KR20090047289A - Exosome and composition of cancer vaccine containing it - Google Patents

Abstract

The present invention provides an exosome containing a lot of proteins that can be extracted from the heat-treated tumor cells to enhance the anti-tumor specific immune response and cancer vaccine composition that can be applied to MHC independently as a main component thereof.

Since the cancer vaccine composition of the present invention is not subject to major histocompatibility (MHC), it can be widely used for autologous and allogeneic individuals.

Tumor Cells, Heat Treatment, Exosomes, Cancer Vaccines

Description

Exosome and cancer vaccine composition containing same {EXOSOME AND COMPOSITION OF CANCER VACCINE CONTAINING IT}

The present invention relates to an exosome and a cancer vaccine composition containing the same, and more particularly, an exosome which is extracted from heat treated tumor cells and secretes a lot of proteins that can enhance an anti-tumor immune response, and contains the same to MHC. Dependably applicable cancer vaccine compositions.

Cancer deaths are the number one cause of death in Korea and one of the most important causes of death in developed countries. To date, efforts to treat cancer have been developed and used until the 1950s, surgical surgery, radiation therapy in the 1960s, chemotherapy in the 1970s. In the 1980s, with the rapid development of basic sciences, especially immunology and molecular biology, immunotherapies and gene therapies began to emerge, and although much research is currently being conducted, they are still insufficient to achieve breakthroughs. It is true.

Cancer vaccines are a type of active immunotherapy that uses specific antigens from tumor cells as immunogens and administers humoral or cellular immune mechanisms to cancer-specific antigens by administering them to cancer patients in a similar process to viral vaccines. It is a treatment method that takes full advantage of the immune function in patients involved in killing cancer cells.

 Exosomes are membrane vesicles secreted from different types of cells, including major histocompatibility (MHC) and heat shock proteins (HSP), which are immunologically important proteins. It is known to induce strong antitumor immune responses.

The immunological role of heat shock protein 70 (HSP70) secreted extracellularly is a potent innate immunity activator, inducing an inflammatory immune response.

Conventional cancer treatments include Surgery (1950s) and Radiotherapy (1960s) as local therapies, and Chemotherapy (1970s) and Immunotherapy (Immunotherapy; 1990s) as systemic therapies. There is. More than 50 cancer treatment drugs are currently registered around the world. Most of them are anti-cancer chemotherapy agents, and cancer cell-specific therapeutic agents are still under development.

On the other hand, the existing anti-cancer drugs used in chemotherapy mainly developed using only the growth rate difference between normal cells and cancer cells, and works on both fast dividing and proliferating cells. Thus, despite being normal cells, cell division may damage active bone marrow cells, gastric and intestinal epithelial cells, hair follicle cells, etc., and may cause fatal side effects such as decreased immunity, gastrointestinal disorders, hair loss, pain, vomiting, and cardiotoxicity.

Cancer vaccines are an active immunotherapy regimen that utilizes specific antigens of tumor cells as immunogens and administers humoral or cellular immune mechanisms to cancer specific antigens by administering them to cancer patients in a similar process to viral vaccines. It is a therapeutic method that makes full use of immune function in patients involved in cancer cell killing by activating. Currently, cancer-related research institutes such as the US, Canada, and Japan are emerging as the most suitable next-generation cancer treatment methods. On the other hand, cancer vaccines have a difference as shown in Table 1 below in comparison with a general virus vaccine.

[Table 1] Comparison of characteristics of cancer vaccine and antivirus

Cancer vaccine Antivirus Vaccine administration After tumor formation Before virus infection Structure of immunogen Complicated, diverse simple Type of immunogen Infinity Limited Host's immune status Low (for immunity) normal

The advantages and disadvantages of the cancer vaccine described above and the problems to be solved are described in Table 2.

Table 2 Pros and Cons of Cancer Vaccines

Advantages Disadvantages One Selectively destroy only cells that express cancer specific antigens If cancer antigens are not sufficiently exposed, cancer cells can continue to proliferate without being detected by immune cells 2 Effectively suppresses cancer recurrence or metastasis by utilizing memory function of immune cells The struggle between cancer cells and the host immune system that have persisted in cancer patients has concerns about a reduction in efficiency at the application level. 3 Compared to conventional drug treatment or radiation therapy, the market share is higher economically and expected to improve the 5-year survival rate solution Use of various immunopotentiators that contribute to the preparation of immunogens that can maximize the sensitization of cancer antigens and the induction of immune activity

On the other hand, cancer vaccines can be classified into cell vaccines, dan protein vaccines, peptide vaccines and exosome vaccines, and the analysis on the effects and manufacturing functionalities for each is described in Table 3.

