JP2018070572A - Immunotherapy system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide immunotherapy systems or cell formulations which exert high therapeutic effect if being using to cancer and have less side effects as well as can be effectively used also in infection prevention/treatment.SOLUTION: The present invention provides a system or a cell formulation for a tumor treatment or microbial infection prevention/treatment produced using an exosome obtained by culturing after introducing into cells an antigenic protein or peptide as a foreign "danger signal", or the gene of a protein or peptide comprising an epitope thereof, and recovering and isolating from the culture supernatant.SELECTED DRAWING: None

Description

  The present invention relates to an immunotherapy system for cancer and the like and a microbial infection prevention / treatment system obtained using exosomes obtained from a gene-transferred cell.

  As immunotherapy for diseases such as cancer, DNA vaccines encoding various tumor-associated antigens (hereinafter TAAs) have been tried, and their effects have been reported, although limited (Non-patent Document 1). However, TAAs are originally antigens of cells in which normal cells of the same individual have become cancerous, and are usually expressed to some extent in normal cells, albeit in different amounts. For this reason, TAAs are generally low in immunogenicity, and it has always been a problem that even when TAAs protein itself or DNA encoding it is administered, sufficient immune response cannot be induced. Treatment of B cell tumor with anti-idiotype vaccine Except for some examples, sufficient effects are not recognized (Non-Patent Document 1).

  On the other hand, in recent years, new cancer diagnosis and treatment methods using exosomes have attracted attention. In particular, since it was reported in 2007 that exosomes contain mRNA and microRNA derived from secretory cells, research on the use of exosomes for cancer diagnosis as cancer carriers and nucleic acid drug carriers has rapidly developed. Exosomes released from tumor cells are known to have tumor-associated antigens (TAAs), and attempts have been made to introduce an anti-tumor immune response using exosomes. However, TAAs are generally low in immunogenicity, and without the combined use of cytokines such as granulocyte monocyte colony stimulating factor (GM-CSF), the effect of inducing an immune response is observed even when exosomes themselves are administered. There was not (nonpatent literature 2). Therefore, tumor cell-derived exosomes modified with cytokine, interleukin-12 (IL-12), were made, and in vitro experiments showed that these have the effect of suppressing immunosuppression (non-patent literature) 3), but the healing effect in the living body has not been confirmed. In addition, immune induction was attempted using exosomes derived from dendritic cells (DC) pulsed with TAAs instead of tumor cell-derived exosomes. However, these DC-derived exosomes show an immunization effect on mature DCs, but no effect was observed on immature DCs (Non-patent Document 4).

  Tumor-associated antigens (TAAs) of primary tumor cells are weakly antigenic as described above, and are often expressed in normal cells, so exosomes released from tumor cells and dendrites pulsed with TAAs are used. Only exosomes derived from cells (DC) are insufficient to elicit anti-tumor immunity, and a sufficient healing effect cannot be obtained. Without the presence of a “foreign danger signal” for activating the immune system, it is difficult to elicit immunity against weakly antigenic TAAs.

  In addition to cancer, infectious diseases are important diseases that continue to kill many people every year. More than 3 million people die each year from AIDS, tuberculosis, and malaria alone, which are called the three major infectious diseases, and it is still one of the leading causes of death in many low- and middle-income countries. Research on infection prevention using exosomes has also become active in recent years. Studies using exosomes secreted from macrophages infected with Mycobacterium tuberculosis (Non-Patent Document 5) and studies on prevention of infection using exosomes from DC stimulated with Toxoplasma antigen (Non-Patent Document 6) have been conducted. .

  However, bacterial infection and stimulation of antigen-presenting cells with isolated toxoplasma antigen require complicated methods, special equipment, and technology, and therefore cultivate easy and versatile methods that can be widely applied in general. Is desired.

  The present inventor has found that tumor-associated antigens (TAAs) of primary tumor cells are weakly antigenic as described above and are often expressed in normal cells. Thought to be insufficient to trigger. Therefore, in order to activate the immune system, it was thought that the presence of a “foreign danger signal” was necessary, and the “danger signal carrying exosome preparation derived from an antigen gene-introduced cell” was devised. That is, it is a preparation that effectively induces and induces immunity using exosomes obtained from cultured cells into which a highly antigenic protein gene has been introduced.

