Vaccines comprising peripheral blood mononuclear cells loaded with natural killer t cell ligands and antigens

Abstract

The present invention relates to an immunoprophylactic and therapeutic vaccine comprising a peripheral blood mononuclear cell loaded with a natural killer T cell ligand and an antigen, and more particularly, to an immunotherapeutic vaccine comprising a peripheral blood mononuclear cell loaded with alpha-galactosylceramide, a natural killer T cell ligand and a glycolipid. The compositions of the present invention are readily available because there is no need to isolate specific cells from peripheral blood mononuclear cells. Besides, the immunization of the peripheral blood mononuclear cells loaded with the natural killer T cell ligands and antigens not only induces the obvious-level activation of the natural killer cells and the natural killer T cells and the response of cytotoxic T lymphocytes, but also has a synergistic treatment effect in the aspect of treating malignant tumors. Therefore, the compound can be beneficially used as an anti-cancer immunotherapeutic agent.

Description

Background Art Field of the Invention

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of Korean Patent No. 10-2022-0116552, filed on September 15, 2022, which is incorporated herein by reference in its entirety.

[0003] Field of the Invention

[0004] The present invention relates to a vaccine comprising peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens (eg cancer antigens).

[0005] Description of Related Technology

[0006] Recent advances in medicine have improved the survival rate of cancer patients, but the incidence of cancer is also increasing due to changes in environmental factors and an increase in life expectancy. So far, a lot of research is being conducted to treat cancer, and new cancer drugs and treatments are being developed, which has greatly improved the treatment effect of cancer patients. However, for malignant tumors, despite the development of microsurgical treatment and radiotherapy, as well as the development of new therapeutic agents for chemotherapy, the treatment effect is still limited, and there are limitations such as side effects and cancer recurrence due to nonspecific anti-cancer effects. Immunotherapy is a treatment method that is currently being actively used or developed to supplement this. Immunotherapy has the characteristics of complementing traditional cancer therapies by inducing tumor-specific toxicity, reducing the side effects of systemic toxicity, and establishing an active memory response to cancer and cancer antigens.

[0007] Immunotherapy, especially immune cell vaccines using antigen presenting cells, can effectively activate CD8+T cells and CD4+T cells, thereby showing excellent anti-cancer effects.

[0008] At present, the most commonly used immune cells in antigen presenting cell vaccines are dendritic cells, which engulf antigens and deliver them to effector cells (such as T cells) together with strong co-stimulatory signals, thereby effectively activating effector cells and inducing a strong immune response. In actual clinical cell therapy using dendritic cells, first, dendritic cells are isolated from the patient's bone marrow or peripheral blood, or their precursor cells (monocytes) are separated, and then proliferated and differentiated into dendritic cells on a large scale, and then antigens are added and activated, and then injected back into the patient's body. Once injected into the body, dendritic cells deliver specific antigen information to T cells and activate them, effectively inducing antigen-specific immune responses. Despite these advantages, dendritic cells have not been widely used in antigen presenting cell vaccines because few dendritic cells can be obtained directly from blood and lymphatic tissues, they are difficult to separate, and when differentiated from monocytes, they must be cultured in vitro for several days.

[0009] In previous studies, the inventors have confirmed the effect of B cells loaded with α-GC on inducing cytotoxic T lymphocyte responses (Korean Patent No. 10-0809873). Since B cells are present in large quantities in lymphoid tissues and blood, and are easily proliferated in vitro, they are effective substitutes for dendritic cells in cell vaccines, and have the advantage of migrating to lymphoid organs after intravenous administration. Despite these advantages, B cells have not yet been widely used in antigen presenting cell vaccines due to their weak immunogenicity.

[0010] On the other hand, recent studies have shown that invariant natural killer T cells (iNKT cells) play a vital role in controlling various immune responses and immunopathological phenomena. Ligand-activated invariant natural killer T cells can induce the activation of T cells, B cells, natural killer cells, and dendritic cells.

[0011] Cancer cells loaded with α-GC (a ligand for natural killer T cells) can induce cancer antigen-specific T cell activity. It is known that mice administered with T cells loaded with α-GC and antigen peptides strongly induced the expression of IFN-γ and granzyme B, and exhibited anticancer therapeutic effects in mice with lung metastatic cancer by activating antigen-specific cytotoxic T lymphocyte responses (Yeonseok Chung et al., OncoImmunology, 1:2, 141-151, 2012).

[0012] The inventors have invented an effective cell therapy vaccine by demonstrating that the major histocompatibility complex of B cells can generate an active immune response against cancer antigens by loading B cells with natural killer T cell ligands, thereby changing the autoimmune tolerance of these cells to immunogenicity and exhibiting anticancer effects.

[0013] Furthermore, the present inventors produced an effective cell therapy vaccine by demonstrating in previous studies that when α-GC-loaded monocytes are loaded with antigenic peptides or adenovirus transducing antigen expression and administered in vivo, antigen-specific immune responses are induced and significant anticancer effects are exhibited (Korean Patent No. 10-1055666).

[0014] However, the cell therapy developed for clinical trials based on the above-mentioned previous research is produced using a manufacturing process that removes T cells from the patient's peripheral blood and leaves B cells and monocytes. The disadvantage of this method is that it is difficult to separate T cells, B cells and monocytes from peripheral blood separately, and the manufacturing process is complicated.

[0015] In order to solve these problems, the present inventors developed an anticancer immunotherapy and preventive vaccine based on the research on the above-mentioned various immune cells by loading natural killer T cell ligands and antigens into peripheral blood mononuclear cells (peripheral blood mononuclear cells are obtained without separating each immune cell).

[0016] Peripheral blood mononuclear cells are a general term for a mononuclear mesenchymal cell population present in the blood, which is mainly composed of monocytes and lymphocytes. Generally, peripheral blood mononuclear cells refer to the white blood cell portion obtained by centrifuging blood by gravity, and polymorphonuclear leukocytes are excluded. Lymphocytes include T cells, B cells and natural killer cells (NK cells). Specifically, the cell therapy agent developed by the inventor uses peripheral blood mononuclear cells as raw materials, and peripheral blood mononuclear cells are cells with round nuclei composed of lymphocytes (T cells, B cells and natural killer cells) and monocytes. The proportion of each type of immune cell in peripheral blood mononuclear cells varies from person to person, but is generally composed of 45%-70% T cells, 5%-15% B cells, 5%-10% natural killer cells and 5%-10% monocytes.

[0017] The present inventors have devoted themselves to solving the problems of dendritic cell vaccines differentiated from dendritic cells and monocytes and the problems of existing inventions using B cells and monocytes. As a result, it was confirmed that vaccines using peripheral blood mononuclear cells have a synergistic effect that is superior to the immune effects of B cells, monocytes and T cells, respectively, and the development of the peripheral blood mononuclear cell vaccine of the present invention was completed.

