Vaccine comprising a ligand for natural killer T cells and natural killer cells loaded with cancer antigens

Abstract

The present invention relates to an immunotherapeutic and prophylactic vaccine comprising a ligand for natural killer T cells and natural killer cells loaded with a cancer antigen. More specifically, the present invention relates to an immunotherapeutic or prophylactic vaccine comprising natural killer cells loaded with α-galactosylceramide (α-GC), which is a ligand for natural killer T cells and a kind of glycolipid. The composition of the present invention is such that natural killer cells are more easily obtainable than dendritic cells, and immunization with natural killer cells loaded with a ligand and antigen for natural killer T cells can be used as an anti-cancer immunotherapeutic agent not only for the therapeutic effect against malignant tumors but also for inducing a significant level of cytotoxic T lymphocyte response and a therapeutic effect against malignant tumors.

Description

Technical Field

[0001] Background of the Invention 1. Field of the Invention The present invention relates to a vaccine comprising a ligand for natural killer T cells and natural killer cells loaded with cancer antigens.

Background Art

[0002] 2. Description of the Related Art Natural killer cells are well-known as important innate immune cells that prevent the occurrence, growth, and metastasis of cancer by eliminating cancer cells and virus-infected cells through degranulation of cytotoxic granules containing perforin and granzyme, apoptosis mediated by cell death receptors, and IFN-γ production (Vivier et al., Science, 331: 44-49, 2011). Natural killer cells are similar to cytotoxic CD8+ T cells in that they cause cytotoxicity to target cells, but different from cytotoxic T cells, they do not have antigen specificity, do not require sensitization or immunization, and can kill cancer cells with low MHC class I expression that cannot be attacked by cytotoxic T cells, thus playing a unique role in anti-cancer immunity (Uzhachenko and Shanker., Frontiers in Immunology, 10: 1906, 2019). Due to the unique characteristics of natural killer cells different from cytotoxic CD8+ T cells, active research has been conducted on anti-cancer immunotherapy focusing on enhancing the cytotoxic ability of natural killer cells such as CAR-NK (chimeric antigen receptor-NK) (Shimasaki et al., Nature Reviews Drug Discovery, 19: 200-218, 2020).

[0003] Invariant natural killer T cells (iNKT) are innate immune cells that express an invariant T cell receptor and are known to play important roles in various immune diseases such as cancer, infectious diseases, and autoimmune diseases mainly through cytokine secretion (Bendelac et al., Annual Review of Immunology, 25:297-336, 2007). α-Galactosylceramide (α-GC), a representative ligand of natural killer T cells, is a glycolipid extracted from the sponge Agelas mauritianus. It is presented by the CD1d molecule, binds to the Vα14-Jα18+ T cell receptor of natural killer T cells, thereby activating invariant natural killer T cells (Kawano et al., Science, 278:1626-1629, 1997). Natural killer T cells activated by α-GC rapidly produce various cytokines such as IL-4 and IFN-γ, activate natural killer cells, and induce an acquired immune response including dendritic cell maturation and activation and differentiation of T cells and B cells (Kaer et al., Nature Reviews Immunology, 5:31-42, 2005).

[0004] Cancer cells loaded with α-GC have been reported to be able to function as antigen-presenting cells that can induce natural killer T cells and natural killer cell responses, as well as cancer antigen-specific T cell immune responses (Shimizu et al., The Journal of Immunology, 178: 2853-2861, 2007 & Chung et al., OncoImmunology, 1:2, 141-151, 2012). α-GC-loaded B cells or T cells delivered with antigens via peptides or viral vectors have also been shown to be able to acquire the characteristics of antigen-presenting cells (Chung et al., Cancer Research, 66:6843-6850, 2006 and Chung et al., OncoImmunology, 1:2, 141-151, 2012). This indicates that the loading of α-GC and the delivery of cancer antigens to cells expressing CD1d molecules can be utilized as a cellular vaccine for cancer treatment.

[0005] Therefore, the inventors have added the function of antigen-presenting cells by changing the immunogenicity of natural killer cells by loading α-GC, a natural killer T cell ligand, onto natural killer cells that have cytotoxic effects and deliver antigens, and confirmed that natural killer cells can induce an anti-cancer immune response, thereby completing the present invention.

