KR20070105662A - B cell-based vaccine loaded with the ligand of natural killer t cell and antigen - Google Patents

Abstract

A B cell-based vaccine loaded with a ligand of natural killer T cell and an antigen is provided to improve easiness of collection, induce cytotoxic T lymphocyte reaction in a similar level to the dendritic cell vaccine, and prevent and treat subcutaneous cancer and metastatic cancer. A B cell-based vaccine contains a B cell loaded with a ligand of natural killer T cell, a B cell loaded with the ligand and an antigen, or a B cell loaded with the ligand and expressing the antigen, wherein the ligand of natural killer T cell is selected from alpha-galactosyl ceramide, alpha-glucuronosyl ceramide, phosphatidylinositol tetramannoside, isoglobotrihexosyl ceramide, ganglioside GD3, phosphatidyl choline, beta-galactosyl ceramide, lipophosphoglycan, glycoinositol phospholipid, alpha-galactosyl ceramide derivatives including beta-anomer galactosyl ceramide and alpha-anomer galactosyl ceramide, bacteria lipid antigen and alpha galactosyl ceramide mutant; and the antigen is derived from pathogenic bacteria, virus and parasite-containing pathogen.

Description

B cell-based vaccine loaded with the ligand of natural killer T cell and antigen

1 is a diagram showing bidirectional activation between alpha-galactosyl ceramide (αGalCer) loaded B cells and natural killer T cells ( i NKT),

Figure 1a is a graph showing the results of measuring the expression of CD19, CD11c and CD1d by flow cytometry after B11 cells were purely isolated from the spleen of the mouse after removing CD11c ⁺ cells,

FIG. 1B shows C57BL / 6 wild-type or Jα281 ^{− / −} mice treated with vehicle alone, αGalCer, αGalCer loaded B cells (B / αGalCer) or dendritic cells (DC / αGalCer), followed by IL-4 or IFN-γ The graph shows the result of ELISPOT analysis of the number of cells producing

1C is a graph showing the results of analyzing CFSE + cells isolated from spleen cells after vehicle or αGalCer loaded B cells were labeled with CFSE and administered to C57BL / 6 mice.

Figure 2 is a diagram showing the activation of peptide-specific CD8 + T cells by B cells loaded with peptide (OVA _257-264 ) and αGalCer,

FIG. 2A shows transplantation of CD8 + T cells specific for CFSE labeled OVA _257-264 (OT-I T cells) into C57BL / 6 mice followed by vehicle alone, B / αGalCer, B / pep, or B / αGalCer / pep After the administration of the OT-I T cell division and cytokine production is a graph showing the results measured by flow cytometry,

FIG. 2B shows that C57BL / 6 mice are injected with BSE alone, B / αGalCer, B / pep or B / αGalCer / pep, and injected with homologous targets labeled with CFSE after 1, 3, or 5 weeks. It is a graph showing the result of measuring cytotoxic T lymphocyte activity in vivo,

CFSE ^high : Target group loaded with peptide

CFSE ^low : No peptide loaded control

FIG. 2C shows the response of CD8 + T cells to produce IFN-γ by separating spleen cells of mice 7 days after administration of the B cell vaccine in vivo (left), and loaded with OVA _257-264 in vitro It is a graph which measured the number of CD8 + T cells which produce IFN- (gamma) which appear when restimulated with allogeneic splenocytes (right).

3 is a diagram showing the comparison of the efficiency of cytotoxic T lymphocyte formation with B cell-mediated cell vaccine and dendritic cell (DC) -mediated cell vaccine.

FIG. 3A shows serially diluting B / αGalCer / pep and DC / αGalCer / pep loaded with OVA _257-264 peptide and administering it to mice followed by measurement of in vivo cytotoxic T lymphocyte activity against OVA _257-264 peptide. It's a graph,

FIG. 3B shows in vivo cytotoxic T lymphocyte activity against OVA _257-264 peptide induced by administration of OVA _257-264 peptide and vehicle loaded B / vehicle / pep and DC / vehicle / pep in this manner It's a graph,

4 is a diagram showing the types of immune cells involved in inducing a cytotoxic T lymphocyte response by a B cell-mediated vaccine.

FIG. 4A is a graph showing in vivo cytotoxic T lymphocyte activity against OVA _257-264 peptide after injection of an antibody that removes specific immune cells before or after administration of B cells loaded with _αGalCer and OVA _257-264 peptide. ego,

Figure 4b is a wild type, Jα281 ^{- / -,} or MHC class II ^{- / -} treated with αGalCer with OVA _257-264 peptide is a B cell loaded with a mouse and a standard in vitro cytotoxicity against OVA _257-264 peptide ⁵¹ It is a graph shown by performing Cr emission analysis,

5 is a diagram showing that B cells loaded with _αGalCer and OVA _257-264 peptide act as direct antigen presenting cells to CD8 + T cells.

FIG. 5a shows the results of measuring the in vivo cytotoxicity of OVA _257-264 peptide after loading _αGalCer and OVA _257-264 peptide together in B cells isolated from wild-type or bm-1 mice, respectively, and administering to the wild-type mice. Is the graph shown,

5B shows OVA ₂₅₇ induced after immunization of C57BL / 6 mice using B cells loaded with _αGalCer and OVA _257-264 peptide or B cells loaded with OVA _257-264 peptide and B cells loaded with αGalCer, respectively. Graph showing in vivo cytotoxicity against _-264 peptide,

6 is a diagram showing that a B cell-mediated vaccine provides prophylactic and therapeutic anticancer immune activity against B16 melanoma that expresses OVA.

6A shows MO-5 cancer cells 7 days after immunizing C57BL / 6 mice with B cells alone, B / αGalCer, B / pep, B / αGalCer / pep, DC / pep, or DC / αGalCer / pep It is a graph showing the result of measuring the tumor size of the mouse transplanted subcutaneously,

FIG. 6B is a graph showing the tumor size measured after tumor transplantation and regeneration of MO-5 cells (2 × 10 ⁵ cells) 70 days after the first tumor was transplanted to mice without tumor formation at 6A,

In Fig. 6c and Fig. 6d is a C57BL / 6 mouse MO-5 tumor cells (2 × 10 ⁵ pieces) implanted in the subcutaneous, and, B cells, alone and after 9 days in 1 day, B / αGalCer, B / pep, B / αGalCer It is a graph showing the results of measuring the tumor size of mice by immunization with / pep, DC / pep, or DC / αGalCer / pep, respectively.

*, p <0.05: Significant difference from 'B cell only' control (FIGS. 6A, 6C and 6D) or group of mice that did not receive age-adjusted vaccine (FIG. 6B)

7 shows that the B cell-mediated vaccine induces an anticancer immune response in tumors expressing Her-2 / neu , which is an actual cancer antigen.

7a shows B cells alone, B cells loaded with _αGalCer , B cells loaded with Her-2 / neu _63-71 (hP63), B cells loaded with αGalCer and hP63, dendritic cells loaded with hP63, and αGalCer. It is a graph showing in vivo cytotoxicity induced after immunizing dendritic cells loaded with hP63 in each BALB / c mouse,

7B and 7C show that the CT-26-Her2 / neu was implanted into the tail vein (FIG. 7B) or subcutaneously (FIG. 7C) and immunized with the B cells or dendritic cells (DC). A graph showing the results of measuring survival rate (FIG. 7B) or tumor size (FIG. 7C).

8 is a diagram confirming whether the B cell vaccine can be applied to the method of transducing the whole antigen into the B cell virus,

FIG. 8a shows the detection of Her-2 / neu expressed on the surface of B cells after infection of B cells with adenovirus (AdHM) capable of expressing extracellular and cell membrane regions of Her-2 / neu , a tumor-associated antigen. Is the graph shown,

FIG. 8B is an experiment showing that B cells transformed with AdHM can activate DN32.D3 cells by loading αGalCer. B cells, B cells loaded with αGalCer, B cells transformed with AdHM, and AdHM IL-2 of the culture supernatant in which B cells loaded with αGalCer and DN32.D3 cells were cultured together was measured and measured by ELISA.

9 is a diagram showing whether B cells transduced with Her-2 / neu , a tumor-associated antigen, effectively induce a cytotoxic immune response in mice.

FIG. 9A shows B cells isolated from splenocytes of BALB / c mice with cells transduced with adenovirus (B / AdHM), additionally cells with αGalCer (B / AdHM / αGalCer), B cells alone and B / αGalCer After immunization with each other, the target cells loaded with antigenic peptides of cytotoxic T cells were transplanted, and then the antigen-specific cytotoxic T lymphocyte response induced in the body was measured.

9B is a graph showing the long-lasting cytotoxic T cell response after immunizing mice with B / AdHM and B / AdHM / αGalCer vaccines, respectively.

FIG. 9C shows that IFN-γ production of CD8 + T cells was measured after immunization of mice with B / AdHM and B / AdHM / αGalCer vaccine, respectively, followed by or without stimulation with cytotoxic T cell peptides in vitro. Is the graph shown,

naive: group of mice not receiving vaccine

10 is a diagram showing the activation of natural killer (NK) cells by B cells (B / AdHM and B / AdHM / αGalCer) vaccines transduced with adenovirus, and cytotoxic T cells work efficiently. ,

Figure 10a is a graph showing the activation of natural killer cells,

FIG. 10B is a graph showing the solubility of target cells induced according to the concentration of cell vaccine to determine the efficiency of inducing cytotoxic T cells between vaccine populations.

FIG. 11 shows the results of B cell transfected with AdHM (B / AdHM and B / AdHM / αGalCer) vaccines showing antibody induction specific to Her-2 / neu .

