KR20090047290A - Exosomes and Cancer Vaccine Compositions Containing the Same - Google Patents

Abstract

The present invention provides a method for introducing a gene for inducing the expression of MHC class II into a cancer cell line and containing the MHC II protein in an exosome isolated from the cell and a cancer vaccine containing the exosome as a main component. To provide a composition.

In particular, the exosomes of the present invention and the cancer vaccine composition containing the same are expressed in the major tissue-adapter II protein in the exosomes compared to the exosomes derived from the normal tumors, and the activity of the dendritic cells which phagocytosed the resulting CD4 helper T lymphocytes. It is more effective in inducing anti-cancer immune response by activating increased action to enhance Th1-type immune response.

Tumor cell, major histocompatibility II, exosomes, cancer vaccine, CIITA gene

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, to a exosome into which a gene for inducing the expression of major histocompatibility II (MHC class II), a method for producing the same, and a main ingredient in the exosome It is more effective for inducing anti-cancer immune responses because TGF-II protein expression and phagocytic dendritic cell activity are increased and consequently the activation of CD4 helper T lymphocytes to enhance Th1-type immune responses. It is about.

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 functional aspects 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.

The present invention has been made to solve the above-mentioned problems, the object of the present invention is to introduce a gene for inducing the expression of major tissue adaptive body II (MHC class II) anti-tumor immune response as compared to exosomes derived from conventional tumors To provide a exosomes and a method for producing the same that secrete a lot of protein that can be improved.

 Another object of the present invention is that anti-cancer immunity is increased because the expression of major histocompatibility II protein in exosomes increases the activity of dendritic cells that phagocytize and thereby activates CD4 helper T lymphocytes. It is to provide a cancer vaccine composition more effective in inducing a response.

In order to achieve the above-mentioned problem, the present invention provides an exosome into which a gene for inducing the expression of major histocompatibility II (MHC class II) is introduced.

The expression inducing gene is preferably MHC class II transactivator (CIITA), and the exosomes may be preferably derived from tumor cells.

The exosomes of the present invention described above were incubated with dendritic cells in an environment similar to the reaction in the body, and the dendritic cells phenotyped with the exosomes increased the major histocompatibility II protein and ultimately the activity of CD4 helper T lymphocytes. to provide.

The present invention comprises the steps of: (1) cloning a gene for inducing the expression of major histocompatibility II in a retroviral vector, (2) introducing the cloned retroviral vector into a tumor cell line; And (3) separating the exosome from the tumor cell line.

The gene is preferably an MHC class II transactivator (CIITA) and the tumor cell line is a B16F1 tumor cell line.

The present invention contains an exosome prepared by the above-described exosomes or a method for preparing the same as an active ingredient, a pharmaceutically acceptable carrier, and may further include an immunoadjuvant.

The cancer vaccine composition enhances cellular immunity.

Exosomes into which the main tissue conformer II (MHC class II) expression gene of the present invention are introduced further enhance the main tissue conformer II and Th1 type immune responses on dendritic cell surfaces obtained by phagocytosis compared to exosomes derived from conventional tumors. It is more effective in inducing anti-cancer immune response by increasing the action of CD4 helper T lymphocytes.

It is also more effective in inducing the maturation and activation of dendritic cells and the activity of T lymphocytes.

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. Belonging 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.

Thus, one embodiment of the present invention is an exosome into which the main tissue adaptive body II (MHC class II) expression gene is introduced, and more specifically, the main tissue adaptive body II (MHC class II) expression gene is injected into tumor cells. It is formed by extracting exosomes from the tumor cells. As a result, compared with the conventional tumor-derived exosomes, the secretion of the major histocompatibility II without affecting the expression level of the tumor antigen.

In more detail, the present invention is injected into the tumor cells of the main tissue adaptive body II (MHC class II) expression gene, and then the exosomes are extracted from the tumor cells. The main tissue adaptive body II (MHC class II) expression gene can be applied without any limitation as long as it can express the main tissue compatible body II, Preferably MHC class II transactivator (CIITA) gene to tumor cells I can import it. The CIITA gene (gene accession number NM_000246) is a transcriptional cofactor that essentially plays a role in the expression of MHC class II proteins.

