Vaccines containing peripheral blood mononuclear cells loaded with natural killer T cell ligands and antigens

Abstract

The present invention relates to immunoprophylactic and therapeutic vaccines comprising peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen, specifically, immunotherapeutic vaccines comprising peripheral blood mononuclear cells loaded with α-galactosylceramide, a natural killer T cell ligand, and a glycolipid. The composition of the present invention can be easily obtained because it does not require the isolation of specific cells from peripheral blood mononuclear cells. In addition, immunization with peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen not only induces significant levels of activation of natural killer cells and natural killer T cells and cytotoxic T lymphocyte responses, but also has a synergistic effect in the treatment of malignant tumors, and therefore can be useful as an anti-cancer immunotherapeutic drug.

Description

[Technical Field]

[0001] Background of the Invention 1. Field of the Invention CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of Korean Patent No. 10-2022-0116552, filed on September 15, 2022, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates to vaccines comprising peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen, such as a cancer antigen.

[0003] 2. Description of Related Art Recent advances in medicine have increased the survival rate of cancer patients, but the incidence of cancer is also increasing due to changes in environmental factors and longer life expectancies. Much research has been conducted on cancer treatment, and new drugs and treatment methods have been developed, significantly improving the effectiveness of cancer treatment for cancer patients. However, in the case of malignant tumors, despite the development of microsurgery and radiation therapy and new therapeutic agents for chemotherapy, therapeutic effects remain limited and there are limitations such as side effects due to nonspecific anticancer effects and cancer recurrence. Immunotherapy is a treatment method that is currently being actively used or developed to complement conventional cancer treatments. Immunotherapy has the properties of complementing conventional cancer treatments by inducing tumor-specific toxicity, reducing systemic toxic side effects, and establishing active memory responses against cancer and cancer antigens.

[0004] Immunotherapy, particularly immune cell vaccines using antigen-presenting cells, can effectively activate CD8+ T cells and CD4+ T cells, and thus exhibits excellent anti-cancer effects.

[0005] Currently, the most commonly used immune cells in antigen-presenting cell vaccines are dendritic cells (DCs), which phagocytose antigens and deliver them to effector cells such as T cells with strong costimulatory signals, thereby efficiently activating the effector cells and inducing a strong immune response. In actual clinical cell therapy using dendritic cells, dendritic cells are first isolated from the patient's bone marrow or peripheral blood, or their precursor monocytes are isolated, then massively expanded, differentiated into dendritic cells, activated by the addition of antigen, and then infused back into the patient. Once infused, dendritic cells transmit specific antigen information to T cells, activating them and effectively inducing antigen-specific immune responses. Despite these advantages, dendritic cells are not widely used in antigen-presenting cell vaccines because there are very few dendritic cells that can be obtained directly from blood and lymphoid tissues, their isolation is difficult, and when differentiated from monocytes, they must be cultured in vitro for several days.

[0006] In a previous study, the inventors confirmed the effectiveness of α-GC-loaded B cells in inducing cytotoxic T lymphocyte responses (Korean Patent No. 10-0809873). B cells are present in large quantities in lymphoid tissues and blood, can be easily proliferated in vitro, and are therefore effective as an alternative to dendritic cells for cellular vaccines. They also have the advantage of migrating to lymphoid organs after intravenous administration. Despite these advantages, B cells are not widely used in antigen-presenting cell vaccines due to their weak immunogenicity.

[0007] Recent studies have demonstrated that invariant natural killer T cells (iNKT cells) play an important role in regulating various immune responses and immunopathological phenomena. Ligand-activated iNKT cells can induce the activation of T cells, B cells, natural killer cells, and dendritic cells.

[0008] Cancer cells loaded with α-GC, a ligand for natural killer T cells, can induce the activation of cancer antigen-specific T cells. It has been shown that administration of T cells loaded with α-GC and antigenic peptides strongly induced the expression of IFN-γ and granzyme B, and that administration of α-GC and antigenic peptides in mice with metastatic lung cancer exhibited anticancer therapeutic effects through the activation of antigen-specific cytotoxic T lymphocyte responses (Yeonseok Chung et al., OncoImmunology, 1:2, 141-151, 2012).

[0009] The inventors have created an effective cell therapy vaccine by demonstrating that loading B cells with natural killer T cell ligands generates an active immune response against cancer antigens presented by the major histocompatibility complex on B cells, converting the self-tolerance of these cells to immunogenicity and exerting anti-cancer effects.

[0010] Furthermore, the present inventors have previously confirmed that when α-GC-loaded monocytes are loaded with an antigen peptide or transduced with an antigen-expressing adenovirus and administered to the body, an antigen-specific immune response is induced and a significant anti-cancer effect is exhibited, thereby producing an effective cell therapy vaccine (Korean Patent No. 10-1055666).

[0011] However, the cell therapy developed from the previous research is produced using a manufacturing process that removes T cells from the patient's peripheral blood and leaves B cells and monocytes. This method has the disadvantage that it is difficult to separate T cells, B cells, and monocytes from peripheral blood, making the manufacturing process complicated.

[0012] To solve these problems, based on the above-mentioned research on various immune cells, the present inventors developed an anti-cancer immunotherapy and prophylactic vaccine by loading peripheral blood mononuclear cells, which can be obtained without isolating each immune cell, with natural killer T cell ligands and antigens.