FIG. 11A shows that BALB / c mice are immunized with B / AdHM and B / AdHM / αGalCer, respectively, and then anti-Her-2 / neu antibodies in serum bind to TAUF cells, mouse cancer cells expressing Her-2 / neu on the surface. It is a graph showing the result of measuring the titer of anti-Her-2 / neu antibody to the extent that,

11B is a graph showing humoral immune responses in each group,

Figure 12 shows the anticancer activity of the B cell vaccine transduced with adenovirus, a graph showing the results of immunizing mice with lung cancer induced B-cell vaccine by administering mouse cancer cells expressing Her-2 / neu .

FIG. 13 shows IL-2, which is an indicator of activation of natural killer T cells, according to the degree and cell number of B cells loaded with αGalCer activating natural killer T cells to induce IL-2 production according to αGalCer concentration. Figure showing the degree of activation of natural killer T cells by B or dendritic cells (DC) loaded with αGalCer,

14 is a graph showing a phenomenon in which specific immune cells are removed by administering an antibody capable of removing CD4 +, CD8 +, or NK1.1 cells in vivo.

Figure 15 is a diagram showing the anticancer immune activity of the cell vaccine mediated by B cells against thymic tumor (EG-7),

*, p <0.001: Statistical significance with group treated with B cell alone

The present invention relates to a vaccine for immune prophylaxis and treatment comprising a ligand of a natural killer T cell and a B cell loaded with an antigen. More specifically, the present invention relates to an alpha-galactosyl ceramide, which is a natural killer T cell ligand and a type of glycolipid. It relates to a vaccine for the prevention and treatment of immune containing a B cell as a medium.

In the human body, antigen-presenting cells are not efficiently presented by antigen-presenting cells, so that immune responses to them are not effectively induced. Cancer vaccines are a novel therapeutic vaccine that destroys cancer cells by inducing a powerful immune response by artificially activating an immune mechanism that specifically acts on cancer cells (eg, introducing cells into antigens).

Among the vaccine approaches available, cell vaccines using antigen-presenting cells (APCs), such as dendritic cells (DCs), are known as a reliable method for generating effective T cell immunity. (Rosenberg, SA et al., Nat. Med., 10, 909-915, 2004). In particular, dendritic cell vaccines are known to efficiently induce antigen-specific effector T cells and memory T cells and have been applied to chemotherapy in many clinical trials (Rosenberg, SA et al., Nat. Med., 10, 909-915, 2004). These dendritic cells (DCs) are ideal antigen-presenting cells for immunotherapy because they have the ability to migrate to lymphoid organs, which are sites that accept antigens and present antigens to corresponding T cells. More importantly, dendritic cells provide strong co-stimulation to T cells (Figdor, CG et al., Nat. Med., 10, 475-480, 2004; and Banchereau, J. et al. , Cell, 106, 271-274, 2001). Although dendritic cell vaccine approaches have been well established experimentally and in clinical trials, the fact that few dendritic cells are present in blood and lymphoid tissues and that they are difficult to multiply ex vivo from mononuclear cells in blood is widely used as vaccines. (Schultze, JL et al., Trends Immunol., 25, 659-664, 2004).

On the other hand, because B cells are present in large amounts in lymphoid tissues or blood and can be easily proliferated in vitro, they are not only effective substitutes for dendritic cells for cellular vaccines (Schultze, JL et al., Trends Immunol., 25, 659). 664, 2004; von Bergwelt-Baildon, MS et al., Blood, 99, 3319-3325, 2002; and Schultze, JL et al., J. Clin. Invest., 100, 2757-2757, 1997) After administration, they move to lymphoid organs.

Despite these advantages, B cells have not been widely used as antigen presenting cell vaccines because of their weak immunogenicity. Indeed, there is evidence that B cells directly induce autoimmune tolerance of CD4 + and CD8 + T cells, due to the lack of costimulation (Bennett, SR et al., J. Exp. Med., 188, 1977-1983, 1998; and Eynon, EE et al., J. Exp. Med., 175, 131-138, 1992), co-stimulation of T cells to activate antigen-specific T cells. Refers to the second signal required along with an antigen that can be recognized. However, the fact that activated B cells can stimulate both CD4 + T cells and CD8 + T cells (von Bergwelt-Baildon, MS et al., Blood, 99, 3319-3325, 2002; Schultze, JL et al., J. Clin Invest., 100, 2757-2765, 1997; Lapointe, R. et al., Cancer Res., 63, 2836-2843, 2003; and Heit, A. et al., J. Immunol., 172, 1501-. 1507, 2004) suggesting that B cells activated by appropriate stimulation can act as antigen presenting cells with immunogenicity to induce antigen specific T cell immunity.

It is well known that invariant spontaneous killer T ( i NKT) cells play a pivotal role in coordinating various immune responses and immune pathologies. Although killed in less than 1% of the total lymphocytes in mice, killer T cells manage the responses to autologous and foreign antigens and determine whether to induce autoimmune or immune responses (Kronenberg, M., Annu. Rev). Immunol., 23, 877-900, 2005; and Park, SH & Bendelac, A., Nature, 406, 788-792, 2000). In addition, these cells have been reported to function to suppress immune responses in cancer, diabetes and immune-privileged sites (Sonoda, KH, et al., J. Exp. Med., 190 , 1215-1226, 1999).

On the other hand, the ligand-activated intact natural killer T cells induce activation of not only dendritic cells but also T, B and spontaneous killer cells. When injecting alpha-galactosyl ceramide (αGalCer), which is a ligand of intact natural killer T cells, It causes anticancer immunity through killer and T cells (Moodycliffe, AM, et al., Nat. Immunol., 1, 521-525, 2000).

Alpha-galactosylceramide (αGalCer) is a glycolipid originally derived from sponges and is a ligand of natural killer T cells that have a Vα14 + T cell receptor (TCR). It is known to be presented by the CD1d molecule in antigen presenting cells (APC) (Kawano et al., Science, 278: 1626, 1997). Activation of natural killer T cells produces large amounts of IFN-γ and IL-4, which can modulate the immune response to certain diseases or infections (Chen et al., J. Immunol., 159: 2240, 1997 Wilson et al., Proc. Natl. Acad. Sci. USA, 100: 10913, 2003).

In mice treated with protein antigen and alpha-galactosyl ceramide, cell-mediated immunity and humoral-mediated immunity, including cytotoxic T cell responses, occur (Hermans, IF, et al., J. Immunol, 171, 5140-5147, 2003; and Stober, D. et al., J. Immunol., 170, 2540-2548, 2003). Furthermore, recent studies have shown that dendritic cells loaded with alpha-galactosylceramide produce a longer-lasting natural killer T cell response compared to native alpha-galactosyl ceramides, resulting in immunity of natural killer T cell ligands. The enhancing effect suggests that it can be increased by targeting professional antigenic cells (professional APC).

Accordingly, the present inventors have confirmed that the natural killing T cell ligands can be loaded on B cells to change their autoimmunity to immunogenicity, thereby generating an active immune response against antigens presented by MHC molecules of B cells. To verify this, B-cells loaded with alpha-galactosyl ceramide (αGalCer) and peptides and B-cells transformed with alpha-galactosylceramide (αGalCer) and adenovirus expressing cancer antigens were antigen-specific. The present invention was completed by confirming that it exhibits an immune response and an anticancer effect.

It is an object of the present invention to present antigens by altering the immune tolerance of B cells to immunogenicity through natural killer T cell ligands, in particular through natural killer T cells activated by alpha-galactosylceramide and B cells loaded with the antigen. It is to provide a vaccine for immune prevention and treatment that can induce antigen-specific immune response through B cells.

The present invention provides a vaccine for immune prevention / treatment and an anticancer vaccine comprising B cells loaded with ligands and antigens of natural killer T cells.

In addition, the present invention provides a vaccine for cancer, which is mediated by B cells that load ligands of natural killer T cells and express cancer antigens.

The present invention also provides a natural killer T cell activator mediated by B cells loaded with alpha-galactosylceramide.

In addition, the present invention provides a cytotoxic response inducer comprising B cells expressing a cancer antigen.

Hereinafter, the present invention will be described in detail.

The present invention provides a vaccine for immune prevention / treatment and an anticancer vaccine comprising a B cell loaded with a ligand of a natural killer T cell or a B cell loaded with a ligand and an antigen of a natural killer T cell.

Ligands of the natural killer T cells are alpha-galacturonosylceramide and alpha-glucuronosylceramide (Sphingomonas spp.) Origin (alpha-glucuronosylceramide, Mattner, J. et. al. Nature 434: 525, 2005, Kinjo, Y. et al. Nature 434: 520, 2005). Phosphatidyl inositol tetramannoside from M. tuberculosis (Phospatidylinositoltetramannoside, Fischer, K. et al. PNAS 101: 10685, 2004)

Autoantigen isoglobotrihexosylceramide (Zhou, D. et al. Science 306: 1786, 2004) and ganglioside GD3, Wu, DY et al. J. Exp. Med. 198: 173, 2003), phosphatidylcholine (J. Immunol. 175: 977, 2005), beta-galactosylceramide (beta-galactosylceramide, betα GalCer, Ortaldo JR et al. J. Immunol. 172: 943), per surface of Leishmania Binding lipophosphoglycans and glycoinositol phospholipids (J. Exp. Med. 200: 895, 2004), beta-anomer galactosyl ceramides, analogs of alpha-galactosyl ceramides (beta-anomeric GalCer) and alpha-anomeric galactosylceramides (alpha.-anomeric GalCer; J. Immunol. 173: 3693, 2004), variants of alpha-galactosylceramide (J. Am. Chem. Soc. 126: 13602, 2004) and glucoder from bacterial lipid antigens such as Nocardia falcinica It comprises: (965, 2000 glucose monomycolate, Moody, D. B. et al J. Exp Med 192...) Monaural cholate.