At this time, the main histocompatibility Ⅱ (MHC class II) expression gene is introduced into the cells, wherein the importable cells can be either normal cells or tumor cells, but more preferably, the tumor cells are used to utilize tumor antigens or the like. do. Meanwhile, the present invention has been described using the B16F1 tumor cell line as an example, but is not limited thereto and may be applied to all tumor cells.

As a method of introducing the expression gene into a cell, all of the conventional gene cloning methods may be used, and preferably, the expression gene may be cloned and introduced into a retroviral vector. In the present invention, a gene was introduced into a cell using a retroviral vector, but other transduction methods (eg, electric shock, liposomes, lentiviral vectors, etc.) are all possible.

The present invention extracts the exosomes from the tumor cells into which the main tissue conformer II (MHC class II) expression gene is introduced, and the extraction method may be applied to a conventional exosome extraction method.

Next, the present invention can inject the above-described exosomes of the present invention into dendritic cells, through which dendritic cells increase the expression of major histocompatibility II and increase the activity of CD4 helper T lymphocytes. As a result, tumor growth can be suppressed, and the survival rate of the mouse was significantly improved. This was dependent on the concentration of the injected exosomes. Preferably, 50 ug or less is injected to prevent excessive immune response.

In conclusion, the present invention secured tumor cell lines expressing MHC class II by introducing MHC class II expressing genes into tumor cells, and when exosomes were purely isolated from the cell lines, MHC class II expression was confirmed.

Subsequently, when the exosomes were injected into the dendritic cells, the activity of the dendritic cells injected with the exosomes was significantly increased, and the activation of the T cells was also increased by using the dendritic cells ingested with the exosomes. .

 Therefore, by injecting exosomes expressing MHC class II, the CD4 helper T lymphocytes that can provide MHC class II repertoir of tumor antigen to the immune system and can further enhance the existing Th1 type immune response. The action of was increased, confirming that it is more effective in the induction of anti-cancer immune response.

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 oral or It may be formulated in parenteral dosage forms. 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. Also typical of parenteral formulations 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 tumor cells to which the main tissue conformer II (MHC class II) expression gene is introduced 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 is to 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, such that the dosage in any way limits the scope of the invention. It is not.

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 : Organization Conformity  Ⅱ ( MHC  Ⅱ) Introduction of Expression Genes into Tumor Cell Lines>

MHCII transactivator (CIITA) genes, which induce MHCII expression in retroviral vectors, were cloned and introduced into B16F1 tumor cell lines.

More specifically, when the introduction process of the gene is described with reference to the cleavage map of the CIITA gene shown in FIG. 1, the BamHI portion of CIITA was first enzymatically ligated to the pRetro vector at 16 ° C and o / n. 293T cells were then seeded at 40-60% confluence and 3 plasmids were transferred (9ug retroviral plasmid, 4.5ug GAP-POL plasmid, 4.5ug VSV plasmid as transit293 transfection reagent). Then, after 2 days of transfection, the media were collected, 4-5 ml of DMEM was added, and the collected media was filtered through a 0.45 um filter. After 2ml aliquots were stored at -80 ℃ and 3 days later the virus was collected again.

After that, we filled our cell with B-50, 40-50%, in a 10 cm dish. 2 ml of virus was added to 6 ml of medium, and the polybrene concentration was 8 ug / ml. After 4-6 hours, 4ml of the medium was added, and after 24 hours, the medium was exchanged, followed by further culture for 2-3 days.

< Example  2 : CⅡTA  In the cell line where the gene was introduced MHC  II and TRP Confirmation of Expression of -2>

The expression of MHC II and the expression of endogenous antigen TRP-2 in the B16F1 tumor cell line into which the CIITA gene was introduced in Example 1 were confirmed by flow cytometry by staining by intracellular stairing method (FIG. 2).

Specifically, ⁵ × 10 ⁵ cells were obtained, suspended in PBS containing 0.1% BSA, and the first antibody was treated with purified anti-TRP-2 antibody and reacted at 4 ° C. for 1 hour. After washing with PBS containing 0.1% BSA, the fluorescent labeled secondary antibody (anti-goat Ig-PE) was reacted for 30 minutes at 4 ℃ and fixed with PBS containing 2% PFA after washing. Fluorescence was measured by flow cytometry (Epics XL. Coulter, Marseille).

