WO2021025388A1 - Exosome derived from irradiated cancer cells, pharmaceutical composition for cancer treatment containing mature dendritic cells obtained using same, and method for producing same - Google Patents

Exosome derived from irradiated cancer cells, pharmaceutical composition for cancer treatment containing mature dendritic cells obtained using same, and method for producing same Download PDF

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WO2021025388A1
WO2021025388A1 PCT/KR2020/010134 KR2020010134W WO2021025388A1 WO 2021025388 A1 WO2021025388 A1 WO 2021025388A1 KR 2020010134 W KR2020010134 W KR 2020010134W WO 2021025388 A1 WO2021025388 A1 WO 2021025388A1
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cells
cancer
dendritic cells
pharmaceutical composition
exosomes
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French (fr)
Korean (ko)
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이동은
김우식
변의백
최대성
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한국원자력연구원
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    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
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    • 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
    • A61K35/13—Tumour cells, irrespective of tissue of origin
    • 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
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4615—Dendritic cells
    • 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
    • A61K39/46—Cellular immunotherapy
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    • A61K39/4622—Antigen presenting cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/46449—Melanoma antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals

Definitions

  • the present invention relates to a pharmaceutical composition for cancer treatment including exosomes derived from irradiated cancer cells, mature dendritic cells obtained by using the same, and a method for preparing the same, and more particularly, to improved immunogenicity using irradiated cancer cells
  • the present invention relates to an anticancer immunotherapy technology for solving the limitations of conventional anticancer treatment techniques by developing a dendritic cell cancer vaccine produced by preparing an exosome and exposing it to dendritic cells (DC).
  • DC dendritic cells
  • Existing cancer treatment methods include surgical surgery, radiation therapy, and chemotherapy using anticancer drugs to remove cancer cells as much as possible. These can be used in a relatively wide range of cancer treatments, but most solid tumors are cancerous. It is mainly effective in the early stage of non-metastatic metastasis, thus limiting the treatment of metastatic cancer.
  • target anticancer drugs have appeared as alternatives to radiation therapy and chemotherapy, but target anticancer drugs such as these have specific genes. And/or it is effective only in cancer cells in which the protein is expressed, and recurrence and resistance occur.
  • anticancer immunotherapy is a method of treating cancer by increasing the immune activity against cancer cells by using the characteristics of immune cells in the body or by suppressing the method of avoiding cancer cells by attacking immune cells.Immune cell therapy , Immune checkpoint inhibitors, therapeutic cancer vaccines, and Therapeutic antibodies.
  • Therapeutic cancer vaccines expose cancer cell-derived antigens or cancer lysates as well as anti-cancer vaccines using intact tumour cells or cancer cell lysate derived from cancer cell lines.
  • DC-based cancer vaccine that uses autologous dendritic cells (DC) produced by the method.
  • DC autologous dendritic cells
  • WO2016-168680 A1 discloses a method for preparing immune cells by delivering extracellular vesicles to immune cells.
  • Conventional methods for preparing tumor lysates for the production of dendritic cell cancer vaccines have been prepared by separating the entire tumor into single cells, pre-treating with strong acid, and then undergoing a freeze-thaw process several times or using chemicals.
  • the disadvantages are the possibility of denaturation of the antigenic protein, the risk of residual toxic chemicals, and the very low immunogenicity of the antigen.
  • one aspect of the present invention is to provide a method for preparing exosomes for preparing mature dendritic cells.
  • Another aspect of the present invention is to provide an exosome having excellent maturation ability of dendritic cells and uses thereof.
  • a vaccine for treating cancer a pharmaceutical composition for treating cancer, and an immunotherapy agent comprising dendritic cells capable of inducing strong activity of T cells.
  • irradiating cancer cells with radiation comprising the step of obtaining an exosome from irradiated cancer cells.
  • an exosome for preparing mature dendritic cells obtained from irradiated cancer cells.
  • irradiating cancer cells with radiation Obtaining exosomes from irradiated cancer cells; And there is provided a method for producing a dendritic cell therapy comprising the step of loading the exosomes on the dendritic cells.
  • a vaccine for cancer treatment a pharmaceutical composition for cancer treatment, and an immunotherapy agent, including dendritic cells carrying exosomes obtained from irradiated cancer cells.
  • Dendritic cells are known as the most powerful antigen-presenting cells, and treatments using them are highly likely to become a new trend in the field of cancer treatment along with anticancer drugs, surgical surgery, and irradiation, which are currently used in most cancer treatments. It is expected that it can be applied to various cancer treatment fields by activating the immune function of Furthermore, according to the present invention, not only can the dendritic cells be effectively activated, but also cancer cell-specific immunity can be effectively induced, and thus remarkably improved anti-cancer immunity can be induced.
  • Figure 1 shows the results of analyzing the Nanoparticle Tracking Analysis (NTA) in order to confirm the amount of exosomes (Fig. 1(a)) and the results of analyzing the exosomes through TEM (Fig. 1(b)).
  • NTA Nanoparticle Tracking Analysis
  • Figure 2 is a result of confirming the presence or absence of cytotoxicity through annexin V and propidium iodide (PI) staining after treating dendritic cells with exosomes (Fig. 2(a)) and stained with CFSE The results of confirming the uptake ability of exosomes in dendritic cells (Fig. 2(b)) are shown.
  • PI propidium iodide
  • Figure 3 shows the effect of exosomes on the maturation of dendritic cells
  • Figure 3 (a) is a flow cytometer confirming whether the expression of CD80, CD86, MHC-I, MHC-II of the dendritic cells
  • Figure 3 ( b) shows the results as a bar graph.
  • FIG. 3(c) is a result of analyzing IL-12p70, an anti-inflammatory cytokine IL-10 through EILISA
  • 3(d) is a result of confirming this phenomenon once again through intracellular staining.
  • Figures 4 (a) and (b) show the effect of inducing maturation of T cells by dendritic cells treated with exosomes
  • Figure 4 (c) is a cytokine secreted from these T cells by applying an ELISA kit ( IFN-g, IL-2, IL-4) is the result of analysis of the secretion.
  • Figure 5(a) shows an exemplary dosing regimen of dendritic cells treated with gamma-irradiated melanoma-derived exosomes
  • Figure 5(b) is a graph showing the tendency of slowing cancer growth when dendritic cells are used as a vaccine. It is represented by
  • the present invention obtains exosomes with significantly improved immunogenicity using radiation-irradiated cancer cells, and develops a mature dendritic cell cancer vaccine in which it is exposed to dendritic cells (DC) to solve the limitations of conventional anticancer treatments. It's about technology.
  • exosome used in the present invention is a membrane-structured vesicle secreted from various types of cells, and it binds to other cells and tissues to transfer membrane components, proteins, RNA, etc. It is known to do.
  • the immature dendritic cells of the present invention can be obtained by isolating DC progenitor cells from a suitable tissue source containing DC progenitor cells or by a method of preparing immature DCs.
  • the method of preparing immature DC refers to a method of producing immature DC by differentiating progenitor cells in vitro, and the progenitor cells may be blood mononuclear cells or hematopoietic stem cells derived from a suitable tissue source,
  • the suitable tissue source may be bone marrow, peripheral blood, cord blood, and the like, more preferably derived from bone marrow cells.
  • Cancer to which the present invention can be applied may also be referred to as a tumor, meaning a malignant neoplasm, and they are used interchangeably.
  • the type of cancer is not particularly limited, and melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, liver cancer, retinoblastoma, astrocytoma, glioblastoma, gingival cancer, tongue cancer, leukemia, neuroblastoma, head cancer, cervical cancer , Breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer, sarcoma, colon cancer, brain tumor, gastric cancer, esophageal cancer, lymphoma, fibrosarcoma, mast cell tumor And/or bladder cancer.
  • the cancer includes, for example, mammary gliomas, complex mammary cancer, mammary malignant mixed tumors, intraductal papillary adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, neuroepithelial tissue tumors such as glioma, ventricular tumor, nerve Cellular tumor, fetal type of extraneurodermal tumor, schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma, gastrointestinal lymphoma, gastrointestinal lymphoma, small to medium cell type lymphoma, cecal cancer, ascending colon cancer, descending Colon cancer, transverse colon cancer, sigmoid colon cancer, rectal cancer, ovarian epithelial cancer, germ cell tumor, stromal cell tumor, pancreatic duct cancer, invasive pancreatic duct cancer, adenocarcinoma of pancreatic cancer, acinar cell carcinoma,
  • cancer to which the present invention can be applied may include cancer that is classified as a solid cancer and/or causes metastasis of cancer cells, and preferably, melanoma, brain cancer, lung cancer, gastric cancer, liver cancer, head cancer, cervical cancer , Prostate cancer, pancreatic cancer, colon cancer, and lymphoma.
  • the exosome according to the present invention is obtained from cancer cells irradiated with radiation, and more specifically, the steps of irradiating the cancer cells with radiation; And it is obtained by a method of producing an exosome for producing mature dendritic cells, comprising the step of obtaining an exosome from irradiated cancer cells.
  • the step of irradiating the cancer cells with radiation may be performed, for example, by diluting the cancer cells in phosphate buffer saline (PBS) or the like and then irradiating radiation.
  • PBS phosphate buffer saline
  • the radiation may have a total dose of 30 Gy to 200 Gy, for example 50 Gy to 150 Gy, preferably 80 Gy to 120 Gy, more preferably 100 Gy.
  • the dose is less than 30 Gy, immunogenicity tends to be low, and when the dose exceeds 200 Gy, the increase rate of immunogenicity is not large compared to the increasing radiation energy.
  • cells irradiated with a dose within the scope of the present invention are co-cultured on dendritic cells, that is, when loading on dendritic cells, maturation of dendritic cells can be effectively induced.
  • an exosome for preparing mature dendritic cells, obtained from irradiated cancer cells, and use of exosomes obtained from irradiated cancer cells, for preparing a dendritic cell therapeutic agent for cancer treatment is provided.
  • the exosome obtained according to the present invention has no cytotoxicity as can be seen in FIG. 2, and induces remarkably improved T cell activity when based on the same amount compared to dendritic cells obtained from cancer cells not irradiated with radiation. can do.
  • the exosomes of the present invention may be used for preparing mature dendritic cells and/or for preparing dendritic cell therapeutic agents for cancer treatment, wherein the mature dendritic cells may be dendritic cell therapeutic agents for cancer treatment.
  • the method of manufacturing a dendritic cell therapy agent of the present invention comprises the steps of irradiating cancer cells with radiation; Obtaining exosomes from irradiated cancer cells; And loading the exosomes on dendritic cells.
  • the steps of irradiating cancer cells and obtaining exosomes from the irradiated cancer cells are as described above, wherein the radiation may be gamma rays, electron rays, ultraviolet rays, or X-rays, preferably gamma rays.
  • the step of obtaining exosomes from the irradiated cancer cells may be performed by centrifugation, but is not particularly limited, and may be obtained by any method known in the art.
  • the irradiated cancer cells are cultured for 24 to 48 hours, then centrifuged at least once at 2000 g to 10000 g, and the supernatant after centrifugation is filtered with, for example, 0.1 to 0.5 ⁇ m filter, and then further at 100 000 g. It can be obtained from pellets obtained by centrifugation.
  • the step of loading the exosomes on the dendritic cells is a step of inducing differentiation of immature dendritic cells into mature dendritic cells by culturing the dendritic cells in a medium containing exosomes.
  • the cultivation may be performed for 24 hours, for example, may be performed for 18 hours to 36 hours.
  • a pharmaceutical composition for cancer treatment such as a vaccine and an immunotherapeutic agent, comprising dendritic cells carrying exosomes obtained from irradiated cancer cells.