Already, it is known that dendritic cells (DC) loaded with alpha-galactosylceramide activate invariant natural killer T ( i NKT) cells (van der Vliet HJ, et al., J Immunol Methods , 1; 247 (1-2): 61-72, 2001). The present inventors confirmed whether B cells loaded with alpha-galactosylceramide exhibited a similar effect to the dendritic cells described above.

B cells expressing CD19 (FIG. 1A) and dendritic cells were isolated from mice and cultured with alpha-galactosyl ceramide and natural killer T cell hybridomas, and then the IL-2 concentration in the culture medium was measured. Both dendritic cells (DC / αGalCer) and B cells (B / αGalCer) loaded with alpha-galactosyl ceramide stimulated natural killer T cell hybridomas to generate IL-2 (see FIG. 13). Therefore, it was confirmed that both B / αGalCer and DC / αGalCer can activate constant natural killer T cells in vitro.

To determine whether B / αGalCer and DC / αGalCer administration could activate immune cells in the body, B / αGalCer or DC / αGalCer prepared using DCs from B cells obtained from C57BL / 6 mice and intravenously in homogeneous mice IL-4 and IFN- [gamma] levels were measured by injection via ELISPOT. Specifically, wild-type C57BL / 6 or Jα281-/-mice were injected intravenously with B cells loaded with vehicle, αGalCer, αGalCer, or dendritic cells loaded with αGalCer. One week later, splenocytes were harvested, made into single cells, and then placed in a plate for elispot, and then stimulated with vehicle or αGalCer. Thereafter, an ELISA test was performed to measure cells producing IL-4 and IFN-γ according to the manufacturer's instructions (IL-4 ELISPOT kit, IFN-γ ELISPOT kit, R & D system).

As a result, the cells producing IL-4 and IFN-γ were significantly induced in both the B / αGalCer and DC / αGalCer groups (FIG. 1B). However, in Jα281-/-mice lacking invariant natural killer T cells, unlike normal mice, cells that produce cytokines are not induced, which is invariant in IFN-γ and IL-4 producing cells. Suggests that it is dependent on natural killer T cells (see FIG. 1B).

In order to find out how B cells are affected by the activation of natural killer T cells induced when B / αGalCer is administered to the body, we label B cells loaded with alpha-galactosyl ceramide with CFSE. The mice were immunized with intravenous naïve mice, and after 24 or 48 hours, costimulatory molecules on CFSE ⁺ cells in immune cells of spleen cells were analyzed by flow cytometry. As a result, CD80 had no change, but CD86 induced expression at high levels (see FIG. 1C) and a slight increase in CD40 and MHC class II molecules. That is, when B / αGalCer was administered into the body, it was confirmed that B cells were activated after 24 hours or 48 hours.

The inventors examined whether epitope peptides of antigens that bind alpha-galactosylceramide and MHC I molecules can be loaded together into B cells to stimulate peptide specific CD8 + T cells to induce antigen specific cell degradation. To this end, OVA-specific CD8 + T cells labeled with CFSE were transplanted into mice, followed by B cells alone, B cells with B / αGalCer, vehicle + peptide (B / pep) or B cells with αGalCer + peptide ( B / αGalCer / pep) was injected intravenously and CD8 + T cell responses were measured from lymphocytes obtained from the spleen and lymph nodes. As a result, BVA alone or B / αGalCer-treated group showed little OVA-specific CD8 + T cell division, whereas B / pep-administered group induced significantly more CD8 + T cell division and B / αGalCer. / pep group showed significant division of cells, more than 40% of the dividing cells produced IL-2, more than 90% produced IFN-γ, and the level was higher than the B / pep group Significantly higher (see FIG. 2A). The results indicate that by loading alpha-galactosyl ceramide and peptide together on B cells, CD8 + T cells can be activated at much higher levels.

We measured cytotoxic T lymphocyte activity to see if mice can induce cytotoxic immune responses after intravenous administration of B cells alone, B / αGalCer, B / pep or B / αGalCer / pep, respectively. As a result, only the B / αGalCer / pep-administered group completely destroyed the target loaded with the peptide (FIG. 2B) and observed a significant increase in CD8 + T cells producing IFN-γ for the peptide ( 2c, left). In addition, when restimulated in vitro with the same antigen, B cells alone or mice administered B / αGalCer normally induce an immune response to induce CD8 + T cells producing peptide specific IFN-γ (FIG. 2c, right), B / pep-treated mice showed no increase of IFN-γ-producing CD8 + T cells, and B / αGalCer / pep-treated mice received B cells alone or B / αGalCer. Compared to CD8 + T cells to respond to significantly more peptides were formed.

We used a cytotoxic T lymphocyte assay to compare the cytotoxic effects of B cell-mediated vaccines with the cytotoxic effects of dendritic cell vaccines to determine the minimum number of cells needed to completely lyse the target. saw. As a result, in consideration of the large surface area of dendritic cells, it was confirmed that the B / αGalCer / pep-administered group caused cytotoxicity in the body with similar efficiency as the DC / αGalCer / pep-administered group. In addition, the effects of cytotoxicity in the body were similar between the DC / pep and DC / αGalCer / pep administration groups, which means that loading of αGalCer on dendritic cells does not increase the vaccine efficacy of the peptide-loaded dendritic cells.

In order to identify the type of immune cells involved in inducing a cytotoxic T lymphocyte response, the inventors immunized with B / αGalCer / pep after administering an antibody that removes each immune cell. As a result, removal of CD4 + T cells or NK1.1 cells did not inhibit the induction of cytotoxic T lymphocyte responses, whereas removal of CD8 + T cells did not destroy target cells (FIG. 4A).

The inventors examined whether B cells loaded with peptides function as antigen presenting cells or whether dendritic cells recover peptides carried by B cells to induce cytotoxic T lymphocyte responses. To this end, CTL epitope peptides from OVA can be loaded onto MHC I, but results from B cells obtained from bm-1 mice in which CD8 + T cells do not recognize MHC I-peptide complexes due to mutations in the H-2K region. In addition, the cells were confirmed to stimulate the activation of natural killer T cells in response to αGalCer. In addition, injection of B / αGalCer / pep prepared from wild-type B cells into wild-type mice completely produced OVA-specific cytotoxicity in the body, but B / αGalCer / pep prepared from B cells of bm-1 mice was injected into wild-type mice. Injection did not produce OVA-specific cytotoxic activity, confirming that B cells function as antigen presenting cells (FIG. 5A).

The inventors then investigated whether αGalCer and peptide could form cytotoxic T lymphocytes when administered separately and when administered together. As a result, it was confirmed that no cytotoxicity was induced in the body in mice administered with B / αGalCer and B / pep together (FIG. 5B). This indicates that the peptide and αGalCer presented by the same B cell are essential for generating OVA specific cytotoxicity.

We investigated whether vaccine administration of B / αGalCer / pep induces anticancer immunity. First, melanoma transduced with OVA and administered with B cell alone, B / αGalCer, B / pep, B / αGalCer / pep, DC / pep, or DC / αGalCer / pep vaccine to test prophylactic anticancer immune activity. Was implanted subcutaneously. As a result, cancer was formed in mice administered with B / αGalCer, whereas cancer did not grow in mice administered with B / αGalCer / pep, DC / pep, or DC / αGalCer / pep (see FIG. 6A). Subsequently, cancer cells were transplanted into surviving mice at the 70th day after the first cancer transplantation, and there was no growth of the cancer, which was established by the B / αGalCer / pep vaccine to establish a memory immune response against the cancer. (See FIG. 6B).

Next, we examined whether cancers that were already present could be eliminated by administering the B / αGalCer / pep vaccine, but mice that received DC / pep, DC / αGalCer / pep or B / αGalCer / pep vaccine were completely Reduction of growth was observed (see FIGS. 6C and 6D).

In order to find out whether B cell-mediated vaccine administration can actually be applied to cancer antigens, the present inventors have administered B cells showing _αGalCer and Her-2 / neu _63-71 using the Her-2 / neu model. In one mouse group, it was confirmed that the cytotoxicity against Her-2 / neu in the body was expressed at a meaningful level (see FIG. 7A). In addition, after injecting Her-2 / neu -expressing cancer cells into the mouse, the B cell vaccine was administered. As a result, when the B / αGalCer or B / pep vaccine was administered, the survival time was slightly longer than that of the B cell alone group. (See FIG. 7b), mice receiving B cells loaded with _αGalCer and Her-2 / neu _63-71 were observed to survive for the entire experimental period. In addition, when immunized mice with a B-cell vaccine or dendritic cell vaccine, the B-cell administration group loaded with _αGalCer and Her-2 / neu _63-71 showed cancer cells expressing Her-2 / neu at a level similar to that of dendritic cell vaccine. It was confirmed that the growth of (refer to Figure 7c).

Therefore, the B cell-mediated vaccine administration of the present invention proved to be as effective as the vaccine based on dendritic cells in the preventive and therapeutic effects of anticancer immunity.

In addition, the present invention provides a vaccine for immune prevention and treatment and an anticancer vaccine comprising B cells that load ligands of natural killer T cells and express antigens.