[0013] Peripheral blood mononuclear cells (PBMCs) are a collective term for mononuclear mesenchymal cells present in the bloodstream, primarily composed of monocytes and lymphocytes. Generally, PBMCs refer to the leukocyte fraction obtained by centrifuging blood and excluding polymorphonuclear leukocytes. Lymphocytes include T cells, B cells, and natural killer cells (NK cells). Specifically, the cell therapy developed by the present inventors uses PBMCs, which are round-nucleated cells composed of lymphocytes (T cells, B cells, and natural killer cells) and monocytes, as raw materials. The proportion of each immune cell type in PBMCs varies greatly 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.

[0014] The present inventors have endeavored to solve the problems associated with dendritic cell vaccines differentiated from dendritic cells and monocytes, as well as the problems associated with prior inventions that use B cells and monocytes. As a result, they have confirmed that vaccines using peripheral blood mononuclear cells have a synergistic effect superior to the immune effects of B cells, monocytes, and T cells, respectively, and have completed the development of the vaccine comprising peripheral blood mononuclear cells of the present invention.

[0015] Furthermore, the cell therapy agent of the present invention has various advantages over previous cell therapy agents, such as increasing the amount of peripheral blood mononuclear cells used as the starting material through a process that does not isolate each type of immune cell. If the process of staining and removing T cells in the manufacturing process of existing cell therapy agents is eliminated, the amount of peripheral blood mononuclear cells used as the starting material will increase, and the final production volume will increase, leading to savings in time, labor, and costs and an increase in production volume. Summary of the Invention

[0016] SUMMARY OF THE INVENTION It is an object of the present invention to provide an immunotherapeutic or prophylactic vaccine comprising peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen. Another object of the present invention is to provide a method for producing an immunotherapeutic or prophylactic vaccine comprising peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen.

[0017] To achieve the above object, the present invention provides an immunotherapeutic or prophylactic vaccine comprising peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen. In another aspect, the present invention provides a method for producing an immunotherapeutic or prophylactic vaccine comprising peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen.

[0018] The present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises, as an active ingredient, peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen. The present invention also provides the use of peripheral blood mononuclear cells loaded with a natural killer T cell ligand and a cancer antigen for the prevention or treatment of cancer. The present invention also provides a method for preventing or treating cancer by administering to a subject an effective amount of peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen. [Effects of the Invention]

[0019] beneficial effects The present invention relates to immunoprophylactic and immunotherapeutic vaccines comprising peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen, particularly immunotherapeutic vaccines comprising peripheral blood mononuclear cells loaded with α-galactosylceramide (α-GC), a natural killer T cell ligand, and a glycolipid. The compositions of the present invention are easily available because they do not require the isolation of specific cells from peripheral blood mononuclear cells. Immunization with peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen not only induces significant levels of natural killer cell and natural killer T cell activation and cytotoxic T lymphocyte responses, but also exhibits synergistic therapeutic effects against malignant tumors, making them effective as anticancer immunotherapeutic agents. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a diagram illustrating a manufacturing process for a peripheral blood mononuclear cell vaccine according to one embodiment of the present invention. [Figure 2a] Figure 2a is a set of graphs showing the activity of natural killer T cells (NKT) and natural killer cells (NK) in the spleens of mice after administration of peripheral blood mononuclear cell vaccines loaded with α-GC and / or transduced with antigen-expressing adenovirus. [Figure 2b-2c] Figure 2b is a graph showing IFN-γ production by the natural killer T cells (NKT) of Figure 2a. Figure 2c is a graph showing IFN-γ production by the natural killer cells (NK) of Figure 2a.

[0021] [Figure 3a] Figure 3a is a diagram showing the experimental setup comparing the activation of cytotoxic T cells in mice following administration of peripheral blood mononuclear cell vaccines loaded with α-GC and / or transduced with adenovirus expressing the antigen (target peptide: MAGE-A3282-290). [Figure 3b-3c] Figure 3b is a set of histograms showing the results of measuring the activity of antigen-specific cytotoxic T cells induced by peripheral blood mononuclear cell vaccines loaded with α-GC and / or transduced with antigen-expressing adenoviruses, and Figure 3c is a graph showing the activity of antigen-specific cytotoxic T cells induced by peripheral blood mononuclear cell vaccines loaded with α-GC and / or transduced with antigen-expressing adenoviruses.

[0022] [Figure 4a] FIG. 4a is a diagram showing the experimental method for confirming the therapeutic efficacy of peripheral blood mononuclear cell vaccines loaded with α-GC and / or transduced with adenoviruses expressing antigens against murine solid tumors. [Figure 4b-4c]Figure 4b is a graph showing the anti-cancer effect (change in tumor size) of peripheral blood mononuclear cell vaccines loaded with α-GC and / or transduced with antigen-expressing adenoviruses. Figure 4c is a graph showing the anti-cancer effect (change in survival rate) of peripheral blood mononuclear cell vaccines loaded with α-GC and / or transduced with antigen-expressing adenoviruses.

[0023] [Figure 5a-5b] Figure 5a is a diagram showing an experimental method for confirming the therapeutic effect of a peripheral blood mononuclear cell vaccine loaded with α-GC and / or transduced with an adenovirus expressing an antigen for lung metastatic cancer. Figure 5b is a set of photographs showing the lungs of a lung metastatic cancer model mouse administered a peripheral blood mononuclear cell vaccine loaded with α-GC and / or transduced with an adenovirus expressing the antigen. [Figure 5c] FIG. 5c is a graph showing the therapeutic effect (number of metastatic nodules) of peripheral blood mononuclear cell vaccines loaded with α-GC and / or transduced with adenovirus expressing an antigen against lung metastatic cancer.