Table 3 Summary of relative benefits of cancer vaccine manufacturing strategy

Non-purified (cell vaccine)  Semi-purified (protein vaccine) Tablet type (peptide vaccine) Tablet type (exosome vaccine) Effective aspect Immunogen Adequacy + + + + + + + + Immunogen Diversity + + + + + + + + Immune response + + + + + +  + + Manufacturing functional aspect Purity + + + + + + + + + productivity + + + + + + + + + Quality control + + + + + + + + + Remarks Most effective · Long time trials · Tissue compatibility issues · Dendritic cell vaccines Immune sources; Tumor specific antigen / immune enhancer extracted from cancer cells Immune sources; Synthesized peptide for specific MHC It is easy to manufacture and largely induces immune response by separating exosomes from culture by mass cultivating certain cancer cell lines already established.

As a result, the exosome vaccine, which is evaluated to be the most effective among the conventional cancer vaccines, also does not produce a sufficient immune response when administered in a cancer patient whose immune system is lowered, so that the effect as a cancer vaccine has a very small problem.

Furthermore, in the case of the conventional cancer vaccine, it was possible to apply only to cells having the same MHC type, and there was a problem that application was difficult when the MHC types were different.

The present invention has been made to solve the above-described problems, the object of the present invention is extracted from heat treated tumor cells and exo-secreting a lot of protein that can improve the anti-tumor immune response as compared to conventional tumor-derived exosomes Is to provide some.

Another object of the present invention is to provide a cancer vaccine composition containing exosomes extracted from heat treated tumor cells, which is applicable independently to MHC.

Exosome according to a feature of the present invention for achieving the above-described problem is extracted from heat treated tumor cells.

The heat treatment may be preferably performed at 40 to 45 ° C. for 15 to 60 minutes.

The tumor cells are preferably applicable to all tumor cells that can produce exosomes.

The exosomes secrete more proteins that enhance the immune response than exosomes extracted from untreated heat treated tumor cells, and the proteins are applicable to HSP70, HSP70, HSP90, HSP60 and GP96.

Cancer vaccine composition according to another feature of the present invention contains the exosome of the present invention described above as an active ingredient, and comprises a pharmaceutically acceptable carrier.

The cancer vaccine composition enhances cellular immunity and further comprises an immune adjuvant.

The cancer vaccine composition is MHC independent.

Exosomes extracted from the heat treated tumor cells of the present invention can be utilized as a cancer vaccine composition by secreting more proteins that can enhance the antitumor immune response as compared to exosomes derived from conventional tumors. In addition, the cancer vaccine composition of the present invention is not subject to major histocompatibility (MHC), it can be widely used in autologous and allogeneic individuals. Furthermore, exosomes obtained after heat treatment are more effective in inducing maturation and activation of dendritic cells and inducing Th1 immune responses.

Although the present invention has been described in detail only with respect to the embodiments described, it will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the present invention, and such modifications and variations are included in the appended claims. Belongs to

It is natural.

Hereinafter, the present invention will be described in more detail.

Conventional cancer vaccine compositions do not produce a sufficient immune response when administered in cancer patients with a reduced immune system, and thus have a very small effect as cancer vaccines.

The exosomes according to an embodiment of the present invention is extracted from the heat treated tumor cells. This results in a lot of protein secretion that can enhance the antitumor immune response compared to conventional tumor-derived exosomes.

On the other hand, the heat treatment may be preferably carried out for 15 to 60 minutes at 40 ~ 45 ℃. If the temperature is less than 40 ° C., the effect of heat treatment is insignificant. If the temperature is higher than 45 ° C., the survival rate of the cells may be sharply reduced. Furthermore, if the heat treatment time is less than 15 minutes, the heat treatment effect is insignificant, and if the heat treatment time exceeds 60 minutes, the survival rate of the cells may decrease rapidly.

The heat treatment method is preferably performed in a state where the cells are attached to the bottom of the test tube, and takes a manner of immersing in a preheated water bath.

The present invention can extract exosomes by applying heat treatment to all the tumor cells, but in the present invention described CT26 tumor cells, but not limited to this is applicable to any tumor cells capable of secreting exosomes.