In contrast to cellular vaccines loaded with peptides, cellular vaccines that incorporate whole antigens through viruses are applicable to all humans, not limited to haplotypes of major histocompatibility complexes, and induce specific immune responses to multiple epitopes. In particular, there is an advantage that can induce a humoral immune response and a cellular immune response at the same time. Tumor-associated antigen, Her-2 / neu for the target result of the infection of B cells by adenovirus (AdHM) with a gene expressing region and the membrane area outside of the Her-2 / neu cells, B three carriages alpha-go It was confirmed that the lactosyl ceramide did not have any influence on the present (see FIG. 8B). In addition, degradation of target cells could not be observed in the group of mice immunized with B and B / αGalCer at one week after the immunization, whereas cytotoxic T cells in the group of mice immunized with B / AdHM and B / AdHM / αGalCer. The reaction could be confirmed (see FIGS. 9A and 9C).

B cells (B / AdHM / αGalCer) transduced with adenovirus loaded with alpha-galactosyl ceramide (αGalCer) have a somewhat higher cytotoxic T cell response than B cells (B / AdHM) introduced with adenovirus. Alpha-galactosylceramide loading plays a decisive role, unlike peptide-loaded B cell vaccines, which induce a cytotoxic T-cell response, as long-term tendency tends to be prolonged but no significant difference appears. It does not seem to do. However, administration of B-cell vaccines loaded with alpha-galactosyl ceramide to mice stimulates NKT cells in the body and, as a result, activates natural killer cells unlike the transduced B-cell vaccine administration group (Fig. 10a).

In order to measure the efficiency of B cell vaccine inducing cytotoxic T cells, the present inventors compared the degree of lysis of target cells induced when the cell vaccine was administered below the level of inducing complete cytotoxic T cells. As a result, in the case of B cells (B / AdHM) transduced with adenovirus, only about 20% of the total cell number would function as antigen presenting cells. Although it is necessary to administer a large number of B cells compared to the loaded B cell vaccine, it is believed that it can efficiently induce an antigen specific cytotoxic T cell response. In addition, when a small number of B cells were administered, it was found to be somewhat advantageous for lysing target cells when NKT was aided by the loading of alpha-galactosyl ceramide.

The virus introduced into B cells for cancer antigen expression may be adenovirus, retrovirus, vaccinia virus, pox virus, Sindbis virus, etc., but is not limited thereto. In addition to the virus-based method, antigen gene transfer is applicable to: 1) binding DNA to liposomes and transducing them to protect the DNA from enzymatic degradation or to absorb it into endosomes; 2) consisting of proteins in DNA A method of enhancing DNA transfer efficiency to cells by binding a molecular conjugate or a synthetic ligand (eg, asialoglycoprotein, transferrin, polymeric IgA) 3) Protein (PTD) A new DNA delivery system using a transduction domain) to deliver antigen genes by increasing the efficiency of DNA delivery to cells [eg Mph-1] 4) In addition to the above methods, peptides or antigen proteins are applied to B cells by B The cells can be presented to the antigen.

The present invention also provides a natural killer T cell activator mediated by B cells loaded with alpha-galactosylceramide.

As described above, B cells loaded with alpha-galactosyl ceramide of the present invention stimulate IL-derived T-cell hybridomas in vitro as well as dendritic cells to produce IL-2 (see FIG. 13) and are invariant in vivo. Natural killer T cells were activated, resulting in the activation of immune cells (see FIG. 1B). Therefore, B cells loaded with alpha-galactosyl ceramide of the present invention can be used as a natural killer T cell activator like conventional dendritic cells.

In addition, the present invention provides a cytotoxic response inducer comprising B cells expressing a cancer antigen.

The B cell vaccine transduced with adenovirus has the advantage of being able to induce a cellular immune response and a humoral immune response, unlike a B cell vaccine loaded with peptides to induce a cellular immune response. To determine if B cell vaccines transduced with AdHM could induce Her-2 / neu specific antibodies, mice were immunized with B / AdHM and B / AdHM / αGalCer, respectively, and then anti-Her-2 / in serum. As a result of measuring the activity of neu antibody, B / B and AdHM / AdHM / αGalCer two groups wherein all long -Her-2 / neu antibody was observed remains horizontal high reverse (see Fig. 11a), B cells (B) Unlike the B / αGalCer and the B / AdHM and B / AdHM / αGalCer vaccine administration group, it was confirmed that the humoral immune response is generated (see Figure 11b).

We measured the survival rate by intravenously injecting Her-2 / neu -expressing mouse cancer cells (TAUF) on day 7 after immunizing mice with B-cell vaccine, and compared them to mice treated with only B cells. In the group of mice receiving B-cell (B / αGalCer) loaded with lactosyl ceramide, the survival time was slightly extended, and the anti-cancer effect was excellent in mice immunized with B-cell vaccine transduced with adenovirus expressing cancer antigen. It could be confirmed (see FIG. 12A). Therefore, it was found that the immune response induced by the B cell vaccine loaded with alpha-galactosyl ceramide and transduced with adenovirus effectively prevented cancer.

Next, in order to confirm the therapeutic effect of the B cell vaccine, BALB / c mice were immunized with B, B / αGalCer, B / AdHM, B / AdHM / αGalCer, respectively, and cancer cells (TAUF) were injected into the tail vein. As a result of the induction, the group of mice that received only B cells alone showed the shortest survival period, and the group of B / αGalCer vaccine was observed in a similar trend. The mice immunized with the B / AdHM vaccine had a longer survival compared to the B and B / αGalCer administration groups, but did not completely inhibit cancer production. On the other hand, the B / AdHM / αGalCer-administered group showed a significantly increased survival compared to the other-administered groups, and thus showed an excellent effect compared to the anticancer effect induced by immunization of B, B / αGalCer or B / AdHM (FIG. 12B). Reference).

The vaccine of the present invention may contain one or more active ingredients exhibiting the same or similar functions in addition to the ligands and B cells of natural killer T cells. The vaccine may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredient for administration. Pharmaceutically acceptable carriers may be used in combination with saline solution, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, and antioxidants, buffers, bacteriostatic agents, etc., as necessary. Other conventional additive components may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA.

The vaccine of the present invention may be administered parenterally, and parenteral administration may be by subcutaneous injection, intravenous injection, intramuscular injection or intrathoracic injection injection. In order to formulate into a parenteral formulation, B cells loaded with the ligand of the natural killer T cell of the present invention, B cells loaded with the ligand and peptide of the natural killer T cell, or B cells infected with a virus expressing a cancer antigen are stabilizers. Or mixed with buffers to prepare solutions or suspensions and formulate them in unit dosage forms of ampoules or vials (see criteria for viral vectors applied for transduction of existing dendritic cells).

The vaccine can be administered in an amount effective to stimulate an immune response in the patient. For example, the vaccine may be administered to a human once to several circuits, the dosage is a cell number 1x10 ³ dogs / kg ~ 1x10 ⁹ / kg, preferably from 1x10 ⁴ pcs / kg ~ 1x10 ⁸ dogs / kg. When preparing a B cell vaccine loaded with alpha-galactosyl ceramide, culture is performed using a medium containing 1-2 μg / ml of alpha-galactosyl ceramide per 1x10 ⁶ to 1x10 ⁷ cells / ml of B cells. When constructing a B cell vaccine loaded with alpha-galactosyl ceramide and peptide, a medium containing 1-2 μg / ml of alpha-galactosyl ceramide per ⁷ cells / ml of B cells 1 × 10 ⁶ to 1 × ¹⁰ was used, and the peptide was used. The B cells 1x10 ⁶ ~ 1x10 ⁷ / ml incubated in the medium containing 1-10μg / ml peptide and loaded on the cells.

Alpha-galactosylceramide has not been shown to induce toxicity in rodents and monkeys (Nakata et al., Cancer Res., 58: 1202-1207, 1998). No adverse effects have been reported in mice injected with 2200 μg / Kg of αGalCer (Giaccone et al., Clin. Cancer Res., 8: 3702, 200). In ongoing clinical trials, side effects such as minor headaches have been reported by systemic administration of αGalCer (Mie Nieda et al., Blood, 103: 383-389, Giaccone et al., Clin. Cancer Res., 8: 3702, 200) paracetamol could be prevented and mild systemic side effects were not necessarily seen in these subjects (Giaccone et al., Clin. Cancer Res., 8: 3702, 200). In the present invention, αGalCer does not cause dose-limiting toxicity (50-4800 μg / m ² ) and shows resistance in dose escalation studies, and thus, αGalCer is considered a safe substance.

The peptides include antigen peptides derived from viruses, bacteria, fungi, parasites and cancers, and antigens induced by viral expression include antigens derived from viruses, bacteria, fungi, parasites and cancers.

An "antigen" is any substance that can be recognized by the host's immune system and cause an immune response when it enters the host's body (eg, proteins, peptides, cancer cells, glycoproteins, glycolipids, live viruses, saviruses, DNA). Etc.).

The antigen may also be provided in purified or unpurified form, preferably provided in purified form.

The present invention can also be applied to antigens including proteins, recombinant proteins, peptides, polysaccharides, glycoproteins, lipopolysaccharides and DNA molecules (polynucleotides), cancer cells, live viruses, and saviruses of pathogens.