[0006] Summary of the Invention An object of the present invention is to provide a vaccine for immunotherapy or prophylaxis comprising natural killer cells loaded with a ligand of natural killer T cells and a cancer antigen.

[0007] To achieve the above object, the present invention provides a vaccine for immunotherapy or prophylaxis comprising natural killer cells loaded with a ligand of natural killer T cells and a cancer antigen.

[0008] Advantageous Effects The present invention relates to immunotherapeutic and prophylactic vaccines comprising ligands for natural killer T cells and natural killer cells loaded with cancer antigens, and more specifically, to immunotherapeutic or prophylactic vaccines comprising natural killer cells loaded with α-galactosylceramide (hereinafter, α-GC), which is a ligand for natural killer T cells and a type of glycolipid. The composition of the present invention is such that natural killer cells are more easily obtainable than dendritic cells, and immunization with natural killer cells loaded with ligands and antigens for natural killer T cells can be used as an anti-cancer immunotherapeutic agent not only for the therapeutic effect on malignant tumors but also for inducing a significant level of cytotoxic T lymphocyte response and a therapeutic effect on malignant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Brief Description of the Drawings

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Figure 1b-1c

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Figure 2a

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Figure 5a-5b

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[0014] Description of Preferred Embodiments Hereinafter, the present invention will be described in detail. It is well known that dendritic cells (DCs) loaded with α-galactosylceramide (α-GC) activate invariant natural killer T (NKT) cells (van der Vliet HJ, et al., J Immunol Methods., 1;247(1-2):61-72, 2001). The present inventors confirmed the effect of inducing a cytotoxic T lymphocyte response in B cells, monocytes, and immature myeloid cells loaded with α-GC (Korean Patent Publication No. 10-2007-0105662, Korean Patent Publication No. 10-2009-0051598), and confirmed whether a similar effect is observed in natural killer cells loaded with α-GC.

[0015] Therefore, the present inventors produced a vaccine by changing the immunogenicity of natural killer cells through α-GC loading and antigen delivery to natural killer cells having a cytotoxic effect.

[0016] Antigen delivery using viral vectors can efficiently deliver cancer antigens to cells and is suitable for the large-scale production of cell therapy drugs targeting cancers that express specific cancer antigens. When a virus capable of expressing the above antigen is used, the entire antigen can be introduced, and it is applicable to all people without being restricted to specific haplotypes of the major histocompatibility complex, and has the advantage that it can induce not only cellular immune responses but also humoral immune responses. On the other hand, viral vectors are restricted in their application to individualized cancer treatment.

[0017] The antigen delivery method through peptide loading, when used clinically, is restricted by the haplotype of the major histocompatibility complex (MHC) of an individual, so it cannot be universally used and has the drawback of presenting only a single epitope. However, neoepitopes, which are peptide sequences created by highly tumor-specific mutations that exist only in cancer cells and not in normal cells and can induce cancer cell-specific immune responses, are known as ideal targets for individualized anti-cancer immunotherapy for cancer patients. Anti-cancer immunotherapy using immune cell vaccines that deliver neoepitopes discovered for individualized treatment of cancer patients is expected to induce the generation of more cancer cell-specific T cells than conventional immunotherapy, minimize the side effects of anti-cancer treatment by preventing damage to normal cells, and at the same time induce a strong anti-cancer treatment effect.

[0018] Therefore, natural killer cells were isolated from mice, loaded with α-GC, and a natural killer cell vaccine was constructed by delivering the antigen in two ways (either transferring the antigen gene into a viral vector expressing the antigen or loading the peptide) (see Example 1).

