상 세포로부터 유래된 지질 분자의 세포막을 포함하는 쉘;을 포함하는 나노구조체로서,nanoparticle core; And a shell comprising a cell membrane of lipid molecules derived from dendritic cells; As a nanostructure comprising, 상기 쉘은 αPD-1 단일클론 항체를 포함하는 것을 특징으로 하는 면역관문 억제용 수지상세포 모방 나노구조체.The shell is a dendritic cell-mimicking nanostructure for immune checkpoint inhibition, characterized in that it comprises an αPD-1 monoclonal antibody. 제 1항에 있어서,According to claim 1, 상기 αPD-1 단일클론 항체는 지질 분자와 컨쥬게이션된 것인, 수지상세포 모방 나노구조체.Wherein the αPD-1 monoclonal antibody is conjugated with a lipid molecule, a dendritic cell-mimicking nanostructure. 제 1항에 있어서,According to claim 1, 상기 나노구조체는 항원이 펄싱(pursing)된 것인, 수지상세포 모방 나노구조체.The nanostructure is an antigen-pulsed, dendritic cell-mimicking nanostructure. 제 1항에 있어서,According to claim 1, 상기 항원은 종양 항원 유래 펩타이드 또는 단백질인, 수지상세포 모방 나노구조체.The antigen is a tumor antigen-derived peptide or protein, dendritic cell-mimicking nanostructure. 제 1항에 있어서,According to claim 1, 상기 나노구조체는 20 ㎚ 내지 50 ㎛의 평균입경을 가지는, 수지상세포 모방 나노구조체.The nanostructure is a dendritic cell mimicking nanostructure having an average particle diameter of 20 nm to 50 μm. 제 1항에 있어서,According to claim 1, 상기 나노구조체는 20 ㎚ 내지 1000 ㎚의 평균입경을 가지는, 수지상세포 모방 나노구조체.The nanostructure is a dendritic cell mimicking nanostructure having an average particle diameter of 20 nm to 1000 nm. 제 1항에 있어서,According to claim 1, 상기 쉘의 표면은 생리활성 고분자, 생리활성 성분 또는 단백질이 표지된 것인, 수지상세포 모방 나노구조체.The surface of the shell is a physiologically active polymer, physiologically active component or protein labeled, dendritic cell-mimicking nanostructure. 제 1항에 따른 면역관문 억제용 수지상세포 모방 나노구조체를 포함하는 암 치료용 약학적 조성물.A pharmaceutical composition for cancer treatment comprising the dendritic cell-mimicking nanostructure for inhibiting immune checkpoints according to claim 1. (S10) 수지상세포로부터 세포막을 정제하는 단계;(S10) purifying cell membranes from dendritic cells; (S20) 상기 세포막에 에너지를 가하여 세포막 현탁액을 형성하는 단계;(S20) forming a cell membrane suspension by applying energy to the cell membrane; (S30) 상기 세포막 현탁액으로부터 리포좀을 수득하는 단계;(S30) obtaining liposomes from the cell membrane suspension; (S40) 나노입자 및 상기 리포좀을 혼합한 후, 필터 압축하여 상기 세포막이 도입된 나노입자를 수득하는 단계; 및(S40) mixing the nanoparticles and the liposomes, and then compressing the filter to obtain the nanoparticles into which the cell membrane is introduced; and (S50) 지질 분자와 컨쥬게이션된 αPD-1 단일클론 항체를 상기 세포막이 도입된 나노입자에 도입하는 단계;(S50) introducing an αPD-1 monoclonal antibody conjugated with a lipid molecule into the nanoparticle into which the cell membrane is introduced; 를 포함하는 면역관문 억제용 수지상세포 모방 나노구조체의 제조방법.Method for producing a dendritic cell-mimicking nanostructure for inhibiting immune checkpoints comprising a. 제 9항에 있어서,According to claim 9, 상기 (S40) 단계 이후, 상기 세포막이 도입된 나노입자에 항원을 펄싱하는 단계;를 더 포함하는, 면역관문 억제용 수지상세포 모방 나노구조체의 제조방법.After the step (S40), pulsing an antigen to the nanoparticle into which the cell membrane has been introduced; a method for producing a dendritic cell-mimicking nanostructure for suppressing immune checkpoints.
PCT/KR2022/020602 2021-12-16 2022-12-16 DENDRITIC CELL-MIMETIC FUNCTIONAL NANOSTRUCTURE COMPRISING αPD-1, AND PREPARATION METHOD THEREFOR Ceased WO2023113545A1 (en)

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