Vaccines comprising natural killer cells loaded with natural killer t cell ligands and cancer antigens

Abstract

The present invention relates to a vaccine for immunoprophylaxis and treatment comprising natural killer cells loaded with natural killer T cell ligands and antigens, and more particularly, to a vaccine for immunotherapy comprising natural killer cells loaded with natural killer T cell ligands and antigens. The vaccine comprises a natural killer T cell ligand alpha-galactosylceramide (alpha-GC) loaded with natural killer T cells and a natural killer cell of a glycolipid. The composition according to the present invention can be used as an anti-cancer immunotherapy because natural killer cells can be obtained more easily than dendritic cells, and immunization with natural killer cells loaded with natural killer T cell ligands and antigens induces a significant level of cytotoxic T lymphocyte response, and the medicine also has a treatment effect on malignant tumors.

Description

Technical Field

[0001] The present invention relates to a vaccine comprising natural killer cells loaded with natural killer T cell ligands and cancer antigens. Background Art

[0002] It is well known that natural killer cells are important innate immune cells that eliminate cancer cells and virus-infected cells through degranulation of cytotoxic granules containing perforin and granzymes, death receptor-mediated apoptosis and IFN-γ production, thereby preventing the development, growth and metastasis of cancer (Vivier et al., Science, 331:44-49, 2011). + T cells are similar in that they produce cytotoxicity to target cells, but unlike cytotoxic T cells, natural killer cells do not have antigen specificity, do not require sensitization or immunization, and can kill cancer cells with low MHC class I expression, which cytotoxic T cells cannot attack, thus playing a unique role in anti-cancer immunity (Uzhachenko and Shanker., Frontiers in Immunology, 10:1906, 2019). Because natural killer cells are different from cytotoxic CD8 + The unique characteristics of T cells are currently being actively studied for anticancer immunotherapy, with a focus on enhancing the cytotoxic capacity 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 invariant T cell receptors and are known to play an important role 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) is a glycolipid extracted from the sponge Agelasmauritianus and is a representative ligand for natural killer T cells. It is presented by the CD1d molecule and binds to the Vα14-Jα18 of natural killer T cells. +T cell receptor binding, thereby activating invariant natural killer T cells (Kawano et al., Science, 278: 1626-1629, 1997). α-GC activated natural killer T cells rapidly produce various cytokines, such as IL-4 and IFN-γ, which activate natural killer cells and induce acquired immune responses, including maturation of dendritic cells and activation and differentiation of T cells and B cells (Kaer et al., Nature Reviews Immunology, 5: 31-42, 2005).

[0004] It is reported that cancer cells loaded with α-GC can also function as antigen presenting cells, which 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 and Chung et al., OncoImmunology, 1: 2, 141-151, 2012). It has also been shown that B cells or T cells loaded with α-GC delivered antigens by peptide or viral vectors can also 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 loading α-GC with cancer antigens and delivering them to cells expressing CD1d molecules can be used as cell vaccines for anticancer therapy.

[0005] Therefore, the present inventors changed the immunogenicity of natural killer cells by loading the natural killer T cell ligand α-GC on natural killer cells with cytotoxic effects and delivering antigens, thereby increasing the function of antigen presenting cells, and confirmed that natural killer cells can induce anti-cancer immune responses, thereby completing the present invention. Summary of the invention

[0006] The object of the present invention is to provide a vaccine for immunotherapy or prevention, which comprises natural killer cells loaded with natural killer T cell ligands and cancer antigens.

[0007] In order to achieve the above object, the present invention provides a vaccine for immunotherapy or prevention, which comprises natural killer cells loaded with natural killer T cell ligands and cancer antigens.