[0019] First, the inventors constructed a natural killer cell vaccine loaded with α-GC and delivered by a viral vector expressing an antigen, and confirmed that administration of the vaccine could activate natural killer T cells and natural killer cells. As a result, in mice that received natural killer cells loaded with α-GC and natural killer cells loaded with α-GC and adenovirus-delivered cancer antigens GP100 and MAGE-A3, activation of natural killer T cells and natural killer cells was confirmed (see FIGS. 1a to 1c). In addition, an in vivo CTL assay was performed to determine whether the vaccine could induce a cytotoxic immune response by activating antigen-specific cytotoxic T lymphocytes. As a result, it was confirmed that an effective cytotoxic response was induced by a natural killer cell vaccine loaded with α-GC and delivering a cancer antigen (GP100 and MAGE-A3 or HPV16 / 18 E6E7) via an adenovirus (see FIGS. 2a to 2e). In addition, the anti-cancer effect of the vaccine was confirmed using HPV16 / 18 E6E7. As a result, it was confirmed that the vaccine exhibited a remarkable anti-cancer effect such as suppressing tumor size and increasing the survival rate of mice (see FIGS. 3a to 3c).

[0020] Next, the inventors constructed a natural killer cell vaccine loaded with α-GC and an antigen peptide, and determined whether administration of the vaccine could induce a cytotoxic T cell response. As a result, α-GC and OVA 257-264It was confirmed that a natural killer cell vaccine loaded with a peptide induced an effective cytotoxic response (see FIGS. 4a to 4c). In addition, the anti-cancer effect of the vaccine was investigated, and it was confirmed that the vaccine exhibited a remarkable anti-cancer effect of suppressing tumor size and increasing the survival rate of mice (see FIGS. 5a to 5c). The natural killer cells of the present invention have a cytotoxic function, not only have an anti-cancer effect by themselves, but also load a ligand of natural killer T cells, particularly α-GC, and perform antigen delivery, thereby obtaining the property of an antigen-presenting cell that activates an antigen-specific cytotoxic T cell response, thereby improving the therapeutic effect against malignant tumors. Therefore, the vaccine composition containing the above cells can be used as a cancer treatment agent.

[0021] The ligands of the natural killer T cells described above include α-galactosylceramide (α-GC), α-glucuronosylceramide, phosphatidylinositol tetramannoside, isoglobotrihexosylceramide, ganglioside GD3, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sulfide, β-galactosylceramide, lipophosphoglycan, glycoinositol phospholipid, β-anomer galactosylceramide and α-anomer galactosylceramide which are analogs of α-galactosylceramide, bacterial lipid antigens, and mutants of α-galactosylceramide.

[0022] The above antigen can be used as a vaccine and can be any antigen that can induce an immune response, and includes antigens derived from pathogens including pathogenic bacteria, viruses, and parasites, or cancer antigens, and can be the full length or a fragment of the above antigen.

[0023] Antigens derived from pathogenic bacteria include Bordetella pertussis antigens (pertussis toxin, filamentous hemagglutinin, and pertactin), tetanus toxoid, diphtheria toxoid, Helicobacter pylori antigens (capsular polysaccharides of serogroups A, B, C, Y, and W-135), pneumococcal antigens (pneumococcal type 3 capsular polysaccharide), tuberculosis antigens, cholera antigens (cholera toxin B subunit), staphylococcal antigens (staphylococcal enterotoxin B), Shigella antigens (Shigella polysaccharide), Borrelia antigens, Candida albicans antigens, and malaria parasite antigens.

[0024] Antigens derived from viruses include influenza virus antigens (hemagglutinin and neuraminidase), human papillomavirus (HPV) antigens (glycoproteins), vesicular stomatitis virus antigens (vesicular stomatitis virus glycoprotein), cytomegalovirus (CMV) antigens, hepatitis virus antigens (hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis G virus (HGV) antigens) (core antigen and surface antigen), respiratory syncytial virus (RSV) antigens, herpes simplex virus antigens, human immunodeficiency virus (HIV) antigens (GP-120, GP-160, p18, Tat, Gag, Pol, Env), and combinations thereof.

[0025] Cancer antigens include gp100, melanoma antigen gene (MAGE), human papillomavirus (HPV) E6 / E7, tyrosinase, tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), murine globulin 1 (MUC-1), carcinoembryonic antigen (CEA), p53, alpha-fetoprotein, breast cancer protein expressed by Her-2 / neu, proteinase 3, WT-1, PAP, PSA, PSMA, G250, BAGE, GAGE, NY-ESO-1, MART-1, MC1R, Ig idiotype, CDK4, caspase-8, beta-catenin, CIA, BCR / ABL, EBV LMP2a, HCV, HHV-8, 5T4, neoantigens derived from tumor-specific mutations, and combinations thereof.