The following list of antigens may be provided as a means of inducing an immune response in place of the cancer antigens of the examples, including but not limited to: influenza virus antigens (haemagglutinin and neuraminidase antigens), Bordetella pertussis antigens (pertussis toxin, filamentous haemagglutinin, pertactin), human papilloma virus (HPV) antigen, glycoprotein, Helicobacter pylori antigen (capsula polysaccharides of serogrup A, B, C, Y and W-135 ), Tetanus toxoid, diphtheria toxoid, pneumococcal antigen (Streptococcus pnemoniae type 3 capsular polysaccharide), tuberculosis antigen, human immunodeficiency virus, HIV) antigens (GP-120, GP-160, p18, Tat, Gag, Pol, Env), cholera antigens (cholera toxin B subunit), staphylococcal antigens (staphylococcal enter) otoxin B), shigella polysaccharides, vesicular stomatitis virus antigens (vesicular stomatitis virus glycoprotein), cytomegalovirus (CMV) antigens, hepatitis antigens (hepatitis) A (HAV), B (HBV), C (HCV), D (HDV) and G (HGV) antigens (core antigen and surface antigen), respiratory synctytial virus (RSV) antigens, herpes simplex (herpes simplex) antigen or combinations thereof (e.g. diphtheria, pertussis and derivatives, DPT), Borrelia sp. antigen (e.g. OspA, OspB, OspC antigen), Candida albicans antigen (e.g. MP65), Plasmodium antigen (e.g. CS protein)

Cancer antigens are produced as a result of somatic mutations in normal genes and occupy most of the cancer-specific antigens and cancer antigens, which are products resulting from genetic instability of cancer cells, and are antigens with increased expression in cancer cells. Proteins that are transiently expressed or expressed only in specific tissues include cancer covalent antigens that are autoantigens normally present in the body. Examples of cancer specific antigens include cancer antigens caused by cancer viruses such as HPV E6 / E7. Examples of cancer covalent antigens include gp100, tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase (TRP-2). -related protein-2), MAGE, MUC-1, arcinoembryonic antigen (CEA), p53, alpha-fetoprotein and Her-2 / neu (Rosenberg SA., Nature, 17; 411). 6835): 380-4, 2001).

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Bidirectional Activation Between B Cells Loaded with Alpha-Galactosylceramide and Natural Killing T Cells

<1-1> Comparison of Natural Killer T Cell Activation Effects of B Cells and Dendritic Cells

It is known that dendritic cells (DC) loaded with alpha-galactosylceramide (Kim, S., et a., Synthesis, 847, 2004) activate invariant natural killer T ( i NKT) cells. Therefore, in the present invention, it was first confirmed whether the B cells loaded with such alpha-galactosyl ceramide had a similar effect to the dendritic cells.

In the present invention, 6-10-week-old female C57BL / 6 and BALB / c were used. OT-I and C57BL / 6 bm1 (bm1) mice were purchased from The Jackson Laboratory, and Jα281-/-and MHC II-/-mice were respectively studied by Kim JH et al., Am J Pathol., 167 (5): 1231-41, 2005) and Professor Park Se-ho (Park, SH, et al., J. Exp. Med. 193: 893, 2001). All mice were conserved under specific pathogen-free conditions at the Seoul National University Pharmacy Animal Center. In addition, the antibodies were GK1.5 (anti-CD4: ATCC number: TIB-207), 2.43 (anti-CD8; ATCC number: TIB-210) and PK136 (anti-NK1.1; ATCC number: HB-191) high. Obtained from bridoma, 150 μg per mouse was administered intraperitoneally to remove lymphocytes for each in vivo.

 Specifically, in order to purely separate B cells from each mouse, spleens were removed from the mice and then homogenized to remove CD11c + cells from anti-CD11c microbeads (Miltenyibiotec) from the spleen cells, followed by anti-B220 microbeads. B220 + cells were isolated using (Miltenyibiotec). The cells are purely isolated B cells, wherein at least 99% express CD19 (see FIG. 1A).

To separate dendritic cells from the spleen, the spleen extracted from the mice was reacted for 30 minutes at 37 ° C. using 7 ml of medium containing collagenase D (1 mg / ml, Roche) and DNase I (50 μg / ml, Sigma Aldrich). Then EDTA (final concentration 10 mM, pH 7.2) was added and reacted for 5 minutes. Dendritic cells were isolated from cells isolated from the spleen using a 15.5% Accudenz density gradient (Accurate Chemical & Scientific).

Cells isolated as described above (B cells or dendritic cells) were incubated for 14 hours in a carbon dioxide incubator with the prepared alpha-galactosyl ceramide (1ug / ml) or solvent (0.5% polysorbate), and then killed naturally. Incubated with cell hybridoma (DN32.D3) (Claire Forestier et al., The Journal of Immunology, 171: 4096, 2003) for 24 hours. Subsequently, IL-2 concentration, which is an indicator of activation of natural killer T cell hybridomas in the culture, was measured by the sandwich ELISA method.

As a result, dendritic cells (DC / αGalCer) loaded with alpha-galactosyl ceramide stimulated DN32.D3 to produce IL-2. Similarly, B cells loaded with alpha-galactosylceramide (B / αGalCer) also efficiently stimulated DN32.D3 to produce IL-2. The IL-2 level produced by the B cell group in which hybridomas and antigen-presenting cells were mixed at the same rate was the same as the amount of IL-2 produced in the dendritic cell group, but when mixed at a lower rate, the IL-2 level was lower than the IL-2 level in the dendritic cell group. Small (see FIG. 13).

<1-2> Confirmation of activation of natural killer T cell dependent immune response

In the present invention, intravenous injection of B / αGalCer or DC / αGalCer prepared using B cells obtained from C57BL / 6 mice and DCs to homogeneous mice to increase IL-4 and IFN-γ levels through ELISPOT. Measured. Specifically, wild-type C57BL / 6 or Jα281-/-mice were injected intravenously with vehicle, αGalCer, αGalCer loaded B cells or αGalCer loaded dendritic cells. One week later, splenocytes were harvested and made into single cells, which were then placed in a plate for elispot, incubated for 6 hours with vehicle or αGalCer. After that, according to the manufacturer's (IL-4 ELISPOT kit, IFN-γ ELISPOT kit, both R & D system) instructions were carried out Elisot test to measure the cells producing IL-4 and IFN-γ.

As a result, the cells producing IL-4 and IFN-γ were significantly induced in both the B / αGalCer and DC / αGalCer groups (FIG. 1B). However, in Jα281-/-mice lacking invariant natural killer T cells, unlike normal mice, cells that produce cytokines are not induced, which is invariant in IFN-γ and IL-4 producing cells. It suggests that natural killer T cells are induced (see FIG. 1B).

<1-3> Confirmation of B cell activation due to natural killer T cell activation

The present inventors analyzed the co-stimulatory molecules in CFSE + cells after intravenous administration of B / αGalCer labeled with CFSE (Carboxyfluorescein Succinimidyl Ester) to determine what changes in vivo occur when B cells are injected. Specifically, single cells were obtained from spleen and lymph nodes of C57BL / 6, and then CD11c + cells were removed from lymphocytes isolated from spleen and lymph organs using anti-CD11c microbeads (Miltenyibiotec), and anti-B220 microbeads (Miltenyibiotec). After B cells were isolated, the cells were labeled with 10 μM of CFSE and introduced into the tail vein of allogeneic mice. CFSE labeling was carried out by diluting CFSE (Molecular Probe) at an appropriate concentration in RPMI medium, incubating for 15 minutes at 37 ° C, and then washing three times with RPMI medium to remove residual CFSE. 24 and 48 hours after labeling, CFSE + isolated from lymph nodes or spleen of the mouse Costimulatory molecules of the cells were analyzed by flow cytometry.

As a result, it was observed that within 24 hours CD80 did not change, but CD86 induced a high level of expression and a slight increase in the expression of CD40 and MHC II (FIG. 1C). No increase in CD80 was observed after 48 hours post-injection, but the remainder was mostly comparable to the 24 hour level. That is, it was confirmed that B cells administered by natural killer T cells activated by B / αGalCer within 24 hours were also activated.

According to the results of Examples <1-1>, <1-2> and <1-3>, both B / αGalCer and DC / αGalCer can activate natural killer T cells, and B cells are alpha-galacto As activated by natural killer T cells presented with silceramide, it was confirmed that the activation was bidirectional.

Example 2 Promoting Activation of Peptide Specific CD8 + T Cells by B Cells Loaded with Peptides and αGalCer

The present inventors examined whether alpha-galactosyl ceramide (αGalCer) and MHC I peptides were loaded on B cells together to stimulate peptide-specific CD8 + T cells to induce OVA-specific cytotoxicity.

To this end, first, CFSE-labeled OVA-specific CD8 T cells (OT-I) were implanted into the C57BL / 6 mouse by a tail vein and then cultured in a vehicle-containing medium (B cells) one day later. , B cells loaded with αGalCer (B / aGarCer), B cells loaded with OVA _257-264 peptide (B / pep) and B cells loaded with αGalCer and OVA _257-264 peptide (B / aGarCer / pep) Injection. After 48 hours single cells were obtained from the spleen and then the division of OT-I cells was observed. Separately, incubated for 4 hours with 1 μM OVA _257-264 peptide and 1 μL / mL GolgiPlug, OT-I cells producing IL-2 and IFN-γ were prepared using Cytoperm / Cytofix kit (BD Pharmingen). Tested by my cytokine staining.

As a result, the division of OVA-specific CD8 + T cells, that is, OT-I cells, was hardly induced in the group of B cells alone or B / αGalCer. In particular, the group administered B / αGalCer showed a slight division of OT-I cells (FIG. 3A, 1.2 ± 0.5% vs. 5.4 ± 1.8%), which is considered to be an antigen-specific change. On the other hand, B / pep administration group induced a considerable number of OT-I cell division. However, when restimulated with the same peptide in vitro, only a few of these cells produced IL-2 (<4%) and produced relatively few IFN-γ (38%). In contrast, the B / αGalCer / pep group showed marked division of OT-I cells, at least 40% of the dividing cells produced IL-2, and at least 90% produced IFN-γ, The level was much higher than that of the B / pep group (Fig. 2a).