[0018] In addition, compared with previous cell therapeutic agents, the cell therapeutic agent of the present invention has various advantages, including increasing the amount of peripheral blood mononuclear cells as raw materials by a method that does not separate each type of immune cell. If the process of staining and removing T cells in the existing cell therapeutic agent manufacturing process is eliminated, the peripheral blood mononuclear cells used as raw materials will increase, and the final output will increase, thereby saving time, labor and cost, and increasing the manufacturing volume. Summary of the invention

[0019] The object of the present invention is to provide an immunotherapeutic or preventive vaccine comprising peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens.

[0020] Another object of the present invention is to provide a method for producing an immunotherapeutic or preventive vaccine comprising peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens.

[0021] In order to achieve the above object, the present invention provides an immunotherapeutic or preventive vaccine, which comprises peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens.

[0022] In another aspect of the present invention, the present invention provides a method for producing an immunotherapeutic or prophylactic vaccine comprising peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens.

[0023] The present invention also provides a pharmaceutical composition for preventing or treating cancer, wherein the composition contains peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens as active ingredients.

[0024] The present invention also provides the use of peripheral blood mononuclear cells loaded with natural killer T cell ligands and cancer antigens in preventing or treating cancer.

[0025] In addition, the present invention provides a method for preventing or treating cancer, comprising administering to a subject an effective amount of peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens.

[0026] Beneficial Effects

[0027] The present invention relates to a vaccine for immunoprophylaxis and immunotherapy, which comprises peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens, and in particular to an immunotherapy vaccine, which comprises peripheral blood mononuclear cells loaded with α-galactosylceramide (α-GC), natural killer T cell ligands and a glycolipid. The composition of the present invention is easy to obtain because it is not necessary to separate specific cells from peripheral blood mononuclear cells, and the immunization of peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens not only induces significant levels of activation of natural killer cells and natural killer T cells and cytotoxic T lymphocyte responses, but also exhibits a synergistic therapeutic effect on malignant tumors, and can therefore be effectively used as an anticancer immunotherapeutic agent.

[0028] BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1is a schematic diagram showing a process for producing a peripheral blood mononuclear cell vaccine according to an embodiment of the present invention.

[0030] Figure 2a is a set of graphs showing the activities of natural killer T cells (NKT) and natural killer cells (NK) in the spleen of mice after administration of a peripheral blood mononuclear cell vaccine transduced with adenovirus loaded with α-GC and / or expressing antigen.

[0031] Figure 2b Yes Display Figure 2a Figure 2. IFN-γ production by natural killer T cells (NKT).

[0032] Figure 2c Yes Display Figure 2a Figure 2 shows the production of IFN-γ by natural killer (NK) cells.

[0033] Figure 3a It is shown that by administering α-GCs loaded with GCs and / or expressing antigens (target peptide: MAGE-A3 282-290 Schematic diagram of the experimental method for comparing the activation of cytotoxic T cells in mice using adenovirus-transduced peripheral blood mononuclear cell vaccine.

[0034] Figure 3b is a set of histograms showing the measurement of antigen-specific cytotoxic T cell activity induced by vaccines of peripheral blood mononuclear cells transduced with adenovirus loaded with α-GC and / or expressing antigen.

[0035] Figure 3c The graph shows the antigen-specific cytotoxic T cell activity induced by adenovirus-transduced peripheral blood mononuclear cell vaccines loaded with α-GC and / or expressing antigens.

[0036] Figure 4a Schematic diagram of the experimental method for confirming the therapeutic effect of adenovirus-transduced peripheral blood mononuclear cell vaccine loaded with α-GC and / or expressing antigen on solid cancer in mice.

[0037] Figure 4b The graph shows the anticancer effect (change in tumor size) of a peripheral blood mononuclear cell vaccine transduced with adenovirus loaded with α-GC and / or expressed antigen.

[0038] Figure 4c The graph shows the anticancer effect (change in survival rate) of a peripheral blood mononuclear cell vaccine transduced with adenovirus loaded with α-GC and / or expressed antigen.

[0039] Figure 5a Schematic diagram of the experimental method for confirming the therapeutic effect of adenovirus-transduced peripheral blood mononuclear cell vaccine loaded with α-GC and / or expressed antigen on lung metastatic cancer.

[0040] Figure 5b is a set of photographs showing the lungs of mice in a lung metastasis cancer model to which a peripheral blood mononuclear cell vaccine transduced with adenovirus loaded with α-GC and / or expressing antigens was administered.

[0041] Figure 5c The graph shows the therapeutic effect (number of metastatic nodules) of a peripheral blood mononuclear cell vaccine transduced with adenovirus loaded with α-GC and / or expressed antigen on lung metastatic cancer.

[0042] Figure 6a Schematic diagram of the experimental method for confirming the therapeutic effect of adenovirus-transduced peripheral blood mononuclear cell vaccine loaded with α-GC and / or expressed antigen on metastatic cancer.

[0043] Figure 6b The graph shows the anticancer effect (change in survival rate) of a peripheral blood mononuclear cell vaccine transduced with adenovirus loaded with α-GC and / or expressed antigen.

[0044] Figure 7a It is confirmed that α-GC and HER2 are co-loaded 63-71 Peptide (target peptide: HER2 63-71 )'s peripheral blood mononuclear cell vaccine to activate antigen-specific cytotoxic T cells.

[0045] Figure 7b A set of histograms showing the co-loading of α-GC and HER2 63-71 Peptide-based peripheral blood mononuclear cell vaccines induce antigen-specific cytotoxic T cell activity.

[0046] Figure 7c It is a graph showing the antigen-specific cytotoxic T cell activity induced by peripheral blood mononuclear cells co-loaded with α-GC and HER263-71 peptide.

[0047] Figure 8a The purpose is to confirm the efficacy of peripheral blood mononuclear cell vaccines loaded with α-GC and / or cancer antigen peptides on mouse solid cancer (target peptide: HER2 63-71 ) is a schematic diagram of the experimental method for evaluating the therapeutic effect of .

[0048] Figure 8b It is a graph showing the anticancer effect (change in tumor size) of a peripheral blood mononuclear cell vaccine loaded with α-GC and / or a cancer antigen peptide.

[0049] Figure 9a The purpose is to compare the efficacy of adenovirus-transduced peripheral blood mononuclear cell vaccine loaded with α-GC and / or expressed antigen and B cell / monocyte vaccine (target peptide: GP100 25-33) Schematic diagram of the experimental method for activating antigen-specific cytotoxic T cells.

[0050] Figure 9b is a set of histograms showing the activity of antigen-specific cytotoxic T cells induced by adenovirus-transduced peripheral blood mononuclear cell vaccines and B cell / monocyte virus vaccines loaded with α-GC and / or expressing antigens.