[0026] In addition, the antigen may be directly loaded onto natural killer cells in the form of a peptide, lipopolysaccharide, polysaccharide, glycoprotein, or polynucleotide including DNA and RNA, or may be transduced and expressed in natural killer cells by a recombinant virus. Compared with a cell vaccine loaded with a peptide, a cell vaccine that introduces the entire antigen via a virus is not restricted by the haplotype of the major histocompatibility complex gene complex, is applicable to all people, can induce an immune response specific to multiple epitopes, and in particular, has the advantage that it can simultaneously induce a humoral immune response and a cellular immune response.

[0027] The antigen can be expressed by introduction through a recombinant virus. The viruses introduced into natural killer cells for antigen expression as described above include, but are not limited to, adenovirus, retrovirus, vaccinia virus, poxvirus, and Sindbis virus. In addition to the method using a virus, the following methods can be applied for the delivery of the antigen gene: 1) a method of binding DNA to liposomes to protect DNA from enzymatic degradation or to introduce DNA by uptake into endosomes, 2) a method of linking a molecular conjugate consisting of a protein or synthetic ligand to DNA to increase the efficiency of DNA delivery into cells (e.g., asialoorosomucoid, transferrin, polymeric IgA), 3) a method of delivering the antigen gene by increasing the efficiency of DNA delivery into cells through a novel DNA delivery system using PTD (protein transduction domain) (e.g., Mph-1), and 4) in addition to the above methods, a peptide can be used, or an antigen protein can be applied to natural killer cells to present the antigen to natural killer cells.

[0028] The vaccine of the present invention may further include one or more active ingredients having the same or similar effects as natural killer T cell ligands and natural killer cells in addition to natural killer T cell ligands and natural killer cells. The vaccine may also include, in addition to the above-described active ingredients, one or more pharmaceutically acceptable carriers for administration. The pharmaceutically acceptable carrier may be selected or prepared by mixing more than one component selected from the group consisting of physiological saline, Ringer's solution, buffered physiological saline, dextrose solution, maltodextrin solution, glycerol, and ethanol. In addition, common additives such as antioxidants, buffers, and bactericides may be added. For preparing injection solutions such as aqueous solutions, suspensions, and emulsions, diluents, dispersants, surfactants, binders, and lubricants may be further added. The vaccine of the present invention may be further prepared in a suitable form for each disease or according to the components by following the methods described in Remington’s Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA.

[0029] The vaccine of the present invention can be administered parenterally, and parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection, and intrathoracic injection. To prepare the vaccine as a parenteral preparation, natural killer cells loaded with the ligand of natural killer T cells of the present invention, natural killer cells loaded with the ligand of natural killer T cells and peptides, or natural killer cells infected with a virus expressing a cancer antigen are mixed with a stabilizer or buffer to produce a solution or suspension, which is then formulated as an ampule or vial.

[0030] The vaccine of the present invention can be formulated into various forms according to the administration route. For example, the vaccine of the present invention can be prepared in the form of a sterilized injection solution or suspension, or in the form of a lyophilized preparation using lyophilization technology. The lyophilized vaccine of the present invention is typically maintained at about 4°C and can be reconstituted in a stabilizing solution with or without adjuvants such as physiological saline and / or HEPES.

[0031] To achieve the present invention, the effective dose of the vaccine to be administered is determined in consideration of the administration method, administration frequency, specific disease during treatment, disease severity, medical history, whether the patient is undergoing combined treatment with other drugs, the patient's age, height, weight, health status, and other physical conditions, but is not necessarily limited thereto. Generally, as the weight of the patient during treatment increases, it is preferable to increase the dose of this preparation.