[0008] Beneficial effects

[0009] The present invention relates to a vaccine for immunotherapy and prevention, comprising natural killer cells loaded with natural killer T cell ligands and cancer antigens, and more particularly, to a vaccine for immunotherapy or prevention, comprising natural killer cells loaded with natural killer T cell ligand α-galactosylceramide (hereinafter referred to as α-GC) and a glycolipid. The composition of the present invention can be used as an anti-cancer immunotherapy drug, because natural killer cells are more easily obtained than dendritic cells, and immunization with natural killer cells loaded with natural killer T cell ligands and antigens induces a significant level of cytotoxic T lymphocyte response and a therapeutic effect on malignant tumors.

[0010] BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1a It is a graph showing natural killer T cells (NKT) and IFN-γ production capacity of natural killer cells induced in spleen by vaccines comprising natural killer cells (NK) loaded with α-GC and / or cross-linked with adenovirus expressing antigen.

[0012] Figure 1b It is shown Figure 1a Figure 3. Graph of IFN-γ production by natural killer T cells (NKT).

[0013] Figure 1c It is shown Figure 1a Figure 3. Graph of IFN-γ production by natural killer (NK) cells.

[0014] Figure 2a is a diagram showing an experimental method for confirming the cytotoxic T cell response activity of a vaccine comprising natural killer cells loaded with α-GC and / or cross-linked with an adenovirus expressing an antigen.

[0015] Figure 2b Figure 2 shows the administration of NK / α-GC, NK / Adk35GM or NK / α-GC / Adk35GM and injection of GP100 loaded with CFSE labeled 25-33 A set of histograms showing the results of in vivo measurements of cytotoxic T cell activity after targeting of peptides to cells.

[0016] Figure 2c It is shown Figure 2b Graphical representation of cytotoxic T cell activity.

[0017] Figure 2d Figure 2 shows the administration of NK / α-GC / Ad-E6E7 and the injection of HPV16 E6 loaded with CFSE labeling. 49-57 and HPV16 E7 49-57 A set of histograms showing the results of in vivo measurements of cytotoxic T cell activity after targeting of peptides to cells.

[0018] Figure 2e It is shown Figure 2d Graphical representation of cytotoxic T cell activity.

[0019] Figure 3a It is a diagram showing an experimental method for confirming the anticancer effect of a vaccine comprising natural killer cells loaded with α-GC and cross-linked with an adenovirus expressing an antigen.

[0020] Figure 3b It is a graph showing the anticancer effect (change in tumor size) of a vaccine comprising natural killer cells loaded with α-GC and cross-linked with an adenovirus expressing an antigen (NK / α-GC / Ad-E6E7).

[0021] Figure 3c It is a graph showing the anticancer effect (mouse survival rate) of a vaccine comprising natural killer cells loaded with α-GC and cross-linked with an adenovirus expressing an antigen (NK / α-GC / Ad-E6E7).

[0022] Figure 4a : is a diagram showing an experimental method for confirming the activation of antigen-specific cytotoxic T cells by a vaccine comprising natural killer cells co-loaded with α-GC and a peptide.

[0023] Figure 4b is a set of histograms showing in vivo cytotoxic T cell activity induced by a vaccine comprising natural killer cells loaded with α-GC (NK / α-GC), the vaccine comprising ovalbumin loaded with 257-264 peptide-containing natural killer cells (NK / OVApep), or the vaccine contains co-loaded α-GC and ovalbumin 257-264 Peptide for natural killer cells (NK / α-GC / OVApep).

[0024] Figure 4c It is shown Figure 4b Graphical representation of cytotoxic T cell activity.

[0025] Figure 5a It is a diagram showing an experimental method for confirming the anticancer effect of a vaccine comprising natural killer cells co-loaded with α-GC and a peptide.

[0026] Figure 5b It is a graph showing the anticancer effect (change in tumor size) of a vaccine comprising natural killer cells co-loaded with α-GC and a peptide (NK / α-GC / OVA pep).

[0027] Figure 5c It is a graph showing the anticancer effect (mouse survival rate) of a vaccine comprising natural killer cells co-loaded with α-GC and a peptide (NK / α-GC / OVA pep). DETAILED DESCRIPTION

[0028] Hereinafter, the present invention is described in detail.