[0051] Fig.9c It is a graph showing the antigen-specific cytotoxic T cell activity induced by adenovirus-transduced peripheral blood mononuclear cell vaccine loaded with α-GC and / or expressing antigen and B cell / monocyte virus vaccine.

[0052] Fig.10a The present invention is to compare the efficacy of adenovirus-transduced peripheral blood mononuclear cell vaccine, B cell / monocyte vaccine and T cell vaccine (target peptide: GP100) loaded with α-GC and / or expressed antigen. 25-33 ) Schematic diagram of the experimental method for activating antigen-specific cytotoxic T cells.

[0053] Fig.10b is a set of histograms showing the activity of antigen-specific cytotoxic T cells induced by adenovirus-transduced peripheral blood mononuclear cell vaccines, B cell / monocyte virus vaccines, and T cell vaccines loaded with α-GC and / or expressing antigens.

[0054] Fig.10c It is a graph showing the antigen-specific cytotoxic T cell activity induced by adenovirus-transduced peripheral blood mononuclear cell vaccine loaded with α-GC and / or expressing antigen, B cell / monocyte virus vaccine and T cell vaccine.

[0055] Fig.11a Schematic diagram of the experimental method for comparing the therapeutic effects of peripheral blood mononuclear cell vaccines and B cells / monocytes transduced with adenovirus loaded with α-GC and / or expressed antigens on solid cancers.

[0056] Fig.11b is a set of histograms showing the anticancer effects (changes in tumor size) of peripheral blood mononuclear cell vaccines and B cell / monocyte virus vaccines transduced with adenovirus loaded with α-GC and / or expressed antigens. DETAILED DESCRIPTION

[0057] Hereinafter, the present invention will be described in detail.

[0058] The present invention provides an immunotherapeutic or preventive vaccine for cancer, which comprises peripheral blood mononuclear cells loaded with natural killer T cell ligands and cancer antigens.

[0059] Peripheral blood mononuclear cells refer to cells with a round nucleus, including lymphocytes (T cells, B cells and natural killer cells) and monocytes. The proportion of each type of immune cell in peripheral blood mononuclear cells varies from person to person, but generally consists of 45%-70% T cells, 5%-15% B cells, 5%-10% natural killer cells and 5%-10% monocytes.

[0060] Herein, the inventors have developed an anti-cancer therapeutic cell vaccine, which can change the immunogenicity of immune cells by loading them with α-GC (a natural killer T cell ligand) and delivering antigens to immune cells, thereby enhancing the function of antigen presenting cells and significantly enhancing the anti-cancer immune response.

[0061] Neo-epitopes are peptide sequences generated by highly tumor-specific mutations that exist only in cancer cells but not in normal cells and can induce cancer cell-specific immune responses. They are considered to be ideal targets for personalized anti-cancer immunotherapy for cancer patients. Anti-cancer immunotherapy uses an immune cell vaccine to deliver neo-epitopes discovered for personalized treatment of cancer patients. Compared with traditional immunotherapy, anti-cancer immunotherapy using immune cell vaccines is expected to induce more cancer cell-specific T cells, thereby minimizing the side effects of anti-cancer therapy by preventing damage to normal cells while inducing a strong anti-cancer therapeutic effect.

[0062] α-Galactosylceramide (α-GC) is an immunopotentiator that induces various anti-cancer immune responses by stimulating NKT cells. In particular, it induces T cell immune responses by allowing B cells and monocytes to stimulate T cells with the same efficiency as dendritic cells.

[0063] It is well known that dendritic cells (DCs) loaded with α-galactosylceramide (α-GC) activate constant natural killer T (NKT) cells (van der Vliet HJ et al., J Immunol Methods, 1; 247 (1-2): 61-72, 2001). The present inventors have 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). In addition, it has been confirmed that natural killer cells loaded with α-GC activate natural killer T (NKT) cells and induce cytotoxic T lymphocyte responses (Korean Patent No. 10-2022-0072485).

[0064] Therefore, the present inventors changed the immunogenicity of immune cells by loading α-GC and delivering antigens to immune cells, thereby increasing the function of antigen presenting cells and producing an anti-cancer vaccine with enhanced immune function.

[0065] Meanwhile, antigen delivery using viral vectors is suitable for mass production of cell therapeutic agents targeting cancer, which target cancers expressing specific cancer antigens by efficiently delivering cancer antigens to cells. When using a virus capable of expressing the above antigen, the entire antigen can be introduced, so it is suitable for all people, not limited to a specific monomer type 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.

[0066] When used clinically, the method of delivering antigens via peptide loading is limited to the monomeric form 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, neo-epitopes are peptide sequences generated by highly tumor-specific mutations that are present only in cancer cells and not in normal cells, and can induce cancer cell-specific immune responses, and are known to be ideal targets for personalized anti-cancer immunotherapy for cancer patients. Compared with traditional immunotherapy, anti-cancer immunotherapy using immune cell vaccines (which deliver new epitopes discovered for personalized treatment of cancer patients) is expected to induce the production of more cancer cell-specific T cells.

[0067] The ligands of the above-mentioned natural killer T cells include the group consisting of α-galactosylceramide, α-glucuronylceramide, phosphatidylinositol tetramethyl glycoside, isoglobin trihexosylceramide, ganglioside GD3, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sulfitide, β-galactosylceramide, lipophosphoglycan, glycoinositol phospholipids, β-anomeric galactosylceramide, α-anomeric galactosylceramide, bacterial lipid antigens and variants of α-galactosylceramide, wherein the β-anomeric galactosylceramide and α-anomeric galactosylceramide are analogs of α-galactosylceramide.

[0068] The above-mentioned antigen can be any antigen that can be used as a vaccine and induce an immune response, and includes antigens or cancer antigens derived from pathogens including pathogens, viruses and parasites. Antigens derived from the pathogens 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 (Streptococcus pneumoniae type 3 capsular polysaccharides), tuberculosis antigens, cholera antigens (cholera toxin B subunit), Staphylococcus antigens (staphylococcal enterotoxin B), Shigella antigens (Shigella polysaccharides), Borrelia antigens, Candida albicans antigens and Plasmodium antigens and combinations thereof.

[0069] 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. 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 (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, EBVLMP2a, HCV, HHV-8, 5T4 and tumor-specific mutation-derived neoantigens and combinations thereof.

[0070] Vaccines can be used to treat or prevent cancer.

[0071] The above-mentioned cancers may include liver cancer, thyroid cancer, testicular cancer, bone cancer, glioblastoma, oral cancer, ovarian cancer, brain tumor, multiple myeloma, gallbladder cancer, bile duct cancer, colon cancer, head and neck cancer, lymphoma, bladder cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, breast cancer, cervical cancer, prostate cancer, rectal cancer, spinal cord tumor, pancreatic cancer, salivary gland cancer, lung cancer, skin cancer, laryngeal cancer, melanoma, acute myeloid leukemia, neuroblastoma, retinoblastoma and colorectal cancer, etc.