  • the term "vaccine” refers to a biological preparation containing an antigen that gives immunity to a living body, and refers to an immunogen or antigenic substance that induces immunity to a living body by injection or oral administration to a person or animal to prevent infection. .
  • the pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. It refers to any component suitable for delivering an antigenic substance to the in vivo site, for example, water, saline, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solution, Hans solution, and other aqueous physiological equilibrium solutions. , Oils, esters and glycols, but are not limited thereto.
  • the carrier for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like.
  • the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol.
  • Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • the carrier of the present invention may contain an auxiliary component suitable for enhancing chemical stability and isotonicity, and by including a stabilizer such as trehalose, glycine, sorbitol, lactose, or monosodium glutamate (MSG), It can protect the vaccine composition against.
  • the vaccine composition of the present invention may contain a suspension liquid such as sterile water or saline (preferably buffered saline).
  • the pharmaceutical composition of the present invention may contain any adjuvant in an amount sufficient to enhance the immune response to the immunogen.
  • Suitable adjuvants are described in Takahashi et al. (1990) Nature 344:873-875, for example, aluminum salts (aluminum phosphate or aluminum hydroxide, Squalene mixture (SAF-1), muramyl ) Peptides, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunit (cholera toxin B subunit), polyphosphazene and derivatives, and immuno-stimulating complexes (ISCOMs), but are not limited thereto.
  • the immunologically effective amount of an immunogen must be determined empirically, in which case factors that can be considered include the immunogenicity, route of administration, and number of immunizations administered. It can also be adjusted according to the patient's cancer progression and metastasis, the type of formulation, the patient's age, sex, weight, health status, diet, administration time, and administration method.
  • the antigenic substance is contained in a concentration necessary to induce formation of an appropriate level of antibody in vivo.
  • the pharmaceutical composition for cancer treatment may be an anti-tumor therapeutic vaccine formulated for intravenous administration, and in this case, the dendritic cells are 10 6 to 10 9 cells, for example, 10 7 to 10 in humans. It can be included in the amount of 8 cells. Also, in the case of a mouse, it may be included in an amount of 10 4 to 10 6 cells, for example, 10 5 to 10 6 cells.
  • the anti-tumor therapeutic vaccine may be administered at intervals of 1 to 28 days, for example, at intervals of 2 to 7 days, 2 or more times, for example, 2 to 10 times, preferably 3 times To 5 times, for example 3 times.
  • cells containing dendritic cells which are active ingredients of the pharmaceutical composition prepared according to the present invention, are inoculated into the human body as a therapeutic vaccine, it is also possible to eliminate cell proliferation in order to increase safety. For example, selectively, in order to use it more safely as a cell vaccine, it is treated with heat treatment, radiation treatment, or mitomycin C (MMC) treatment, and the proliferative property can be eliminated while leaving the function as a vaccine. have. For example, when using X-ray irradiation, it can be irradiated with a total radiation dose of 1000 to 3300 Rad.
  • MMC mitomycin C
  • mitomycin C treatment method for example, 25 to 50 ⁇ g/ml of mitomycin C may be added to dendritic cells, followed by heat retention at 37°C for 30 to 60 minutes.
  • heat treatment may be performed at 50°C to 65°C for 20 minutes.
  • the mature dendritic cells prepared according to the method of the present invention exhibit a cancer treatment effect, and thus may be provided as a pharmaceutical composition for preventing or treating cancer. Accordingly, according to the present invention, there is provided a pharmaceutical composition for cancer treatment comprising dendritic cells carrying exosomes obtained from irradiated cancer cells.
  • an immunotherapeutic agent comprising dendritic cells carrying exosomes obtained from irradiated cancer cells.
  • the mature dendritic cells that can be used in the present invention are not particularly limited, and may include both autologous or allogenic dendritic cells.
  • the vaccine, pharmaceutical composition or immunotherapy of the present invention may further include a pharmaceutically acceptable carrier.
  • the term "administration" means introducing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is to be administered through any general route as long as they can reach the target tissue.
  • the vaccine, pharmaceutical composition, or immunotherapy of the present invention can be administered to mammals including humans by any method.
  • it can be administered orally or parenterally, and parenteral administration methods are not limited thereto, but intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal , Intranasal, intestinal, topical, sublingual or rectal administration.
  • the vaccine for treating cancer of the present invention may be administered intravenously.
  • the pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration according to the route of administration as described above.
  • one or more buffers e.g., saline or PBS
  • antioxidants e.g., bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g., aluminum hydroxide). Side
  • suspending agents e.g., thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.
  • Solid preparations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions or capsules, and the like, and such solid preparations include at least one excipient in the pharmaceutical composition of the present invention.
  • starch including corn starch, wheat starch, rice starch, potato starch, etc.
  • calcium carbonate sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol
  • It may be prepared by mixing cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose or gelatin.
  • tablets or dragees can be obtained by blending the active ingredient with a solid excipient, pulverizing it, adding a suitable auxiliary, and processing into a granule mixture.
  • Liquid preparations for oral use include suspensions, liquid solutions, emulsions, or syrups, but may include various excipients, such as wetting agents, sweetening agents, fragrances, or preservatives, in addition to water or liquid paraffin, which are simple diluents commonly used. .
  • cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and an anti-coagulant, a lubricant, a wetting agent, a fragrance, an emulsifier and a preservative may be additionally included.
  • parenterally When administered parenterally, it may be formulated according to a method known in the art in the form of an injection, transdermal administration, and nasal inhalation together with a suitable parenteral carrier. Injections must be sterile and protected from contamination by microorganisms such as bacteria and fungi.
  • suitable carriers for injections are, but are not limited to, water, ethanol, polyols, such as glycerol, propylene glycol, and liquid polyethylene glycols, mixtures thereof and/or solvents or dispersion media containing vegetable oils. have.
  • suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used.
  • PBS phosphate buffered saline
  • isotonic solutions such as 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used.
  • various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like may be additionally included.
  • the injection may further include an isotonic agent such as sugar or sodium chloride in most cases.
  • transdermal administration In the case of transdermal administration, ointments, creams, lotions, gels, external solutions, pasta, liniment, and air rolls are included.
  • transdermal administration' means that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin by topically applying the pharmaceutical composition to the skin.
  • pressurized packs using the mature dendritic cell culture of the present invention and a suitable propellant e.g. dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas
  • a suitable propellant e.g. dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas
  • the dosage unit can be determined by providing a valve that delivers a metered amount.
  • gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.
  • the vaccine, pharmaceutical composition or immunotherapeutic agent of the present invention may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, or methods using biological response modifiers.
  • treatment refers to a clinical procedure to change the natural process of an individual or cell to be treated, and may be performed to prevent clinical pathology. Desirable effects of treatment include suppression of the occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, improvement of the disease state, improvement, alleviation or improved prognosis, etc. Include. In addition, the term'prevention' means any action that suppresses the onset or delays the progression of a disease.
  • the dendritic cells be effectively activated, but also cancer cell-specific immunity can be effectively induced, and thus remarkably improved anti-cancer immunity can be induced.
  • melanoma cell line B16-BL6 Using melanoma cell line B16-BL6, 1.8 ⁇ 10 6 cells cultured in DMEM (10% FBS, 100 U/ml penicillin/streptomycin) were diluted in PBS (2 mL) and then gamma irradiated (Gammacell). 220 60Cogammairradiator), after exposure to 100 Gy of gamma rays, and cultured for 24 hours under 10 mL of serum free culture medium in a 100 mm culture plate.
  • DMEM 50% FBS, 100 U/ml penicillin/streptomycin
  • non-gamma-irradiated melanoma control
  • 1.8 ⁇ 10 6 cell cells were cultured in a 100 mm culture plate in 10 mL of serum-free culture medium for 24 hours.
  • Example 1 After 24 hours of culturing according to Example 1 and Comparative Example 1, all the cell culture media were collected, centrifuged at ⁇ 2,000 g for 20 minutes, and then the supernatant was separated again and centrifuged at ⁇ 10,000 g for 45 minutes. The supernatant obtained after centrifugation was filtered using a 0.2 ⁇ m filter, and then centrifuged at ⁇ 100,000g for 150 minutes. After centrifugation, the supernatant was discarded and the pellets were washed with PBS (150 minutes at ⁇ 100,000 g).
  • Nanoparticle Tracking Analysis to determine the amount of exosomes of the thus obtained gamma-irradiated melanoma-inducing exosomes (hereinafter referred to as'Gamma-exo') and non-gamma-irradiated melanoma-inducing exosomes (hereinafter, also referred to as'Naive-exo').
  • NTA gamma-irradiated melanoma-inducing exosomes
  • 1(b) shows the results of analyzing exosomes through TEM.
  • the differentiation of mouse dendritic cells using bone marrow-derived cells is induced by differentiation through GM-CSF/IL-4 (dendritic cell growth factor).
  • GM-CSF/IL-4 dendritic cell growth factor
  • femoral bone marrow was collected from C57BL/6 mice using bone marrow collection injection, and the collected bone marrow was washed and red blood cells were removed using ammonium chloride.
  • the isolated cells were cultured for 8 days by adding RPMI 1640 (10% FBS, 100 U/ml penicillin/streptomycin, 20 ng/ml GM-CSF, 0.5 ng/ml IL-4) in a 6-well plate.
  • BMDC dendritic cells
  • PI propidium iodide
  • dendritic cells During the activation of dendritic cells, CD80, CD86, MHC-I and MHC-II, which are expressed on the surface, play an important role in the interaction with T cells and induce T cell activity. Based on this, the dendritic cells were treated with Gamma-exo of Example 1 and Naive-exo of Comparative Example 1 at concentrations of 5 and 25 ⁇ g for 24 hours. 100 ng/ml of LPS was used as a positive control for the maturation of dendritic cells.
  • the most important feature of mature dendritic cells in vivo is to induce T lymphocyte activity.
  • An experiment was performed to confirm whether the dendritic cells treated with Gamma-exo of Example 1 can enhance cytotoxic T lymphocyte (CTL) response and induce differentiation into Th1-type cells.
  • CTL cytotoxic T lymphocyte
  • CD4 + and CD8 + T cells isolated from the spleen of BALB / C mice to perform the MLR (Allogenic mixed lymphocyte reaction) technique were derived from C57BL/6 of Example 1 Gamma-exo and Comparative Example 1 After incubation with Naive-exo-treated dendritic cells, the degree of proliferation of T cells was measured.
  • the Gamma-exo-treated dendritic cells of Example 1 effectively induced the maturation of naive CD4 and CD8 T cells, but derived from Naive-exo of Comparative Example 1. It was confirmed that the exosome-treated dendritic cells inhibited the maturation of T cells rather than the non-treated dendritic cells.
  • mice 5 ⁇ 10 5 melanoma cells were injected subcutaneously in mice (C57BL/6). Three days after the injection, gamma-ray and non-gamma-ray irradiated melanoma-derived exosome-treated dendritic cells were intravenously injected three times at three-day intervals (Fig. 5(a)). Immature dendritic cells were used as a negative control.

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Abstract

The present invention pertains to: an exosome derived from irradiated cancer cells; a pharmaceutical composition containing mature dendritic cells obtained using same; and a method for producing same. Provided are: a method for producing an exosome for preparing mature dendritic cells, the method comprising the steps of irradiating cancer cells with radiation, and obtaining an exosome from the irradiated cancer cells; an exosome for preparing mature dendritic cells, the exosome being obtained from irradiated cancer cells; a use of the exosome; and a method for producing a dendritic cell therapeutic agent, the method comprising the steps of irradiating cancer cells with radiation, obtaining an exosome from irradiated cancer cells, and loading the exosome onto dendritic cells; and a cancer treatment vaccine, a cancer treatment pharmaceutical composition, and an immunotherapeutic agent, which contain dendritic cells loaded with an exosome obtained from irradiated cancer cells.