The results show that by loading B / pep and alpha-galactosylceramide together, CD8 + T cells can be activated at much higher levels.

Example 3 Induction of Cytotoxic T Cell Responses with Persistence by B / αGalCer / pep and Overcoming Immune Tolerance

<1-1> Induction of Sustained Cytotoxic T Cell Responses by B / αGalCer / peptide

We measured intracellular cytotoxic T lymphocyte activity after intravenous administration of B cells alone, B / αGalCer, B / pep, or B / αGalCer / pep to C57BL / 6 mice, respectively. To see if it can be induced. In addition, it was examined whether the cytotoxic immune response induced by the B cell vaccine was continuously maintained. Specifically, C57BL / 6 mice were immunized with B only, B / αGalCer, B / pep and B / αGalCer / pep, respectively, and then 1 week, 3 weeks, 5 weeks later in vivo CTL assay (in vivo CTL assay). ) Was performed. First, the spleen single cells of allogeneic unimmunized mice were divided into two groups, one group loaded with OVA _257-264 peptide and then labeled with 20 μM CFSE (CFSE ^high ) and the other group with 2 μM without peptide load. Labeled with CFSE to control cells (CFSE ^low ). The two groups of cells were injected in the same amount and injected into immunized mice. After one day, spleen single cells were extracted and the CFSE ^high and CFSE ^low cell populations were measured by flow cytometry. At this time, the lower the ratio of CFSE ^high means a greater cytotoxic immune response.

As a result, only peptide-loaded targets were completely destroyed only in the group to which B / αGalCer / pep was administered, and specifically cytotoxic to 5 weeks after one vaccination. On the other hand, both mice treated with B / αGalCer or B / pep did not induce peptide-specific cytotoxic T cell responses, and CD8 producing IFN-γ for peptide only in the group administered with B / αGalCer / pep. A significant increase in T cells was observed (FIG. 2C, left).

<1-2> Overcoming Immune Tolerance by B / αGalCer / pep

In general, the immune tolerance is induced when only the antigen is administered, and the present inventors examined whether the immune tolerance induced by administration of the antigen alone can be overcome when αGalCer is loaded on the B cell together with the peptide. Specifically, C57BL / 6 mice were immunized as described in <1-1> of Example 3, and spleen single cells were extracted one week after, and then stimulated with spleen single cells loaded with OVA for one week. CD8 + T cells producing IFN- [gamma] against stimulated spleen single cells were tested by the above intracellular cytokine staining method.

As a result, in the case of B cells alone or B / αGalCer-administered mice, an immune response was normally induced to induce CD8 T + cells producing peptide-specific IFN-γ (FIG. 2C, right). Mice administered did not show an increase in CD8 + T cells producing IFN-γ, which is believed to indicate that the mice exhibit 'immune tolerance' to the peptide.

In contrast, mice that received B / αGalCer / pep formed significantly more CD8 + T cells that responded to peptides compared to B cells alone or B / αGalCer. Here, in the case of B alone or B / αGalCer-treated mice, no antigen-specific immune response was induced since no antigen was administered. In vitro re-stimulation of spleen single cells loaded with OVA resulted in the initial immune response. to be. On the other hand, in the case of mice administered B / αGalCer / pep, the in vitro restimulation of spleen single cells loaded with OVA becomes a recall response because the antigen has already been administered to the mice. In conclusion, it was confirmed that B / αGalCer / pep overcomes immunotolerance and exhibits immunogenicity and shows strong reactivity.

Example 4 Induction of Production of Cytotoxic T Lymphocytes of B Cell Vaccine with Efficacy Similar to Dendritic Cell Vaccine

We compared the cytotoxic effects of B cell mediated vaccines with the cytotoxic effects of dendritic cell vaccines.

To this end, in vivo cytotoxic T lymphocyte assays were used to determine the minimum cell number needed to completely lyse the target. Specifically, B cells or dendritic cells were incubated with αGalCer or vehicle for 16 to 18 hours, followed by 1 μg / ml of OVA _257-264 peptide for 1 hour. Cells were then serially diluted and intravenously injected into homologous mice one week after, followed by in vivo cytotoxic T lymphocyte analysis. That is, as in the above example, the target cells to which the peptide was applied were labeled with CFSE ^high , and the control cells without the peptide were labeled with CFSE ^low and injected into the mouse in the same amount, and then the splenocytes of the mouse were flown for a predetermined time. Analysis was performed by an analyzer to calculate the CFSE ^low : CFSE ^high ratio to measure the lysis of antigen-specific target cells.

As a result, the DC / αGalCer / pep administration group completely lysed the target cells with only 16000 cells, while the B / αGalCer / pep administration group completely peptide-specific lysis with 80000 cells, and the DC / αGalCer / Administration of the same number of pep (16000) B / αGalCer / pep was observed to cause moderate cytotoxicity in the body (Fig. 3a). However, considering that the surface area of dendritic cells is much larger than that of B cells, it can be said that B / αGalCer / pep causes cytotoxicity in the body as effectively as DC / αGalCer / pep. In addition, DC / pep caused OVA specific efficient cytotoxicity, whereas B / pep administration did not induce cytotoxicity regardless of the number of cells (FIG. 3B). In particular, it was important that the tendency of cytotoxicity in the body was very similar between the DC / pep and DC / αGalCer / pep administration groups, which means that loading of αGalCer on dendritic cells does not increase the vaccine efficiency of the peptide-loaded dendritic cells. do.

Example 5 Confirmation of Requirements for Cytotoxic T Lymphocyte Formation of B / αGalCer / pep

To identify the type of immune cells involved in inducing a cytotoxic T lymphocyte response, the present inventors used to remove each immune cell 4 days before or 4 days after B / αGalCer / pep administration to C57BL / 6 mice. Intracellular cytotoxic T lymphocyte analysis was performed after injection of an antibody (anti CD4 eliminating antibody: GK1.5, anti CD8 eliminating antibody: 2.43, anti NK1.1 eliminating antibody: PK136).

As a result, irrespective of the timing of immune cell removal (both before and after B / αGalCer / pep administration), CD4 + T cells or NK1.1 cell removal did not inhibit the induction of cytotoxic T lymphocyte responses (FIG. 4A). On the other hand, elimination of CD8 + T cells did not cause destruction of the target cells. Similar to the above results, it was confirmed that MHC II-/-(CD4 + T cell deficient) mice normally induced a cytotoxic T lymphocyte response, whereas Jα281-/-(deficient natural T cell deficient) mice were not induced. (FIG. 4B).

Example 6 Antigen Presentation of B Cells and Confirmation of Induction of Cytotoxic T Lymphocytes

We know whether B cells loaded with peptides function as antigen presenting cells or act only as a carrier for peptides, that is, whether dendritic cells recover peptides carried by B cells to induce cytotoxic T lymphocyte responses. saw. For this purpose, we used bm-1 mice that could load OVA peptides on MHC I, but CD8 + T cells did not recognize MHC I-peptide complexes due to mutations in the H-2K region (Norbury, CC et al., Science 304, 1318-1321, 2004). The B cells obtained from such bm-1 normally express CD1d, and react with αGalCer to stimulate the activation of natural killer T cells. Thus, in the interaction between bm-1 B cells and natural killer T cells, You can see that there is no problem.

Once again, when B / αGalCer / pep prepared from wild-type B cells was injected into wild-type mice, it was confirmed that OVA-specific cytotoxicity was completely produced in the body. On the contrary, when B / αGalCer / pep prepared from B cells of bm-1 mice was injected into wild-type mice, it was found that wild-type mice did not produce OVA-specific cytotoxic activity. These results indicate that dendritic cells or other specialized antigen presenting cells of recipient mice do not contribute to the formation of cytotoxic T lymphocytes (FIG. 5A).

The present inventors then investigated whether cytotoxic T lymphocytes could be formed when administered separately with αGalCer and peptide. C57BL / 6 mice were intravenously injected with B / αGalCer + B / pep or B / αGalCer / pep alone, followed by in vivo cytotoxic T lymphocyte analysis.

As a result, it was confirmed that cytotoxicity was not induced at all in the mice administered B / αGalCer + B / pep (FIG. 5B). These results suggest that loading of peptides and αGalCer on the same B cell is essential for generating OVA specific cytotoxicity.

Example 7 Confirmation of Anticancer Effect of B / αGalCer / pep

<7-1> In vivo experiment using OVA model

We investigated whether vaccine administration of B / αGalCer / pep induces anticancer immunity. First, in order to test prophylactic anticancer immunity, a single dose of B cell alone, B / αGalCer, B / pep, B / αGalCer / pep, DC / pep, or DC / αGalCer / pep vaccine was used. Introduced B16 melanoma (MO-5) (Dr. Kenneth Rock, University of Massachusetts: Falo, LD, et al., Nat. Med. 1: 649, 1995) was implanted subcutaneously. As a result, in mice administered B / αGalCer, cancer growth tended to be slightly delayed, but finally, cancer was formed (FIG. 6A), whereas B / αGalCer / pep, DC / pep, or DC / αGalCer / Cancer did not grow in mice administered pep.

Subsequently, MO-5 cancer cells were transplanted subcutaneously into the surviving mice 70 days after the first cancer transplantation to confirm whether these mice can maintain long-term anticancer activity. As a result, there was no growth of cancer in these mice (FIG. 6B), suggesting that a memory immune response to the cancer was established by administration of the B / αGalCer / pep vaccine. Even when performed using thymic tumors (EG-7) transduced with OVA, a result consistent with the above was obtained (FIG. 15).