[0032] The vaccine can be administered in an effective dose to induce an immune response in the patient. For example, the vaccine is 1×10 3 ~1×10 9 cells / kg, more preferably 1×10 4 cells / kg~1×10 8 cells / kg, and can be administered to humans once or several times a day. To prepare a natural killer cell vaccine loaded with α-galactosylceramide, the medium needs to be supplemented with α-galactosylceramide at a concentration of 1 - 2 μg / ml per 1×10 6 ~1×10 7 natural killer cells / ml. To prepare a natural killer cell vaccine loaded with α-galactosylceramide and peptide, the medium needs to be supplemented with α-galactosylceramide at a concentration of 1 - 2 μg / ml per 1×10 6 ~1×10 7 natural killer cells / ml, and loaded with peptide at a concentration of 1 - 10 μg / ml per 1×10 6 ~1×10 7 natural killer cells / ml.

[0033] α-Galactosylceramide does not seem to induce toxicity in rodents and monkeys (Nakata et al., Cancer Res 58:1202-1207, 1988). No side effects were reported even when 2200 μg / kg of αGalCer was injected into mice (Giaccone et al., Clin Cancer Res 8:3702, 2002). In ongoing clinical trials, some side effects such as mild headache have been reported with systemic administration of αGalCer (Mie Nieda et al., Blood 103:383-389, Giaccone et al., Clin Cancer Res 8:3702, 2002), but these can be prevented by administration of paracetamol and not all subjects necessarily experience mild systemic side effects (Giaccone et al., Clin Cancer Res 8:3702, 2002).

[0034] Hereinafter, the present invention will be described in detail by way of examples. However, the following experimental examples are only for illustrating the present invention and the content of the present invention is not limited thereto.

[0035] Example 1: Construction of natural killer cell vaccine <1-1> Construction of natural killer cell vaccine that delivers an antigen by a viral vector expressing the antigen To isolate natural killer cells from mice, the spleens and bone marrows of mice were collected and then homogenized. After lysing red blood cells using ACK lysis buffer (Gibco), T cells, B cells, and neutrophils expressing CD3ε, CD19, or Ly6G on the cell surface were removed using microbeads (Miltenyibiotec), and then CD49b+ cells were obtained using anti-CD49b microbeads (Miltenyibiotec), and pure natural killer cells were isolated using a BD FACSAria III instrument. The isolated and purified natural killer cells were placed in a medium containing α-GC (1 μg / ml), solvent (DMSO), and / or adenovirus (100 MOI) in addition to serum and IL-2 (5 ng / mL) for gene delivery of cancer antigens (GP100 and MAGE-A3 cancer antigens, or E6 and E7 cancer antigens of human papillomavirus types 16 and 18). After centrifuging at 2000 rpm for 90 minutes at 20°C in a cell culture plate, the cells were cultured in a CO2 incubator for 14 - 15 hours (37°C, relative humidity 80 - 95%, CO2 concentration 5%) to prepare natural killer cells loaded with α-GC, natural killer cells expressing cancer antigens transformed with adenovirus, and natural killer cells loaded with α-GC and expressing cancer antigens transformed with adenovirus. The prepared natural killer cell vaccine was washed three times with Dulbecco's phosphate buffered saline (DPBS, Welgene), dissolved in DPBS, and administered to the tail vein of mice.

[0036] <1-2> Construction of natural killer cell vaccine loaded with antigen peptide Viral vectors are suitable gene delivery vehicles for large-scale production of cell therapy drugs targeting cancer cells expressing specific cancer antigens because they can deliver cancer antigens common to cancer cells with high efficiency. However, since viral vectors have limitations in the application of individualized treatment to cancer patients using neoepitopes, the present inventors constructed a natural killer cell vaccine using antigen delivery via peptide loading.

[0037] Specifically, natural killer cells isolated from C57BL / 6 mice by the method of Example <1-1> were placed in a medium containing serum and IL-2 (5 ng / mL), α-GC (1 μg / mL), solvent (DMSO), and / or ovalbumin 257-264 (OVA 257-264 ) peptide (2 μg / mL), and cultured in a CO2 incubator for 15 - 16 hours (37°C, relative humidity 80 - 95%, CO2 concentration 5%) to prepare natural killer cell vaccine loaded with α-GC, natural killer cell vaccine loaded with OVA 257-264 peptide, or natural killer cell vaccine loaded with α-GC and OVA 257-264 peptide. The prepared natural killer cell vaccine was washed 3 times with DPBS, dissolved in DPBS, and administered to the tail vein of mice.