Description

방사선 조사된 암세포 유래 엑소좀, 이를 이용하여 획득된 성숙 수지상 세포를 포함하는 암 치료용 약학 조성물 및 이의 제조방법Exosomes derived from cancer cells irradiated with radiation, pharmaceutical composition for cancer treatment including mature dendritic cells obtained by using the same, and method for preparing the same
본 발명은 방사선 조사된 암세포 유래 엑소좀, 이를 이용하여 획득된 성숙 수지상 세포를 포함하는 암 치료용 약학 조성물 및 이의 제조방법에 관한 것으로, 보다 상세하게는 방사선 조사된 암세포를 이용하여 면역원성이 개량된 엑소좀을 제조하고, 이를 수지상 세포(DC)에 노출시켜 생성한 수지상 세포 암 백신을 개발하여 종래 항암치료기법의 한계성 해결하기 위한 항암면역 치료기술에 관한 것이다.The present invention relates to a pharmaceutical composition for cancer treatment including exosomes derived from irradiated cancer cells, mature dendritic cells obtained by using the same, and a method for preparing the same, and more particularly, to improved immunogenicity using irradiated cancer cells The present invention relates to an anticancer immunotherapy technology for solving the limitations of conventional anticancer treatment techniques by developing a dendritic cell cancer vaccine produced by preparing an exosome and exposing it to dendritic cells (DC).
기존의 암 치료 방법으로는 외과적인 수술, 방사선 요법 및 항암제를 이용한 화학 요법 등으로 최대한 암세포를 제거하는 방법이 있으며, 이들은 비교적 광범위하게 암 치료에 사용할 수 있으나, 대부분의 고형암(solid tumor)에서는 암이 전이되지 않은 초기에 주로 효과가 있어 전이암 치료에 한계를 나타낸다. Existing cancer treatment methods include surgical surgery, radiation therapy, and chemotherapy using anticancer drugs to remove cancer cells as much as possible. These can be used in a relatively wide range of cancer treatments, but most solid tumors are cancerous. It is mainly effective in the early stage of non-metastatic metastasis, thus limiting the treatment of metastatic cancer.
또한, 방사선 요법이나 화학 요법의 경우 암세포 이외의 정상 세포에도 손상을 주어 부작용을 나타내는 문제가 있으므로, 이와 같은 방사선 요법과 화학요법의 대안으로 표적 항암제가 등장하였으나, 이와 같은 표적 항암제의 경우 특이적인 유전자 및/또는 단백질이 발현되는 암세포에만 효과가 있고 재발 및 내성이 발생하는 단점이 있다. In addition, since radiation therapy or chemotherapy damages normal cells other than cancer cells and causes side effects, target anticancer drugs have appeared as alternatives to radiation therapy and chemotherapy, but target anticancer drugs such as these have specific genes. And/or it is effective only in cancer cells in which the protein is expressed, and recurrence and resistance occur.
이러한 종래 암 치료법의 한계를 극복하기 위해 최근 항암 면역 치료 연구가 활발히 진행되고 있으며 최근 국내외적으로 임상에서의 사용 허가가 이루어지고 있다. 그러나 이러한 항암 면역 치료법은 제조 비용이 고가이며, 개인 별 복잡한 면역체계로 인한 어려움이 있다. 상세하게, 항암 면역 치료는 체내 면역세포의 특성들을 이용하여 암세포에 대한 면역 활성을 높이거나 암세포가 면역세포의 공격으로 회피하는 방법을 억제시킴으로써 암을 치료하는 방법으로 면역세포요법(Immune cell therapy), 면역관문억제제(Immune checkpoint inhibitors), 치료용 암 백신(Therapeutic cancer vaccines), 항체 치료제(Therapeutic antibodies) 등이 있다.In order to overcome the limitations of such conventional cancer treatments, research on anticancer immunotherapy has been actively conducted in recent years, and approval for clinical use has been made both domestically and overseas. However, these anticancer immunotherapy treatments are expensive to manufacture, and there are difficulties due to individual complex immune systems. Specifically, anticancer immunotherapy is a method of treating cancer by increasing the immune activity against cancer cells by using the characteristics of immune cells in the body or by suppressing the method of avoiding cancer cells by attacking immune cells.Immune cell therapy , Immune checkpoint inhibitors, therapeutic cancer vaccines, and Therapeutic antibodies.
암 백신(Therapeutic cancer vaccines)은 자생적으로 생긴 암 조직(intact tumour cell)이나 암세포주에서 유래한 암 용해물(tumor cell lysate)을 이용한 항암백신(cancer vaccine)과 암세포 유래 항원이나 암 용해물을 노출시켜 생성시킨 자가 유래 수지상 세포(autologous dendritic cell, DC)를 이용한 DC 암 백신(DC-based cancer vaccine)이 있다. 예를 들어 WO2016-168680 A1는 세포외 소포를 면역 세포에 전달함으로써 면역 세포를 준비하기 위한 방법을 개시하고 있다. 종래 수지상 세포 암 백신 생산을 위한 종양 용해물의 제조방법은 전체 종양을 단일 세포들로 분리시키고 강한 산으로 전 처리한 후 여러 번에 걸쳐 동결-해동과정을 거치거나 화학물질을 이용하여 제조하였으나, 항원 단백질의 변성의 가능성, 잔류 독성 화학 물질의 위험, 및 항원의 면역원성이 매우 낮다는 단점이 있다. Therapeutic cancer vaccines expose cancer cell-derived antigens or cancer lysates as well as anti-cancer vaccines using intact tumour cells or cancer cell lysate derived from cancer cell lines. There is a DC-based cancer vaccine that uses autologous dendritic cells (DC) produced by the method. For example, WO2016-168680 A1 discloses a method for preparing immune cells by delivering extracellular vesicles to immune cells. Conventional methods for preparing tumor lysates for the production of dendritic cell cancer vaccines have been prepared by separating the entire tumor into single cells, pre-treating with strong acid, and then undergoing a freeze-thaw process several times or using chemicals. The disadvantages are the possibility of denaturation of the antigenic protein, the risk of residual toxic chemicals, and the very low immunogenicity of the antigen.
따라서, 수지상 세포 암 백신의 복잡한 제조 단계의 보완, 암 항원성의 손실 극복 및 면역 억제 인자들의 제거를 통하여 최상의 항암면역을 유도할 수 있는 기술이 제공되는 경우 관련 분야에서 널리 적용될 수 있을 것으로 기대된다. Therefore, it is expected that it can be widely applied in related fields when a technique capable of inducing the best anticancer immunity through supplementation of the complex manufacturing steps of a dendritic cell cancer vaccine, overcoming the loss of cancer antigenicity, and removing immune suppressor factors is provided.
이에, 본 발명의 한 측면은 성숙 수지상 세포 제조용 엑소좀의 제조방법을 제공하는 것이다. Accordingly, one aspect of the present invention is to provide a method for preparing exosomes for preparing mature dendritic cells.
본 발명의 다른 측면은 수지상 세포의 성숙화능이 우수한 엑소좀 및 이의 용도를 제공하는 것이다.Another aspect of the present invention is to provide an exosome having excellent maturation ability of dendritic cells and uses thereof.
본 발명의 또 다른 측면에 의하면, T 세포의 강한 활성을 유도할 수 있는 수지상 세포치료제의 제조방법을 제공하는 것이다.According to another aspect of the present invention, it is to provide a method for producing a dendritic cell therapeutic agent capable of inducing a strong activity of T cells.
본 발명의 또 다른 측면에 의하면, T 세포의 강한 활성을 유도할 수 있는 수지상 세포를 포함하는 암 치료용 백신, 암 치료용 약학 조성물 및 면역치료제를 제공하는 것이다. According to another aspect of the present invention, it is to provide a vaccine for treating cancer, a pharmaceutical composition for treating cancer, and an immunotherapy agent comprising dendritic cells capable of inducing strong activity of T cells.
본 발명의 일 견지에 의하면, 암세포에 방사선을 조사하는 단계; 및 방사선 조사된 암세포로부터 엑소좀을 획득하는 단계를 포함하는, 성숙 수지상 세포 제조용 엑소좀의 제조방법이 제공된다.According to one aspect of the present invention, irradiating cancer cells with radiation; And there is provided a method for producing an exosome for preparing mature dendritic cells, comprising the step of obtaining an exosome from irradiated cancer cells.
본 발명의 다른 견지에 의하면, 방사선 조사된 암세포로부터 획득된, 성숙 수지상 세포 제조용 엑소좀이 제공된다.According to another aspect of the present invention, there is provided an exosome for preparing mature dendritic cells obtained from irradiated cancer cells.
본 발명의 다른 견지에 의하면, 방사선 조사된 암세포로부터 획득된 엑소좀의 암 치료용 수지상 세포치료제 제조를 위한 용도가 제공된다.According to another aspect of the present invention, there is provided a use of exosomes obtained from irradiated cancer cells for the manufacture of a dendritic cell therapy for cancer treatment.
본 발명의 또 다른 견지에 의하면, 암세포에 방사선을 조사하는 단계; 방사선 조사된 암세포로부터 엑소좀을 획득하는 단계; 및 상기 엑소좀을 수지상 세포에 담지(loading)하는 단계를 포함하는, 수지상 세포치료제의 제조방법이 제공된다.According to another aspect of the present invention, irradiating cancer cells with radiation; Obtaining exosomes from irradiated cancer cells; And there is provided a method for producing a dendritic cell therapy comprising the step of loading the exosomes on the dendritic cells.
본 발명의 또 다른 측면에 의하면, 방사선 조사된 암세포로부터 획득된 엑소좀이 담지된 수지상 세포를 포함하는, 암 치료용 백신, 암 치료용 약학 조성물 및 면역치료제가 제공된다.According to another aspect of the present invention, there is provided a vaccine for cancer treatment, a pharmaceutical composition for cancer treatment, and an immunotherapy agent, including dendritic cells carrying exosomes obtained from irradiated cancer cells.
수지상 세포는 가장 강력한 항원제시 세포로 알려져 있으며, 이를 이용한 치료법은 현재 대부분의 암 치료에 이용되고 있는 항암제, 외과적 수술, 방사선 조사 등과 함께 암 치료 분야에 새로운 트랜드로 자리 잡을 가능성이 높을 뿐 아니라 인체의 면역 기능을 활성화시킴으로서 다양한 암 치료 분야에도 적용할 수 있을 것으로 전망된다. 나아가, 본 발명에 의하면 수지상 세포를 효과적으로 활성화시킬 수 있을 뿐만 아니라 암세포 특이적 면역을 효과적으로 유도할 수 있어 현저하게 향상된 항암 면역을 유도할 수 있을 것으로 기대된다.Dendritic cells are known as the most powerful antigen-presenting cells, and treatments using them are highly likely to become a new trend in the field of cancer treatment along with anticancer drugs, surgical surgery, and irradiation, which are currently used in most cancer treatments. It is expected that it can be applied to various cancer treatment fields by activating the immune function of Furthermore, according to the present invention, not only can the dendritic cells be effectively activated, but also cancer cell-specific immunity can be effectively induced, and thus remarkably improved anti-cancer immunity can be induced.