[0029] 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 cytotoxic T lymphocyte responses in B cells, monocytes and immature myeloid cells loaded with α-GC (Korean Patent Publication No. 10-2007-0105662 and Korean Patent Publication No. 10-2009-0051598), and confirmed whether similar effects were observed in natural killer cells loaded with α-GG.

[0030] Therefore, the present inventors produced a vaccine by altering the immunogenicity of natural killer cells by loading α-GC and delivering antigens to natural killer cells with cytotoxic effects.

[0031] Antigen delivery using viral vectors can efficiently deliver cancer antigens to cells, making it suitable for large-scale production of cell therapies targeting cancers that express specific cancer antigens. When using a virus that can express the above antigen, the entire antigen can be introduced, so it is applicable to all people, not limited to a specific haplotype of the major histocompatibility complex, and has the advantage of being able to induce not only cellular immune responses but also humoral immune responses. On the other hand, the application of viral vectors in personalized cancer therapy is limited.

[0032] In clinical use, the method of delivering antigens via peptide loading is limited to the haplotype of an individual's major histocompatibility complex (MHC), and therefore cannot be used universally, and has the disadvantage of presenting only a single epitope. However, it is known that neo-epitopes are ideal targets for personalized anti-cancer immunotherapy for cancer patients, i.e., peptide sequences generated by highly tumor-specific mutations that are present only in cancer cells, not in normal cells, and can induce cancer cell-specific immune responses. Anti-cancer immunotherapy using immune cell vaccines (which deliver neo-epitopes found for personalized treatment of cancer patients) is expected to induce the generation of more cancer cell-specific T cells than traditional immunotherapy, thereby minimizing the side effects of anti-cancer therapy by preventing damage to normal cells, while inducing a powerful anti-cancer therapeutic effect.

[0033] Therefore, natural killer cells were isolated from mice loaded with α-GC, and natural killer cell vaccines were constructed by delivering antigens in two ways (transferring antigen genes into viral vectors expressing antigens or loading with peptides) (see Example 1).

[0034] First, the present inventors constructed a natural killer cell vaccine loaded with α-GC and delivered with a viral vector expressing an antigen, and confirmed that administration of the vaccine could activate natural killer T cells and natural killer cells. The results confirmed that natural killer T cells and natural killer cells were activated in mice receiving natural killer cells loaded with α-GC and natural killer cells loaded with α-GC and cancer antigens GP100 and MAGE-A3 delivered by adenovirus (see Figure 1a to Figure 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. The results confirmed that the natural killer cell vaccine loaded with α-GC and cancer antigens (GP100 and MAGE-A3 or HPV16 / 18E6E7) delivered by adenovirus induced an effective cytotoxic response (see Figures 2a to 2e ). In addition, the anti-cancer effect of the vaccine was confirmed using HPV16 / 18E6E7. The results confirmed that the vaccine showed significant anti-cancer effects by inhibiting tumor size and improving mouse survival (see Figures 3a to 3c ).

[0035] Next, the inventors constructed a natural killer cell vaccine loaded with α-GC and antigen peptides and determined whether administration of the vaccine induced a cytotoxic T cell response. 257-264 The peptide-based natural killer cell vaccine induced a potent cytotoxic response (see Figures 4a to 4c ). In addition, the anticancer effect of the vaccine was studied and confirmed to show significant anticancer effect, inhibiting tumor size and improving mouse survival rate (see Figures 5a to 5c ). The natural killer cells of the present invention not only have cytotoxic functions and thus have anti-cancer effects themselves, but also acquire the characteristics of antigen presenting cells, which activate antigen-specific cytotoxic T cell responses by loading natural killer T cell ligands (particularly α-GC) and antigen delivery, thereby improving the therapeutic effect on malignant tumors. Therefore, the vaccine composition containing the above cells can be used as a cancer therapeutic drug.