As a result, as shown in FIG. 2, the expression rate of MHC II was significantly improved on the cell surface of the cells (right, B16F1 - CIITA), which had not been introduced to the CIITA gene (left, B16F1). Expression of 2 remained at the equivalent level. When CIITA was introduced, MHC class II protein was expressed on the cell surface.

At this time, it did not affect the expression level of the tumor antigen TRP-2.

< Example  3: CⅡTA  In the cell line where the gene was introduced Exosomes  Separation>

Exosomes were isolated from the cell cultures of the cell line into which the CIITA gene of Example 1 was introduced and from the cell line without the same. Specifically, first, the medium containing 10% petal bovine serum (central bovine serum) was centrifuged at 100,000 xg for 16 hours or more using an ultracentrifuge to remove bovine-derived exosomes. Prepare.

 After the cell culture, the culture solution was obtained two days later and filtered using a 45Ti rotor (Beckman, Coulter Instruments, Fullerton, Calif.) For 5 minutes at 300 x g, 20 minutes at 1,200 x g, and 30 minutes at 10,000 x g. The filter medium was centrifuged at 100,000 x g for 1 hour to obtain pellets, which were dissolved in PBS. The exosomes thus obtained were quantified using the Bradford method (Bio-rad) method.

   To obtain more purified exosomes, resuspend in 2.5 mM sucrose in 20 mM Hepes buffer (pH7.4) and load them into sw41 tubes using a sucrose gradient method, and load them onto 2M sucrose / 20 mM Hepes buffer. Gradients were prepared by loading 0.25M sucrose / 20mM Hepes buffer sequentially. The exosomes were separated by ultracentralization (O / N) at 100,000 x g to obtain 12 fragments of 1 ml of each fragment separated according to a gradient.

In Figure 3 a picture is a photograph of the exosomes observed by electron microscopy.

In Figure 3 b is a biochemical analysis of the separated exosomes, specifically, the labeling expression was identified by Western blot from the fraction obtained by centrifugation of the isolated exosomes (sucrose gradient). . Specifically, cells and exosome lysate were denatured with SDS-sample buffer at 95 ° C. for 5 minutes, loaded with 20 ug of protein in 12% SDS-PAGE, followed by electrophoresis, and polyvinylidene difluolide (PVDF). ) Was transferred to membrane. The membrane was treated with 5% skim milk / TTBS at room temperature for 1 hour, and the primary antibodies (anti-Tsg101, anti-GM1) were reacted at room temperature for 1 hour, and the secondary antibody with HRP was attached at room temperature for 30 minutes. Reacted. This labeled protein was identified by the band using the ECL kit.

As a result, Tsg101 and GM1, previously known as exosome-specific substances, could be obtained from the same fragment. This means that the exosomes can be purely separated from the cell line into which the CIITA gene is introduced by a conventional exosome separation method.

< Example 5: Immunobiochemical Analysis of Cell Line Self-Protein and Exosomes Isolated from It > It was observed by Western blot whether MHC class II was expressed in exosomes isolated from cell lines into which the CIITA gene was introduced. More specifically, Example 4 in the a nti-Tsg101 and anti-GM1 method Same as the primary antibody at different only (that use of any antibody to please specifically described) to observe the expression in anti-MHC class II.

4B shows the expression of MHC class I and II on the surface of exosomes through FACS results. Specifically, 5x10 ⁵ cells were prepared in the same manner as in the FACS method of TRP-2 of Example 2 described above, resuspended in 1% BSA / PBS, and the fluorescently labeled anti-MHC class I and anti-MHC class II antibodies were 4 The reaction was carried out for 1 hour at ℃ and the degree of fluorescence expression was confirmed by flow cytometry.

As a result, the expression of MHC class II was significantly increased in the exosomes obtained from the cell line into which MHC class II was introduced, and the expression of the tumor antigen TRP2 was also increased. In addition, expressed MHC class II could be observed on the surface of exosomes. This means that the MHC II protein, which was not present in the tumor exosomes, is well expressed.