도 1은 엑소좀 양을 확인하기 위하여 Nanoparticle Tracking Analysis (NTA)를 분석한 결과(도 1(a)) 및 TEM을 통하여 엑소좀을 분석한 결과(도 1(b))를 나타낸 것이다. Figure 1 shows the results of analyzing the Nanoparticle Tracking Analysis (NTA) in order to confirm the amount of exosomes (Fig. 1(a)) and the results of analyzing the exosomes through TEM (Fig. 1(b)).
도 2는 수지상 세포에 엑소좀을 처리한 후 아넥신(annexin) V와 프로피디움 요오드화물(Propidium Iodide, PI) 염색을 통해 세포독성 유무를 확인한 결과(도 2(a)) 및 CFSE로 염색된 엑소좀들의 수지상 세포 내 흡수(uptake) 능력을 확인한 결과(도 2(b))를 나타낸 것이다. Figure 2 is a result of confirming the presence or absence of cytotoxicity through annexin V and propidium iodide (PI) staining after treating dendritic cells with exosomes (Fig. 2(a)) and stained with CFSE The results of confirming the uptake ability of exosomes in dendritic cells (Fig. 2(b)) are shown.
도 3은 수지상 세포의 성숙에 대한 엑소좀의 영향을 나타낸 것으로, 도 3(a)은 수지상 세포의 CD80, CD86, MHC-I, MHC-II의 발현 여부를 유세포 분석기로 확인한 것이며, 도 3(b)는 그 결과를 막대 그래프로 도시한 것이다. 한편, 도 3(c)는 IL-12p70, 항-염증사이토카인 IL-10을 EILISA를 통하여 분석한 결과이며, 3(d) 이러한 현상을 세포 내 염색을 통하여 다시 한번 확인한 결과이다.Figure 3 shows the effect of exosomes on the maturation of dendritic cells, Figure 3 (a) is a flow cytometer confirming whether the expression of CD80, CD86, MHC-I, MHC-II of the dendritic cells, Figure 3 ( b) shows the results as a bar graph. Meanwhile, FIG. 3(c) is a result of analyzing IL-12p70, an anti-inflammatory cytokine IL-10 through EILISA, and 3(d) is a result of confirming this phenomenon once again through intracellular staining.
도 4(a) 및 (b)는 엑소좀을 처리한 수지상 세포가 T 세포의 성숙을 유도하는 효과를 나타낸 것이며, 도 4(c)는 ELISA 키트를 적용하여 이들 T세포에서 분비하는 사이토카인(IFN-g, IL-2, IL-4) 분비량을 분석한 결과이다. Figures 4 (a) and (b) show the effect of inducing maturation of T cells by dendritic cells treated with exosomes, and Figure 4 (c) is a cytokine secreted from these T cells by applying an ELISA kit ( IFN-g, IL-2, IL-4) is the result of analysis of the secretion.
도 5(a)는 감마선 조사 흑색종 유래 엑소좀을 처리한 수지상 세포들의 예시적인 투여요법을 나타낸 것이고, 도 5(b)는 수지상 세포를 백신으로 사용하였을 때 암의 성장이 늦춰지는 경향을 그래프로 나타낸 것이다.Figure 5(a) shows an exemplary dosing regimen of dendritic cells treated with gamma-irradiated melanoma-derived exosomes, and Figure 5(b) is a graph showing the tendency of slowing cancer growth when dendritic cells are used as a vaccine. It is represented by
이하, 첨부된 도면을 참조하여 본 발명의 바람직한 실시 형태를 설명한다. 그러나, 본 발명의 실시 형태는 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 이하 설명하는 실시 형태로 한정되는 것은 아니다. Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.
본 발명은 방사선 조사된 암세포를 이용하여 면역원성이 현저히 향상된 엑소좀을 획득하고, 이를 수지상 세포(DC)에 노출시킨 성숙 수지상 세포 암 백신을 개발하여 종래 항암 치료의 한계를 해결하기 위한 항암 면역 치료기술에 관한 것이다. The present invention obtains exosomes with significantly improved immunogenicity using radiation-irradiated cancer cells, and develops a mature dendritic cell cancer vaccine in which it is exposed to dendritic cells (DC) to solve the limitations of conventional anticancer treatments. It's about technology.
본 발명에서 사용된 용어, "엑소좀(exosome)"이란 여러 종류의 세포들로부터 분비되는 막 구조의 소낭체로, 다른 세포 및 조직에 결합하여 막 구성요소, 단백질, RNA를 전달하는 등 다양한 역할을 하는 것으로 알려져 있다.The term "exosome" used in the present invention is a membrane-structured vesicle secreted from various types of cells, and it binds to other cells and tissues to transfer membrane components, proteins, RNA, etc. It is known to do.
본 발명의 미성숙 수지상 세포는 DC 전구 세포를 함유하는 적합한 조직 공급원으로부터 DC 전구 세포를 분리하거나 또는 미성숙 DC를 제조하는 방법에 의해 취득할 수 있다. 미성숙 DC를 제조하는 방법이란, 전구세포를 시험관내에서 분화시켜 미성숙 DC를 생산하는 방법을 말하며, 상기 전구세포는 적합한 조직공급원으로부터 유래한 혈액 단일핵 세포(mononuclear cell) 또는 조혈모세포일 수 있으며, 상기 적합한 조직 공급원이란 골수, 말초 혈액 및 제대혈 등일 수 있으며, 보다 바람직하게는 골수 세포로부터 유래된 것이다.The immature dendritic cells of the present invention can be obtained by isolating DC progenitor cells from a suitable tissue source containing DC progenitor cells or by a method of preparing immature DCs. The method of preparing immature DC refers to a method of producing immature DC by differentiating progenitor cells in vitro, and the progenitor cells may be blood mononuclear cells or hematopoietic stem cells derived from a suitable tissue source, The suitable tissue source may be bone marrow, peripheral blood, cord blood, and the like, more preferably derived from bone marrow cells.
본 발명이 적용될 수 있는 암은 종양이라고도 지칭될 수 있으며, 악성 신생물을 의미하고, 이들은 상호호환적으로 사용된다. 상기 암은 그 종류가 특히 제한되는 것은 아니며, 흑색종, 비소세포 폐암, 소세포 폐암, 폐암, 간암, 망막모세포종, 성상아교세포종, 아교모세포종, 치은암, 설암, 백혈병, 신경모세포종, 두부암, 경부암, 유방암, 췌장암, 전립선암, 신장암, 골암, 고환암, 난소암, 중피종, 자궁경부암, 위장관암, 림프종, 뇌암, 결장암, 육종, 대장암, 뇌종양, 위암, 식도암, 림프종, 섬유육종, 비만세포종 및/또는 방광암을 포함할 수 있다.Cancer to which the present invention can be applied may also be referred to as a tumor, meaning a malignant neoplasm, and they are used interchangeably. The type of cancer is not particularly limited, and melanoma, non-small cell lung cancer, small cell lung cancer, lung cancer, liver cancer, retinoblastoma, astrocytoma, glioblastoma, gingival cancer, tongue cancer, leukemia, neuroblastoma, head cancer, cervical cancer , Breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, ovarian cancer, mesothelioma, cervical cancer, gastrointestinal cancer, lymphoma, brain cancer, colon cancer, sarcoma, colon cancer, brain tumor, gastric cancer, esophageal cancer, lymphoma, fibrosarcoma, mast cell tumor And/or bladder cancer.
나아가, 상기 암에는 예를 들면 유선암, 복합형 유선암, 유선 악성 혼합 종양, 유관내 유두상선암, 폐선암, 편평상피암, 소세포암, 대세포암, 신경상피 조직성 종양인 신경아교종, 뇌실상의종, 신경세포성 종양, 태아형의 신경외 배엽성 종양, 신경초종, 신경섬유종, 수막종, 만성형 림프구성 백혈병, 림프종, 소화관형 림프종, 소화기형 림프종, 소~중 세포형 림프종, 맹장암, 상행 결장암, 하행 결장암, 횡행 결장암, S상 결장암, 직장암, 난소상피암, 배세포종양, 간질세포종양, 췌장관암, 침윤성 췌장관암, 췌장암의 선암, 선방세포암, 선편평상피암, 거세포종, 췌장관내 유두점액성 종양, 점액성 낭포선암, 췌장모세포종, 장액성 낭포선암, 고체 유두상암, 가스트린종, 글루카콘종, 인슐린종, 다발성 내분비선종증1(Wermer 증후군), 비기능성 도세포종, 소마토스타틴종, VIP 산생 종양이 포함되지만 이들에 한정되지 않는다.Further, the cancer includes, for example, mammary gliomas, complex mammary cancer, mammary malignant mixed tumors, intraductal papillary adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, neuroepithelial tissue tumors such as glioma, ventricular tumor, nerve Cellular tumor, fetal type of extraneurodermal tumor, schwannoma, neurofibroma, meningioma, chronic lymphocytic leukemia, lymphoma, gastrointestinal lymphoma, gastrointestinal lymphoma, small to medium cell type lymphoma, cecal cancer, ascending colon cancer, descending Colon cancer, transverse colon cancer, sigmoid colon cancer, rectal cancer, ovarian epithelial cancer, germ cell tumor, stromal cell tumor, pancreatic duct cancer, invasive pancreatic duct cancer, adenocarcinoma of pancreatic cancer, acinar cell carcinoma, acinar squamous cell carcinoma, giant cell tumor, papillary mucinous tumor in the pancreatic duct , Myxoid cystic adenocarcinoma, pancreatoblastoma, serous cystic adenocarcinoma, solid papillary carcinoma, gastrinoma, glucaconoma, insulinoma, multiple endocrine adenoma 1 (Wermer syndrome), nonfunctional islet cell tumor, somatostatinoma, VIP-producing tumors. However, it is not limited to these.
특히, 본 발명이 적용될 수 있는 암은 고형암으로 분류되거나 및/또는 암세포의 전이를 유발하는 암을 포함할 수 있으며, 바람직하게는, 흑색종, 뇌암, 폐암, 위암, 간암, 두부암, 자궁경부암, 전립선암, 췌장암, 대장암, 림프종을 포함할 수 있다.In particular, cancer to which the present invention can be applied may include cancer that is classified as a solid cancer and/or causes metastasis of cancer cells, and preferably, melanoma, brain cancer, lung cancer, gastric cancer, liver cancer, head cancer, cervical cancer , Prostate cancer, pancreatic cancer, colon cancer, and lymphoma.
본 발명에 의한 엑소좀은 방사선을 조사한 암세포로부터 획득되는 것이며, 보다 상세하게는 암세포에 방사선을 조사하는 단계; 및 방사선 조사된 암세포로부터 엑소좀을 획득하는 단계를 포함하는, 성숙 수지상 세포 제조용 엑소좀의 제조방법에 의해 획득되는 것이다. The exosome according to the present invention is obtained from cancer cells irradiated with radiation, and more specifically, the steps of irradiating the cancer cells with radiation; And it is obtained by a method of producing an exosome for producing mature dendritic cells, comprising the step of obtaining an exosome from irradiated cancer cells.
상기 암세포에 방사선을 조사하는 단계는 예를 들어 암세포를 PBS(phosphate buffer saline) 등에 희석한 후 방사선을 조사하여 수행될 수 있다. The step of irradiating the cancer cells with radiation may be performed, for example, by diluting the cancer cells in phosphate buffer saline (PBS) or the like and then irradiating radiation.