[0036] The above-mentioned natural killer T cell ligands include α-galactosylceramide (α-GC), α-glucuronylceramide, phosphatidylinositol tetramethyl glycoside, isoglobin trihexosylceramide, ganglioside GD3, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sulfatide, β-galactosylceramide, lipophosphoglycolide, glycoinositol phospholipids, β-anomeric galactosylceramide, α-anomeric galactosylceramide, bacterial lipid antigens and variants of α-galactosylceramide, wherein the β-anomeric galactosylceramide and the α-anomeric galactosylceramide are analogs of α-galactosylceramide.

[0037] The above antigen may be any antigen that can be used as a vaccine and induce an immune response, and includes antigens derived from pathogens including pathogenic bacteria, viruses and parasites, or cancer antigens, and may be the full length or fragments of the above antigens.

[0038] 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 (capsular polysaccharide of Streptococcus pneumoniae type 3), 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.

[0039] Antigens derived from viruses include 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.

[0040] 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), mouse globulin 1 (MUC-1), carcinoembryonic antigen (CEA), p53, α-fetoprotein, Her-2 / neu expressed breast cancer protein, proteinase 3, WT-1, PAP, PSA, PSMA, G250, BAGE, GAGE, NY-ESO-1, MART-1, MCIR, Ig idiotype, CDK4, caspase-8, β-catenin, CIA, BCR / ABL, EBV LMP2a, HCV, HHV-8, 5T4, tumor-specific mutation-derived neoantigens, and combinations thereof.

[0041] In addition, the antigen antigen can be directly loaded into the natural killer cells in the form of peptides, lipopolysaccharides, polysaccharides, glycoproteins or polynucleotides (including DNA and RNA), and can be transfected into the natural killer cells by recombinant viruses and expressed and loaded. Compared with cell vaccines loaded with peptides, cell vaccines introduced by viruses together with the entire antigen are suitable for all populations, are not limited to the haplotypes of the major histocompatibility complex, and can induce immune responses specific to multiple epitopes, and in particular, have the advantage of being able to induce humoral immune responses and cellular immune responses at the same time.

[0042] The antigen can be expressed by introducing the antigen through a recombinant virus. The viruses mentioned above for introduction into natural killer cells for antigen expression include adenovirus, retrovirus, vaccinia virus, poxvirus and Sindbis virus, but are not always limited thereto. In addition to the method using viruses, the following methods can also be applied to antigen gene delivery: 1) a method of transducing DNA by combining DNA with liposomes to protect DNA from enzymatic degradation or to allow it to be absorbed into endosomes, 2) a method of improving the efficiency of DNA delivery into cells by combining a molecular conjugate composed of a protein or a synthetic ligand with DNA [e.g., asialoglycoprotein, transferrin, polymeric IgA], 3) a method of delivering antigen genes by improving the efficiency of DNA delivery into cells using a novel DNA delivery system using PTD (protein transduction domain) [e.g., Mph-1], and 4) in addition to the above methods, peptides or antigen proteins can be applied to natural killer cells to allow natural killer cells to present antigens.

[0043] In addition to natural killer T cell ligands and natural killer cells, the vaccine of the present invention may also include one or more active ingredients having the same or similar effects as them. In addition to the above-mentioned active ingredients, the vaccine may also include one or more pharmaceutically acceptable delivery vehicles. Pharmaceutically acceptable carriers can be selected or prepared by mixing more than one component selected from the group consisting of saline, Ringer's solution, buffered saline, glucose solution, maltose solution, glycerol and ethanol. Other general additives such as antioxidants, buffer solutions, antibacterial agents, etc. may be added. In order to prepare injectable solutions such as aqueous solutions, suspensions and emulsions, diluents, dispersants, surfactants, adhesives and lubricants may be additionally added. The vaccine of the present invention can also be prepared in a suitable form for each disease or according to the ingredients by following the method represented in Remington's Pharmaceutical Science (latest edition) of Mack Publishing Company, Easton, Pennsylvania.