  It was confirmed that exosomes obtained from cultured cells introduced with the gene of this highly antigenic protein elicit high cellular immunity when administered to a living body, and a remarkable tumor growth inhibitory effect was observed.

  However, exosomes are very fine particles, and are already a mixture of large and small particles when they are secreted. In addition, the shape and component properties are not constant depending on the cells used, production conditions, purification techniques, and the like. For this reason, it is not easy to prepare a preparation for direct administration to a living body.

J Biomed Biotech, 2010 (2010), Article ID 596432, 13 pages. Molecular Therapy (2008) 16 4, 782-790 Int J Mol Med. 2010 May; 25 (5): 695-700. J Immunol 2004; 172, 2126-2136 PLoS One. 2008 Jun 18; 3 (6): e2461 INFORMATION AND IMMUNITY, July 2004, 4127-4137

  Exosome preparations are attracting widespread attention because of their high potential, but production conditions, purification methods, etc. have not been established, and production is highly heterogeneous and difficult to reproduce. It is not easy to use these widely as general therapeutic means.

  As a result of earnest research to solve the above drawbacks, as described above, the exosome obtained by introducing the gene of the antigen protein into the parent cell is not directly administered directly into the living body, but the cell of the immune system In addition to activating this, the immune system cells are then administered (returned) in vivo to achieve the desired immune prevention. It was found that a therapeutic effect can be obtained. That is, the present invention relates to a method of introducing a protein gene with high antigenicity into cultured tumor cells and using the immune system cells obtained by adding exosomes as antitumor immunity preparations and infection prevention / treatment preparations.

  When tumor cells are used as parental cells, the secreted exosome is a powerful cancer vaccine that combines both “danger signals” and TAAs. In fact, using a DNA complex that encodes the gene of Mycobacterium tuberculosis antigen protein as a danger signal, after introducing the "anger signal" gene into tumor cells, the culture was continued and the exosomes were secreted, recovered and isolated After adding exosomes to cultured dendritic cells, the dendritic cells were administered into the tumor of a cancer model mouse. Surprisingly, the therapeutic effect was significantly higher than that of dendritic cells to which exosomes derived from tumor cells without gene transfer (having no danger signal) were added. The excellent effect of immune cell activation by the “supported exosome” was confirmed. At the same time, the importance of “danger signal” in anti-tumor immune activation was confirmed.

  The immune cells to be administered in the present invention have high accumulation in tumor tissues and regional lymph nodes, and since the toxicity of the cells is low and there are few side effects, local administration, intraperitoneal administration, intraarterial administration, intravenous administration Various administrations such as intracranial administration are possible. Intravenous administration of dendritic cells supplemented with angersome-bearing exosomes confirmed high antitumor effects and was also effective in the treatment of metastatic cancer.

  Surprisingly, we added exosomes generated by transfecting a danger signal into a different type of tumor cell from the diseased tumor, and exosomes generated by transducing a danger signal into normal cells as parent cells. Dendritic cells also showed a high tumor growth inhibitory effect. This is probably because tumor cells are phagocytically active and are likely to take up exosomes, and the antigens or epitopes of the taken up exosomes can be rebound to their major histocompatibility complex. That is, it was shown that the exosome derived from the cell into which the antigen gene was introduced can be used effectively against other types of tumors, whether the parent cell is another family or normal cell. .

  In addition, there are various types of TAAs and fragments, such as exosomes with TAAs secreted from tumor cells, TAAs themselves, or cell fragments containing them, in the living body of the patient to be treated. ing. It seems that an antitumor effect appeared by exerting a concerted effect on the immune system cells to which they were administered. Alternatively, exosomes with “danger signals” themselves may have the effect of activating immune system cells as powerful adjuvants.