Exosomes of the present invention secrete more proteins to enhance the immune response compared to exosomes extracted from untreated heat treated tumor cells, the protein corresponds to HSP70, HSP70, HSP90, HSP60 and GP96.

Eventually, when heat treated as described in the present invention for tumor cells, the expression of inducible HSP70 is induced (FIG. 1), and the induced HSP70 is returned to some exosomes (FIG. 2). At this time, the amount of exosomes secreted increases with time (Fig. 3). On the other hand, exosomes (HS Exo) secreted from heat treated tumor cells showed a difference in protein pattern compared to exosomes (CNTL Exo) secreted from untreated heat treated tumor cells, especially the amount of inducible HSP70. Increases (FIGS. 4, 5).

Furthermore, for antigen-presenting cells of autologous and allogeneic systems, HS Exo enhances the expression of group 2 MHC (MHC class II) on the cell surface and enhances the cell culture medium through AKT phosphorylation and degradation of I-kB in antigen-presenting cells. Increasing the amount of inflammatory cytokine secreted (FIG. 6). When the exosomes of the present invention were injected into mice, it was observed that tumor size of the HS Exo vaccine was significantly reduced compared to CNTL Exo vaccine in both autologous and allogeneic tumors. When analyzing the mediated immune response, HS Exo strongly induced Th1-directed immune responses such as the increase of IgG2a and IFN-gamma production (Figs. 7 and 8). Taken together, exosomes extracted from heat treated tumors contain more HSP70, etc., which can enhance the Th1 immune response required to remove cancer cells, and this HS exo is a potential MHC-independent cell-free cancer therapeutic vaccine. Suggest that In other words, the cancer vaccine composition of the present invention can be applied regardless of the MHC type, so it is MHC independent.

Therefore, the cancer vaccine composition according to the second embodiment of the present invention contains the exosome of the present invention described above as an active ingredient, includes a pharmaceutically acceptable carrier, may further include a conventional immune adjuvant, various It may be formulated in oral or parenteral dosage form. Formulations for oral administration include, for example, tablets, pills, hard soft capsules, solutions, suspensions, emulsifiers, syrups, granules, etc. These formulations may contain diluents (e.g. lactose, dextrose, sucrose) in addition to the active ingredients. , Mannitol, sorbitol, cellulose and / or glycine), glidants such as silica, talc, stearic acid and magnesium or calcium salts and / or polyethylene glycols) and other pharmaceutically acceptable carriers. In addition, a typical parenteral formulation is an injectable formulation, preferably an isotonic aqueous solution or suspension containing physiological saline, edible oil, or the like as an injection medium.

The cancer vaccine composition of the present invention may be sterilized and may contain auxiliaries such as preservatives, stabilizers, hydrating or emulsifiers, salts and / or buffers for the control of osmotic pressure and other therapeutically useful substances, Can be formulated accordingly.

The cancer vaccine composition comprises exosomes extracted from heat treated tumor cells as an active ingredient in an amount of 0.025 to 0.05 mg / kg body weight for mammals including humans. It may be administered via the oral or parenteral route, preferably by the oral or parenteral route in an amount of 0.025 to 0.05 mg / kg body weight, and may be further immunized twice at intervals of 40 to 57 weeks. . However, it should be understood that the actual dosage of the cancer vaccine composition should be determined in light of several related factors such as the route of administration, the age, sex and weight of the patient, and therefore the dosage should in no way limit the scope of the invention. It is not limited.

Hereinafter, the present invention will be described in more detail with reference to the following examples. only. The following examples are only for illustrating the present invention, but the scope of the present invention is not limited thereto.

< Example  One : Thermal shock  Production of Tumor Cells>

(1) CT26 tumor cells were heat shocked at 43 ° C. for 30 minutes

CT26 tumor cells derived from the mouse colon carcinoma cell line attached to the bottom of the flask in a 37 ° C. incubator were heat treated at 43 ° C. for 45 minutes in a water bath.