[0044] The vaccine of the present invention can be administered parenterally, and parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection and intrathoracic injection. In order to prepare a vaccine as a parenteral preparation, natural killer cells loaded with natural killer T cell ligands, natural killer cells loaded with natural killer T cell ligands and peptides, or natural killer cells infected with a virus expressing a cancer antigen of the present invention are mixed with a stabilizer or a buffer to produce a solution or a suspension, which is then formulated into an ampoule or a vial.

[0045] The vaccine of the present invention can be formulated in various forms according to the route of administration. For example, the vaccine of the present invention can be prepared in the form of a sterile solution or suspension for injection, or in the form of a freeze-dried formulation using freeze-drying technology. The freeze-dried vaccine of the present invention should generally be maintained at about 4° C. and can be reconstituted in a stable solution containing or not containing an adjuvant (such as saline or / and HEPES).

[0046] For the purpose of the present invention, the effective dose of the vaccine to be administered is determined by taking into account the method of administration, the frequency of administration, the specific disease being treated, the severity of the disease, the medical history, whether the patient is treated with other drugs in combination, the age, height, weight, health status and other physical conditions of the patient, but is not always limited thereto. In general, the dose of such a preparation is preferably increased as the weight of the patient being treated increases.

[0047] The vaccine may be administered in an effective dose to induce an immune response in a patient. For example, a dose of 1x10 3 -1x10 9 cells / kg, more preferably 1x10 4 cells / kg to 1x10 8 To prepare a natural killer cell vaccine loaded with α-galactosylceramide, 1x10 6 -1x10 7 To prepare a natural killer cell vaccine loaded with α-galactosylceramide and peptide, it is necessary to supplement the culture medium with α-galactosylceramide at a concentration of 1-2 μg / ml per 1x10 natural killer cells. 6 -1x10 7 The medium was supplemented with α-galactosylceramide at a concentration of 1-2 μg / ml per 1x10 natural killer cells. 6 -1x10 7 Natural killer cells were supplemented with peptides at a concentration of 1-10 μg / ml.

[0048] α-Galactosylceramide does not seem to induce toxicity in rodents and monkeys (Nakata et al., Cancer Res 58: 1202-1207, 1988). No adverse reactions were reported even in mice injected with 2200 μg / ml of αGalCer (Giaccone et al., Clin Cancer Res 8: 3702, 2002). In ongoing clinical trials, some side effects of systemic administration of αGalCer, such as mild headaches, have been reported (Mie Nieda et al., Blood 103: 383-389, Giaccone et al., Clin Cancer Res 8: 3702, 2002), but these side effects can be prevented by administering acetaminophen, and mild systemic side effects do not necessarily occur in these subjects (Giaccone et al., Clin Cancer Res 8: 3702, 2002).

[0049] Hereinafter, the present invention will be described in detail by way of the following examples.

[0050] However, the following examples are only for illustrating the present invention, and the contents of the present invention are not limited thereto.

[0051] Example 1: Construction of natural killer cell vaccine

[0052] <1-1> Construction of a natural killer cell vaccine in which the antigen is delivered via a viral vector expressing the antigen

[0053] To isolate natural killer cells from mice, mouse spleen and bone marrow 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 natural killer cells separated and purified as described above were placed in a medium containing serum and IL-2 (5ng / mL) together with α-GC (1μg / ml) for gene delivery of cancer antigens (GP100 and MAGE-A3 cancer antigens or E6 and E7 cancer antigens of human papillomavirus HPV types 16 and 18), solvent (DMSO) and / or adenovirus (100MOI). After centrifugation 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 transformed with adenovirus to express cancer antigens, and natural killer cells loaded with α-GC and transformed with adenovirus to express cancer antigens. The prepared natural killer cell vaccine was washed three times with Dulbecco's phosphate buffered saline (DPBS, Welgene), dissolved in DPBS, and injected into the tail vein of mice.