Next, we examined whether cancer that already exists could be eliminated by administering the B / αGalCer / pep vaccine. To this end, the present inventors have proposed the following two treatment models. That is, the mice were administered with the vaccine at the time when cancer was promoted i) 1 day after subcutaneous transplantation or ii) 9 days.

As a result, in the one-day model, the growth of cancer was almost completely suppressed when the DC / pep or DC / αGalCer / pep vaccine was administered. In particular, mice treated with the B / αGalCer / pep vaccine also completely prevented cancer growth. Reduction was observed (FIG. 6C). The 9-day model did not completely reduce growing cancer in any of the vaccine groups. This is probably because B16 melanoma has very active growth. In contrast, mice treated with the B / αGalCer / pep vaccine showed decreased cancer growth compared to the B cell alone group and the B / αGalCer and B / pep administration groups, and DC / pep or DC / αGalCer / pep. It was confirmed that they appear similar to the vaccine administration group (FIG. 6D).

<7-2> Her-2 / neu  In vivo experiments using models

In order to find out whether B cell-mediated vaccine administration can actually be applied to cancer antigens, the present inventors have previously characterized Her-2 / neu expressing cancer cells with well-characterized and known cytotoxic T lymphocyte epitopes. The same experiment as in Example <7-1> was performed with TAUF (Penichet ML et al., Lab Anim Sci., 49: 179-88, 1999). As a result, the present inventors confirmed that, once again, the cytotoxicity against Her-2 / neu in the body was shown to be meaningful in the group of mice treated with B cells presenting _αGalCer and Her-2 / neu _63-71 peptides. (FIG. 7A).

Also, after the injection of cancer cells (TAUF) which express Her-2 / neu in order to analyze the anti-cancer activity in the model with intravenous and subcutaneous in BALB / c mice, a stacking αGalCer and Her-2 / neu _63-71 As a result of administering one B cell vaccine, when the cancer was injected into the vein and the B / αGalCer or B / pep vaccine was administered, it was confirmed that the survival time was slightly extended compared with the B cell alone administration group (FIG. 7B). In contrast, all mice treated with B / αGalCer / pep were observed to survive during the entire experimental period. Inhibition of Her-2 / neu -expressing cancer cells (TAUF) was shown to be suppressed even when the mice survived. I could confirm that. Similar results were also observed in the subcutaneous cancer growth model (FIG. 7C).

Therefore, the B cell-mediated vaccine administration of the present invention proved to be as effective as the vaccine based on dendritic cells in preventing and treating anticancer immunity.

Example 8 Protein Introduction Using Viral Vector Expressing Antigen

Peptide-loaded cell vaccines are not universally available because of the limited use of peptides in the haplotype of a major histocompatibilty complex (MHC) in clinical use, and only a single epitope. It has some disadvantages. On the other hand, the introduction of the whole antigen through the virus can be applied to all humans without being limited to the haplotype of the major histocompatibility complex. In particular, the immune response specific to several epitopes, in particular humoral immune response and cellular immunity The reaction can be induced simultaneously. On the basis of this, the virus was transduced with the whole antigen into the B cells and confirmed whether the immune response can be effectively induced. First, adenoviruses (AdHMs) with genes expressing the extracellular and cell membrane regions of Her-2 / neu (Her-2 / ne extracellular domain + transmembrane domain) (HM), which are tumor-associated antigens, were prepared and used in mouse cancer models. Applied.

Specifically, B cells isolated from splenocytes of BALB / c mice were adenovirus (AdHM, Viromed) 100 MOI expressing the extracellular and cell membrane regions of Her-2 / neu for 90 minutes in medium without serum. Incubated in a 37 ° C. incubator, supplemented with serum and incubated for 8, 24, and 48 hours, respectively, followed by staining with PE-labeled anti-Her-2 / neu antibody (BD biosciences # 340552). After the analysis, the degree of Her-2 / neu expression on the surface (adenovirus transduction efficiency to B cells, ratio of PE + cells in total B cells) was measured.

As a result, when the B cells were incubated with the adenovirus for 8 hours, 24 hours and 48 hours, it was confirmed that the efficiency of about 20% or more in all culture conditions (Fig. 8a). That is, it can be seen that when B cells are incubated with AdHM for 8 hours or longer, Her-2 / neu can be sufficiently expressed on the B cell surface transfected with AdHM. In addition, Her-2 / neu expression in B cells did not change even after incubation with the virus and incubation with alpha-galactosyl ceramide, and B cells had no effect on the presentation of alpha-galactosyl ceramide. It was confirmed that no (Fig. 8b). As mentioned above, when alpha-galactosyl ceramide is presented to DN32.D3 cells by CD1d molecules of B cells, the activated DN32.D3 cells secrete IL-2, which are included in the culture medium. By measuring this IL-2 concentration, it was indirectly confirmed that B cells can present alpha-galactosyl ceramide.

Example 9 Confirming Cytotoxic T Cell Response Induced by B Cells Transduced with Adenovirus

B cells isolated from splenocytes of BALB / c mice were incubated at 37 ° C for 60 minutes in medium without serum with adenovirus (AdHM) 100 MOI expressing the extracellular and membrane regions of Her-2 / neu . Was then incubated with serum supplemented medium for 24 hours to produce B / AdHM cells. In addition, B cells transformed with AdHM were cultured for 23 hours in a medium supplemented with serum containing 1-2 Gg / ml of αGalCer to prepare B cells loaded with αGalCer (B / AdHM / αGalCer). Completed B cells were washed three or more times with RPMI medium to remove free adenovirus and αGalCer and used for the experiment.

The present inventors have intravenously injected mice to investigate whether B cells transduced with adenovirus (AdHM) expressing the extracellular and cell membrane regions of Her-2 / neu can induce a cytotoxic immune response in mice. B cell vaccine was administered. Mice that received B cells alone (B) and B cells (B / αGalCer) loaded with only alpha-galactosyl ceramide were used as negative controls and transfected with B cells (B / AdHM) and AdHM transduced with AdHM. Her-2 / neu specific cytotoxic T cell responses induced by B cells (B / AdHM / αGalCer) loaded with alpha-galactosylceramide were measured. After immunization, target cells loaded with epitope peptides (Her-2 / neu _63-71 , _Anigen ) of cytotoxic T cells were injected to confirm degradation of target cells in the body.

As a result, the degradation of target cells could not be observed in the group of mice immunized with B and B / αGalCer at one week after the immunization, whereas cytotoxic T was observed in the group of mice immunized with B / AdHM and B / AdHM / αGalCer. The cellular response could be confirmed (FIG. 9A). Since B cells transduced with adenovirus are activated, they are thought to function as antigen presenting cells capable of effectively inducing cytotoxic T cells in the body without the help of natural killer T cells. Cytotoxic T cell responses induced by B / AdHM and B / AdHM / αGalCer tended to decrease gradually with time, but cytotoxic T cell responses remained significant until week 7 after immunization (FIG. 9B). . In addition, as a result of confirming the production of IFN-γ in CD8 + T cells with cytotoxic T cells 1 week after immunization, mice in the group immunized with B / AdHM and B / AdHM / αGalCer compared to the mice treated with only B cells. The number of activated CD8 + T cells was increased, especially in the B / AdHM / αGalCer group compared to B / AdHM (FIG. 9C).

Example 10 Induction of Activation of Natural Killing Cells by Loading of αGalCer and Confirmation of Promoting Effect of B Cell Vaccine Transduced with Adenovirus

<10-1> Confirmation of activation of natural killer cells through B cells loaded with αGalCer

Activated natural killer cell populations were calculated based on natural killer cells producing IFN-γ. Briefly, B cells isolated from the spleen of the mouse were incubated for 5 hours under 1 ug / ml GolgiPlug, and after fixing and permeating the cells, anti-mouse IFN-γ: APC, CD3: PE, and CD49b: Each was stained with FITC antibody (both Biolegend). The natural killer cells did not express CD3, and the cell population expressing CD49b, which is a marker of natural killer cells, was used.

B cells loaded with alpha-galactosyl ceramide (αGalCer) and transduced with adenovirus (B / AdHM / αGalCer) have somewhat more cytotoxic T cell responses compared to B cells (B / AdHM) transduced with adenovirus. Alpha-galactosylceramide loading plays a decisive role in contrast to peptide-loaded B cell vaccines in inducing cytotoxic T cell responses, as they show a prolonged long-term tendency but no significant difference. It does not seem to be in charge. However, it was confirmed that administration of B-cell vaccine loaded with alpha-galactosyl ceramide to mice stimulates natural killer T cells and, consequently, activates natural killer cells, unlike the transduced B-cell vaccine group. (FIG. 10A). The activation of these natural killer cells is thought to contribute to the anticancer effects along with the induction of cytotoxic T cell responses.