[0024] [Figure 6] Figure 6a is a diagram showing the experimental method for confirming the therapeutic effect of a peripheral blood mononuclear cell vaccine loaded with α-GC and / or transduced with an adenovirus expressing an antigen against metastatic cancer. Figure 6b is a graph showing the anti-cancer effect (change in survival rate) of a peripheral blood mononuclear cell vaccine loaded with α-GC and / or transduced with an adenovirus expressing an antigen.

[0025] [Figure 7a] FIG. 7a is a diagram showing an experimental method for confirming activation of antigen-specific cytotoxic T cells by a peripheral blood mononuclear cell vaccine co-loaded with α-GC and the HER263-71 peptide (target peptide: HER263-71). [Figure 7b-7c]Figure 7b is a set of histograms showing the activity of antigen-specific cytotoxic T cells induced by peripheral blood mononuclear cell vaccines co-loaded with α-GC and the HER263-71 peptide, and Figure 7c is a graph showing the activity of antigen-specific cytotoxic T cells induced by peripheral blood mononuclear cells co-loaded with α-GC and the HER263-71 peptide.

[0026] [Figure 8a] FIG. 8a is a diagram showing the experimental method for confirming the therapeutic effect of a peripheral blood mononuclear cell vaccine loaded with α-GC and / or a cancer antigen peptide (target peptide: HER263-71) against mouse solid tumors. [Figure 8b] FIG. 8b is a graph showing the anti-cancer effect (changes in tumor size) of peripheral blood mononuclear cell vaccines loaded with α-GC and / or cancer antigen peptides.

[0027] [Figure 9a] Figure 9a is a diagram showing the experimental method for comparing the activation of antigen-specific cytotoxic T cells by peripheral blood mononuclear cell vaccines and B cell / monocyte vaccines (target peptide: GP10025-33) loaded with α-GC and / or expressing adenovirus and / or antigen. [Figure 9b] Figure 9b is a set of histograms showing the activity of antigen-specific cytotoxic T cells induced by peripheral blood mononuclear cell vaccines and B cell / monocyte vaccines loaded with adenovirus expressing α-GC and / or antigen. [Figure 9c] Figure 9c is a graph showing the activity of antigen-specific cytotoxic T cells induced by peripheral blood mononuclear cell vaccines and B cell / monocyte vaccines loaded with α-GC and / or transduced with antigen-expressing adenovirus.

[0028] [Figures 10a-10b]Figure 10a is a diagram showing the experimental method for comparing the activation of antigen-specific cytotoxic T cells by peripheral blood mononuclear cell vaccines, B cell / monocyte vaccines, and T cell vaccines (target peptide: GP10025-33) loaded with α-GC and / or transduced with antigen-expressing adenovirus. Figure 10b is a set of histograms showing the activity of antigen-specific cytotoxic T cells induced by peripheral blood mononuclear cell vaccines, B cell / monocyte vaccines, and T cell vaccines loaded with α-GC and / or transduced with antigen-expressing adenovirus. [Figure 10c] Figure 10c is a graph showing the activity of antigen-specific cytotoxic T cells induced by peripheral blood monocyte vaccines, B cell / monocyte vaccines, and T cell vaccines loaded with α-GC and / or transduced with antigen-expressing adenovirus.

[0029] [Figure 11] Figure 11a is a diagram showing an experimental method for comparing the therapeutic effects of peripheral blood mononuclear cell vaccines loaded with α-GC and / or transduced with antigen-expressing adenoviruses and B cell / monocyte vaccines against solid cancers. Figure 11b is a set of graphs showing the anti-cancer effects (changes in tumor size) of peripheral blood mononuclear cell vaccines loaded with α-GC and / or transduced with antigen-expressing adenoviruses and B cell / monocyte vaccines. DETAILED DESCRIPTION OF THE INVENTION

[0030] Description of the Preferred Embodiments The present invention will now be described in detail. The present invention provides an immunotherapeutic or prophylactic vaccine against cancer, comprising peripheral blood mononuclear cells loaded with a natural killer T cell ligand and a cancer antigen. Peripheral blood mononuclear cells (PBMCs) are cells with round nuclei that consist of lymphocytes (T cells, B cells, and natural killer cells) and monocytes. The proportion of each type of immune cell in PBMCs varies greatly from person to person, but generally, they are composed of 45-70% T cells, 5-15% B cells, 5-10% natural killer cells, and 5-10% monocytes.

[0031] Herein, the present inventors developed an anti-cancer therapeutic cellular vaccine that can induce significantly enhanced anti-cancer immune responses by adding the function of antigen-presenting cells through altering the immunogenicity of immune cells through loading with α-GC, a natural killer T cell ligand, and antigen delivery to immune cells.

[0032] However, neoepitopes, peptide sequences created by highly tumor-specific mutations that are present only in cancer cells but not in normal cells and can induce cancer cell-specific immune responses, are known to be ideal targets for personalized anti-cancer immunotherapy for cancer patients. Anti-cancer immunotherapy using immune cell vaccines that deliver the discovered neoepitopes for personalized treatment of cancer patients is expected to induce the generation of more cancer cell-specific T cells than conventional immunotherapy, and to induce potent anti-cancer therapeutic effects while minimizing the side effects of anti-cancer treatment by preventing damage to normal cells.

[0033] Alpha-galactosylceramide (α-GC) is an immunopotentiator that induces various anti-cancer immune responses by stimulating NKT cells, particularly by enabling B cells and monocytes to stimulate T cells as efficiently as dendritic cells.

[0034] 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 have confirmed the effect of α-GC loading on inducing cytotoxic T lymphocyte responses in B cells, monocytes, and immature myeloid cells (Korean Patent Publication Nos. 10-2007-0105662 and 10-2009-0051598). It has also been confirmed that α-GC-loaded natural killer cells activate natural killer T (NKT) cells and induce cytotoxic T lymphocyte responses (Korean Patent Publication No. 10-2022-0072485).