[0054] <1-2> Construction of natural killer cell vaccine loaded with antigen peptide

[0055] Viral vectors are suitable gene delivery vehicles for large-scale production of cell therapy targeting cancer cells expressing specific cancer antigens because they deliver cancer antigens common to cancer cells with high efficiency. However, viral vectors have limitations in applying personalized treatment to cancer patients using new epitopes, so the present inventors have constructed a natural killer cell vaccine using antigen delivery via peptide loading.

[0056] Specifically, natural killer cells isolated from C57BL / 6 mice by the method of Example <1-1> were incubated with α-GC (1 μg / mL), solvent (DMSO) and / or ovalbumin. 257-264 (OVA 257-264 ) peptide (2 μg / mL) was placed in a medium containing serum and IL-2 (5 ng / 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 vaccines loaded with α-GC and OVA 257-264 Peptide-based natural killer cell vaccines or loaded with α-GC and OVA 257-264Peptide-based natural killer cell vaccine The prepared natural killer cell vaccine was washed three times with DPBS, dissolved in DPBS, and injected into the tail vein of mice.

[0057] Experimental Example 1: Demonstration of the effectiveness of a natural killer cell vaccine in which the antigen is delivered via a viral vector expressing the antigen deliver

[0058] <1-1> Confirmation of induction of activation of natural killer T cells and natural killer cells

[0059] The present inventors have demonstrated whether natural killer T cells and natural killer cells in vivo can be activated by administration of a natural killer cell vaccine.

[0060] Specifically, natural killer cells obtained from C57BL / 6 mice were used to generate natural killer cells loaded with α-GC (NK / α-GC), natural killer cells transduced with adenovirus Adk35GM for cancer antigen (human GP100 and MAGE-A3) delivery (NK / Adk35GM), and natural killer cells loaded with α-GC and antigen delivered using adenovirus Adk35GM (NK / α-GC / Adk35GM). Then, 8.5×10 4 After 17 hours, the levels of natural killer T cells and IFN-γ production in natural killer cells in the spleen were measured by flow cytometry.

[0061] The results confirmed that Figures 1a to 1c As shown, unlike the administration of transduced cells (NK / Adk35GM), the administration of α-GC-loaded natural killer cells (NK / α-GC and NK / α-GaC / Adk35GM) to mice stimulated natural killer T cells in vivo, thereby activating natural killer cells.

[0062] It is thought that activation of these NK T cells and natural killer cells may contribute to anticancer effects while inducing cytotoxic T cell responses.

[0063] <1-2> Demonstration of induction of cytotoxic T cell responses

[0064] <1-2-1> Evaluating the efficacy of a natural killer cell vaccine containing GP100 and MAGE-A3

[0065] To determine whether adenovirus-transduced natural killer cell vaccines could induce antigen-specific cytotoxic T cell immune responses, an in vivo CTL assay was performed.

[0066] Specifically, natural killer cell vaccines loaded with α-GC or adenovirus Adk35GM antigen delivery were constructed using natural killer cells obtained from C57BL / 6 mice, the vaccines were immunized into C57BL / 6 mice, and cytotoxicity assays were performed 10 days later ( Figure 2a). First, the spleen cells of the same mouse were divided into two groups of equal number. 25-33 The target cells of the peptide were stained with 3 μM CFSE (fluorescein diacetate succinimidyl ester) (CFSE high ) and control cells without peptide were labeled with 0.3 μM CFSE (CFSE low ) were labeled and then injected into vaccinated mice in equal amounts. One day later, CFSE in mouse spleen cells was counted by flow cytometry high :CFSE low ratio to measure target cell lysis ( Figure 2a ). CFSE loaded with antigen peptide high The percentage of cells is lower, indicating a higher antigen-specific cytotoxic T cell immune response.