  In addition, exosomes secreted from cells into which pathogen antigen genes have been introduced contain these pathogen antigens or epitopes thereof as “danger signals”. Therefore, it was found to induce strong cellular immunity against these antigens. This shows that the immune system cell to which the exosome of the present invention is added is very effective in preventing and curing microorganism infection.

  Immune system cells to which the exosome of the present invention is added have high accumulation in tumor tissues and regional lymph nodes and low toxicity, so local administration, intraperitoneal administration, intraarterial administration, intravenous administration, intracranial administration, etc. Can be administered in various ways. In addition, many preparation, purification, and storage methods from progenitor cells have already been established, and handling is simple. Furthermore, in one embodiment of the present invention, the curative effect can be improved by using in combination with other antitumor immunotherapy preparations.

  The antitumor effect by administering the dendritic cell which added the exosome obtained from B16 cell which introduce | transduced the gene of the protein with high antigenicity to the mouse | mouth transplanted with B16 cell is shown.   The antitumor effect by administering the dendritic cell which added the exosome obtained from the CHO cell or fibroblast which introduce | transduced the gene of the protein with high antigenicity to the mouse | mouth transplanted with B16 cell is shown.   FIG. 5 shows the effect of inducing cellular immunity against tumor cells by administering to mice mice dendritic cells added with exosomes obtained from B16 cells or fibroblasts introduced with highly antigenic protein genes.   The effect of inducing cellular immunity against the microbial antigen by administering to a mouse dendritic cells added with exosomes obtained from B16 cells or fibroblasts introduced with microbial genes is shown.

  As a danger signal gene to be introduced into a cell according to the present invention, any anti-tumor effect can be expected. It is possible to use a gene of a protein or peptide containing the above-mentioned epitope. Since the effect as a danger signal is derived from its high antigenicity, if a high effect is expected, a highly antigenic protein or peptide, or a protein or peptide gene containing these epitopes should be used. Is desirable. For example, antigenic protein genes of viruses, bacteria, protozoa, heterologous organisms, and heterologous animals can be used.

  As long as an anti-infection effect is expected, antigen proteins, peptides, or proteins containing the epitope of the pathogen of the target disease, peptide genes, or proteins having similar structures, peptide genes can be used. In addition, if used as an adjuvant for activation of immune system cells, highly antigenic proteins, peptides, or proteins and peptides containing these epitopes can all be used.

  In the present invention, the cells that introduce the danger signal gene and secrete exosomes can be cultured cells, regardless of whether they are cultivated or not, tumor cells, fibroblasts, blood cells, DCs, macrophages. Any animal / plant / microbe cells can be used.

  As a method for introducing a gene into a cell according to the present invention, a method using a virus, a method using calcium phosphate, a polycation, a cationic lipid or an aggregate thereof, an electroporation method, a microinjection method and the like can be used. In Examples, the same DNA complex as described in Non-Patent Document 8 was used. The characteristics of the composite are described below. However, the description of this feature is for explaining the present invention and does not limit the present invention.

  Even if a gene is introduced into a cultured cell using an artificial vector (polycation, cationic lipid, etc.), the gene expression period is short, and the expression usually disappears after 3 to 4 days. Also, when many DNA complexes are added to cells to increase expression, the cells become weak due to their toxicity and die within a day or two. In order to efficiently prepare exosomes carrying a danger signal, conditions for secreting exosomes for a long period of time while expressing genes introduced by cells are necessary. We have developed a DNA ternary complex system in which a polyanion is added to the conventional DNN / polycation (or cationic lipid) complex in order to obtain a gene transfer system that is highly expressed in vivo. However, various effects have been achieved (Patent Document 1). This DNA ternary complex system is suitable for the efficient preparation of exosomes because gene expression lasts for a relatively long period of time (Non-patent Document 7) and has low cytotoxicity (Non-patent Document 8).

PCT / JP2007 / 060002

J Pharmaceutical Sciences, 103, 179-184, 2014 Pharmaceuticals 2015, 7, 165-174.

  Cells transfected with various methods can be cultured for several days, and exosomes carrying the desired danger signal can be easily obtained from the supernatant by ordinary methods such as ultracentrifugation and exosome isolation reagents.