The heat shocked tumor cells were cell cultured at normal temperature (37 ° C.), and the expression of heat shock proteins in cells over time was compared using western blot. Specifically, after incubation at 37 degrees after heat shock, the protein was separated from the cells (0, 1, 4, 6.5, 12, 24 hr) after a certain time (reaction for 1 hour on ice in tween 20-containing PBS) After extracting only the supernatant by centrifugation), the protein is quantified by the BCA method, and then electrophoresed with the same amount (20 ug) of protein per sample. Proteins isolated by size can be transferred to a membrane such as nitrocellulose and then bound to a primary antibody (anti-hsp60, anti-ihsp70) or anti- hsp90, anti-gp96) is reacted at room temperature for 1 hour. After reacting the secondary antibody (anti-goat Ig-HRP, anti-mouse Ig-HRP) capable of binding the primary antibody for 30 minutes, the enzyme (Horse Radish Peroxidase (HRP)) finally attached to the secondary antibody A reactive substrate (H 2 O 2) is added and the black band formed by exposing the membrane to the X-ray film is observed through a developer and a fixer.

 Through this, it was confirmed that significant expression of inducible hsp70 (inducible hsp70, iHSP70) occurred in the heat shocked tumor cells (FIG. 1). In the figure, the band of iHSP70 was observed from 4 hours after incubation at 37 degrees after thermal shock, indicating that expression was induced.

< Example  2> induced HSP70 this To exosomes Regression

 In Example 1, it was observed at the cellular level through FACS using the antibodies against HSP70 and the exosome label LAMP1, 3 whether HSP70 induced expression after heat shock was returned to exosomes and secreted (FIG. 2). ).

 After thermal shock, the cells were incubated at 37 ° C., and after a certain time, the cells were rolled and reacted with the primary antibody against each protein. One hour after the reaction, the secondary antibody labeled with a fluorescent material capable of binding the primary antibody was bound for another 30 minutes, and then the intensity of the fluorescent material was measured by flow cytometry.

As a result, over time, the expression of LAMP1, LAMP3, etc., proteins specific for multivesicular bodies (MVBs), which are the source of Hsp70 and exosomes, have increased on the cell surface over time. Increasing LAMP1, 3 at the cell surface suggests that the frequency of fusion with the cell surface membranes has increased. That is, the heat treatment increases the amount of exosomes secreted out of the cell through the fusion of the polyhedron and the cell surface membrane, and HSP70, whose expression is increased by heat treatment, is also secreted out of the cells through the exosomes. It offers the possibility.

< Example  3> Exosomes  Yield measurement

The cell culture solution of Example 1 was secured and analyzed for exosome production through ELISA using an antibody of exosome-labeled protein (FIG. 3).

Specifically, the CT26-MUC1 cells were laid on a 6 well plate, and after 2 days, the cell culture solution was collected, and only the supernatant was taken through a series of centrifugation processes (300g, 1200g, and 10000g). The supernatant taken above was bound to the ELISA plate coated with antibodies to MUC1 and LAMP3, which are contained in exosomes, throughout the day. Next, CTXb (cholera toxin subunit b) capable of binding to GM1 of exosomes was reacted. After one hour, the color development developed by adding a substrate for the enzyme attached to the CTXb was measured using an ELISA reader. As a result of ELISA using MUC1, LAMP3, GM1, etc., which are included in exosomes, it was confirmed that the amount of exosome secretion increased when the heat shock was not given (control).

< Example  4> Heat Shock Protein Analysis

After 24 hours of heat shock, the cell cultures were collected and exosomes were separated through an ultracentrifuge. The separated exosomes were analyzed using normal exosomes and SDS-PAGE, and various heat shock proteins were analyzed using Western blot (FIG. 4).

Specifically, the exosomes separated from the ultracentrifuge in Example 3 were electrophoresed in a predetermined amount of protein according to the protein quantification method, and the Western blot method was performed in the same manner as described in FIG. Proteins that you want to see with control proteins exosome-specific proteins (CD71, tsg101), cell specific and exosome-free proteins (calnexin, beta-actin), cells, exosomes common protein (alpha-tubulin), here hsc70, hsp70 Western blot was performed using antibodies against hsp90, hsp60, gp96.

As a result, it was confirmed that some proteins other than proteins secreted from normal tumor exosomes exist in heat shock tumor exosomes, and the inducible HSP70 protein was expressed in exosomes as in cells through Western blot.

< Example  5> Measurement of immunostimulatory capacity

To compare the immunostimulatory capacity of heat shock exosomes with HSP70 and normal tumor exosomes, 24 hours after normal tumor exosomes and heat shock tumor exosomes were added to dendritic cells derived from bone marrow cells MHC class II, CD80, CD86, CD40, etc., molecules related to immune response enhancement that express dendritic cell activation on cell surface were analyzed by FACS using fluorescently labeled antibodies (FIG. 5, CNTL: heat shock note). Exosomes isolated from non-tumored tumor cells, HS: exosomes isolated from heat shock semi-tumor cells, PBS: solvent control).