[0035] Therefore, the present inventors have produced an anti-cancer vaccine with enhanced immune function by adding the function of antigen-presenting cells through altering the immunogenicity of immune cells through α-GC loading and antigen delivery to immune cells.

[0036] On the other hand, antigen delivery using viral vectors is suitable for the large-scale production of cell therapy agents targeting cancers expressing specific cancer antigens by delivering cancer antigens to cells with high efficiency. When a virus capable of expressing the antigen is used, the entire antigen can be introduced, which has the advantage of being applicable to everyone without being restricted to a specific haplotype of the major histocompatibility complex, and of inducing not only cellular but also humoral immune responses. On the other hand, viral vectors are limited in their application to personalized cancer therapy.

[0037] Clinically, antigen delivery via peptide loading has the disadvantage that it cannot be universally used and only presents a single epitope because it is limited by an individual's major histocompatibility complex (MHC) haplotype. However, neoepitopes, peptide sequences created by highly tumor-specific mutations that are present only in cancer cells but not in normal cells and can induce cancer cell-specific immune responses, are known to be ideal targets for personalized anti-cancer immunotherapy in cancer patients. Anti-cancer immunotherapy using immune cell vaccines that deliver discovered neoepitopes for personalized treatment of cancer patients is expected to induce the generation of more cancer cell-specific T cells than conventional immunotherapy.

[0038] The above-mentioned natural killer T cell ligands include α-galactosylceramide (α-GC), α-glucuronosylceramide, phosphatidylinositol tetramannoside, isoglobotrihexosylceramide, ganglioside GD3, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sulfitides, β-galactosylceramide, lipophosphoglycan, glycoinositol phospholipids, β-anomeric galactosylceramide and α-anomeric galactosylceramide, which are analogs of α-galactosylceramide, bacterial lipid antigens, and α-galactosylceramide mutants.

[0039] The antigen may be any antigen that can be used as a vaccine and that can induce an immune response, and may be a full-length or fragment of the antigen, including antigens derived from pathogens, including pathogenic bacteria, viruses, and parasites, or cancer antigens.

[0040] Antigens 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), Streptococcus pneumoniae antigens (pneumococcal type 3 capsular polysaccharide), tuberculosis antigens, cholera antigens (cholera toxin B subunit), Staphylococcus antigens (Staphylococcal enterotoxin B), Shigella antigens (Shigella polysaccharide), Borrelia antigens, Candida albicans antigens, and Plasmodium antigens.

[0041] 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 and surface antigens), 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.

[0042] 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), muringlobulin 1 (MUC-1), carcinoembryonic antigen (CEA), p53, alpha-fetoprotein, breast cancer protein expressed by Her-2 / neu, proteinase 3, WT-1, PAP, PSA, PSMA, G250, BAGE, GAGE, NY-ESO-1, MART-1, MCIR, Ig idiotype, CDK4, caspase-8, beta-catenin, CIA, BCR / ABL, EBV LMP2a, HCV, HHV-8, 5T4, neoantigens derived from tumor-specific mutations, and combinations thereof.

[0043] The vaccine may be used to treat or prevent cancer. The above cancers may include all types of cancer, including liver cancer, thyroid cancer, testicular cancer, bone cancer, glioblastoma, oral cancer, ovarian cancer, brain tumor, multiple myeloma, gallbladder cancer, biliary tract 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, colorectal cancer, etc. The cancer may be a solid tumor or a metastatic tumor.

[0044] Antigens may also be directly loaded into peripheral blood mononuclear cells in the form of peptides, lipopolysaccharides, polysaccharides, glycoproteins, or polynucleotides including DNA and RNA, or may be transfected into peripheral blood mononuclear cells by recombinant viruses, which then express and load the antigens.Compared to peptide-loaded cellular vaccines, cellular vaccines that introduce whole antigens via viruses have the advantages of being applicable to everyone, not being limited by major histocompatibility complex haplotypes, and being able to induce immune responses specific to multiple epitopes, particularly being able to simultaneously induce humoral and cellular immune responses.

[0045] In addition to the natural killer T cell ligand and peripheral blood mononuclear cells, the vaccine of the present invention may additionally contain one or more active ingredients having the same or similar effects.

[0046] In addition to the above-mentioned active ingredients, the vaccine may also contain one or more pharmaceutically acceptable carriers for administration. The pharmaceutically acceptable carrier may be selected or prepared by mixing one or more components selected from the group consisting of saline, Ringer's solution, buffered saline, dextrose solution, maltodextrose solution, glycerol, and ethanol. Other common additives, such as antioxidants, buffers, and bactericides, may be added. To prepare injection solutions, such as aqueous solutions, suspensions, and emulsions, diluents, dispersants, surfactants, binders, and lubricants may be further added. The vaccine of the present invention may be further prepared in a suitable form for each disease or depending on the ingredients, by following the methods described in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA.

[0047] The vaccine of the present invention can be administered parenterally, and parenteral administration includes subcutaneous injection, intravenous injection, intramuscular injection, and intrathoracic injection.To prepare the vaccine as a formulation for parenteral administration, the peripheral blood mononuclear cells loaded with natural killer T cell ligand, the peripheral blood mononuclear cells loaded with natural killer T cell ligand and peptide, or the peripheral blood mononuclear cells infected with the virus that expresses the cancer antigen of the present invention are mixed with a stabilizer or buffer to produce a solution or suspension, which is then formulated into an ampoule or vial.

