KR20210016995A - Exosome derived from radiated cancer cell, cancer vaccine comprising dendritic cell prepared by using the same and preparing method thereof - Google Patents

Abstract

The present invention relates to: an exosome derived from irradiated cancer cells; a cancer treatment vaccine comprising mature dendritic cells prepared by using the same; and a preparing method thereof. Provided are: a method for producing an exosome for preparing mature dendritic cells, wherein the method comprises the steps of irradiating cancer cells with radiation, and obtaining an exosome from the irradiated cancer cells; an exosome for preparing mature dendritic cells, which is obtained from irradiated cancer cells; a method for producing a dendritic cell therapeutic agent, which comprises 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

Exosome derived from radiated cancer cell, cancer vaccine comprising dendritic cell prepared by using the same and preparing method thereof, including exosomes derived from irradiated cancer cells, mature dendritic cells obtained using the same }

The present invention relates to a vaccine for cancer treatment including exosomes derived from irradiated cancer cells, mature dendritic cells obtained by using the same, and to a method for manufacturing the same, and more particularly, to a method for producing the same, immunogenicity using irradiated cancer cells It relates to an anticancer immunotherapy technology to solve the limitations of conventional anticancer treatment techniques by developing a dendritic cell cancer vaccine produced by preparing an improved exosome and exposing it to dendritic cells (DC).

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.

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.

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 are difficult 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.

Therapeutic cancer vaccines are anticancer vaccines using intact tumour cells or cancer cell lysate derived from cancer cell lines, and antigens or cancer lysates derived from cancer cells. There is a DC-based cancer vaccine using autologous dendritic cells (DC) produced by exposure. For example, WO2016-168680 A1 discloses a method for preparing immune cells by delivering extracellular vesicles to immune cells. In the conventional method for producing a tumor lysate for dendritic cell cancer vaccine production, the entire tumor was separated into single cells, pretreated with a strong acid, and then freeze-thawed several times or prepared 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.

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.

Accordingly, 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 ability to mature dendritic cells.

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.

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.

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.

According to another aspect of the present invention, there is provided an exosome for preparing mature dendritic cells obtained from irradiated cancer cells.

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.

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.

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 activated effectively, but also cancer cell-specific immunity can be effectively induced, and thus remarkably improved anticancer 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)).
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.
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.
Figures 4 (a) and (b) show the effect of inducing the maturation of T cells of 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 analyzing the secretion amount.
5(a) shows an exemplary dosing regimen of exosome-treated dendritic cells derived from gamma-ray irradiation melanoma, and FIG. 5(b) is a graph showing the tendency of slowing cancer growth when dendritic cells are used as a vaccine. will be.

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.

The present invention obtains exosomes with remarkably improved immunogenicity using radiation-irradiated cancer cells, and develops a mature dendritic cell cancer vaccine in which it is exposed to dendritic cells (DC), thereby solving the limitations of conventional anticancer treatments. It is about treatment techniques.

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.

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.

Further, the cancer includes, for example, mammary cancer, complex mammary cancer, mammary malignant mixed tumor, intraductal papillary adenocarcinoma, lung adenocarcinoma, squamous cell carcinoma, small cell carcinoma, large cell carcinoma, neuroepithelial tissue tumors such as glioma, ventricular tumor, neuro 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.

In particular, cancer to which the present invention can be applied may be classified as a solid cancer or may include cancer that 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 exosomes according to the present invention are obtained from cancer cells irradiated with radiation, and more particularly, the steps of irradiating 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.

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 the cells irradiated with a dose falling within the scope of the present invention are co-cultured on the dendritic cells, that is, loading on the dendritic cells, the maturation of the dendritic cells can be effectively induced.

As described above, according to the present invention, there is provided an exosome for preparing mature dendritic cells obtained from irradiated cancer cells. 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, wherein the mature dendritic cells may be a dendritic cell therapy agent for cancer treatment.

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. In more detail, 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. 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.

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.

Furthermore, according to the present invention, there is provided a vaccine for treating cancer, comprising dendritic cells carrying exosomes obtained from irradiated cancer cells.

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. .

The vaccine composition of the present invention may include 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 of the present invention may contain suitable auxiliary ingredients and preservatives to enhance chemical stability and isotonicity, and may include a stabilizer such as trehalose, glycine, sorbitol, lactose or monosodium glutamate (MSG) to change or freeze temperature. The vaccine composition can be protected against drying. The vaccine composition of the present invention may contain a suspension liquid such as sterile water or saline (preferably buffered saline).

The vaccine 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.