[0072] The cancer can be a solid cancer or a metastatic cancer.

[0073] In addition, antigens can be directly loaded into peripheral blood mononuclear cells in the form of peptides, lipopolysaccharides, polysaccharides, glycoproteins or polynucleotides (including DNA and RNA), and can be transfected into peripheral blood mononuclear cells by recombinant viruses and expressed and loaded. Compared with cell vaccines loaded with peptides, cell vaccines imported by viruses with the entire antigen are suitable for all populations, are not limited to the monomeric type of the major histocompatibility complex, and can induce immune responses specific to multiple epitopes, especially, have the advantage of being able to induce humoral immune responses and cellular immune responses simultaneously.

[0074] In addition to natural killer T cell ligands and peripheral blood mononuclear cells, the vaccine of the present invention may also include one or more active ingredients having the same or similar effects as them.

[0075] In addition to the above-mentioned active ingredients, the vaccine can 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 can be added, such as antioxidants, buffer solutions, antibacterial agents, etc. In order to prepare injectable solutions, such as aqueous solutions, suspensions and emulsions, diluents, dispersants, surfactants, adhesives and lubricants can be added additionally. Vaccine of the present invention can be further prepared in the appropriate form for each disease or according to the composition by following the method represented in Remington's Pharmaceutical Science (latest edition) of Mack Publishing Company, Easton, Pennsylvania.

[0076] The vaccine of the present invention can be administered parenterally, and parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection and thoracic injection. In order to prepare a vaccine as a parenteral preparation, peripheral blood mononuclear cells loaded with natural killer T cell ligands, peripheral blood mononuclear cells loaded with natural killer T cell ligands and peptides, or peripheral blood mononuclear cells infected with viruses expressing cancer antigens of the present invention are mixed with stabilizers or buffers to produce solutions or suspensions, which are then formulated into ampoules or vials.

[0077] 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).

[0078] In order to implement the present invention, the effective dose of the vaccine to be administered is administered 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, as the weight of the patient being treated increases, the dose of such a preparation is preferably increased.

[0079] The vaccine can be administered at an effective dose to induce an immune response in a patient. For example, a dose of 1×10 3 -1x10 9 cells / kg, more preferably 1×10 4 cells / kg to 1×10 8 To prepare the peripheral blood mononuclear cell vaccine loaded with α-galactosylceramide, the culture medium must be supplemented with 1×10 6 -1×10 7 In order to prepare the peripheral blood mononuclear cell vaccine loaded with α-galactosylceramide and polypeptide, the culture medium must be supplemented with 1×10 6 -1×10 7 Peripheral blood mononuclear cells were treated with 1-2 μg / ml α-galactosylceramide and 1×10 6 -1×10 7 The polypeptide was expressed at a concentration of 1 to 10 μg / ml per peripheral blood mononuclear cell.

[0080] 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 taking acetaminophen, and mild systemic side effects do not necessarily occur in these subjects (Giaccone et al., Clin Cancer Res 8: 3702, 2002).

[0081] The present invention also provides a pharmaceutical composition for preventing and treating cancer, wherein the composition comprises peripheral blood mononuclear cells loaded with natural killer T cell ligands and cancer antigens as active ingredients.

[0082] The present invention also provides the use of peripheral blood mononuclear cells loaded with natural killer T cell ligands and cancer antigens in preventing or treating cancer.

[0083] The present invention also provides a method for preventing or treating cancer, comprising administering to a subject an effective amount of peripheral blood mononuclear cells loaded with natural killer T cell ligands and cancer antigens.

[0084] In addition, the present invention provides a method for producing an anti-cancer immunotherapeutic or preventive vaccine comprising peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens.

[0085] The method comprises the following steps:

[0086] (a) a step of obtaining peripheral blood mononuclear cells;

[0087] (b) a step of removing red blood cells; and

[0088] (c) A step of delivering natural killer T cell ligands and antigens to peripheral blood mononuclear cells.

[0089] In step (a), for example, peripheral blood mononuclear cells can be obtained using known leukocyte removal techniques. Here, peripheral blood mononuclear cells can include T cells, B cells, natural killer cells, monocytes and some red blood cells, but may not include neutrophils, eosinophils or basophils.

[0090] In step (b), red blood cells may be removed by any method known in the art, such as using a suitable lysis buffer.

[0091] In step (c), the natural killer T cell ligand can be added to a culture medium (e.g., a culture medium for peripheral blood mononuclear cells) and delivered to the peripheral blood mononuclear cells by culture. In step (c), the antigen can be delivered by a recombinant virus. Viruses introduced into peripheral blood mononuclear cells for antigen expression include retroviruses, vaccinia viruses, poxviruses, and Sindbis viruses, but are not always limited thereto.

[0092] In addition to methods using viruses, the following methods can also be used for the delivery of antigen genes: 1) methods of transducing DNA by binding it to liposomes to protect the DNA from enzymatic degradation or allow it to be taken up into endosomes, 2) methods of improving the efficiency of DNA delivery into cells by binding a molecular conjugate composed of a protein or a synthetic ligand to DNA (for example: asialoglycoprotein, transferrin, polymeric IgA), 3) methods of improving the efficiency of DNA delivery into cells by using a novel DNA delivery system using a PTD (protein transduction domain) (for example: Mph-1), and 4) in addition to the above methods, peptides or antigen proteins can be applied to peripheral blood mononuclear cells to allow the peripheral blood mononuclear cells to present antigens.

[0093] Unlike existing methods that use each immune cell, the above preparation method does not require the removal of other immune cells to obtain each immune cell, which reduces the time and cost of vaccine manufacturing and increases manufacturing volume.

[0094] The present inventors changed the immunogenicity of peripheral blood mononuclear cells by loading α-GC and delivering antigens to peripheral blood mononuclear cells, thereby making the peripheral blood mononuclear cells present antigens, thereby producing a vaccine with significantly improved immune function.

[0095] Antigen delivery using viral vectors is suitable for mass production of cell therapeutic agents targeting cancers expressing specific cancer antigens by efficiently delivering cancer antigens to cells. When using a virus capable of expressing 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.

[0096] When used clinically, the method of delivering antigens through peptide loading is limited to the monomeric type of the individual major histocompatibility complex (MHC), so it cannot be used universally, and has the disadvantage of presenting only a single epitope. However, new epitopes are peptide sequences generated by highly tumor-specific mutations, only present in cancer cells, not in normal cells, and can induce cancer cell-specific immune responses, and are known to be ideal targets for personalized anti-cancer immunotherapy for cancer patients. Compared with traditional immunotherapy, anti-cancer immunotherapy using immune cell vaccines (which are new epitopes discovered for personalized treatment delivery for cancer patients) is expected to induce more cancer cell-specific T cells. It can also induce a powerful anti-cancer therapeutic effect while minimizing the side effects of anti-cancer therapy by preventing damage to normal cells.