상기 방사선은 총 선량이 30 Gy 내지 200Gy일 수 있으며, 예를 들어 50 Gy 내지 150Gy, 바람직하게는 80Gy 내지 120Gy, 더욱 바람직하게는 100Gy일 수 있다. 상기 선량이 30Gy 미만인 경우에는 면역원성(immunogenicity)이 낮은 경향이 있고, 200Gy를 초과하는 경우에는 증가하는 방사선 에너지에 비해 면역원성 증가율이 크지 않다. 반면 본 발명의 범위에 속하는 선량을 조사한 세포를 수지상세 포에 공배양(co-culture), 즉 수지상 세포에 담지(loading)하는 경우 수지상 세포의 성숙이 효과적으로 유도될 수 있다.The radiation may have a total dose of 30 Gy to 200 Gy, for example 50 Gy to 150 Gy, preferably 80 Gy to 120 Gy, more preferably 100 Gy. When the dose is less than 30 Gy, immunogenicity tends to be low, and when the dose exceeds 200 Gy, the increase rate of immunogenicity is not large compared to the increasing radiation energy. On the other hand, when cells irradiated with a dose within the scope of the present invention are co-cultured on dendritic cells, that is, when loading on dendritic cells, maturation of dendritic cells can be effectively induced.
이와 같이 본 발명에 의하면, 방사선 조사된 암세포로부터 획득된, 성숙 수지상 세포 제조용 엑소좀 및 방사선 조사된 암세포로부터 획득된 엑소좀의 암 치료용 수지상 세포치료제 제조를 위한 용도가 제공된다. 본 발명에 따라 획득되는 엑소좀은 도 2에서 확인할 수 있는 바와 같이 세포 독성이 없으며, 방사선이 조사되지 않은 암세포로부터 획득된 수지상 세포에 비하여 동일한 양을 기준으로 할 때 현저하게 향상된 T 세포 활성을 유도할 수 있다. As described above, according to the present invention, there is provided an exosome for preparing mature dendritic cells, obtained from irradiated cancer cells, and use of exosomes obtained from irradiated cancer cells, for preparing a dendritic cell therapeutic agent for cancer treatment. The exosome obtained according to the present invention has no cytotoxicity as can be seen in FIG. 2, and induces remarkably improved T cell activity when based on the same amount compared to dendritic cells obtained from cancer cells not irradiated with radiation. can do.
상기 본 발명의 엑소좀은 성숙 수지상 세포 제조 용도 및/또는 암 치료용 수지상 세포치료제 제조를 위한 용도로 사용될 수 있으며, 이때 상기 성숙 수지상 세포는 암 치료용 수지상 세포치료제일 수 있다.The exosomes of the present invention may be used for preparing mature dendritic cells and/or for preparing dendritic cell therapeutic agents for cancer treatment, wherein the mature dendritic cells may be dendritic cell therapeutic agents for cancer treatment.
본 발명에 의하면, 상술한 본 발명의 엑소좀을 이용한 수지상 세포치료제의 제조방법이 제공된다. 보다 상세하게, 본 발명의 수지상 세포치료제의 제조방법은 암세포에 방사선을 조사하는 단계; 방사선 조사된 암세포로부터 엑소좀을 획득하는 단계; 및 상기 엑소좀을 수지상 세포에 담지(loading)하는 단계를 포함하는 것이다. According to the present invention, there is provided a method for preparing a dendritic cell therapeutic agent using the exosomes of the present invention described above. More specifically, the method of manufacturing a dendritic cell therapy agent of the present invention comprises the steps of irradiating cancer cells with radiation; Obtaining exosomes from irradiated cancer cells; And loading the exosomes on dendritic cells.
암세포에 방사선을 조사하는 단계 및 방사선 조사된 암세포로부터 엑소좀을 획득하는 단계는 상술한 바와 같으며, 이때 상기 방사선은 감마선, 전자선, 자외선 또는 X-선일 수 있고, 바람직하게는 감마선인 것이다. The steps of irradiating cancer cells and obtaining exosomes from the irradiated cancer cells are as described above, wherein the radiation may be gamma rays, electron rays, ultraviolet rays, or X-rays, preferably gamma rays.
상기 방사선 조사된 암세포로부터 엑소좀을 획득하는 단계는 원심분리에 의해 수행될 수 있으나, 특히 제한되는 것은 아니며, 당해 기술 분야에 알려진 어떠한 방법에 의해서도 획득될 수 있다. 바람직하게는 방사선 조사된 암세포를 24 시간 내지 48 시간 배양한 후 2000g 내지 10000g에서 1회 이상 원심분리하고, 원심분리 후 상층액을 예를 들어 0.1 내지 0.5 μm 필터에 의해 필터링한 후 100000g에서 추가로 원심분리하여 획득된 펠렛으로부터 획득될 수 있다. The step of obtaining exosomes from the irradiated cancer cells may be performed by centrifugation, but is not particularly limited, and may be obtained by any method known in the art. Preferably, the irradiated cancer cells are cultured for 24 to 48 hours, then centrifuged at least once at 2000 g to 10000 g, and the supernatant after centrifugation is filtered with, for example, 0.1 to 0.5 μm filter, and then further at 100 000 g. It can be obtained from pellets obtained by centrifugation.
한편, 상기 엑소좀을 수지상 세포에 담지하는 단계는 엑소좀을 포함하는 배지에서 수지상 세포를 배양하여 미성숙 수지상 세포를 성숙 수지상 세포로 분화 유도하는 단계인 것이다. 이때, 상기 배양은 24 시간 동안 수행되는 것일 수 있으며, 예를 들어 18 시간 내지 36 시간 동안 수행되는 것일 수 있다.Meanwhile, the step of loading the exosomes on the dendritic cells is a step of inducing differentiation of immature dendritic cells into mature dendritic cells by culturing the dendritic cells in a medium containing exosomes. At this time, the cultivation may be performed for 24 hours, for example, may be performed for 18 hours to 36 hours.
나아가, 본 발명에 의하면, 방사선 조사된 암세포로부터 획득된 엑소좀이 담지된 수지상 세포를 포함하는, 암 치료용 약학 조성물, 예를 들어 백신 및 면역치료제가 제공된다.Further, according to the present invention, there is provided a pharmaceutical composition for cancer treatment, such as a vaccine and an immunotherapeutic agent, comprising dendritic cells carrying exosomes obtained from irradiated cancer cells.
본 발명에서 용어, "백신"은 생체에 면역을 주는 항원을 함유한 생물학적인 제제로서, 감염증의 예방을 위하여 사람이나 동물에 주사하거나 경구 투여함으로써 생체에 면역이 생기게 하는 면역원 또는 항원성 물질을 말한다. In the present invention, the term "vaccine" refers to a biological preparation containing an antigen that gives immunity to a living body, and refers to an immunogen or antigenic substance that induces immunity to a living body by injection or oral administration to a person or animal to prevent infection. .
본 발명의 약학 조성물에는 약제학적으로 허용되는 담체를 포함할 수 있다. 항원 물질을 생체 내 부위에 전달하는데 적합한 임의의 성분을 의미하며, 예를 들어, 물, 식염수, 인산염 완충 식염수, 링거 용액, 덱스트로스 용액, 혈청 함유 용액, 한스 용액, 기타 수용성의 생리학적 평형 용액, 오일, 에스테르 및 글리콜 등이 포함되나, 이에 한정되지 않는다. 예컨데, 경구 투여용 담체는 락토스, 전분, 셀룰로스 유도체, 마그네슘 스테아레이트, 스테아르산 등을 포함할 수 있다. 또한 비경구 투여용 담체는 물, 적합한 오일, 식염수, 수성 글루코스 및 글리콜 등을 포함할 수 있다. 또한, 안정화제 및 보존제를 추가로 포함할 수 있다. 적합한 안정화제로는 아황산수소나트륨, 아황산나트륨 또는 아스코르브산과 같은 항산화제가 있다. 적합한 보존제로는 벤즈알코늄 클로라이드, 메틸- 또는 프로필-파라벤 및 클로로부탄올이 있다. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. It refers to any component suitable for delivering an antigenic substance to the in vivo site, for example, water, saline, phosphate buffered saline, Ringer's solution, dextrose solution, serum-containing solution, Hans solution, and other aqueous physiological equilibrium solutions. , Oils, esters and glycols, but are not limited thereto. For example, the carrier for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. In addition, the carrier for parenteral administration may include water, suitable oil, saline, aqueous glucose and glycol. In addition, it may further comprise a stabilizer and a preservative. Suitable stabilizers include antioxidants such as sodium hydrogen sulfite, sodium sulfite or ascorbic acid. Suitable preservatives are benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
본 발명의 담체는 화학적 안정성 및 등장성을 증진시키기 위해 적합한 보조 성분을 포함할 수 있으며, 트레할로스, 글라이신, 솔비톨, 락토오스 또는 모노소듐 글루타메이트(MSG)와 같은 안정화제를 포함시켜 온도 변화 또는 동결건조에 대해 백신 조성물을 보호할 수 있다. 본 발명의 백신 조성물은 멸균수 또는 식염수(바람직하게는 완충된 식염수)와 같은 현탁 액체를 포함할 수 있다.The carrier of the present invention may contain an auxiliary component suitable for enhancing chemical stability and isotonicity, and by including a stabilizer such as trehalose, glycine, sorbitol, lactose, or monosodium glutamate (MSG), It can protect the vaccine composition against. The vaccine composition of the present invention may contain a suspension liquid such as sterile water or saline (preferably buffered saline).
본 발명의 약학 조성물은 면역원에 대한 면역반응을 향상시키기에 충분한 양의 임의의 애쥬번트(adjuvant)를 함유할 수 있다. 적합한 애쥬번트는 문헌 Takahashi et al. (1990) Nature 344:873-875에 기술되어 있으며, 예컨대, 알루미늄염(알루미늄 포스페이트(Aluminium phosphate) 또는 알루미늄 히드록시드(Aluminium hydroxide), 스쿠알렌(Squalene) 혼합물(SAF-1), 무라밀(muramyl) 펩티드, 사포닌(Saponin) 유도체, 마이코박테리아(mycobacterium) 세포벽 제조물, 모노포스포릴(monophosphoryl) 지질 A, 미콜산(mycolic acid) 유도체, 비이온성 블록 공중합체 계면활성제, Quil A, 콜레라 독소 B 서브유닛(cholera toxin B subunit), 폴리포스파젠(polyphosphazene) 및 유도체, 및 면역자극 복합체(immun-stimulating complexes, ISCOMs)를 포함하나, 이에 한정되지는 않는다.The pharmaceutical composition of the present invention may contain any adjuvant in an amount sufficient to enhance the immune response to the immunogen. Suitable adjuvants are described in Takahashi et al. (1990) Nature 344:873-875, for example, aluminum salts (aluminum phosphate or aluminum hydroxide, Squalene mixture (SAF-1), muramyl ) Peptides, saponin derivatives, mycobacterium cell wall preparations, monophosphoryl lipid A, mycolic acid derivatives, nonionic block copolymer surfactants, Quil A, cholera toxin B subunit (cholera toxin B subunit), polyphosphazene and derivatives, and immuno-stimulating complexes (ISCOMs), but are not limited thereto.
다른 모든 약학 조성물과 마찬가지로, 면역원의 면역학적 유효량은 경험적으로 결정되어야 하며, 이 경우 고려될 수 있는 인자는 면역원성, 투여 경로 및 투여되는 면역 투여 회수를 들 수 있다. 또한 환자의 암의 진행과 전이 상태, 제형의 종류, 환자의 연령, 성별, 체중, 건강 상태, 식이, 투여 시간 및 투여 방법에 따라 조절될 수 있다. 통상적으로, 상기 항원물질이 생체 내에서 적절한 수준의 항체 형성을 유도하기에 필요한 농도로 포함한다. As with all other pharmaceutical compositions, the immunologically effective amount of an immunogen must be determined empirically, in which case factors that can be considered include the immunogenicity, route of administration, and number of immunizations administered. It can also be adjusted according to the patient's cancer progression and metastasis, the type of formulation, the patient's age, sex, weight, health status, diet, administration time, and administration method. Typically, the antigenic substance is contained in a concentration necessary to induce formation of an appropriate level of antibody in vivo.