As with all other vaccine 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 doses of the immunity administered. In addition, it can 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. For example, in the vaccine for treating cancer, the dendritic cells may be included in an amount of 10 ⁶ to 10 ⁹ cells, for example, 10 ⁷ to 10 ⁸ cells in the case of humans. For example, 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.

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.

Since cells containing dendritic cells, which are the active ingredients of the vaccine produced by 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.

In addition, since the mature dendritic cells prepared according to the method of the present invention exhibit a cancer treatment effect, it 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.

In addition, according to the present invention, there is provided 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 pharmaceutically acceptable carrier may further include, for example, a carrier for oral administration or a carrier for parenteral administration. Carriers 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.

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 dendritic cell vaccine of the present invention is administered through any general route as long as they can reach the target tissue. Can be. 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.

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 hides) Rockside), suspending agents, 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. 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.

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. .

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. .

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.

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.

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.

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.

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.

According to the present invention, not only can the dendritic cells be activated effectively, but also cancer cell-specific immunity can be effectively induced, and thus remarkably improved anticancer immunity is expected to be induced.

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.

Example

One. Cancer cells Induced through gamma irradiation Exosome Separation and analysis

Example One

Using melanoma cell line B16-BL6, 1.8×10 ⁶ cells cultured with DMEM (10% FBS, 100 U/ml penicillin/streptomycin) were diluted in PBS (2 mL), and then gamma-ray 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.

Comparative example One

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.

Experimental example One: Exosome Separation and analysis

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).

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. Derived from gamma irradiation melanoma Exosome Dendritic cells Confirmation of cytotoxicity and uptake ability induced during treatment

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.

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).

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. Derived from gamma irradiation melanoma Exosomes And Non-gamma rays Investigation melanoma origin Exosome Dendritic cells Maturity Confirm

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.

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. Derived from gamma-irradiated melanoma Exosomes And Non-gamma rays Investigation melanoma origin Exosomes Analysis of the effect of treated dendritic cells on the proliferation, activity, and differentiation of T cells

The most important feature of mature dendritic cells in vivo is to induce the activity of T lymphocytes. 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.

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.

In addition, as a result of analyzing the secretion of cytokines (IFN-g, IL-2, IL-4) secreted from T cells by applying an ELISA kit, as shown in Fig. 4(c), Gamma-exo treatment of Example 1 It was confirmed that dendritic cells can induce the activity 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.

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. Gamma rays and Non-gamma rays Investigation melanoma origin Exosomes Efficacy evaluation of treated dendritic cells as therapeutic vaccines

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 injection, three gamma-ray and non-gamma-ray irradiated melanoma-derived exosome-treated dendritic cells were injected intravenously (Fig. 5(a)). Immature dendritic cells were used as a negative control.

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.

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 (15)

Irradiating cancer cells with radiation; And
Obtaining exosomes from irradiated cancer cells
Containing, a method for producing an exosome for preparing mature dendritic cells.
The method of claim 1, wherein the radiation has a total dose of 30 Gy to 200 Gy.
Exosomes for preparing mature dendritic cells obtained from irradiated cancer cells.
According to claim 3, wherein the mature dendritic cells is a dendritic cell therapy for cancer treatment, mature dendritic cell production exosomes.
Irradiating cancer cells with radiation;
Obtaining exosomes from irradiated cancer cells; And
A method for producing a dendritic cell therapy comprising the step of loading the exosomes on dendritic cells.
The method of claim 5, wherein the radiation is gamma rays, electron rays, ultraviolet rays or X-rays.
The method of claim 5, wherein the step of obtaining the exosome is performed by centrifugation.
The method of claim 5, wherein the step of supporting the exosomes on the dendritic cells is the step of inducing the differentiation of immature dendritic cells into mature dendritic cells by culturing dendritic cells in a medium containing exosomes.
The method of claim 8, wherein the culture is performed for 18 hours to 36 hours.
A vaccine for cancer treatment comprising dendritic cells carrying exosomes obtained from irradiated cancer cells.
The vaccine for cancer treatment according to claim 10, wherein the cancer treatment vaccine is formulated for intravenous administration.
The vaccine for cancer treatment according to claim 10, wherein the dendritic cells are contained in an amount of 10 ⁶ to 10 ⁹ cells.
The vaccine for cancer treatment according to claim 10, wherein the cancer treatment vaccine is administered two or more times at intervals of 1 to 28 days.
A pharmaceutical composition for cancer treatment, comprising dendritic cells carrying exosomes obtained from irradiated cancer cells.
Immunotherapy comprising dendritic cells carrying exosomes obtained from irradiated cancer cells.

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