  Immune system cells can be easily collected, isolated and cultured from blood, from ascites, water supply, lymph tissue, spleen, and the like by conventional methods. In addition, immune system cells such as dendritic cells can be obtained by easily differentiating and culturing from undifferentiated cells such as monocytes in blood by an ordinary method.

  The obtained immune system cells with added exosomes carrying the Danger signal can be administered to a living body by intra-tumor, para-tumor, subcutaneous, intravenous, intra-arterial, etc. Easier to administer.

  The present invention will be described more specifically with reference to examples. In addition, these Examples are for demonstrating this invention, Comprising: This invention is not limited at all.

Control sky from exosomes (ES-B16-Ex) from B16 melanoma cells transfected with the gene of Early Secret Antigen Target-6 (ESAT-6), an antigenic protein of Mycobacterium tuberculosis, and B16 melanoma cells not transfected Isolation of Exosome (Empty-B16-Ex) A plasmid encoding ESAT-6 (ESAT-6 plasmid) and PEI “Max” were the same as those described in Non-Patent Document 8. As the HA, hyaluronic acid “derived from Tritosaka” manufactured by Seikagaku Corporation was used. The PBS used was a solution of Phosphate Buffered Salts (Tablet) manufactured by Roman Industries in distilled ion-exchanged water. The same applies to the following embodiments.

Operating procedure (1) Three days before gene introduction, 1.0 × 10 ⁴ B16 melanoma cells per well are plated in a 24-well multiplate, 10% fetal bovine serum (FBS), penicillin (100 units / mL) And Eagle's Minimum Essential Medium (EMEM) medium containing streptomycin (0.1 mg / mL).
(2) The cultured medium was removed, and 500 μl of EMEM medium containing 10% exosome-free FBS, penicillin (100 units / mL), and streptomycin (0.1 mg / mL) was added to the wells.
(3) After 4.84 μl of an aqueous solution containing 1.5 μg of ESAT-6 plasmid was mixed with 1.78 μl of CS solution (5 mg / ml) so that the DNA / CS ratio (charge molar ratio) was 8, 88 μl of PEI “Max” aqueous solution (5 mg / ml) was mixed with DNA / PEI “Max” ratio (charge molar ratio) to 12, and after 20 minutes, 7.5 μl of 2-fold concentrated PBS was added. .
(4) 15 μL of the DNA complex suspension prepared in (3) was added to the well (1.5 μg / well of ESAT-6 plasmid).
(5) The plate was incubated at 37 ° C. and 5% CO 2 -95% air for 5 hours.
(6) The medium was replaced with 1 ml of EMEM containing fresh 10% exosome-free FBS, 25 U penicillin and 25 μg streptomycin, and incubated at 37 ° C. for 72 hours.
(7) After 72 hours of incubation, the supernatant in the well was collected.
(8) After centrifugation (3000 × g, 15 minutes), the supernatant was collected, and half the volume of Total Exosome Isolation (from the cell culture media (The Thermo Fisher Scientific, Inc., USA, USA). ).) Was added and left at 4 ° C. overnight.
(9) After centrifugation (10000 × g, 1 hour), the precipitated exosome was suspended in 0.16 ml of PBS. The exosome suspension was stored at 4 ° C. and used within a week.
(10) A similar experiment was carried out on B16 cells into which no gene had been introduced to prepare a control empty exosome (Empty-B16-Ex) suspension.

Results Both exogenous and non-transfected ones were mixed with Exosome Isolation (from cell culture media) and centrifuged overnight to obtain white exosome precipitates. Exosomes in the resuspended solution are stained with Exo-green or Exo-red (both System Biosciences (San Francisco, Inc., CA, USA)) and ExoQuick-TC (System Francisco, Inc., San Francisco, Inc., San Francisco, Inc.). USA)) and observed with a fluorescence microscope, it was confirmed that a bright spot with a diameter of several tens of nanometers was floating at the same position, and that an exosome suspension was indeed obtained. It was.