< Example  6: by dendritic cells Exosomes  Intake>

 When exosomes were added to dendritic cells, the exosomes were labeled with fluorescent material and then dendritic cell line DC2.4 [Kenneth, USA] to see if MHC class II containing exosomes could be delivered to dendritic cells. (kenneth) Professor's team generated DC from the spinal cord of C57bl / 6 mice, and then introduced them into cell lines by introducing oncogenes such as ras. How taken up by dendritic cells was observed by fluorescence microscopy (FIG. 5). Specifically, fluorescently labeled exosomes were added as a result of mixing DC2.4 cells on a 6 well plate the day after mixing with N-Rh-PE for one day, and after 15, 30, 60 minutes, As observed by the fluorescence microscope, the development of fluorescence by dendritic cells means that the exosomes labeled with the fluorescent substance were ingested by the dendritic cells, and the intake levels were the exosomes added with CIITA and the exosomes not added. This was the same level.

5 is an electron micrograph comparing the degree of ingestion of exosomes by dendritic cells, and it can be seen that ingestion of exosomes is made by dendritic cells through which MHC class II proteins possessed by exosomes are delivered to dendritic cells. It means you can be. Thus, it can be expected that dendritic cells readily acquire MHC class II epitopes for tumor antigens, thereby broadening the range of immune responses to tumors and inducing improved anticancer immune responses.

< Example  7: MHC class Ⅱ  Expression On exosomes  Activation of dendritic cells

Activation of dendritic cells by MHC class II expression exosomes was compared by FACS centering on the expression of MHC class II and CD86, the activation markers on dendritic cell surface (FIG. 6). More specifically, after 24 hours after laying DC2.4 cells, the cells were collected and after 24 hours, the cells were coated with antibodies to MHC class II and CD86 with fluorescence, and their fluorescence was analyzed by flow cytometry. Fluorescence levels were graphed. Below PBS (phosphate-buffered saline) means an exosome solvent, B16 Exo is a B16-derived exosome, CII Exo is an exosome isolated from the cell CIITA introduced into B16.

As a result, it can be seen that the expression of activation markers is significantly increased by the exosome (B16-CIITA Exo) in which the CIITA gene is introduced. This means that injection of CIITA has increased the ability of exosomes to stimulate dendritic cells .

< Example  8 : On exosomes  Activation of dendritic cells Cytokine  Comparative measurement with yield>

 The degree of activation of dendritic cells by exosomes was measured by cytokine production (FIG. 7). Specifically, the next day after laying DC2.4 cells, each sample was given and after 24 hours, the cells were collected, 1 ml of trizol was added, chloroform was added after 5 minutes, centrifuged for 15 minutes, and the upper layer was extracted. After isopropanol was mixed, centrifugation was performed again. 75% ethanol was added to the resulting pellet and centrifuged once more. The resulting pellets were dried in air to within 10 and finally dissolved in RNAse free DW. After quantification with a spectrophotometer, 3 ug of RNA was used to make cDNA. 3 ug RNA is mixed with dNTP, oligo dT, reverse-trasncriptase set and reacted at 37 degrees. After 1 hour, PCR is performed using 1 ul of each sample as a template. PCR reaction mixture (specific primer, dNTP, Taq polymerase set). The product generated after PCR was confirmed by staining with EtBr after electrophoresis.

 As a result, mRNA levels of the inflammatory cytokine TNFa and dendritic cell marker CCR7 were increased by the CIITA-induced cell-derived exosomes, and the amount of Th1-directed cytokine IL-12 was significantly increased. It appears in CCR7 upon activation of terrestrial cells. Proteins such as TNFa and IL-12 were further increased by CIITA injected exosomes.

< Example  9: measuring activation of T cells>

When exosomes from CIITA-introduced cell lines are mediated through dendritic cells, the activation of T cells is increased, or each exosome is injected into dendritic cells, co-cultured with T cells, and when T cells are activated in the culture medium. The amount of cytokine secreted IL-2 was measured (FIG. 8). Specifically, each exosome was put in DC2.4 and co-cultured with naive T cells after 4 hours. Culture cultures were obtained and some were subjected to ELISA for IL-2, and some were used for the culture of CTLL-2D, an IL-2-dependent cell line. IL-2 is a cytokine produced when T cells are activated, and CTLL-2D is required for IL-2 for cell division, and cell proliferation rate varies according to the degree of IL-2 production.

 As a result, it was confirmed that the activation of T cells was further increased by the cell line-derived exosomes introduced with the CIITA gene.