[0048] The vaccine of the present invention can be formulated into various forms depending on the route of administration.For example, the vaccine of the present invention can be prepared in the form of a sterile injectable solution or suspension, or in the form of a freeze-dried preparation using freeze-drying technology.The freeze-dried vaccine of the present invention is typically kept at about 4°C, and can be reconstituted in a stabilizing solution containing or not containing an adjuvant such as saline or / and HEPES.

[0049] To achieve the present invention, the effective dose of the vaccine to be administered is determined taking into consideration, but not necessarily limited to, the method of administration, the frequency of administration, the specific disease being treated, the severity of the disease, medical history, whether the patient is undergoing concomitant treatment with other drugs, the age, height, weight, health condition, and other physical conditions of the patient. Generally, it is preferable to increase the dose of this formulation as the weight of the patient being treated increases.

[0050] The vaccine may be administered at a dose effective to induce an immune response in a patient. For example, the vaccine may be administered at a dose of 1×10 3 ~1×10 9 cells / kg, more preferably 1 x 10 4 cells / kg~1×10 8 The dose of α-galactosylceramide-loaded peripheral blood mononuclear cells (PBMCs) can be administered to humans once or several times daily. To prepare the α-galactosylceramide-loaded PBMCs, the medium is filled with 1 x 10 6 ~1×10 7 The medium should be supplemented with α-galactosylceramide at a concentration of 1-2 μg / ml per PBMC / ml. To prepare PBMC vaccines loaded with α-galactosylceramide and peptides, the medium should be supplemented with 1 x 10 PBMCs. 6 ~1×10 7 α-galactosylceramide at a concentration of 1-2 μg / ml per 1 x 10 peripheral blood mononuclear cells / ml, and 1 x 10 6 ~1×10 7 The peptide should be supplemented at a concentration of 1-10 μg / ml per PBMC / ml.

[0051] α-Galactosylceramide does not appear to induce toxicity in rodents and monkeys (Nakata et al., Cancer Res 58:1202-1207, 1988). No side effects were reported when mice were injected with 2200 μg / kg of α-GalCer (Giaccone et al., Clin Cancer Res 8:3702, 2002). Ongoing clinical trials have reported some side effects, such as mild headaches, with systemic administration of α-GalCer (Mie Nieda et al., Blood 103:383-389; Giaccone et al., Clin Cancer Res 8:3702, 2002). However, these can be prevented by administration of paracetamol, and mild systemic side effects do not necessarily occur in these subjects (Giaccone et al., Clin Cancer Res 8:3702, 2002).

[0052] The present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises, as an active ingredient, peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen. The present invention also provides the use of peripheral blood mononuclear cells loaded with a natural killer T cell ligand and a cancer antigen for the prevention or treatment of cancer.

[0053] The present invention also provides a method for preventing or treating cancer by administering to a subject an effective amount of peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen. The present invention also provides a method for producing an anti-cancer immunotherapeutic or prophylactic vaccine comprising peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen.

[0054] The method involves the following steps: (a) obtaining peripheral blood mononuclear cells; (b) removing red blood cells; and (c) delivering a natural killer T cell ligand and an antigen to peripheral blood mononuclear cells; Includes. In step (a), peripheral blood mononuclear cells can be obtained, for example, using known leukapheresis techniques. As used herein, peripheral blood mononuclear cells may include T cells, B cells, natural killer cells, monocytes, and some red blood cells, but may not include neutrophils, eosinophils, or basophils.

[0055] In step (b), red blood cells may be removed by any method known in the art, for example using a suitable lysis buffer. In step (c), the natural killer T cell ligand may be added to a culture medium (e.g., a culture medium for peripheral blood mononuclear cells) and delivered to the peripheral blood mononuclear cells through the culture. In step (c), the antigen may be delivered by a recombinant virus. Viruses introduced into peripheral blood mononuclear cells for antigen expression include, but are not limited to, adenovirus, retrovirus, vaccinia virus, poxvirus, and Sindbis virus.

[0056] In addition to virus-based methods, the following methods can be applied to deliver antigen genes: 1) transduction of DNA by binding it to liposomes to protect it from enzymatic degradation or by endocytosis into endosomes; 2) increasing the efficiency of intracellular DNA delivery by linking molecular conjugates consisting of proteins or synthetic ligands to DNA (e.g., asialoglycoprotein, transferrin, polymeric IgA); 3) increasing the efficiency of intracellular DNA delivery through novel DNA delivery systems using PTDs (protein transduction domains) (e.g., Mph-1); and 4) in addition to the above methods, peptides can be used, or antigen proteins can be applied to peripheral blood mononuclear cells to induce antigen presentation in peripheral blood mononuclear cells.

[0057] The above preparation method differs from existing methods that use individual immune cells, eliminating the need for a process to remove other immune cells in order to obtain individual immune cells, which reduces the time and cost of vaccine production and increases production volume.

[0058] The present inventors have produced a vaccine with significantly improved immune function by altering the immunogenicity of peripheral blood mononuclear cells through α-GC loading and antigen delivery to peripheral blood mononuclear cells, thereby enabling them to present antigens.

[0059] Antigen delivery using viral vectors allows for highly efficient delivery of cancer antigens to cells, making it suitable for the large-scale production of cell therapy agents targeting cancers expressing specific cancer antigens. When viruses capable of expressing the antigens are used, the entire antigen can be introduced, which has the advantages of being applicable to everyone without being restricted to a specific haplotype of the major histocompatibility complex, and of inducing not only cellular but also humoral immune responses. However, viral vectors are limited in their application to personalized cancer therapy.