[0038] Experimental Example 1: Confirmation of the effectiveness of natural killer cell vaccine in which antigen is delivered by a virus vector expressing the antigen <1-1> Confirmation of induction of activation of natural killer T cells and natural killer cells The present inventors confirmed whether natural killer T cells and natural killer cells in the body can be activated through administration of the natural killer cell vaccine.

[0039] Specifically, using natural killer cells obtained from C57BL / 6 mice, natural killer cells loaded with α-GC (NK / α-GC), natural killer cells into which adenovirus Adk35GM was introduced for delivery of cancer antigens (human GP100 and MAGE-A3) (NK / Adk35GM), and natural killer cells loaded with α-GC and having antigens delivered by adenovirus Adk35GM (NK / α-GC / Adk35GM) were generated. Then, 8.5×10 4 individual natural killer cells were administered intravenously. 17 hours later, the levels of IFN-γ production in natural killer T cells and natural killer cells in the spleen were measured by flow cytometry.

[0040] As a result, as shown in FIGS. 1a to 1c, when natural killer cells loaded with α-GC (NK / α-GC and NK / α-GC / Adk35GM) were administered to mice, unlike the case where transduced cells (NK / Adk35GM) were administered, natural killer T cells in the body were stimulated, and as a result, it was confirmed that natural killer cells were activated. The activation of these natural killer T cells and natural killer cells is considered to potentially contribute to the anti-cancer effect along with the induction of cytotoxic T cell responses.

[0041] <1-2> Confirmation of induction of cytotoxic T cell response <1-2-1> Evaluation of the effectiveness of natural killer cell vaccine containing GP100 and MAGE-A3 To determine whether a natural killer cell vaccine introduced with an adenovirus can induce an antigen-specific cytotoxic T cell immune response, a CTL assay was performed in vivo.

[0042] Specifically, using natural killer cells obtained from C57BL / 6 mice, an α-GC-loaded or adenovirus Adk35GM antigen delivery natural killer cell vaccine was constructed, immunized into C57BL / 6 mice, and a cytotoxicity assay was performed 10 days later (FIG. 2a). First, spleen cells from the same mice were equally divided into two groups. GP100 25-33 Target cells loaded with the peptide were labeled with 3 μM of CFSE (carboxyfluorescein diacetate succinimidyl ester) (CFSE high )), and control cells without the peptide were labeled with 0.3 μM of CFSE (CFSE low ), and then an equal amount was injected into the vaccinated mice. One day later, the CFSE high :CFSE low ratio in mouse spleen cells was calculated by flow cytometry, and the lysis of target cells was measured (FIG. 2a). CFSE loaded with the antigen peptide highThe lower the percentage of cells, the higher the antigen-specific cytotoxic T cell immune response.

[0043] As a result, as shown in FIGS. 2b and 2c, an effective cytotoxic T cell immune response was observed when immunizing with natural killer cells (NK / Adk35GM, NK / α-GC / Adk35GM) into which the antigen was introduced via an adenovirus vector.

[0044] <1-2-2> Evaluation of the effectiveness of a natural killer cell vaccine containing E6 and E7 antigens of human papillomavirus (HPV) types 16 and 18 To determine whether natural killer cell-mediated vaccination can be applied to other cancer antigens, the inventors evaluated the effectiveness of a natural killer cell vaccine containing the E6 and E7 cancer antigens of human papillomavirus (HPV) types 16 and 18.

[0045] Specifically, 6.5×10 5 natural killer cells designed to be loaded with α-GC and express HPV16 / 18 E6E7 via the adenovirus Ad-E6E7 were used to immunize C57BL / 6 mice, and then a cytotoxic assay was performed in vivo 8 days later. Target cells loaded with HPV16 E6 49-57 and E7 49-57 peptides were labeled with CFSE high while control cells were labeled with CFSE low without loading with peptides, equal amounts of cells from both groups were mixed and injected into the immunized mice. One day later, single spleen cells were collected and the ratio of the CFSE high cell population of the CFSE low cell population was measured by flow cytometry.