[0097] In the present invention, peripheral blood mononuclear cells are isolated from mice, loaded with α-GC, and antigens are delivered in two ways (antigen gene transfer to a viral vector expressing the antigen or loaded with a peptide) to produce a peripheral blood mononuclear cell vaccine.

[0098] First, a peripheral blood mononuclear cell vaccine (wherein the peripheral blood mononuclear cell vaccine is loaded with α-GC and the antigen gene is delivered by an adenovirus vector expressing the antigen) was produced, and whether the administration of the vaccine can activate natural killer T cells and natural killer cells in the body was confirmed. The results confirmed that natural killer T cells and natural killer cells were activated in mice administered with peripheral blood mononuclear cells loaded with α-GC and peripheral blood mononuclear cells loaded with α-GC and cancer antigens GP100 and MAGE-A3 delivered by adenovirus (see Figure 2a to Figure 2c ). 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. Thus, it was confirmed that the peripheral blood mononuclear cell vaccine loaded with α-GC and adenoviral-delivered cancer antigens (GP100 and MAGE-A3) induced a potent cytotoxic response (see Figure 3a to Figure 3c ). In addition, the vaccine's efficacy in treating solid cancers has also been demonstrated. The results confirmed that the vaccine showed significant anti-cancer effects, inhibited tumor size, and increased mouse survival (see Figures 4a to 4c ).

[0099] In addition, the therapeutic effect of the above vaccine on lung metastatic cancer was studied. As a result, almost no cancer tissue was observed in the two cell groups into which the cancer antigen was introduced by adenovirus (see Figure 5a to Figure 5c ), the survival rate of mice was significantly improved (see Figure 6a and Figure 6b ).

[0100] Next, the present inventors produced a peripheral blood mononuclear cell vaccine loaded with α-GC and antigen peptides, and confirmed whether the administration of the vaccine induced a cytotoxic T cell response. 63-71 The peptide-containing peripheral blood mononuclear cell vaccine induced a potent cytotoxic response (see Figures 7a to 7c ).

[0101] In addition, the anticancer effect of a peripheral blood mononuclear cell vaccine loaded with α-GC and cancer antigen peptides was studied, and the results showed that tumor size was inhibited (see Figure 8a and 8b ).

[0102] The anticancer effects of the vaccine composed of the peripheral blood mononuclear cells of the present invention and the vaccine composed of other immune cells were compared. First, the inventors compared the ability of the peripheral blood mononuclear cell vaccine and the B cell / monocyte vaccine to induce cytotoxic T cell immune responses. As a result, cancer antigen-specific cytotoxicity was observed in both groups administered with the above two vaccines, and slightly higher cytotoxicity ( Figures 9a to 9c ). In addition, the ability of peripheral blood mononuclear cell vaccines, B cell / monocyte vaccines, and T cell vaccines to induce cytotoxic T cell immune responses was compared. The results confirmed that cytotoxic T cell immune responses specific to cancer antigens were induced not only by peripheral blood mononuclear cells, B cells / monocytes, but also by T cells (see Fig.10a and 10c In particular, it was demonstrated that the peripheral blood mononuclear cell vaccine exhibited a strong anticancer therapeutic effect of sustained inhibition of cancer growth compared with the B cell / monocyte vaccine ( Fig.11a and 11b ). This appears to be because the various immune cells present in PBMCs act complementary to each other to induce an enhanced anti-cancer therapeutic response.

[0103] Hereinafter, the present invention will be described in detail through the following experimental examples.

[0104] However, the following experimental examples are only used to illustrate the present invention, and the contents of the present invention are not limited thereto.

[0105] Example 1: Production of peripheral blood mononuclear cell vaccine

[0106] <1-1> Isolation and purification of mouse T cells, B cells / monocytes and natural killer cells

[0107] In order to separate the cells of each composition of mouse peripheral blood mononuclear cells, the mouse spleen was collected and then homogenized. After lysing the red blood cells using ACK lysis buffer (Gibco), microbeads (Miltenyibiotec) were used to separate T cells expressing CD4 or CD8α on the cell surface and B cells / monocytes expressing B220 or CD11b. After separating T cells and B cells / monocytes, CD49b+ natural killer cells were obtained from the remaining spleen cells using anti-CD49b microbeads (Miltenyibiotec). Since the number of immune cells obtained from mouse blood is limited, immune cells are isolated from the spleen. However, since the composition of immune cells in blood and spleen is different, in order to match the composition of blood, each immune cell is mixed (T cells: B cells / monocytes: natural killer cells = 5:4:1) and arranged in proportion to make it similar to human peripheral blood mononuclear cells.

[0108] <1-2> Construction of peripheral blood mononuclear cell vaccines in which antigens are delivered via viral vectors expressing the antigens

[0109] The peripheral blood mononuclear cells separated and purified as described above are placed in a culture medium containing serum and α-GC (1 μg / mL), solvent (DMSO) and / or adenovirus (200 MOI) for cancer antigen gene delivery. The cells are centrifuged in a cell culture plate (2,000 rpm, 20° C., 90 minutes) and cultured in a CO2 incubator (37° C., 80% to 95% relative humidity, 5% CO2 concentration) to produce a peripheral blood mononuclear cell vaccine. The peripheral blood mononuclear cell vaccine is washed 3 times with Dulbecco's phosphate buffered saline (DPBS, Welgene), and the washed peripheral blood mononuclear cells are dissolved in DPBS and injected into the tail vein of mice.

[0110] <1-3> Construction of peripheral blood mononuclear cell vaccine loaded with antigen peptides

[0111] Viral vectors are suitable gene delivery vehicles for large-scale production of cell therapies 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 treatments to cancer patients using new epitopes, so the inventors have constructed a peripheral blood mononuclear cell vaccine using antigen delivery via peptide loading.

[0112] Specifically, peripheral blood mononuclear cells isolated from mice by the method of Example <1-1> were treated with α-GC (1 μg / mL) or solvent (DMSO), placed in a serum-containing culture medium, and cultured in a CO2 incubator for 15 hours (37°C, 80% to 95% relative humidity, 5% CO2 concentration). Then, by adding HER2 63-71 The peptide (2 μg / mL) was further cultured in a CO2 incubator at 37°C for 2 hours to prepare a peripheral blood mononuclear cell vaccine. The prepared peripheral blood mononuclear cell vaccine was washed three times with DPBS, dissolved in DPBS, and injected into the tail vein of the mouse.