예를 들어 상기 암 치료용 약학 조성물은 정맥 내 투여용으로 제제화된 항종양 치료 백신일 수있으며, 이 경우에 있어서 상기 수지상 세포는 인간의 경우 10 6 내지 10 9 cell, 예를 들어 10 7 내지 10 8 cell의 양으로 포함될 수 있다. 또한 마우스의 경우 10 4 내지 10 6 cell, 예를 들어 10 5 내지 10 6 cell의 양으로 포함될 수 있다.For example, the pharmaceutical composition for cancer treatment may be an anti-tumor therapeutic vaccine formulated for intravenous administration, and in this case, the dendritic cells are 10 6 to 10 9 cells, for example, 10 7 to 10 in humans. It can be included in the amount of 8 cells. Also, in the case of a mouse, it may be included in an amount of 10 4 to 10 6 cells, for example, 10 5 to 10 6 cells.
한편, 상기 항종양 치료 백신은 1일 내지 28일 간격으로, 예를 들어 2일 내지 7일 간격으로, 2회 이상 투여되는 것일 수 있으며, 예를 들어 2회 내지 10회, 바람직하게는 3회 내지 5회, 예를 들어 3회 투여될 수 있다. On the other hand, the anti-tumor therapeutic vaccine may be administered at intervals of 1 to 28 days, for example, at intervals of 2 to 7 days, 2 or more times, for example, 2 to 10 times, preferably 3 times To 5 times, for example 3 times.
본 발명에 의해 제조되는 약학 조성물의 유효성분인 수지상 세포를 포함하는 세포는, 인간 체내에 치료용 백신으로서 접종하기 때문에, 안전성을 높이기 위해 세포 증식성을 없애 두는 것도 가능하다. 예를 들면, 선택적으로 세포 백신으로서 보다 안전하게 이용하기 위해, 가열처리, 방사선처리, 또는 마이토마이신 C(mitomycin C, MMC) 처리 등으로 처리하고, 백신으로서의 기능을 남긴 채, 증식성을 없앨 수 있다. 예를 들면, X선 조사를 이용하는 경우, 총방사선량 1000 내지 3300 Rad로 조사할 수 있다. 마이토마이신 C 처리법은, 예를 들면, 수지상세포에 25~50 ㎍/㎖의 마이토마이신 C를 첨가하여, 37℃, 30분 내지 60분간 보온처리할 수 있다. 열에 의한 세포처리방법은, 예를 들면, 50℃ 내지 65℃에서 20분간 가열처리를 행할 수 있다.Since cells containing dendritic cells, which are active ingredients of the pharmaceutical composition prepared according to the present invention, are inoculated into the human body as a therapeutic vaccine, it is also possible to eliminate cell proliferation in order to increase safety. For example, selectively, in order to use it more safely as a cell vaccine, it is treated with heat treatment, radiation treatment, or mitomycin C (MMC) treatment, and the proliferative property can be eliminated while leaving the function as a vaccine. have. For example, when using X-ray irradiation, it can be irradiated with a total radiation dose of 1000 to 3300 Rad. In the mitomycin C treatment method, for example, 25 to 50 µg/ml of mitomycin C may be added to dendritic cells, followed by heat retention at 37°C for 30 to 60 minutes. In the cell treatment method by heat, for example, heat treatment may be performed at 50°C to 65°C for 20 minutes.
이와 같이, 본 발명의 방법에 따라 제조된 성숙 수지상 세포는 암 치료 효과를 보이기 때문에, 암 예방 또는 치료용 약학적 조성물로 제공될 수 있다. 이에 본 발명에 의하면, 방사선 조사된 암세포로부터 획득된 엑소좀이 담지된 수지상 세포를 포함하는, 암 치료용 약학 조성물이 제공된다.As described above, the mature dendritic cells prepared according to the method of the present invention exhibit a cancer treatment effect, and thus may be provided as a pharmaceutical composition for preventing or treating cancer. Accordingly, according to the present invention, there is provided a pharmaceutical composition for cancer treatment comprising dendritic cells carrying exosomes obtained from irradiated cancer cells.
또한, 본 발명에 의하면, 방사선 조사된 암세포로부터 획득된 엑소좀이 담지된 수지상 세포를 포함하는 면역치료제가 제공된다.In addition, according to the present invention, there is provided an immunotherapeutic agent comprising dendritic cells carrying exosomes obtained from irradiated cancer cells.
본 발명에 사용될 수 있는 성숙 수지상 세포는 특별히 한정되는 것은 아니고, 자가 유래 (autologous) 또는 동종유래 (allogenic)의 수지상 세포를 모두 포함할 수 있다.The mature dendritic cells that can be used in the present invention are not particularly limited, and may include both autologous or allogenic dendritic cells.
본 발명의 백신, 약학적 조성물 또는 면역치료제는 약학적으로 허용 가능한 담체를 더 포함할 수 있다.The vaccine, pharmaceutical composition or immunotherapy of the present invention may further include a pharmaceutically acceptable carrier.
본 발명에서 용어, "투여"는 어떠한 적절한 방법으로 환자에 소정의 물질을 도입하는 것을 의미하며, 본 발명의 약학 조성물의 투여경로는 이들이 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 한편, 본 발명의 백신, 약학적 조성물 또는 면역치료제는 인간을 비롯한 포유동물에 어떠한 방법으로도 투여할 수 있다. 예를 들어, 경구 또는 비경구로 투여할 수 있으며, 비경구적인 투여방법으로는 이에 제한되는 것은 아니나, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장내 투여일 수 있다. 바람직하게, 본 발명의 암 치료용 백신은 정맥 내 투여될 수 있다.In the present invention, the term "administration" means introducing a predetermined substance to a patient by any suitable method, and the administration route of the pharmaceutical composition of the present invention is to be administered through any general route as long as they can reach the target tissue. I can. Meanwhile, the vaccine, pharmaceutical composition, or immunotherapy of the present invention can be administered to mammals including humans by any method. For example, it can be administered orally or parenterally, and parenteral administration methods are not limited thereto, but intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal , Intranasal, intestinal, topical, sublingual or rectal administration. Preferably, the vaccine for treating cancer of the present invention may be administered intravenously.
본 발명의 약학적 조성물은 상술한 바와 같은 투여 경로에 따라 경구 투여용 또는 비경구 투여용 제제로 제형화 할 수 있다. 제형화할 경우에는 하나 이상의 완충제(예를 들어, 식염수 또는 PBS), 항산화제, 정균제, 킬레이트화제(예를 들어, EDTA 또는 글루타치온), 충진제, 증량제, 결합제, 아쥬반트(예를 들어, 알루미늄 하이드록사이드), 현탁제, 농후제 습윤제, 붕해제 또는 계면활성제, 희석제 또는 부형제를 사용하여 조제될 수 있다.The pharmaceutical composition of the present invention can be formulated into a formulation for oral administration or parenteral administration according to the route of administration as described above. When formulated, one or more buffers (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g., aluminum hydroxide). Side), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.
경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 액제, 겔제, 시럽제, 슬러리제, 현탁액 또는 캡슐제 등이 포함되며, 이러한 고형제제는 본 발명의 약학적 조성물에 적어도 하나 이상의 부형제 예를 들면, 전분(옥수수 전분, 밀 전분, 쌀 전분, 감자 전분 등 포함), 칼슘카보네이트(calcium carbonate), 수크로스(sucrose), 락토오스(lactose), 덱스트로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨 말티톨, 셀룰로즈, 메틸 셀룰로즈, 나트륨 카르복시메틸셀룰로오즈 및 하이드록시프로필메틸-셀룰로즈 또는 젤라틴 등을 섞어 조제될 수 있다. 예컨대, 활성성분을 고체 부형제와 배합한 다음 이를 분쇄하고 적합한 보조제를 첨가한 후 과립 혼합물로 가공함으로써 정제 또는 당의정제를 수득할 수 있다. Solid preparations for oral administration include tablets, pills, powders, granules, liquids, gels, syrups, slurries, suspensions or capsules, and the like, and such solid preparations include at least one excipient in the pharmaceutical composition of the present invention. For example, starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, It may be prepared by mixing cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose or gelatin. For example, tablets or dragees can be obtained by blending the active ingredient with a solid excipient, pulverizing it, adding a suitable auxiliary, and processing into a granule mixture.
단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제 또는 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물 또는 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제 또는 보존제 등이 포함될 수 있다.In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, or syrups, but may include various excipients, such as wetting agents, sweetening agents, fragrances, or preservatives, in addition to water or liquid paraffin, which are simple diluents commonly used. .
또한, 경우에 따라 가교결합 폴리비닐피롤리돈, 한천, 알긴산 또는 나트륨 알기네이트 등을 붕해제로 첨가할 수 있으며, 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant, and an anti-coagulant, a lubricant, a wetting agent, a fragrance, an emulsifier and a preservative may be additionally included. .
비경구적으로 투여하는 경우 적합한 비경구용 담체와 함께 주사제, 경피 투여제 및 비강 흡입제의 형태로 당 업계에 공지된 방법에 따라 제형화될 수 있다. 주사제의 경우에는 반드시 멸균되어야 하며 박테리아 및 진균과 같은 미생물의 오염으로부터 보호되어야 한다. 주사제의 경우 적합한 담체의 예로는 이에 한정되지는 않으나, 물, 에탄올, 폴리올, 예를 들어, 글리세롤, 프로필렌 글리콜 및 액체 폴리에틸렌 글리콜 등, 이들의 혼합물 및/또는 식물유를 포함하는 용매 또는 분산매질일 수 있다. 보다 바람직하게는, 적합한 담체로는 행크스 용액, 링거 용액, 트리에탄올 아민이 함유된 PBS (phosphate buffered saline) 또는 주사용 멸균수, 10% 에탄올, 40% 프로필렌 글리콜 및 5% 덱스트로즈와 같은 등장 용액 등을 사용할 수 있다. 상기 주사제를 미생물 오염으로부터 보호하기 위해서는 파라벤, 클로로부탄올, 페놀, 소르빈산, 티메로살 등과 같은 다양한 항균제 및 항진균제를 추가로 포함할 수 있다. 또한, 상기 주사제는 대부분의 경우 당 또는 나트륨 클로라이드와 같은 등장화제를 추가로 포함할 수 있다.When administered parenterally, it may be formulated according to a method known in the art in the form of an injection, transdermal administration, and nasal inhalation together with a suitable parenteral carrier. Injections must be sterile and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections are, but are not limited to, water, ethanol, polyols, such as glycerol, propylene glycol, and liquid polyethylene glycols, mixtures thereof and/or solvents or dispersion media containing vegetable oils. have. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, isotonic solutions such as 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like may be additionally included. In addition, the injection may further include an isotonic agent such as sugar or sodium chloride in most cases.
경피 투여제의 경우 연고제, 크림제, 로션제, 겔제, 외용액제, 파스타제, 리니멘트제, 에어롤제 등의 형태가 포함된다. 상기에서 경피 투여'는 약학적 조성물을 국소적으로 피부에 도포하여 약학적 조성물에 함유된 유효한 양의 활성성분이 피부 내로 전달되는 것을 의미한다. In the case of transdermal administration, ointments, creams, lotions, gels, external solutions, pasta, liniment, and air rolls are included. In the above,'transdermal administration' means that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin by topically applying the pharmaceutical composition to the skin.