[0046] As a result, as shown in FIGS. 2d and 2e, it was confirmed that cytotoxicity against HPV16 E6E7 was induced in vivo in the group of mice that received natural killer cells designed to be loaded with α-GC and express HPV16 / 18 E6E7.

[0047] Based on the results of the above Experimental Examples <1-1> and <1-2>, administration of the natural killer cell vaccine loaded with α-GC and with antigen delivery by an adenovirus vector induces both a natural immune response that is activation of natural killer T cells and natural killer cells, and an acquired immune response that is a cytotoxic T cell immune response, whereby it can be inferred that a strong anti-cancer therapeutic effect can be exhibited.

[0048] <1-3> Evaluation of the anti-cancer effect of the natural killer cell vaccine The present inventors investigated whether administration of the natural killer cell vaccine induces anti-cancer immunity. For this purpose, 4 days after subcutaneously implanting 2×10 5 TC-1 cancer cells into the flanks of C57BL / 6 mice, the C57BL / 6 mice were immunized with 2.6×10 5 NK / α-GC or NK / α-GC / Ad-E6E7 (FIG. 3a). Subsequently, changes in tumor size and the survival rate of the mice were measured. The tumor size was measured using calipers for the width, length, and height of the tumor, and calculated using the following formula. (Tumor size) = (width) × (length) × (height) × (π / 6)

[0049] As a result, as shown in FIGS. 3b and 3c, when the natural killer cell vaccine (NK / α-GC / Ad-E6E7) was administered, cancer growth was suppressed and a high survival rate was observed. In contrast, in the mice administered with NK / α-GC, tumor growth was hardly inhibited and a low survival rate was observed. Through the above results, it was confirmed that the anti-cancer therapeutic effect could not be induced by activating natural killer T cells and natural killer cells loaded only with α-GC.

[0050] Experimental Example 2: Confirmation of the effectiveness of natural killer cell vaccine loaded with antigen peptide <2-1> Confirmation of induction of cytotoxic T cell response The present inventors investigated whether the natural killer cell vaccine loaded with the antigen peptide of Example <1-2> could induce a cytotoxic T cell response. C57BL / 6 mice were immunized by intravenous administration of natural killer cells loaded with α-GC (NK / α-GC), natural killer cells loaded with OVA 257-264 peptide (NK / OVA pep), or natural killer cells loaded with both α-GC and OVA 257-264 peptide (NK / α-GC / OVA pep), and a cytotoxic assay was performed in vivo 8 days later (Figure 4a).

[0051] As a result, as shown in Figures 4b and 4c, only in the group of mice administered with NK / α-GC / OVA pep, the CFSE 257-264 target cells loaded with OVA peptide were mostly lysed. This confirmed that cytotoxic T cells could also be activated by the natural killer cell vaccine loaded with α-GC and peptide. high

[0052] However, in Experimental Example <1-2>, unlike the group administered with natural killer cells that delivered only the antigen via an adenovirus vector not containing α-GC (Figure 2c), no cytotoxic T cell response was observed in the group administered with NK / OVA pep loaded with only the peptide (Figure 4c). This suggests that delivering the entire antigen via an adenovirus and inducing a diverse immune response specific to multiple epitopes is superior to presenting only a single peptide in inducing a cytotoxic T cell immune response.

[0053] <2-2> Evaluation of the anti-cancer effect of natural killer cell vaccine The present inventors investigated whether a natural killer cell vaccine loaded with an antigen peptide could induce an anti-cancer therapeutic effect (Figure 5a). For this purpose, 2 days after transplanting 1×10 5 B16F10-OVA cancer cells subcutaneously into mice, C57BL / 6 mice were immunized with 2.5×10 5 NK / OVA pep or NK / α-GC / OVA pep.

[0054] As a result, as shown in Figures 5b and 5c, when the natural killer cell vaccine (NK / α-GC / OVA pep) was administered, cancer growth was significantly suppressed and a high survival rate was observed. In contrast, in mice administered with NK / OVA pep, tumor growth was hardly inhibited and a low survival rate was observed. Therefore, it became clear that simply loading the peptide could not induce a sufficient anti-cancer therapeutic effect.