[0067] Results, such as Figure 2b and 2c As shown, when immunization was performed with natural killer cells (NK / Adk35GM, NK / α-GC / Adk35GM) into which antigens were introduced via adenoviral vectors, effective cytotoxic T cell immune responses were observed.

[0068] <1-2-2> Evaluation of natural killer cells containing E6 and E7 antigens of human papillomavirus (HPV) types 16 and 18 Vaccine efficacy

[0069] To determine whether NK cell-mediated vaccination could be applied to other cancer antigens, the present inventors evaluated the efficacy of NK cell vaccines containing E6 and E7 cancer antigens of human papillomavirus (HPV) types 16 and 18.

[0070] Specifically, C57BL / 6 mice were treated with 6.5x10 5 NK cells were loaded with α-GC and transformed with adenovirus Ad-E6E7 to express HPV16 / 18E6E7, and then in vivo cytotoxicity assay was performed 8 days later. 49-57 and E7 49-57 CFSE high , while control cells were stained with CFSE without peptide loading. low Labeled, and equal amounts of cells from both groups were mixed and injected into immunized mice. One day later, single splenocytes were collected and CFSE was measured by flow cytometry. high CFSE low The proportion of the cell population.

[0071] Results, such as Figure 2d and 2eAs shown, in a group of mice that received natural killer cells loaded with α-GC and engineered to express HPV16 / 18 E6E7, induction of cytotoxicity against HPV16 E6E7 was demonstrated.

[0072] Based on the results of the above experimental examples <1-1> and <1-2>, it can be inferred that by administering a natural killer cell vaccine loaded with α-GC and antigens delivered by adenovirus vectors, innate immune responses, activation of natural killer T cells and natural killer cells, as well as acquired immune responses and cytotoxic T cell immune responses can be induced, thereby showing a powerful anti-cancer therapeutic effect.

[0073] <1-3> Evaluating the anticancer effects of natural killer cell vaccines

[0074] The present inventors investigated whether administration of a natural killer cell vaccine induces anti-cancer immunity.

[0075] In view of this, when converting 2x10 5 TC-1 cancer cells were implanted subcutaneously into one flank of C57BL / 6 mice 4 days later and 2.6x10 5 C57BL / 6 mice were immunized with NK / α-GC or NK / α-GC / Ad-E6E7 ( Figure 3a ). Then, the tumor size change and mouse survival rate were measured. The tumor size was calculated using the following equation by measuring the width, length, and height of the tumor using a caliper.

[0076] (Tumor size) = (width) x (length) x (height) x (π / 6)

[0077] Results, such as Figure 3b and 3c As shown, when the natural killer cell vaccine (NK / α-GC / Ad-E6E7) was administered, cancer growth was inhibited and a high survival rate was observed. In contrast, in mice administered with NK / α-GC, tumor growth was hardly inhibited and a low survival rate was observed.

[0078] From the above results, it was confirmed that the anticancer therapeutic effect could not be induced by activating natural killer T cells and natural killer cells loaded with α-GC alone.

[0079] Experimental Example 2: Confirmation of the effectiveness of natural killer cell vaccines loaded with antigen peptides

[0080] <2-1> Demonstration of induction of cytotoxic T cell responses

[0081] The present inventors investigated whether the natural killer cell vaccine loaded with antigen peptides of Example <1-2> can induce cytotoxic T cell response.

[0082] Through intravenous administration of natural killer cells (NK / α-GC) loaded with α-GC and OVA 257-264 Natural killer cells loaded with peptide (NK / OVApep) or α-GC and OVA 257-264 C57BL / 6 mice were immunized with natural killer cells (NK / α-GC / OVApep) and in vivo cytotoxicity assay was performed 8 days later ( Figure 4a ).