<10-2> Confirmation of the Enhancement of Effect of B Cell Vaccine Transduced with Adenovirus by Loading αGalCer

In order to measure the efficiency of B cell vaccine inducing cytotoxic T cells, the present inventors compared the degree of lysis of target cells induced when the cell vaccine was administered below the level of inducing complete cytotoxic T cells. To this end, B cells (B / αGalCer / pep) loaded with alpha-galactosyl ceramide and cytotoxic T cell epitope peptides were used as a positive control, and one week after each immunization of each B cell vaccine. Cytotoxic T cell induction efficiency induced by B / AdHM and B / AdHM / αGalCer administration was measured (FIG. 10B). In the case of B / αGalCer / pep, it is presumed that peptides were applied so that most B cells can present antigens, but as mentioned above, only about 20% of B cells transfected with adenoviruses. It is thought to function as an antigen presenting cell. Given this, B-cell vaccines incorporating antigens as adenoviruses can efficiently induce antigen-specific cytotoxic T cell responses, despite the need for a greater number of B cell doses than peptide-loaded B-cell vaccines. I think that. In addition, when a small number of B cells were administered, it was confirmed that the loading of alpha-galactosyl ceramide was somewhat advantageous for lysing the target cells when assisted with natural killer T cells.

Example 11 B Cell Vaccine Transduced with Adenovirus Inducing Antigen-Specific Antibody Responses

The B cell vaccine transduced with adenovirus has the advantage of being able to induce a cellular immune response and a humoral immune response, unlike a B cell vaccine loaded with peptides to induce a cellular immune response. To determine if B cell vaccines transduced with AdHM can induce Her-2 / neu specific antibodies, BALB / c mice were immunized with B / AdHM and B / AdHM / αGalCer, respectively, and then anti-Her in serum. In order to measure the titer of the −2 / neu antibody, the degree of binding Her-2 / neu to TAUF cells, which are mouse cancer cells expressing the cell surface, was confirmed by flow cytometry.

Blood was extracted with orbital blood collection at 1, 3, 5, and 7 weeks after immunization with B cell vaccine to measure antibody response. The blood of the mouse was left at room temperature for 2 hours, and then serum was separated by centrifugation at 8000 rpm for 10 minutes. To determine the titer of anti-Her-2 / neu antibody in serum, dilution with buffer (1% FBS, PBS containing 0.09% azide) twice from 1: 50 and then Her-2 / neu expressing cancer cells Incubated with phosphorus TAUF cells at 4 ° C. for 60 minutes. TAUF cells cultured with mouse serum with buffer were washed, and then mouse antibodies bound to TAUF cells were stained using a goat antibody which binds to a mouse antibody labeled with FITC as a secondary antibody. Subsequently, the amount of mouse antibody bound to TAUF cells was measured using a flow cytometer (FACSCaliber, BD Biosciences), and the positive fluorescence intensity was increased by 1.8 times based on blood of unimmunized mice. The antibody titer was calculated.

As a result, it was observed that anti-Her-2 / neu antibodies remained at high titers for at least 7 weeks after immunization in both B / AdHM and B / AdHM / αGalCer populations similar to inducing cytotoxic T cells (FIG. 11a), unlike B cells (B) and B / αGalCer, it was confirmed that humoral immune responses were generated in both the B / AdHM and B / AdHM / aGarCer vaccine administration groups (FIG. 11B).

Example 12 Confirmation of Prevention and Treatment of Cancer Using Immunization of B Cell Vaccine Loaded with Alpha-galactosylceramide and Transduced with Adenovirus

In the Her-2 / neu tumor model, 2 x 10 ⁵ TAUF cells were administered to BALB / c mice by tail vein to allow tumor development in mice. Thereafter, the mice bearing tumors were immunized with B cell vaccine, respectively, and then survival rates were measured.

In the present invention, it was investigated whether the cellular and humoral immune responses induced with the B cell vaccine transformed with adenovirus ultimately show effective anticancer activity. After intravenous injection of mouse cancer cells (TAUF) expressing Her-2 / neu , lung cancer-induced mice were confirmed to have a prophylactic and therapeutic effect of B cell vaccine. After 7 days of immunization with 2 × 10 ⁶ B-cell vaccines, the survival rate of mice intravenously injected with 2 × 10 ⁵ cancer cells was measured. As a result, alpha-galactosyl ceramide was loaded in comparison with the group of mice receiving only B cells (B). Survival was observed in mice that were immunized with B cells administered with B cells (B / aGarCer) and with B cells (B / AdHM) transformed with adenovirus expressing cancer antigens. Intravenous administration of a B-cell vaccine (B / AdHM / aGarCer) loaded with alpha-galactosyl ceramide and expressing cancer antigens observed that mice survived during the entire experimental period, resulting in B / aGarCer or B / AdHM vaccines. Compared with the B / AdHM / aGarCer vaccine showed a high anticancer effect (Fig. 12a). Therefore, it was found that the immune response induced by the B cell vaccine effectively prevented cancer.

Next, to confirm the therapeutic effect of the B cell vaccine, BALB / c mice were immunized with 1 × 10 ⁶ B, B / aGarCer, B / AdHM, and B / AdHM / aGarCer, respectively, and after 3 days, 5 × 10 ⁴ cancer cells ( Lung cancer was induced by injection into the tail vein. The group of mice receiving B cells alone showed the shortest survival time, and the group of B / aGarCer vaccine was observed with similar trend. Mice immunized with B / AdHM vaccine had a slightly longer survival compared to B and B / aGarCer groups. On the other hand, the B / AdHM / aGarCer-administered group showed a significant increase in survival compared to other experimental groups, similar to that observed in the prophylactic model. It can be seen that (Fig. 12B).

Preparation Example Preparation of Injection Solution

Injection solution of the anticancer vaccine of the present invention was prepared by the following method.

Alpha-galactosylceramide 1 ~ 2μg / ml, B cells 5x10 ⁶ / ml, peptide 1-2μg / ml, 5'-chloro-3,2'-dihydroxychalcone or 5'-chloro-2, 1 g of 3'-dihydroxychalcone hydrochloride, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

Ligands of natural killer T cells of the present invention, in particular alpha-galactosyl ceramide (αGalCer) and antigen-loaded B cells, not only induce similar levels of cytotoxic T lymphocyte responses compared to conventional dendritic cell vaccines, but also subcutaneously B cells transformed with adenovirus expressing cancer antigens, which have a prophylactic and inhibitory effect on cancer and metastatic cancer, induce an antigen-specific immune response, and thus a vaccine including the cells can be used as a cancer prevention and treatment agent. In addition, since the vaccine of the present invention induces an immune response even in the absence of CD4 + T cells, the number of CD4 + T cells may be significantly reduced, and thus may be used to immunize HIV patients with immunodeficiency.

Claims (11)

A vaccine for immunotherapy and prevention comprising a B cell loaded with a ligand of a natural killer T cell, a B cell loaded with the ligand and an antigen or a B cell loaded with the ligand and expressing an antigen. The ligand of claim 1, wherein the ligand of the natural killer T cell is alpha-galactosyl ceramide, alpha-glucuronosyl ceramide, phosphatidylinositol tetramannoside, isoglobotrihexylceramide, ganglioside GD3, phosphatidylcholine, Beta-anomer galactosylceramide and alpha-anomeric galactosylceramide, bacterial lipid antigens and analogs of beta-galactosylceramide, lipophosphoglycan, glycoinositol phospholipid, alpha-galactosylceramide Vaccine for immunotherapy and prevention, characterized in that it is selected from the group consisting of variants of alpha galactosyl ceramide. The vaccine for immunotherapy and prevention of claim 1, wherein the antigen is an antigen or a cancer antigen derived from a pathogen including pathogenic bacteria, viruses, and parasites. The antigen of claim 3, wherein the antigen from the pathogen is Bortella pertussis antigen (pertussis toxin, filamentous haemagglutinin, pertactin), tetanus toxoid, diphtheria antigen (diphtheria toxoid), Helicobacter pylori (Hycobacter pylori) Helicobacterpylori antigen (capsula polysaccharides of serogrup A, B, C, Y and W-135), pneumococcal antigen (Streptococcus pnemoniae type 3 capsular polysaccharide), tuberculosis antigen, cholera antigen (cholera toxin B) subunit, staphylococcal antigen (staphylococcal enterotoxin B), shigella polysaccharides, Borrelia sp. antigen, Candida albicans antigen, and Plasmodium antigen The vaccine for immunotherapy and prevention, characterized in that the selected from. The antigen of claim 3, wherein the virus-derived antigen is an influenza virus antigen (haemagglutinin and neuraminidase antigens), human papilloma virus (HPV) antigen (glycoprotein), vesicular stomatitis virus ) Antigens (vesicular stomatitis virus glycoprotein), cytomegalovirus (CMV) antigens, hepatitis virus antigens (hepatitis A (HAV), B (HBV), C (HCV), D (HDV) and G (HGV)) (Core antigen and surface antigen), respiratory synchtial virus (RSV) antigen, herpes simplex virus antigen, human immunodeficiency virus (HIV) antigen (GP- 120, GP-160, p18, Tat, Gag, Pol, Env) and combinations thereof. The cancer antigen of claim 3, wherein the cancer antigen is HPV E6 / E7, gp100, tyrosinase, tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), MAGE, MUC-1 , Vaccines for immunotherapy and prophylaxis, characterized in that it is selected from the group consisting of CEA (carcinoembryonic antigen), p53, alpha-fetoprotein and Her-2 / neu . The vaccine of claim 1, wherein the antigen is in the form of a peptide, lipopolysaccharide, polysaccharide, glycoprotein, or polynucleotide. The vaccine for immunotherapy and prevention according to claim 1, wherein the expression of the antigen is introduced and expressed by a recombinant virus. The vaccine of claim 8, wherein the recombinant virus is an adenovirus, a retrovirus, a vaccinia virus, a pox virus, or a Sindbis virus into which a gene expressing an antigen is introduced. Natural killer T cell activator mediated by B cells loaded with alpha-galactosylceramide. A cytotoxic response inducer comprising B cells carrying alpha-galactosyl ceramide and expressing cancer antigens.

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