Isolation of exosomes (Ag-B16-Ex) from B16 melanoma cells into which the gene of Mycobacterium tuberculosis Major Major Protein Protein 85B (Ag85B), which is an antigenic protein of Mycobacterium tuberculosis, was introduced in the same manner as in Example 1, B16 melanoma After the Ag85B gene was introduced into the B16 melanoma cell-derived exosome (Ag-B16-Ex) into which the Ag85B gene had been introduced was isolated from the culture supernatant.

Isolation of exosomes (AD-B16-Ex) from B16 melanoma cells into which the gene for adenovirus antigen protein (ADP), an adenovirus antigen protein, has been introduced In the same manner as in Example 1, the ADP gene was transferred to B16 melanoma cells. After the introduction, B16 melanoma cell-derived exosome (AD-B16-Ex) introduced with the ADP gene was isolated from the culture supernatant.

Isolation of Exosome (OVA-B16-Ex) from B16 Melanoma Cells Introduced with Ovalbumin (OVA) Gene After introducing the OVA gene into B16 melanoma cells in the same manner as in Example 1, An exosome derived from B16 melanoma cells (OVA-B16-Ex) into which the OVA gene was introduced was isolated from the culture supernatant.

Isolation of exosomes (ES-CHO-Ex) from Chinese hamster ovary cells (CHO cells) introduced with the gene for early secretory antigenic 6 kDa (ESAT-6), an antigenic protein of Mycobacterium tuberculosis, the same method as in Example 1 The CHO cell derived from the culture supernatant after introducing the ESAT-6 gene into the CHO cell, and the exosome derived from the CHO cell into which the gene has not been introduced (ES-CHO-Ex) (Empty-CHO-Ex) was isolated.

Isolation of exosomes (ES-Fb-Ex) from fibroblasts introduced with the gene of early secretory antigenic 6 kDa (ESAT-6), an antigenic protein of Mycobacterium tuberculosis , collected under anesthesia, shaved after ethanol sterilization The mouse ear pieces were cut into approximately 3 mm × 3 mm and treated with collagenase D and pronase to obtain fibroblasts. After culturing this and introducing the ESAT-6 gene in the same manner as in Example 1, fibroblast-derived exosomes (ES-Fb-Ex) introduced with the ESAT-6 gene were isolated from the culture supernatant. did.

Dendritic Cell Differentiation Culture Immature dendritic cells were obtained from the peripheral blood of mice using monocyte-derived dendritic cell differentiation induction kits.

Antitumor effect of dendritic cells added with exosomes secreted from cells introduced with a danger signal gene (1)
(1) B16 cells were transplanted subcutaneously into C57BL / 6 mice (male, 5 weeks old) to prepare a cancer model mouse.
(2) When the diameter of the tumor exceeded 4 mm, the dendritic cells obtained by adding each exosome suspension (200 μL) obtained in Examples 1 to 4 and culturing overnight were placed in the tumor three times in 3 days. After administration, changes in tumor size were examined.

Results The results are shown in FIG. The vertical axis of the figure is the average value of tumor size. Dendritic cells cultured with the addition of exosome, Empty-B16-Ex, obtained from B16 cells not introduced with a danger signal gene did not show a significant antitumor effect, whereas a danger signal gene was introduced. Dendritic cells cultured overnight with exosomes, AD-B16-Ex, Ag-B16-Ex, ES-B16-Ex, OVA-B16-Ex obtained from B16 cells showed a clear antitumor effect .

Antitumor effect of dendritic cells added with exosomes secreted from cells introduced with a danger signal gene (2)
(1) B16 cells were transplanted subcutaneously into C57BL / 6 mice (male, 5 weeks old) to prepare a cancer model mouse.
(2) When the diameter of the tumor exceeded 4 mm, the dendritic cells obtained by adding each exosome suspension (200 μL) obtained in Examples 5 and 6 and culturing overnight were placed in the tumor three times in 3 days. After administration, changes in tumor size were examined.