흡입 투여제의 경우, 본 발명의 성숙 수지상 세포 배양물과 적합한 추진제, 예를 들면, 디클로로플루오로메탄, 트리클로로플루오로메탄, 디클로로테트라플루오로에탄, 이산화탄소 또는 다른 적합한 기체를 사용하여, 가압 팩 또는 연무기로부터 에어로졸 스프레이 형태로 편리하게 전달할 수 있다. 가압 에어로졸의 경우, 투약 단위는 계량된 양을 전달하는 밸브를 제공하여 결정할 수 있다. 예를 들면, 흡입기 또는 취입기에 사용되는 젤라틴 캡슐 및 카트리지는 화합물, 및 락토즈 또는 전분과 같은 적합한 분말 기제의 분말 혼합물을 함유하도록 제형화 할 수 있다.For inhalation dosages, pressurized packs using the mature dendritic cell culture of the present invention and a suitable propellant, e.g. dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas Alternatively, it can be conveniently delivered from a nebulizer in the form of an aerosol spray. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.
본 발명의 백신, 약학적 조성물 또는 면역치료제는 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 또는 생물학적 반응조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The vaccine, pharmaceutical composition or immunotherapeutic agent of the present invention may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, or methods using biological response modifiers.
본 명세서에서 '치료'란 치료되는 개체 또는 세포의 자연적 과정을 변경시키려는 임상적 시술을 의미하며, 임상적 병리의 예방을 위해서도 수행될 수 있다. 치료의 바람직한 효과는 질병의 발생 또는 재발 억제, 증상의 완화, 질병의 임의의 직접 또는 간접적인 병리학적 결과의 감소, 질병 진행 속도의 감소, 질병 상태의 개선, 호전, 완화 또는 개선된 예후 등을 포함한다. 또한, 용어 '예방'은 질병의 발병을 억제시키거나 진행을 지연시키는 모든 행위를 의미한다.In the present specification, "treatment" refers to a clinical procedure to change the natural process of an individual or cell to be treated, and may be performed to prevent clinical pathology. Desirable effects of treatment include suppression of the occurrence or recurrence of the disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, improvement of the disease state, improvement, alleviation or improved prognosis, etc. Include. In addition, the term'prevention' means any action that suppresses the onset or delays the progression of a disease.
본 발명에 의하면 수지상 세포를 효과적으로 활성화 시킬 수 있을 뿐만 아니라 암세포 특이적 면역을 효과적으로 유도할 수 있어 현저하게 향상된 항암 면역을 유도할 수 있을 것으로 기대된다.According to the present invention, not only can the dendritic cells be effectively activated, but also cancer cell-specific immunity can be effectively induced, and thus remarkably improved anti-cancer immunity can be induced.
이하, 구체적인 실시예를 통해 본 발명을 보다 구체적으로 설명한다. 하기 실시예는 본 발명의 이해를 돕기 위한 예시에 불과하며, 본 발명의 범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through specific examples. The following examples are only examples to aid understanding of the present invention, and the scope of the present invention is not limited thereto.
실시예Example
1. 암세포에 대한 감마선 조사를 통하여 유도되는 1. Induced through gamma irradiation of cancer cells 엑소좀의Exosome 분리 및 분석 Separation and analysis
실시예Example 1 One
흑색종(melanoma cell line B16-BL6)을 이용하여 DMEM(10% FBS, 100 U/ml 페니실린/스트렙토마이신)으로 배양된 1.8×10 6 세포를 PBS (2 mL)에 희석한 후 감마선 조사기(Gammacell 220 60Cogammairradiator)를 통하여 100 Gy의 감마선에 노출 시킨 후 100 mm 배양 플레이트에서 10 mL의 무혈청 배양액(serum free) 배양 배지 조건 하에서 24 시간 배양하였다. Using melanoma cell line B16-BL6, 1.8×10 6 cells cultured in DMEM (10% FBS, 100 U/ml penicillin/streptomycin) were diluted in PBS (2 mL) and then gamma irradiated (Gammacell). 220 60Cogammairradiator), after exposure to 100 Gy of gamma rays, and cultured for 24 hours under 10 mL of serum free culture medium in a 100 mm culture plate.
비교예Comparative example 1 One
비감마선 조사 흑색종(control)의 경우는 1.8×10 6cell 세포를 100 mm 배양 플레이트에 10 mL의 무혈청 배양액 배양 배지 조건에서 24 시간 배양하였다. In the case of non-gamma-irradiated melanoma (control), 1.8×10 6 cell cells were cultured in a 100 mm culture plate in 10 mL of serum-free culture medium for 24 hours.
실험예Experimental example 1: One: 엑소좀의Exosome 분리 및 분석 Separation and analysis
실시예 1 및 비교예 1 각각에 따른 배양 24 시간 후 세포 배양 배지들을 모두 수거 후 ×2,000g에서 20분간 원심분리 후 상층액을 다시 분리하여 ×10,000g에서 45분간 원심분리를 진행하였다. 원심 분리 후 얻어진 상층액을 0.2 μm 필터를 이용하여 필터 후 ×100,000g에서 150분간 원심분리를 진행하였다. 원심분리 후 상층액을 버리고 PBS로 펠렛(pellet)들을 세척(×100,000g에서 150분간) 하였다. After 24 hours of culturing according to Example 1 and Comparative Example 1, all the cell culture media were collected, centrifuged at × 2,000 g for 20 minutes, and then the supernatant was separated again and centrifuged at × 10,000 g for 45 minutes. The supernatant obtained after centrifugation was filtered using a 0.2 μm filter, and then centrifuged at ×100,000g for 150 minutes. After centrifugation, the supernatant was discarded and the pellets were washed with PBS (150 minutes at × 100,000 g).
이렇게 얻어진 감마선 조사 흑색종 유도 엑소좀(이하 'Gamma-exo'라고도 지칭함) 및 비감마선 조사 흑색종 유도 엑소좀(이하 'Naive-exo'라고도 지칭함)들의 엑소좀 양을 확인하기 위하여 Nanoparticle Tracking Analysis (NTA)를 분석한 결과 흑도 1(a)에서 확인할 수 있는 바와 같이 흑색종의 감마선 조사를 통해 엑소좀의 양이 거의 2배에 이르도록 현저하게 높아지는 것으로 확인되었다. 도 1(b)는 TEM을 통하여 엑소좀을 분석한 결과를 나타낸 것이다. Nanoparticle Tracking Analysis (Nanoparticle Tracking Analysis) to determine the amount of exosomes of the thus obtained gamma-irradiated melanoma-inducing exosomes (hereinafter referred to as'Gamma-exo') and non-gamma-irradiated melanoma-inducing exosomes (hereinafter, also referred to as'Naive-exo'). As a result of analyzing NTA), it was confirmed that the amount of exosomes was remarkably increased to almost double through gamma irradiation of melanoma, as can be seen in blackness 1(a). 1(b) shows the results of analyzing exosomes through TEM.
2. 감마선 조사 흑색종 유래 2. Derived from gamma irradiation melanoma 엑소좀의Exosome 수지상세포Dendritic cells 처리 시 유도되는 세포 독성 및 흡수(uptake)능 확인 Confirmation of cytotoxicity and uptake ability induced during treatment
골수유래 세포를 이용한 마우스의 수지상 세포의 분화 방법은 GM-CSF/IL-4 (수지상세포 성장 인자)를 통하여 분화 유도된다. 수지상 세포의 분화는 C57BL/6 마우스로부터 골수 채취용 주사를 이용해 대퇴부 골수를 채취하였고, 채취한 골수를 세척한 후 적혈구를 염화암모늄을 이용하여 제거하였다. 분리한 세포를 6-웰 플레이트에서 RPMI 1640 (10% FBS, 100 U/ml 페니실린/스트렙토마이신, 20 ng/ml GM-CSF, 0.5 ng/ml IL-4)를 첨가하여 8일 동안 배양하였다. The differentiation of mouse dendritic cells using bone marrow-derived cells is induced by differentiation through GM-CSF/IL-4 (dendritic cell growth factor). For differentiation of dendritic cells, femoral bone marrow was collected from C57BL/6 mice using bone marrow collection injection, and the collected bone marrow was washed and red blood cells were removed using ammonium chloride. The isolated cells were cultured for 8 days by adding RPMI 1640 (10% FBS, 100 U/ml penicillin/streptomycin, 20 ng/ml GM-CSF, 0.5 ng/ml IL-4) in a 6-well plate.
배양 후 수지상세포(BMDC)에 실시예 1의 Gamma-exo 및 비교예 1의 Naive-exo를 각각 5 및 25 μg의 농도로 24 시간 동안 처리하였다. 처리 후 아넥신(annexin) V와 프로피디움 요오드화물(Propidium Iodide, PI) 염색 후 세포독성 유무를 확인하였다. 그 결과 도 2(a)에서 확인할 수 있는 바와 같이 모든 그룹들에서 독성이 유도되지 않는 것이 확인되었다. After incubation, dendritic cells (BMDC) were treated with Gamma-exo of Example 1 and Naive-exo of Comparative Example 1 at concentrations of 5 and 25 μg, respectively, for 24 hours. After treatment, annexin V and propidium iodide (PI) were stained, and then cytotoxicity was confirmed. As a result, it was confirmed that toxicity was not induced in all groups, as can be seen in FIG. 2(a).
추가 적으로 CFSE로 염색된 엑소좀들의 수지상세포 내 흡수(uptake) 능력을 확인하고 그 결과를 도 2(b)에 나타내었다. Additionally, the ability of exosomes stained with CFSE to uptake in dendritic cells was confirmed, and the results are shown in FIG. 2(b).
3. 감마선 조사 흑색종 유래 3. Derived from gamma irradiation melanoma 엑소좀Exosomes 및 And 비감마선Non-gamma rays 조사 흑색종 유래 Investigation melanoma origin 엑소좀의Exosome 수지상 세포 Dendritic cells 성숙화능Maturity 확인 Confirm
수지상 세포의 활성 시 표면에서 발현되는 CD80, CD86, MHC-I, MHC-II는 T 세포와의 상호작용에서 중요한 역할을 하면서 T 세포의 활성을 유도한다. 이를 바탕으로 수지상 세포에 실시예 1의 Gamma-exo 및 비교예 1의 Naive-exo를 5 및 25 μg의 농도로 24 시간 동안 처리하였다. 100 ng/ml의 LPS는 수지상 세포의 성숙에 대한 양성 대조군(positive control)로서 사용하였다. 처리 24 시간 후 세포들을 anti-CD11c-PE-Cy7, anti-CD80-FITC, anti-CD86-FITC, anti-MHC-I-PE, anti-MHC-II-PE 항체를 이용하여 염색한 후 유세포분석기 FACSverse로 분석하였다. During the activation of dendritic cells, CD80, CD86, MHC-I and MHC-II, which are expressed on the surface, play an important role in the interaction with T cells and induce T cell activity. Based on this, the dendritic cells were treated with Gamma-exo of Example 1 and Naive-exo of Comparative Example 1 at concentrations of 5 and 25 μg for 24 hours. 100 ng/ml of LPS was used as a positive control for the maturation of dendritic cells. After 24 hours of treatment, cells were stained with anti-CD11c-PE-Cy7, anti-CD80-FITC, anti-CD86-FITC, anti-MHC-I-PE, anti-MHC-II-PE antibodies, and then flow cytometry Analyzed by FACSverse.