As a result, it can be seen that heat shock exosomes have a higher ability to induce the expression of immunostimulants in dendritic cells derived from bone marrow cells than normal exosomes.

< Example  6> MHC type Heat shock protein analysis for two different dendritic cell lines

Experiments were carried out in the same manner as in Example 4 using macrophages, RAW264.7 and dendritic cell lines of different MHC types (FIG. 6).

Specifically, Figure 6a shows the expression level of MHC class II on the surface of macrophage RAW264.7 cell, Figure 6b shows the expression level of MHC class II on the surface of dendritic cell line DC2.4 cell, Figure 6c shows the dendritic cell line DC2 RT-PCR results of cytokines in .4 are shown, and finally FIG. 6D shows the production of IL-12 in cell culture of dendritic cell line DC2.4. In all the results, it can be seen that heat shock exosomes have better immune stimulation than normal exosomes.

< Example  7> Measuring tumor treatment effect

After injecting cancer cells expressing MUC1 protein into mice, when tumors grew to about 1 cm ² , exosomes were injected twice with CpG, and tumor size was measured over time (FIG. 7).

In more specifically with, Babl / c hanhu form a tumor by injecting CT26-MUC1 cells in mice, 100 mm ² scans the respective exo-bit semi-mixed with CpG every two weeks twice when the extent size, and time Tumor size was then measured using a caliper.

   As a result, it was confirmed that heat shock protein enhanced exosomes (HS Exo) is superior to tumor growth inhibition function than exosomes (CNTL Exo) without heat shock.

< Example  8> lymphocyte Activity level

Exosomes mixed with CpG were injected two times at two-week intervals into naive mice. After two weeks, lymphocytes were extracted from the spleens of immunized mice, RNA was extracted, and cytokine activation was assessed. Observed by RT-PCR for (IL-2, perforin) (FIG. 8).

 As a result, since the expression of activated cytokines is further increased in lymphocytes of mice injected with heat shock exosomes, it can be confirmed that the activity of lymphocytes is higher by heat shock exosomes.

< Example  9> Major organizational adequacy  Validation of potential as a cancer vaccine

To confirm the potential as a cancer vaccine that is not subject to major histocompatibility, the spinal cord of C57bl / 6 mice from a dendritic cell line derived from allogenic mice of different primary histocompatibility, DC2.4 or DC1 (Kenneth, USA) Generation of DC and then oncogenes such as ras into cell lines-Dr. Johns Hopkins University, USA T.C. 24 hours after smeared from Professor Wu, proteins were isolated from the cells reacted in the same manner as in FIG. 1, and after electrophoresis of a certain amount of protein, the Western culture of proteins related to intracellular signaling pathways The degree of activation of dendritic cells was measured while performing the blot (FIG. 9).

Figure 9 shows the analysis of intracellular signaling pathways induced by exosomes, the phosphorylation of AKT protein and degradation of I-kB, which is a phenomenon that occurs during intracellular survival signal transmission from about 15 minutes after injection of heat shock exosomes It became. This means that the activity of the cell is promoted by the heat shock protein. Through this, it was confirmed that the heat shock exosome of the present invention was utilized as a cancer vaccine regardless of main histocompatibility.

< Example  10> Allogeneic ( allogenic Observation of Cancer Treatment Effect in Mouse Model

The effect of cancer treatment in the allogenic mouse model was observed. Specifically, after injecting tumors to allogeneic mice, when grown to about 1 cm ² , exo was injected and tumor growth was measured over time.

Specifically, when B16-MUC1 cells were injected into C57bl / 6 mice and the size of the tumor grew to about 1 cm ² , 10 ug of each exosome was injected twice per mouse, followed by two week intervals. The size was measured.

As a result, it can be confirmed that tumor growth inhibition ability is more excellent in mice injected with heat shock exosomes.

< Example  11> Analysis of Spleen Cells Isolated from Mice

(1) Antigen-specific immunoglobulin (antibody) production in serum ELISA Analyzed with

To analyze the T cells required for anti-cancer effects and the resulting immune response mechanisms, 10 ug of exosomes thermally shocked in C57bl / 6 mice were injected twice at two week intervals, one week after each injection. Serum was isolated from mice. Serum was added to the exo-coated ELISA plate and reacted. Induce the color reaction by inserting a secondary antibody that can recognize immunoglobulin in mouse serum, and then adding a substrate for the enzyme bound to the secondary antibody. Immunoglobulin (antibody) production was analyzed by ELISA (FIG. 11A).