[0060] Clinically, antigen delivery via peptide loading has the disadvantage of being limited to an individual's major histocompatibility complex (MHC) haplotype, making it infeasible for universal use and presenting only a single epitope. However, neoepitopes, peptide sequences created by highly tumor-specific mutations that are present only in cancer cells but not in normal cells and can induce cancer-cell-specific immune responses, are known to be ideal targets for personalized anticancer immunotherapy in cancer patients. Anticancer immunotherapy using immune cell vaccines delivering discovered neoepitopes for personalized treatment of cancer patients is expected to induce the generation of more cancer-specific T cells than conventional immunotherapy. Furthermore, by preventing damage to normal cells, it may induce potent anticancer drug treatment effects while minimizing side effects of anticancer treatment.

[0061] In this study, peripheral blood mononuclear cells (PBMCs) were isolated from mice and loaded with α-GC. Antigens were delivered by two methods (antigen gene transfer into antigen-expressing viral vectors or peptide loading) to produce PBMC vaccines.

[0062] First, we produced a peripheral blood mononuclear cell (PBMC) vaccine loaded with α-GC and delivered the antigen gene via an adenoviral vector expressing the antigen. We confirmed that administration of the vaccine could activate natural killer T cells and natural killer cells in vivo. Results showed that NKT cells and natural killer cells were activated in mice administered with α-GC-loaded PBMCs and in mice administered with α-GC-loaded PBMCs and adenovirally delivered cancer antigens GP100 and MAGE-A3 (see Figures 2a-c). Furthermore, in vivo CTL assays were performed to determine whether the vaccine could induce cytotoxic immune responses by activating antigen-specific cytotoxic T lymphocytes. Results confirmed that the PBMC vaccine loaded with α-GC and adenovirally delivered cancer antigens (GP100 and MAGE-A3) induced effective cytotoxic responses (see Figures 3a-c). The efficacy of the vaccine in treating solid cancers was also confirmed. As a result, the vaccine exhibited significant anticancer effects, suppressing tumor size and increasing mouse survival rates (see Figures 4a to 4c).

[0063] The therapeutic effect of the vaccine against lung metastatic cancer was also investigated. In both cell groups in which the cancer antigen was introduced via adenovirus, almost no cancer tissue was observed (see Figures 5a-c), and the survival rate of mice was significantly improved (see Figures 6a and 6b).

[0064] Next, we produced a peripheral blood mononuclear cell vaccine loaded with α-GC and antigenic peptides, and confirmed whether administration of the vaccine would induce a cytotoxic T cell response. 63-71It was confirmed that the peptide-loaded peripheral blood mononuclear cell vaccine induced an effective cytotoxic response (see Figs. 7a to 7c).

[0065] We also investigated the anti-cancer effect of a peripheral blood mononuclear cell vaccine loaded with α-GC and cancer antigen peptides, and confirmed that tumor size was suppressed as a result (see Figures 8a and 8b).

[0066] We compared the anti-cancer effects of the vaccine composed of peripheral blood mononuclear cells of the present invention with those of vaccines composed of other immune cells. First, we compared the ability of peripheral blood mononuclear cell vaccines and B cell / monocyte vaccines to induce cytotoxic T cell immune responses. Cancer antigen-specific cytotoxicity was observed in both groups administered with the two vaccines, with slightly higher cytotoxicity observed in the group administered with the peripheral blood mononuclear cell vaccine (Figures 9a-9c). We also compared the ability of peripheral blood mononuclear cell vaccines, B cell / monocyte vaccines, and T cell vaccines to induce cytotoxic T cell immune responses. We confirmed that cancer antigen-specific cytotoxic T cell immune responses were induced not only by peripheral blood mononuclear cells and B cells / monocytes, but also by T cells (Figures 10a and 10c). In particular, we confirmed that the PBMC vaccine exhibited a stronger anticancer therapeutic effect, demonstrating sustained suppression of cancer growth, compared with the B cell / monocyte vaccine (Fig. 11a and 11b). This is thought to be due to the complementary interactions of various immune cells present in the PBMCs, which induce an enhanced anticancer therapeutic response.

[0067] The present invention will now be described in more detail by the following experimental examples. However, the following experimental examples are merely for the purpose of illustrating the present invention, and the contents of the present invention are not limited thereto.

[0068] Example 1: Production of peripheral blood mononuclear cell vaccines <1-1> Isolation and purification of mouse T cells, B cells / monocytes, and natural killer cells To isolate peripheral blood mononuclear cells (PBMCs), mouse spleens were harvested and homogenized. After lysis of red blood cells using ACK lysis buffer (Gibco), T cells expressing CD4 or CD8α on their cell surface and B cells / monocytes expressing B220 or CD11b were isolated using microbeads (Miltenyibiotec). Following isolation of T cells and B cells / monocytes, CD49b+ natural killer cells were obtained from the remaining spleen cells using anti-CD49b microbeads (Miltenyibiotec). Because the amount of immune cells obtainable from mouse blood is limited, we isolated immune cells from the spleen. However, because the immune cell composition differs between blood and spleen, we mixed each immune cell type (T cells: B cells / monocytes: natural killer cells = 5:4:1) to match the blood composition and distribute them similarly to human PBMCs.

[0069] <1-2> Construction of peripheral blood mononuclear cell vaccines in which antigens are delivered by viral vectors expressing the antigens Peripheral blood mononuclear cells isolated and purified as described above were placed in serum-containing medium with α-GC (1 μg / mL), vehicle (DMSO), and / or adenovirus (MOI) for cancer antigen gene delivery (200 MOI). The cells were centrifuged in a cell culture plate (2,000 rpm, 20°C, 90 min) and cultured in a CO2 incubator (37°C, 80-95% relative humidity, and 5% CO2) to produce the peripheral blood mononuclear cell vaccine. The peripheral blood mononuclear cell vaccine was washed three times with Dulbecco's phosphate-buffered saline (DPBS, Welgene), dissolved in DPBS, and administered into the tail vein of mice.