< Example  10: in serum Tumor specificity  Antibody Test>

In order to analyze the immune response generated by immunization with each exosome, three immunizations were performed with exosomes and serum was separated from rats one week after each immunization. To determine how much tumor antigen specific antibody is present in the isolated serum, the separated serum was serially diluted so that the total antibody production and the type of antibody produced were isotyped against the total antibody production and IgG1 and IgG2a types in the produced antibody. Isotyping was analyzed using a kit capable of testing antibody isotypes (FIG. 9). Specifically, the serially diluted serum is allowed to react on an ELISA dish coated with exosomes. After 1 hour, the anti-mouse antibody was added, and after 1 hour, the substrate for the enzyme attached to the anti-mouse antibody was added to induce the color reaction by the enzyme-substrate reaction, and the color development was measured. In the case of isotyping, an antibody capable of isotyping-specifically distinguishing an anti-mouse antibody is used.

As a result, in the total antibody production, Th2 type IgG1 antibody production, and Th1 type IgG2a antibody production, the highest amount of antibody was produced in the mice injected with the cell line-derived exosomes into which the CIITA gene was introduced, It was shown to be proportional.

< Example  11: Measurement of tumor growth inhibitory effect>

To compare tumor growth inhibitory effects in mice, mice were immunized with each of the exosomes and tumors were injected and tumor growth was compared with the survival rate of the immunized mice (FIG. 10). Specifically, each sample was injected three times at two-week intervals (7 per group), followed by 5 * 10 ^ 5 B16 tumor cells per mouse one week after the last immunization. Tumor size and survival rate of mice (ie, the number of mice remaining undigested after tumor injection / number of experimental mice * 100) were measured over time.

As a result, as shown in Figure 10, when treated with cell line-derived exosomes introduced with the CIITA gene, tumor growth was the slowest and the survival rate of the mice was improved, which was proportional to the concentration of the injected exosomes.

The exosomes of the present invention and cancer vaccine compositions containing the same have an increased effect of CD4 helper T lymphocytes, which can reinforce major histocompatibility II and Th1 type immune responses, as compared to exosomes derived from conventional tumors. It is a very important invention in the medical and health industry because it is more effective in inducing reactions.

1 is a cleavage map of the CIITA gene.

FIG. 2 shows the results of flow cytometry confirming the expression of MHC class II and the expression of endogenous antigen TRP-2 in the cell line into which the CIITA gene was introduced.

Figure 3 is an electron micrograph of the separated exosomes and their biochemical properties are confirmed by Western blot.

4 is a graph analyzing the protein expression in MHC class II expressing cell line and exosomes isolated from the cell line through FACS.

5 is a photograph observing how exosomes are ingested by dendritic cells over time with fluorescence microscopy.

6 is a graph comparing the activation of dendritic cells by MHC class II expressing exosomes by FACS.

7 is a graph comparing the degree of activation of dendritic cells by exosomes with cytokine production.

8a and 8b are graphs showing the degree of activation of T cells by each exosome.

Figure 9 is a graph of the total antibody production and the type of antibody produced is analyzed by using a kit that can test the isotype of the antibody to the total antibody production and IgG1 and IgG2a type in the produced antibody.

10 is a graph comparing the growth rate of tumors and the survival rate of immunized mice after immunizing mice and injecting tumors.

Claims (10)

Exosomes to which MHC class II expression inducing genes have been introduced. The exosome according to claim 1, wherein the expression inducing gene is MHC class II transactivator (CIITA). The exosome according to claim 1, wherein the exosomes are derived from tumor cells. Dendritic cells injected with the exosomes of claim 1. The dendritic cell according to claim 4, wherein the dendritic cell has an increased action of major histocompatibility II and CD4 helper T lymphocytes. (1) cloning a gene that induces the expression of major histocompatibility II in a retroviral vector; (2) introducing the cloned retroviral vector into a tumor cell line; And (3) a method for producing an exosome, comprising the step of separating the exosome from the tumor cell line. The method of claim 6, wherein the gene is MHC class II transactivator (CIITA) and the tumor cell line is a B16F1 tumor cell line. Cancer vaccine composition comprising the exosomes of claim 1 or the exosomes prepared by the method of claim 6 as an active ingredient, and comprises 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.

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