[0083] Results, such as Figure 4b and 4c As shown, only in the group of mice administered with NK / α-GC / OVA pep, OVA was loaded 257-264 Peptide CFSE high Most of the target cells were lysed. This confirmed that cytotoxic T cells can also be activated by natural killer cell vaccines loaded with α-GC and peptides.

[0084] However, in contrast to the group administered with natural killer cells (antigen delivered only by adenoviral vector not containing α-GC) in Experimental Example <1-2>, cytotoxic T cell responses were induced. Figure 2c ), no cytotoxic T cell response was observed in the group administered with NK / OVApep loaded with peptide alone ( Figure 4c ). This suggests that delivering the entire antigen via adenovirus to induce an adverse immune response specific for multiple epitopes is superior to presenting only a single peptide in inducing a cytotoxic T cell immune response.

[0085] <2-2> Evaluating the anticancer effect of natural killer cell vaccines

[0086] The present inventors investigated whether natural killer cell vaccines loaded with antigenic peptides can induce anticancer therapeutic effects ( Figure 5a ).

[0087] In view of this, when converting 1x10 5 Two days after B16F10-OVA cancer cells were implanted subcutaneously into mice, 2.5x10 5 C57BL / 6 mice were immunized with NK / OVApep or NK / α-GC / OVApep.

[0088] Results, such as Figure 5b and 5c As shown, when the natural killer cell vaccine (NK / α-GC / OVApep) was administered, cancer growth was significantly inhibited and a high survival rate was observed. In contrast, in mice administered with NK / OVApep, tumor growth was hardly inhibited and a low survival rate was observed. Therefore, it was shown that the loaded peptide alone could not induce a sufficient anti-cancer therapeutic effect.

[0089] When combined with the above results and the anticancer effect data of Example <1-3> ( Figure 3b and 3c ), which confirmed that the natural killer cell vaccine prepared by loading α-GC and delivering antigens showed stronger anti-cancer effects than cell vaccines loaded with only α-GG or delivered only antigens, inhibiting cancer growth and improving survival rate.

Claims

1. A vaccine for immunotherapy and prevention, comprising natural killer cells loaded with natural killer T cell ligands and antigens.

2. The vaccine for immunotherapy and prevention according to claim 1, wherein the natural killer T cell ligand is selected from the group consisting of α-galactosylceramide, α-glucuronylceramide, phosphatidylinositol tetramethyl glycoside, isoglobin trihexosylceramide, ganglioside GD3, phosphatidylcholine, β-galactosylceramide, lipophosphoglycolysate, glycoinositol phospholipids, β-anomeric galactosylceramide, α-anomeric galactosylceramide, bacterial lipid antigens and variants of α-galactosylceramide, and the β-anomeric galactosylceramide and the α-anomeric galactosylceramide are analogs of α-galactosylceramide.

3. The vaccine for immunotherapy and prevention according to claim 1, wherein the antigen is any antigen derived from a pathogen including pathogenic bacteria, virus and parasite, or a cancer antigen.

4. The vaccine for immunotherapy and prevention according to claim 3, wherein the antigen derived from the pathogen 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 (capsular polysaccharide of Streptococcus pneumoniae type 3), 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.

5. A vaccine for immunotherapy and prevention according to claim 3, wherein the antigen derived from the virus is selected from the group consisting of: influenza virus antigens (hemagglutinin and neuraminidase), human papillomavirus (HPV) antigens (glycoprotein), vesicular stomatitis virus antigens (vesicular stomatitis virus glycoprotein), 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 prevention 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), mouse globulin 1 (MUC-1), carcinoembryonic antigen (CEA), p53, α-fetoprotein, Her-2 / neu expressed breast cancer protein, proteinase 3, WT-1, PAP, PSA, PSMA, G250, BAGE, GAGE, NY-ESO-1, MART-1, MCIR, Ig idiotype, CDK4, caspase-8, β-catenin, CIA, BCR / ABL, EBV LMP2a, HCV, HHV-8, 5T4 and neoantigens derived from tumor-specific mutations.

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

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

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