[0070] <1-3> Construction of peripheral blood mononuclear cell vaccine loaded with antigen peptides Viral vectors are suitable gene delivery vehicles for the large-scale production of cell therapy agents targeting cancer cells expressing specific cancer antigens because they can efficiently deliver cancer antigens common to cancer cells. However, viral vectors are limited in their application to personalized treatment of cancer patients using neoepitopes. Therefore, we constructed a peripheral blood mononuclear cell vaccine using peptide-loaded antigen delivery.

[0071] 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 serum-containing medium, and cultured for 15 hours in a CO2 incubator (37°C, 80-95% relative humidity, and 5% CO2 concentration). 63-71 The peptide (2 μg / mL) was added to the cells to prepare a peripheral blood mononuclear cell vaccine, which was then incubated for 2 hours in a CO2 incubator at 37°C. The prepared peripheral blood mononuclear cell vaccine was washed three times with DPBS, dissolved in DPBS, and administered to the tail vein of mice.

[0072] Comparative Example 1: Production of a B cell / monocyte vaccine To isolate mouse B cells / monocytes, mouse spleens were harvested and homogenized. After lysing red blood cells using ACK lysis buffer (Gibco), B220- or CD11b-expressing B cells / monocytes were isolated using microbeads. Antigens were delivered to the isolated B cells / monocytes using the same methods as in Examples 1-2 and 1-3.

[0073] Comparative Example 2: Production of a T-cell vaccine To isolate mouse T cells, mouse spleens were harvested and homogenized. After lysing red blood cells using ACK lysis buffer (Gibco), T cells expressing CD4 or CD8α on their surface were isolated using microbeads. Antigens were delivered to the isolated T cells using the same methods as in Examples 1-2 and 1-3.

[0074] Example 2: Production of a human peripheral blood mononuclear cell vaccine The manufacturing process of the human peripheral blood mononuclear cell vaccine of the present invention is shown in FIG. Specifically, human peripheral blood mononuclear cells obtained by leukopheresis were lysed with ACK lysis buffer (Lonza) to lyse red blood cells, and then treated with α-GC (1 μg / mL) and an antigen-expressing viral vector (25 MOI). The cells were then cultured in a CO2 incubator at 37°C and 5% CO2 for 15 hours to produce the peripheral blood mononuclear cell vaccine. This eliminates the need for T cell removal from the existing manufacturing process, saving time, manpower, and costs, and increasing production volume.

[0075] Experimental Example 1: Confirmation of the efficacy of peripheral blood mononuclear cell vaccines in which antigens are delivered by viral vectors expressing the antigens <1-1> Confirmation of induction of activation of natural killer T cells and natural killer cells The present inventors confirmed whether natural killer T cells and natural killer cells in the body are activated through administration of a peripheral blood mononuclear cell vaccine. Specifically, peripheral blood mononuclear cells obtained from C57BL / 6 mice were used to generate α-GC-loaded peripheral blood mononuclear cells (PBMC / α-GC), adenovirus-Adk35GM-transduced peripheral blood mononuclear cells (PBMC / Adk35GM) for delivery of cancer antigens (human GP100 and MAGE-A3) (PBMC / Adk35GM), and α-GC-loaded and adenovirus-Adk35GM-transduced peripheral blood mononuclear cells (PBMC / α-GC / Adk35GM). 6 Peripheral blood mononuclear cells were administered intravenously. Six hours later, the levels of IFN-γ production in natural killer T cells and natural killer cells in the spleen were measured by flow cytometry.

[0076] As shown in Figures 2a to 2c, in the mice administered with α-GC-loaded peripheral blood mononuclear cells (PBMC / α-GC and PBMC / α-GC / Adk35GM), natural killer T cells in the body were stimulated and natural killer cells were also activated, unlike in the mice administered with transduced peripheral blood mononuclear cells (PBMC / Adk35GM). Activation of these natural killer T cells and natural killer cells, along with the induction of cytotoxic T cell responses, may contribute to anti-cancer effects.

[0077] <1-2>Evaluation of the ability of peripheral blood mononuclear cells to induce cytotoxic T cell responses To determine whether the adenovirus-transduced peripheral blood mononuclear cell vaccine could induce antigen-specific cytotoxic T cell immune responses, an in vivo CTL assay was performed. Specifically, we constructed PBMC vaccines using α-GC-loaded or adenovirus-delivered Adk35GM antigen-delivered PBMCs obtained from BALB / c mice, immunized them with the vaccines, and performed cytotoxicity assays 7 days later (Figure 3a). First, spleen cells from the same mice were divided into two groups with equal amounts of MAGE-A3. 280-290 Peptide-loaded target cells were incubated with 3 μM CFSE (carboxyfluorescein diacetate succinimidyl ester) (CFSE high ), and peptide-free control cells were labeled with 0.3 μM CFSE (CFSE low ) and then injected into vaccinated mice in equal amounts. One day later, CFSE was detected in mouse splenocytes by flow cytometry. high :CFSE low The ratio was calculated to measure the lysis of target cells (Figure 3b). high A lower percentage of cells indicates a stronger antigen-specific cytotoxic T cell immune response.