11 shows antigen-specific antibody production. As a result of comparing the total antibody production and the production of Th1 type antibody (IgG2a) and Th2 type antibody (IgG1), it was confirmed that the production of IgG1a, a Th1 type antibody, was increased by heat shock protein-enhanced exosomes. This means that exosomes from heat treated tumor cells have a better effect on inducing Th1 type immune responses.

(2) Analysis of Spleen Cells Isolated from Immunized Mice

Expression of IL-4 or IFNgamma cytokines in CD4 or CD8 T cells capable of anti-cancer effects in spleen cells extracted from mice immunized with heat shock protein-enhanced exosomes was analyzed by flow cytometry using antibodies against each. As a result of analysis (Fig. 11b), IFNgamma-producing T cells were most frequently seen in mice immunized with exosomes enhanced with heat shock proteins. This means that the tumor-derived exosomes may have a better effect on the heat treatment of the tumor cells in generating a Th1 immune response that must be generated in terms of preventing cancer cells from recurring.

Exosome of the present invention and cancer vaccine composition containing the same is a very important invention in the medical and health industry because it can be applied to both heterogeneous tissues independent of MHC.

1 is a Western blot result for the expression of heat shock proteins in cells over time.

Figure 2 is a graph observed at the cell level through FACS using the antibody to hsp70 and LAMP1, 3, an exosome marker.

Figure 3 is a graph showing the exosome production of the present invention through the ELISA (ELISA) method using an antibody of the exosome labeling.

Figure 4a is a SDS-PAGE results of the isolated exosomes of the present invention, Figure 4b is a result of analyzing the various heat shock proteins using Western blot.

5 is a fluorescent substance for enhancing immune response-enhancing molecules expressing the activation level of dendritic cells on the cell surface after 24 hours after adding normal tumor exosomes and heat shock tumor exosomes to dendritic cells derived from bone marrow cells. It is the result of analysis by FACS using this labeled antibody.

Figure 6a is a result showing the expression level of MHC class II on the cell surface of the macrophage RAW264.7, Figure 6b is a result showing the expression level of MHC class II on the dendritic cell line DC2.4 cell surface, Figure 6c Shows the RT-PCR results of cytokines in the dendritic cell line DC2.4, Figure 6d means the production of IL-12 in the cell culture of the dendritic cell line DC2.4.

Figure 7 is a graph measuring the size of the tumor over time after the injection of the exosome of the present invention.

Figure 8 is a result of observing the lymphocytes from the spleen of the tumor disappeared mouse, extracting RNA, the activation of lymphocytes by RT-PCR for cytokines (IL-2, perforin).

9 is an electrophoresis result of analyzing intracellular signaling pathways induced by treating exosomes in dendritic cell lines derived from allogeneic mice with different major histocompatibility.

10 shows the results of observing the cancer treatment effect in an allogenic mouse model.

Figure 11a is a graph comparing the total antibody production and production of Th1 type antibody (IgG2a) and Th2 type antibody (IgG1), Figure 11b is the expression of IL-4 or IFN-gamma cytokines in CD4 or CD8 T cells, respectively This is a graph analyzed by flow cytometry using the antibody against.

Claims (11)

Exosome, characterized in that extracted from the heat treated tumor cells. The exosomes of claim 1, wherein the heat treatment is performed at 40 to 45 ° C. for 15 to 60 minutes. According to claim 1, wherein the tumor cells are tumor cells capable of secreting exo The exosomes characterized in that. The exosome of claim 3, wherein the tumor cells are CT-26. The exosome according to claim 1, wherein the exosomes secrete more proteins to enhance the immune response than exosomes extracted from untreated heat treated tumor cells. The exosome according to claim 5, wherein the protein is any one or more selected from the group consisting of HSP70, HSP70, HSP90, HSP60, and GP96. The exosomes according to claim 1, wherein the heat treatment is a hot bath method. A cancer vaccine composition comprising the exosome of claim 1 as an active ingredient and comprising a pharmaceutically acceptable carrier. The cancer vaccine composition of claim 8, wherein the cancer vaccine composition promotes cellular immunity. 9. The cancer vaccine composition according to claim 8, further comprising an immunoadjuvant. The cancer vaccine composition of claim 8, wherein the cancer vaccine composition is MHC independent.

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