[result]
The results are shown in FIG. The vertical axis of the figure is the average value of tumor size. The dendritic cells cultured overnight with the addition of exosomes, Empty-CHO-Ex, obtained from CHO cells not introduced with the danger signal gene did not show a significant antitumor effect, whereas the ESAT-6 gene Dendritic cells cultured overnight with ES-CHO-Ex, an exosome obtained from CHO cells into which CHO has been introduced, have a higher antitumor effect and are effective against different types of tumors. Exosomes have also been shown to induce antitumor effects. Similarly, dendritic cells cultured overnight with ES-Fb-Ex also showed clear antitumor effects, and it was confirmed that exosomes derived from normal cells into which a danger signal gene was introduced also lead to antitumor effects. .

Cellular immunity transduction effect of dendritic cells added with exosomes secreted from cells introduced with a danger signal gene (1)
(1) Dendritic cells obtained by adding the exosome suspension obtained in Example 1 or 6 and cultivating overnight were once applied to the soles of both hind paws of C57BL / 6 mice (male, 5 weeks old) once by 75 μL. Administered.
(2) On the ninth day, lymph nodes on the back of knees of both hind paws were collected, crushed and passed through a mesh, and suspended in RPMI 1640 medium.
(3) The resulting lymphocyte suspension was added to a 96-well culture plate together with mitomycin-treated B16 melanoma cells, incubated at 37 ° C. for 2 days, and then secreted IFN-γ was released into the Mouse IFN-gamma Quantikine ELISA Kit (R & D). Quantified by Systems, Inc. (Minneapolis, MN, USA).

[result]
The results are shown in FIG. The values in the figure represent average values and standard deviations of three or more cases. Dendritic cells cultured by adding exosome ES-B16-Ex obtained from B16 cells introduced with ESAT-6 gene, or exosome obtained from fibroblasts introduced with ESAT-6 gene, ES-Fb-Ex The lymphocytes of mice administered with dendritic cells cultured with the addition of exosomes obtained from B16 cells into which ESAT-6 gene has not been introduced, Empty-B16-Ex, were added and cultured. Compared to the lymphocytes of the administered mice, incubation with B16 cells for 2 days clearly secreted more IFN-γ. From this, it was confirmed that the dendritic cells cultured by adding exosomes obtained from tumor cells into which the danger signal gene was introduced or normal cells had an effect of inducing cellular immunity against the tumor cells.

Cellular immunity transduction effect of dendritic cells added with exosomes secreted from cells introduced with a danger signal gene (2)
(1) Dendritic cells obtained by adding the exosome suspension obtained in Example 1 or 6 and cultivating overnight were once applied to the soles of both hind paws of C57BL / 6 mice (male, 5 weeks old) once by 75 μL. Administered.
(2) On the ninth day, lymph nodes on the back of knees of both hind paws were collected, crushed and passed through a mesh, and suspended in RPMI 1640 medium.
(3) The obtained lymphocyte suspension was added to a Quantferon-TB Gold blood test tube containing 1 μg of ESAT-6, incubated at 37 ° C. overnight, and then secreted IFN-γ was added to Mouse IFN-gamma Quantikine. Quantification was performed by ELISA Kit (R & D Systems, Inc, (Minneapolis, MN, USA)).

[result]
The results are shown in FIG. Here, the values in the figure represent average values and standard deviations of three or more examples. Dendritic cells cultured by adding exosome ES-B16-Ex obtained from B16 cells introduced with ESAT-6 gene, or exosome obtained from fibroblasts introduced with ESAT-6 gene, ES-Fb-Ex The lymphocytes of mice administered with dendritic cells cultured with the addition of exosomes obtained from B16 cells into which ESAT-6 gene has not been introduced, Empty-B16-Ex, were added and cultured. Compared to the lymphocytes of the administered mice, overnight incubation with ESAT-6 antigen protein clearly secreted more IFN-γ. From this, it was confirmed that dendritic cells cultured by adding exosomes obtained from tumor cells into which microbial genes have been introduced or normal cells have the effect of inducing cellular immunity against the microbial antigens.