그 결과, 도 3(a)에서 확인할 수 있는 바와 같이 실시예 1의 Gamma-exo를 처리한 군에서 수지상 세포의 CD80, CD86, MHC-I, MHC-II의 발현이 강력하게 유도됨을 확인하였다. 도 3(b)에는 그 결과를 막대 그래프로 도시하였다. 추가적으로, 염증성 사이토카인 TNF-alpha와 T 세포의 Th1 편향화에 중요한 IL-12p70, 항-염증사이토카인 IL-10을 EILISA를 통하여 분석한 결과 도 3(c)에서 확인할 수 있는 바와 같이 실시예 1의 Gamma-exo 처리 군에서 TNF-alpha와 IL-12p70이 높게 유도됨을 확인하였다. 또한, 이러한 현상을 세포내(intracellular) 염색을 통하여 다시 한번 확인한 결과 도 3(d)에 나타난 바와 같이 ELISA 결과와 동일하게 유도됨을 확인하였다.As a result, it was confirmed that the expression of CD80, CD86, MHC-I, and MHC-II of dendritic cells was strongly induced in the group treated with Gamma-exo of Example 1, as can be seen in FIG. 3(a). The results are shown in a bar graph in FIG. 3(b). Additionally, as shown in FIG. 3(c), as a result of analyzing IL-12p70, which is important for Th1 biasing of T cells with TNF-alpha, an inflammatory cytokine, and IL-10, an anti-inflammatory cytokine, through EILISA, Example 1 It was confirmed that TNF-alpha and IL-12p70 were highly induced in the Gamma-exo treatment group. In addition, as a result of confirming this phenomenon once again through intracellular staining, it was confirmed that it was induced in the same manner as the ELISA result as shown in FIG. 3(d).
4.4. 감마선 조사 흑색종 유래 Derived from gamma-irradiated melanoma 엑소좀Exosomes 및 And 비감마선Non-gamma rays 조사 흑색종 유래 Investigation melanoma origin 엑소좀Exosomes 처리 수지상 세포가 T 세포의 증식, 활성, 분화에 미치는 영향 분석 Analysis of the effect of treated dendritic cells on the proliferation, activity, and differentiation of T cells
성숙 수지상 세포의 생체 내에서의 가장 중요한 특징은 T 림프구의 활성을 유도하는 것이다. 실시예 1의 Gamma-exo가 처리된 수지상 세포가 세포독성 T 림프구 (Cytotoxic T Lymphocyte, CTL) 반응 강화와 Th1형 세포로의 분화를 유도할 수 있는지 유무를 확인하기 위하여 실험을 수행하였다. 상세하게, MLR(Allogenic mixed lymphocyte reaction) 기법을 수행하기 위하여 BALB/C 마우스의 비장으로부터 분리된 CD4 + 및 CD8 + T세포를 C57BL/6로부터 유래된 실시예 1의 Gamma-exo 및 비교예 1의 Naive-exo 처리 수지상 세포와 함께 배양한 후 T 세포의 증식 정도를 측정하였다. The most important feature of mature dendritic cells in vivo is to induce T lymphocyte activity. An experiment was performed to confirm whether the dendritic cells treated with Gamma-exo of Example 1 can enhance cytotoxic T lymphocyte (CTL) response and induce differentiation into Th1-type cells. In detail, CD4 + and CD8 + T cells isolated from the spleen of BALB / C mice to perform the MLR (Allogenic mixed lymphocyte reaction) technique were derived from C57BL/6 of Example 1 Gamma-exo and Comparative Example 1 After incubation with Naive-exo-treated dendritic cells, the degree of proliferation of T cells was measured.
그 결과, 도 4(a) 및 (b)에서 확인할 수 있는 바와 같이 실시예 1의 Gamma-exo 처리 수지상 세포는 naive CD4 및 CD8 T 세포의 성숙을 효과적으로 유도하였지만, 비교예 1의 Naive-exo 유래 엑소좀 처리 수지상 세포는 비-처리된 수지상 세포들 보다 T 세포의 성숙이 오히려 억제되는 것을 확인할 수 있었다. As a result, as can be seen in Figures 4 (a) and (b), the Gamma-exo-treated dendritic cells of Example 1 effectively induced the maturation of naive CD4 and CD8 T cells, but derived from Naive-exo of Comparative Example 1. It was confirmed that the exosome-treated dendritic cells inhibited the maturation of T cells rather than the non-treated dendritic cells.
또한, ELISA 키트를 적용하여 T세포에서 분비하는 사이토카인(IFN-g, IL-2, IL-4) 분비량을 분석한 결과 도 4(c)에서 확인할 수 있는 바와 같이 실시예 1의 Gamma-exo 처리 수지상 세포는 Th1형 세포 및 CD8 T 세포의 활성을 유도할 수 있음이 확인되었다. 반면 비교예 1의 Naive-exo 처리 수지상 세포는 Th1 사이토카인 및 CD8 T 세포의 활성을 오히려 억제하는 것으로 확인되었다. In addition, as a result of analyzing the amount of secretion of cytokines (IFN-g, IL-2, IL-4) secreted from T cells by applying an ELISA kit, Gamma-exo of Example 1 as shown in FIG. 4(c). It was confirmed that the treated dendritic cells can induce the activities of Th1-type cells and CD8 T cells. On the other hand, it was confirmed that the Naive-exo-treated dendritic cells of Comparative Example 1 rather inhibited the activities of Th1 cytokines and CD8 T cells.
이러한 결과를 통하여 실시예 1의 Gamma-exo 처리 수지상세포는 Th1 및 CD8 T 세포의 활성을 강력하게 유도할 수 있음을 확인할 수 있었다. Through these results, it was confirmed that the Gamma-exo-treated dendritic cells of Example 1 can strongly induce the activities of Th1 and CD8 T cells.
5.5. 흑색종 유도 마우스 모델에서 감마선 및 Gamma rays and 비감마선Non-gamma rays 조사 흑색종 유래 Investigation melanoma origin 엑소좀Exosomes 처리 수지상 세포의 치료 백신으로서의 효능 평가 Efficacy evaluation of treated dendritic cells as therapeutic vaccines
C57BL/6 유래 흑색종의 마우스 모델을 구축하기 위하여 5×10 5의 흑색종 세포들을 마우스(C57BL/6) 피하에 주사하였다. 주사 후 3일 뒤 감마선 및 비 감마선 조사 흑색종 유래 엑소좀 처리 수지상 세포들을 정맥으로 3일 간격으로 3번 주사 하였다(도 5(a)). 미성숙 수지상 세포를 음성 대조군(negative control)로 사용하였다. To construct a mouse model of C57BL/6-derived melanoma, 5×10 5 melanoma cells were injected subcutaneously in mice (C57BL/6). Three days after the injection, gamma-ray and non-gamma-ray irradiated melanoma-derived exosome-treated dendritic cells were intravenously injected three times at three-day intervals (Fig. 5(a)). Immature dendritic cells were used as a negative control.
그 결과, 도 5(b)에 나타난 바와 같이 실시예 1의 Gamma-exo 처리 수지상 세포를 백신으로 사용하였을 때 비교예 1의 Naive-exo 처리 수지상 세포보다 암의 성장이 늦춰지는 것을 확인할 수 있었다. As a result, as shown in FIG. 5(b), when the Gamma-exo-treated dendritic cells of Example 1 were used as a vaccine, it was confirmed that the growth of cancer was slowed compared to the Naive-exo-treated dendritic cells of Comparative Example 1.
이상에서 본 발명의 실시예에 대하여 상세하게 설명하였지만 본 발명의 권리범위는 이에 한정되는 것은 아니고, 청구범위에 기재된 본 발명의 기술적 사상을 벗어나지 않는 범위 내에서 다양한 수정 및 변형이 가능하다는 것은 당 기술분야의 통상의 지식을 가진 자에게는 자명할 것이다.Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible without departing from the technical spirit of the present invention described in the claims. It will be obvious to those of ordinary skill in the field.

Claims (13)

  1. 암세포에 방사선을 조사하는 단계; 및 Irradiating cancer cells with radiation; And
    방사선 조사된 암세포로부터 엑소좀을 획득하는 단계Obtaining exosomes from irradiated cancer cells
    를 포함하는, 성숙 수지상 세포 제조용 엑소좀의 제조방법.Containing, a method for producing an exosome for preparing mature dendritic cells.
  2. 제1항에 있어서, 상기 방사선은 총 선량이 30 Gy 내지 200Gy인, 성숙 수지상 세포 제조용 엑소좀의 제조방법.The method of claim 1, wherein the radiation has a total dose of 30 Gy to 200 Gy.
  3. 방사선 조사된 암세포로부터 획득된, 성숙 수지상 세포 제조용 엑소좀.Exosomes for preparing mature dendritic cells obtained from irradiated cancer cells.
  4. 제3항에 있어서, 상기 성숙 수지상 세포는 암 치료용 수지상 세포치료제인, 성숙 수지상 세포 제조용 엑소좀.According to claim 3, wherein the mature dendritic cells is a dendritic cell therapy for cancer treatment, mature dendritic cell production exosomes.
  5. 방사선 조사된 암세포로부터 획득된 엑소좀의 암 치료용 수지상 세포치료제 제조를 위한 용도.Use of exosomes obtained from irradiated cancer cells for manufacturing dendritic cell therapy for cancer treatment.
  6. 암세포에 방사선을 조사하는 단계; Irradiating the cancer cells with radiation;
    방사선 조사된 암세포로부터 엑소좀을 획득하는 단계; 및Obtaining exosomes from irradiated cancer cells; And
    상기 엑소좀을 수지상 세포에 담지(loading)하는 단계를 포함하는, 수지상 세포치료제의 제조방법.A method for producing a dendritic cell therapy comprising the step of loading the exosomes on dendritic cells.
  7. 제6항에 있어서, 상기 방사선은 감마선, 전자선, 자외선 또는 X-선인, 수지상 세포치료제의 제조방법.The method of claim 6, wherein the radiation is gamma rays, electron rays, ultraviolet rays or X-rays.
  8. 제6항에 있어서, 상기 엑소좀을 수지상 세포에 담지하는 단계는 엑소좀을 포함하는 배지에서 수지상 세포를 배양하여 미성숙 수지상 세포를 성숙 수지상 세포로 분화 유도하는 단계인, 수지상 세포치료제의 제조방법.The method of claim 6, wherein the step of supporting the exosomes on the dendritic cells is the step of culturing dendritic cells in a medium containing exosomes to induce differentiation of immature dendritic cells into mature dendritic cells.
  9. 방사선 조사된 암세포로부터 획득된 엑소좀이 담지된 수지상 세포를 포함하는, 암 치료용 약학 조성물.A pharmaceutical composition for cancer treatment, comprising dendritic cells carrying exosomes obtained from irradiated cancer cells.
  10. 제9항에 있어서, 상기 암 치료용 약학 조성물은 정맥 내 투여용으로 제제화된, 암 치료용 약학 조성물.The pharmaceutical composition for cancer treatment according to claim 9, wherein the pharmaceutical composition for cancer treatment is formulated for intravenous administration.
  11. 제9항에 있어서, 상기 수지상 세포는 10 6 내지 10 9 cell 양으로 포함된, 암 치료용 약학 조성물.The pharmaceutical composition for cancer treatment according to claim 9, wherein the dendritic cells are contained in an amount of 10 6 to 10 9 cells.
  12. 제9항에 있어서, 상기 암 치료용 약학 조성물은 1일 내지 28일 간격으로, 2회 이상 투여되는, 암 치료용 약학 조성물.The pharmaceutical composition for cancer treatment according to claim 9, wherein the pharmaceutical composition for cancer treatment is administered two or more times at intervals of 1 to 28 days.
  13. 제9항에 있어서, 상기 약학 조성물은 암 치료용 백신 또는 면역치료제인, 암 치료용 약학 조성물.The pharmaceutical composition for cancer treatment according to claim 9, wherein the pharmaceutical composition is a vaccine or immunotherapy agent for cancer treatment.
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