[0113] Comparative Example 1: Production of B cell / monocyte vaccine

[0114] To separate mouse B cells / monocytes, mouse spleens were collected and then homogenized. After lysing red blood cells using ACK lysis buffer (Gibco), microbeads were used to separate B cells / monocytes expressing B220 or CD11b. Antigens were delivered to separated B cells / monocytes using the same method as in Example <1-2> or <1-3>.

[0115] Comparative Example 2: Production of T cell vaccine

[0116] To isolate mouse T cells, mouse spleens were collected and then homogenized. After lysing red blood cells using ACK lysis buffer (Gibco), microbeads were used to separate T cells expressing CD4 or CD8α on the cell surface. Antigens were delivered to isolated T cells using the same method as in Example <1-2> or <1-3>.

[0117] Example 2: Production of human peripheral blood mononuclear cell vaccine

[0118] The manufacturing process of the human peripheral blood mononuclear cell vaccine of the present invention is as follows Figure 1 shown.

[0119] Specifically, human peripheral blood mononuclear cells obtained by leukocyte removal were lysed with ACK lysis buffer (Lonza) to lyse red blood cells, and treated with α-GC (1 μg / mL) and a viral vector expressing an antigen (25 MOI), and then the cells were cultured in a CO2 incubator at 37°C and 5% CO2 concentration for 15 hours to produce a peripheral blood mononuclear cell vaccine. This eliminates the process of removing T cells from the existing manufacturing process, which has the advantages of saving time, manpower and cost, and increasing production. Experimental example 1: Efficacy of peripheral blood mononuclear cell vaccines in which antigens are delivered by viral vectors expressing the antigens <1-1> Confirmation of activation induction of natural killer T cells and natural killer cells

[0120] The present inventors have demonstrated whether natural killer T cells and natural killer cells in vivo can be activated by administering a peripheral blood mononuclear cell vaccine.

[0121] Specifically, peripheral blood mononuclear cells obtained from C57BL / 6 mice were used to prepare peripheral blood mononuclear cells loaded with α-GC (PBMC / α-GC), peripheral blood mononuclear cells transduced with adenovirus Adk35GM for delivering cancer antigens (human GP100 and MAGE-A3) (PBMC / Adk35GM), and peripheral blood mononuclear cells loaded with α-GC and transduced with adenovirus Adk 35GM. After 6 hours, the production level of IFN-γ in natural killer T cells and natural killer cells in the spleen was measured by flow cytometry.

[0122] Therefore, if Figures 2a to 2c As shown, unlike the mouse group administered with transduced peripheral blood mononuclear cells (PBMC / Adk35GM), the natural killer T cells in the mouse group administered with peripheral blood mononuclear cells loaded with α-GC (PBMC / α-GC and PBMC / β-GC / Adk35GM) were stimulated and the natural killer cells were also activated.

[0123] It is thought that the activation of these NK T cells and natural killer cells, as well as the induction of cytotoxic T cell responses, may contribute to the anti-cancer effects.

[0124] <1-2> Evaluation of the ability of peripheral blood mononuclear cells to induce cytotoxic T cell responses

[0125] To determine whether adenovirus-transduced PBMC vaccines could induce antigen-specific cytotoxic T cell immune responses, in vivo CTL assays were performed.

[0126] Specifically, peripheral blood mononuclear cells obtained from BALB / c mice were used to construct peripheral blood mononuclear cell vaccines loaded with α-GC or adenovirus Adk35GM antigen delivery, which were immunized into BALB / c mice, and cytotoxicity tests were performed 7 days later ( Figure 3a ). First, spleen cells from the same mouse were divided into two groups of equal number. 282-290 The target cells of the peptide were stained with 3 μM CFSE (carboxyfluorescein diacetate succinimidyl ester) (CFSE 高 ) were labeled, and control cells without peptide were labeled with 0.3 μM CFSE (CFSE 低 ) were labeled and then injected into vaccinated mice in equal amounts. One day later, CFSE in mouse spleen cells was counted by flow cytometry 高 :CFSE 低 The ratio of the target cell lysis is measured ( Figure 3b ). CFSE loaded with antigen peptide 高 A lower percentage of cells indicates a higher antigen-specific cytotoxic T cell immune response.

[0127] Results, such as Figure 3c As shown, more than 95% of the target cells were killed by MAGE-A3-specific cytotoxic T cell responses only when the cancer antigen was delivered by Adk35GM adenovirus, thus confirming the cancer antigen-specific cytotoxic T cell immune response.

[0128] Based on the results of the above-mentioned experimental examples <1-1> and <1-2>, it can be inferred that by administering a peripheral blood mononuclear cell vaccine loaded with α-GC and an antigen delivered by an adenovirus vector, both innate immune responses (activation of natural killer T cells and natural killer cells) and acquired immune responses (cytotoxic T cell immune responses) can be induced, thereby showing a powerful anti-cancer therapeutic effect.

[0129] <1-3> Evaluation of the anticancer effect of peripheral blood mononuclear cell vaccines

[0130] <1-3-1> Therapeutic effect of peripheral blood mononuclear cell vaccine on solid cancer

[0131] The present inventors investigated whether administration of peripheral blood mononuclear cell vaccines induces anti-cancer immunity against solid cancers.

[0132] To this end, 1×10 6 B16F10 / GP100 / MAGE-A3 cancer cells were implanted subcutaneously in the flank of C57BL / 6 mice 7 days later and 2×10 6 C57BL / 6 mice were immunized with PBMC / α-GC, PBMC / Adk35GM or PBMC / α-GC / Adk35GM ( Figure 4a ). 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.

[0133] (Tumor size) = (width) × (length) × (height) × (π / 6)

[0134] Results, such as Figure 4b and 4c As shown, when the peripheral blood mononuclear cell vaccine (PBMC / α-GC / Adk35GM) was administered, cancer growth was inhibited, and a high survival rate was observed. In contrast, in mice administered with PBMC / α-GC, cancer growth was hardly inhibited, and a low survival rate was observed.

[0135] The above results confirmed that the anticancer therapeutic effect could not be induced by activating natural killer T cells alone and natural killer cells loaded with α-GC alone.

[0136] <1-3-2> Therapeutic effect of peripheral blood mononuclear cell vaccine on lung metastatic cancer

[0137] The present inventors investigated whether administration of peripheral blood mononuclear cell vaccines induces anti-cancer immunity against lung metastatic cancer.

[0138] To this end, C57BL / 6 mice were injected intravenously with 3 × 10 5 B16F10 / GP100 / MAGE-A3 cancer cells were injected 3 days later with 1.5×10 6 PBMC / α-GC, PBMC / Adk35GM or PBMC / α-GC / Adk35GM. Sixteen days after intravenous injection of cancer cells, the degree of lung metastasis of cancer cells was confirmed ( Figure 5a ).

[0139] Therefore, if Figure 5b and 5c As shown, in the lungs of mice administered with PBMC / Adk35GM and PBMC / α-GC / Adk35GM, two cell populations into which cancer antigens were introduced by adenovirus, almost no cancer tissue was observed.