[0055] Combining the above results with the anti-cancer effect data of Example <1-3> (Figures 3b and 3c), it was confirmed that the natural killer cell vaccine prepared by loading α-GC and delivering the antigen exhibited a stronger anti-cancer effect than the cell vaccine loaded with only α-GC or the cell vaccine delivering only the antigen, inhibited cancer growth, and improved the survival rate.

Claims

**Claim 1** A vaccine for immunotherapy and prophylaxis, comprising a ligand for natural killer T cells and natural killer cells loaded with an antigen. **Claim 2** The vaccine for immunotherapy and prophylaxis according to claim 1, wherein the natural killer T cell ligand is selected from the group consisting of α-galactosylceramide, α-glucuronosylceramide, phosphatidylinositol tetramannoside, isoglobotrihexosylceramide, ganglioside GD3, phosphatidylcholine, β-galactosylceramide, lipophosphoglycan, glycoinositol phospholipid, β-anomer galactosylceramide and α-anomer galactosylceramide which are analogs of α-galactosylceramide, bacterial lipid antigens, and variants of α-galactosylceramide. **Claim 3** The vaccine for immunotherapy and prophylaxis according to claim 1, wherein the antigen is any antigen derived from a pathogen including pathogenic bacteria, viruses, and parasites, or a cancer antigen. **Claim 4** The vaccine for immunotherapy and prophylaxis according to claim 3, wherein the antigen derived from a pathogenic bacterium is selected from the group consisting of Bordetella pertussis antigens (pertussis toxin, filamentous hemagglutinin, and pertactin), tetanus toxoid, diphtheria toxoid, Helicobacter pylori antigens (capsular polysaccharides of serogroups A, B, C, Y, and W-135), pneumococcal antigens (pneumococcal type 3 capsular polysaccharide), tuberculosis antigens, cholera antigens (cholera toxin B subunit), staphylococcal antigens (staphylococcal enterotoxin B), Shigella antigens (Shigella polysaccharide), Borrelia antigens, Candida albicans antigens, and Plasmodium antigens. **Claim 5** The vaccine for immunotherapy and prophylaxis according to claim 3, wherein the virus-derived antigen is selected from the group consisting of influenza virus antigens (hemagglutinin and neuraminidase), human papillomavirus (HPV) antigens (glycoproteins), vesicular stomatitis virus antigens (vesicular stomatitis virus glycoproteins), cytomegalovirus (CMV) antigens, hepatitis virus antigens (hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), hepatitis D (HDV) and hepatitis G (HGV) antigens) (core antigen and surface antigen), respiratory syncytial virus (RSV) antigens, herpes simplex virus antigens, human immunodeficiency virus (HIV) antigens (GP-120, GP-160, p18, Tat, Gag, Pol, Env) and combinations thereof.

6. The vaccine for immunotherapy and prophylaxis according to claim 3, wherein the cancer antigen is selected from the group consisting of gp100, melanoma antigen gene (MAGE), human papillomavirus (HPV) E6 / E7, tyrosinase, tyrosinase-related protein-1 (TRP-1), tyrosinase-related protein-2 (TRP-2), murine globulin 1 (MUC-1), carcinoembryonic antigen (CEA), p53, alpha-fetoprotein, breast cancer protein expressed by Her-2 / neu, proteinase 3, WT-1, PAP, PSA, PSMA, G250, BAGE, GAGE, NY-ESO-1, MART-1, MC1R, Ig idiotype, CDK4, caspase-8, beta-catenin, CIA, BCR / ABL, EBV LMP2a, HCV, HHV-8, 5T4, and neoplasms derived from tumor-specific mutations.

7. The vaccine for immunotherapy and prophylaxis according to claim 1, wherein the antigen is in the form of a peptide, lipopolysaccharide, polysaccharide, glycoprotein, or polynucleotide.

8. The vaccine for immunotherapy and prophylaxis according to claim 1, wherein the antigen is introduced and expressed by a recombinant virus.

9. The vaccine for immunotherapy and prophylaxis according to claim 8, wherein the recombinant virus is an adenovirus, retrovirus, vaccinia virus, poxvirus, or sindbis virus into which a gene expressing the antigen has been introduced.

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