Claims (54)

Prophylactic / therapeutic system created using cell-derived exosomes introduced with highly antigenic protein genes Immune system cells cultured with the addition of cell-derived exosomes that have been introduced with highly antigenic protein genes The cell of claim 2, wherein the immune system cell is a dendritic cell. The system according to claim 1, wherein the highly antigenic protein is a microbial antigen. The cell of claim 2, wherein the highly antigenic protein is a microbial antigen. The cell according to claim 3, wherein the highly antigenic protein is a microbial antigen. The system according to claim 1, wherein the highly antigenic protein is a Mycobacterium tuberculosis antigen. The cell according to claim 2, wherein the highly antigenic protein is a Mycobacterium tuberculosis antigen. The cell according to claim 3, wherein the highly antigenic protein is a Mycobacterium tuberculosis antigen. 2. The system according to claim 1, wherein the highly antigenic protein is the Mycobacterium tuberculosis antigen early security antigenic 6 kDa (ESAT-6). The cell according to claim 2, wherein the highly antigenic protein is the Mycobacterium tuberculosis antigen early secretory antigenic 6 kDa (ESAT-6). 4. The cell according to claim 3, wherein the highly antigenic protein is Mycobacterium tuberculosis antigen early secretory antigenic 6 kDa (ESAT-6). The system according to claim 1, wherein the highly antigenic protein is a Mycobacterium tuberculosis Major Major Protein Protein 85B (Ag85B). The cell according to claim 2, wherein the highly antigenic protein is a Mycobacterium tuberculosis Major Major Protein Protein 85B (Ag85B). The cell according to claim 3, wherein the highly antigenic protein is a Mycobacterium tuberculosis Major Major Protein Protein 85B (Ag85B). The system of claim 1, wherein the highly antigenic protein is ovalbumin (OVA). The cell according to claim 2, wherein the highly antigenic protein is ovalbumin (OVA). The cell according to claim 3, wherein the highly antigenic protein is ovalbumin (OVA). The system of claim 1 for antitumor effects The cell according to claim 2, which has an antitumor effect. The cell according to claim 3, which has an antitumor effect. The system of claim 4 for antitumor effects The cell according to claim 5, which has an antitumor effect. The cell according to claim 6, which has an antitumor effect. The system of claim 7 for antitumor effects The cell according to claim 8, which has an antitumor effect. The cell according to claim 9, which has an antitumor effect. The system of claim 10 for anti-tumor effects 12. The cell of claim 11 for antitumor effects The cell according to claim 12, which has an antitumor effect. 14. The system of claim 13 for antitumor effects 15. The cell of claim 14 for antitumor effect The cell according to claim 15, which has an antitumor effect. The system of claim 16 for antitumor effects The cell of claim 17, which has an antitumor effect. 19. The cell of claim 18 for antitumor effects The system of claim 1 for the purpose of preventing infection and treating infectious diseases The cell according to claim 2 for the purpose of preventing infection or treating infection. The cell according to claim 3 for the purpose of preventing infection or treating infection. The system according to claim 4, which is intended to prevent infection and treat infections. The cell according to claim 5, which has an effect of preventing infection or treating infection. The cell according to claim 6 for the purpose of preventing infection or treating infection. The system according to claim 7 for the purpose of preventing tuberculosis infection and treating infectious diseases 9. The cell according to claim 8, which has an effect of preventing tuberculosis infection and treating infection. The cell according to claim 9, which has an effect of preventing tuberculosis infection and treating infection. The system according to claim 10, which is intended to prevent tuberculosis infection and treat infections. 12. The cell according to claim 11 for the purpose of preventing tuberculosis infection and treating infection. The cell according to claim 12, which is intended to prevent or infect tuberculosis infection. 14. The system of claim 13 for the purpose of preventing tuberculosis infection and treating infectious diseases 15. The cell according to claim 14 for the purpose of preventing tuberculosis infection and treating infection. The cell according to claim 15, which is intended for prevention of tuberculosis infection and treatment of infection. 5. The system according to claim 4, which is intended to prevent infection and treat infection by the microorganism. 6. The cell according to claim 5, which has an effect of preventing infection and treating infection by the microorganism. The cell according to claim 6, which is intended to prevent infection or treat infection by the microorganism.

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