[0140] The present inventors then confirmed the effect of administration of peripheral blood mononuclear cell vaccines on the survival rate of mice with metastatic cancer.

[0141] To this end, C57BL / 6 mice were injected intravenously with 3×10 5 B16F10 / GP100 / MAGE-A3 cancer cells, 3 days later, 2×10 6 PBMC / α-GC, PBMC / Adk35GM or PBMC / α-GC / Adk35GM. Then, the survival rate of mice was followed ( Figure 6a ).

[0142] Results, such as Figure 6b As shown, Figure 5b Similar to the results in , the survival rate of the mouse groups administered with PBMC / Adk35GM and PBMC / α-GC / Adk35GM introduced with cancer antigen was significantly improved, and all mice survived for more than 40 days.

[0143] Combining the results of Experimental Examples <1-2> and <1-3>, it can be seen that the delivery of cancer antigens to peripheral blood mononuclear cells via Adk35GM is sufficient to induce a strong cytotoxic T cell response, thereby producing a powerful anti-cancer therapeutic effect.

[0144] Experimental Example 2: Confirmation of the effectiveness of peripheral blood mononuclear cell vaccine loaded with antigen peptides

[0145] <2-1> Confirmation of induction of cytotoxic T cell response

[0146] The present inventors investigated whether a peripheral blood mononuclear cell vaccine loaded with the antigen peptide of Example <1-2> could induce a cytotoxic T cell response.

[0147] C57BL / 6 mice were intravenously injected with peripheral blood mononuclear cells loaded with α-GC (PBMC / α-GC), loaded with HER2 63-71 Peptide-positive peripheral blood mononuclear cells, simultaneously loaded with α-GC and HER2 63-71 Peptide-containing peripheral blood mononuclear cells (PBMC / α-GC / HER2 pep) were immunized, and in vivo cytotoxicity assay was performed 7 days later ( Figure 7a ).

[0148] Results, such as Figure 7b and 7c As shown, in the group of mice only administered with PBMC / α-GC / HER2 pep, HER2 63-71 Peptide CFSE 高 Most of the target cells were lysed. This confirmed that the peripheral blood mononuclear cell vaccine loaded with α-GC and peptides can also activate cytotoxic T cells.

[0149] However, the cytotoxic T cell response induced in the peripheral blood mononuclear cell administration group in which the antigen was delivered only by adenoviral vector without α-GC in Experimental Example <1-2> was different ( Figure 3c ), no cytotoxic T cell response was observed in the group to which peptide-loaded PBMC / HER2pep was administered alone ( Figure 7c ). This suggests that delivering the entire antigen via adenovirus to induce multiple immune responses against multiple epitopes is superior to presenting only a single peptide in inducing a cytotoxic T cell immune response.

[0150] <2-2> Evaluation of the anticancer effect of peripheral blood mononuclear cell vaccine

[0151] The present inventors investigated whether a peripheral blood mononuclear cell vaccine loaded with antigenic peptides can induce anti-cancer therapeutic effects.

[0152] To this end, 2×10 5 Three days after CT26-HER2 cancer cells were implanted subcutaneously into mice, 2 × 10 6 BABL / c mice immunized with PBMC / α-GC, PBMC / HER2-pep or PBMC / α-GC / HER2-pep ( Figure 8a ).

[0153] Results, such as Figure 8b As shown, when the peripheral blood mononuclear cell vaccine (PBMC / α-GC / HER2-pep) was administered, cancer growth was significantly inhibited. In contrast, in mice administered with PBMC / α-GC or PBMC / HER2 pep, tumor growth was barely inhibited. Therefore, this study suggests that loading peptides alone cannot induce sufficient anticancer therapeutic effects.

[0154] Combined with the above results and the anti-cancer effect data of Examples <1-3> (Figures 4 to 6), it is confirmed that the peripheral blood mononuclear cell vaccine prepared by loading α-GC and delivering antigens shows a stronger anti-cancer effect than the cell vaccine loaded with α-GG alone or delivering antigens alone, inhibiting cancer growth and improving survival rate.

[0155] Experimental Example 3: Compared with vaccines composed of other immune cells, peripheral blood mononuclear cell vaccines have better Cancer efficacy <3-1> Comparison of immune response induced by peripheral blood mononuclear cell vaccine and B cell / monocyte vaccine

[0156] The ability of the PBMC vaccine to induce a cytotoxic T-cell immune response was compared with a conventional anticancer cell vaccine composed of B cells / monocytes.

[0157] To this end, the vaccine was produced by loading α-GC or delivering the antigen to B cells / monocytes obtained from the spleen of C57BL / 6 mice and peripheral blood mononuclear cells through Adk35GM and immunizing C57BL / 6 mice. After 7 days, the target cells (CFSE loaded with GP10025-33 peptide) were 高 Labeled target cells: solvent-treated CFSE 低 Labeled control cells = 1:1) were administered to immunized mice and subjected to in vivo cytotoxicity assay ( Figure 9a One day later, CFSE in mouse spleen cells was counted by flow cytometry. 高 :CFSE 低 Ratio measurement GP100 25-33 Peptide-loaded CFSE 高 Target cell lysis ( Figure 9b ).

[0158] Results, such as Fig.9c As shown, in the group administered with the immune cell vaccine composed of B cells / monocytes (Bmo / α-GC / Adk35GM) and the group administered with the peripheral blood mononuclear cell vaccine (PBMC / α-GC / Adk35GM), more than 80% of the target cells were observed to be killed by the cytotoxic T cell response specific to the cancer antigen GP100, and slightly higher cytotoxicity was confirmed in the group administered with the peripheral blood mononuclear cell vaccine.

[0159] <3-2> Confirmation of immune response induction ability by cellular components of PBMC vaccine

[0160] Next, an in vivo cytotoxicity assay was performed to determine whether each immune cell type comprising the peripheral blood mononuclear cell vaccine could induce an antigen-specific cytotoxic T cell immune response.

[0161] Specifically, T cells, B cells / monocytes and peripheral blood mononuclear cells were obtained from the spleen of C57BL / 6 mice, loaded with α-GC and delivered by Adk35GM antigen to produce immune cell vaccines. Then, C57BL / 6 mice were immunized with the vaccine, and 7 days after immunization, cytotoxicity was detected using the same method as in Example <3-1> ( Fig.10a ).

[0162] Therefore, if Fig.10b and 10c As shown, the cytotoxic response was highest in the group administered with peripheral blood mononuclear cell vaccine (PBMC / α-GC / Adk35GM), followed by the group administered with B cell / monocyte-based immune cell vaccine (Bmo / α-GC / Adk35CM), and the group administered with T cell-based immune cell vaccine (T / α-GC / Adk35GM), in that order.

[0163] The above results show that the tumor antigen GP100-specific cytotoxic T cell immune response is induced not only by B cells / monocytes, which account for about 40% of peripheral blood mononuclear cells, but also by T cells, which account for half of the total number of cells. Combining the results of Experimental Examples <3-1> and <3-2>, it can be seen that T cells, B cells, and monocytes can induce a strong cancer antigen-specific cytotoxic T cell immune response, which account for 90% of the peripheral blood mononuclear cell vaccine loaded with α-GC and delivered by adenovirus vectors.

[0164] <3-3> Comparison of the anticancer therapeutic effects of peripheral blood mononuclear cell vaccines and B cell / monocyte vaccines

[0165] Finally, the anticancer therapeutic effects of peripheral blood mononuclear cell vaccines and B cell / monocyte vaccines were compared.

[0166] To this end, 3×10 5 B16F10 / GP100 / MAGE-A3 cancer cells were subcutaneously implanted into the flank of C57BL / 6 mice. Seven days later, 1.5×10 6 Mice were immunized with peripheral blood mononuclear cells (PBMC / α-GC, PBMC / β-GC / Adk35GM) or B cells / monocytes (Bmo / α-GG, Bmo / α-GC / Adk35GM), and the changes in tumor size were measured ( Fig.11a ).

[0167] Therefore, if Fig.11b As shown, administration of peripheral blood mononuclear cell vaccines (PBMC / α-GC / Adk35GM) and B cells / monocytes (Bmo / α-GC / Adk35GM) inhibited cancer growth. However, when solid cancer in mice administered with Bmo / α-GC / Adk35GM subsequently regenerated, cancer in mice administered with PBMC / α-GC / Adk35GM was suppressed for more than 4 weeks, showing a strong anti-cancer therapeutic effect.

[0168] In conclusion, in Experimental Examples <3-1> and <3-2>, there was no difference in the degree of induction of cytotoxic T cells between the peripheral blood mononuclear cell vaccine and the B cell / monocyte vaccine. However, in Experimental Example <3-3>, it was confirmed that the peripheral blood mononuclear cell vaccine had a stronger anti-cancer therapeutic effect than B cells / monocytes. This suggests that different immune cell populations within peripheral blood mononuclear cells act in a complementary manner to induce an enhanced anti-cancer therapeutic response.

[0169] Industrial Applicability

[0170] The vaccine of the present invention comprising peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens is easy to obtain because it is not necessary to separate specific cells from peripheral blood mononuclear cells, and immunization of peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens not only induces significant levels of activation of natural killer cells and natural killer T cells and cytotoxic T lymphocyte responses, but also exhibits a synergistic therapeutic effect on malignant tumors. Therefore, it can not only prevent cancer, but also be applied to cancer treatment through immunotherapy.

Claims

1. An immunotherapeutic and preventive vaccine comprising peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens.

2. The immunotherapeutic and preventive vaccine according to claim 1, wherein The peripheral blood mononuclear cells are obtained by removing red blood cells from peripheral blood.

3. The immunotherapeutic and preventive vaccine according to claim 1, wherein The peripheral blood mononuclear cells are composed of lymphocytes and monocytes composed of T cells, B cells and natural killer cells.

4. The immunotherapeutic and preventive vaccine 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, phosphatidylethanolamine, phosphatidylinositol, sulfatide, β-galactosylceramide, lipophosphate glycan, glycoinositol phospholipids, β-anomeric galactosylceramide, α-anomeric galactosylceramide, bacterial lipid antigens and variants of α-galactosylceramide, and the β-anomeric galactosylceramide and α-anomeric galactosylceramide are analogs of α-galactosylceramide.

5. The immunotherapeutic and preventive vaccine according to claim 1, wherein The antigen is an antigen derived from a pathogen including pathogenic bacteria, virus and parasite, or a cancer antigen.

6. The immunotherapeutic and preventive vaccine according to claim 5, wherein: The antigens derived from the pathogenic bacteria are 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 polysaccharides 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.

7. The immunotherapeutic and preventive vaccine according to claim 5, wherein the antigen derived from the virus is selected from the following: Influenza virus antigens (hemagglutinin and neuraminidase), human papillomavirus (HPV) antigens (glycoprotein), vesicular stomatitis virus antigens (vesicular stomatitis virus glycoprotein), cytomegalovirus (CMV) antigen, 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) antigen, herpes simplex virus antigen, human immunodeficiency virus (HIV) antigens (GP-120, GP-160, p18, Tat, Gag, Pol, Env) and combinations thereof.

8. The immunotherapeutic and preventive vaccine according to claim 5, wherein: The cancer antigen is selected from at least one of 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 (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.

9. The immunotherapeutic and preventive vaccine according to claim 1, wherein: The antigen is in the form of a peptide, lipopolysaccharide, polysaccharide, glycoprotein or polynucleotide including DNA and RNA.

10. The immunotherapeutic and preventive vaccine according to claim 8, wherein The cancer is selected from the group consisting of liver cancer, thyroid cancer, testicular cancer, bone cancer, glioblastoma, oral cancer, ovarian cancer, brain tumor, multiple myeloma, gallbladder cancer, bile duct cancer, colon cancer, head and neck cancer, lymphoma, bladder cancer, leukemia, esophageal cancer, kidney cancer, stomach cancer, breast cancer, cervical cancer, prostate cancer, rectal cancer, spinal cord tumor, pancreatic cancer, salivary gland cancer, lung cancer, skin cancer, laryngeal cancer, melanoma, acute myeloid leukemia, neuroblastoma, retinoblastoma and colorectal cancer.

11. The immunotherapeutic and preventive vaccine according to claim 1, wherein The antigen is introduced and expressed via a recombinant virus.

12. The immunotherapeutic and preventive vaccine according to claim 11, wherein The recombinant virus is adenovirus, retrovirus, vaccinia virus, poxvirus or Sindbis virus, and a gene for expressing an antigen is introduced into the recombinant virus.

13. A method for producing an anti-cancer immunotherapeutic and preventive vaccine, the method comprising the following steps: (a) a step of obtaining peripheral blood mononuclear cells; (b) a step of removing red blood cells; and (c) A step of delivering natural killer T cell ligands and antigens to peripheral blood mononuclear cells.

14. Anti-cancer immunotherapeutic and preventive vaccines prepared according to the method of claim 13.

15. A pharmaceutical composition for preventing and treating cancer, comprising peripheral blood mononuclear cells loaded with natural killer T cell ligands and cancer antigens as active ingredients.

16. Use of peripheral blood mononuclear cells loaded with natural killer T cell ligands and cancer antigens in preventing or treating cancer.

17. A method for preventing or treating cancer, comprising administering to a subject an effective amount of peripheral blood mononuclear cells loaded with natural killer T cell ligands and cancer antigens.

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