[0078] As a result, as shown in Figure 3c, only with Adk35GM adenovirus-mediated cancer antigen delivery was the MAGE-A3-specific cytotoxic T cell response able to kill more than 95% of target cells, thus confirming the cancer antigen-specific cytotoxic T cell immune response. Based on the results of the above experimental examples <1-1> and <1-2>, it can be inferred that administration of a peripheral blood mononuclear cell vaccine loaded with α-GC and delivered as an antigen by an adenovirus vector induces both an innate immune response, which is the activation of natural killer T cells and natural killer cells, and an adaptive immune response, which is the immune response of cytotoxic T cells, thereby exhibiting a powerful anti-cancer therapeutic effect.

[0079] <1-3>Evaluation of the anti-cancer effect of peripheral blood mononuclear cell vaccine <1-3-1> Therapeutic effect of peripheral blood mononuclear cell vaccine against solid cancers The present inventors investigated whether administration of a peripheral blood mononuclear cell vaccine induces anti-cancer immunity against solid tumors. For this purpose, 1 × 10 6 C57BL / 6 mice were subcutaneously implanted with 2 × 10 B16F10 / GP100 / MAGE-A3 cancer cells expressing GP100 and MAGE-A3 into the flanks of the mice. Seven days later, the mice were cultured in a 2 × 10 6 Mice were immunized with PBMC / α-GC, PBMC / Adk35GM, or PBMC / α-GC / Adk35GM (Figure 4a). The changes in tumor size and survival rates of the mice were then measured. Tumor size was calculated by measuring the width, length, and height of the tumor using a caliper and using the following formula: (Tumor size) = (width) x (length) x (height) x (π / 6)

[0080] As shown in Figure 4b and Figure 4c, administration of the peripheral blood mononuclear cell vaccine (PBMC / α-GC / Adk35GM) suppressed tumor growth and resulted in a high survival rate, whereas administration of PBMC / α-GC showed little inhibition of tumor growth and resulted in a low survival rate. The above results confirmed that activating natural killer T cells and natural killer cells loaded with α-GC alone could not induce anti-cancer therapeutic effects.

[0081] <1-3-2> Therapeutic effect of peripheral blood mononuclear cell vaccine on lung metastatic cancer The present inventors investigated whether administration of a peripheral blood mononuclear cell vaccine induces anti-cancer immunity against lung metastatic cancer. For this purpose, C57BL / 6 mice were injected with 3 × 10 5 B16F10 / GP100 / MAGE-A3 cancer cells were intravenously injected, and 3 days later, 1.5 × 10 6 PBMC / α-GC, PBMC / Adk35GM, or PBMC / α-GC / Adk35GM were administered. 16 days after intravenous injection of cancer cells, the extent of lung metastasis of cancer cells was confirmed (Figure 5a).

[0082] As a result, as shown in Figure 5b and Figure 5c, almost no cancer tissue was observed in the lungs of mice administered PBMC / Adk35GM and PBMC / α-GC / Adk35GM, the two cell populations into which cancer antigens were introduced via adenovirus.

[0083] Next, the present inventors confirmed the effect of administration of peripheral blood mononuclear cell vaccine on the survival rate of mice bearing metastatic cancer. For this purpose, C57BL / 6 mice were injected with 3 × 10 5 B16F10 / GP100 / MAGE-A3 cancer cells were intravenously injected, and 3 days later, 2 × 10 6 Mice were administered PBMC / α-GC, PBMC / Adk35GM, or PBMC / α-GC / Adk35GM, and the survival rate of the mice was then tracked (Figure 6a).

[0084] As a result, as shown in Figure 6b, similar to the results in Figure 5b, the survival rates of the mice administered with PBMC / Adk35GM and the mice administered with cancer antigen-transduced PBMC / α-GC / Adk35GM were significantly improved, and all mice survived for more than 40 days. Combining the results of Experimental Examples <1-2> and <1-3>, it can be seen that delivery of cancer antigens to peripheral blood mononuclear cells via Adk35GM is sufficient to induce a strong cytotoxic T cell response, resulting in a strong anti-cancer therapeutic effect.

[0085] Experimental Example 2: Confirmation of the efficacy of antigen peptide-loaded peripheral blood mononuclear cell vaccine <2-1> Confirmation of induction of cytotoxic T cell responses The present inventors investigated whether the peripheral blood mononuclear cell vaccine loaded with the antigen peptide of Example <1-2> can induce a cytotoxic T cell response. C57BL / 6 mice were treated with α-GC-loaded peripheral blood mononuclear cells (PBMC / α-GC), HER2 63-71 Peptide-loaded peripheral blood mononuclear cells (PBMC / HER2 pep), α-GC and HER2 63-71 Immunization was performed by intravenous administration of peripheral blood mononuclear cells (PBMC / α-GC / HER2 pep) co-loaded with the peptide, and an in vivo cytotoxicity assay was performed 7 days later (FIG. 7a).

[0086] As a result, as shown in Figures 7b and 7c, HER2 was detected only in the mice administered with PBMC / α-GC / HER2 pep. 63-71 Peptide-loaded CFSE high The target cells were largely lysed, confirming that cytotoxic T cells could also be activated by the α-GC and peptide-loaded peripheral blood mononuclear cell vaccine.

[0087] However, unlike the group receiving peripheral blood mononuclear cells to which only the antigen was delivered via an adenovirus vector without α-GC in Experimental Example <1-2>, which induced a cytotoxic T cell response (Fig. 3c), no cytotoxic T cell response was observed in the group receiving PBMC / HER2 pep loaded with only the peptide (Fig. 7c). This suggests that delivery of the entire antigen via adenovirus and induction of diverse immune responses specific to multiple epitopes is superior to presentation of a single peptide in inducing a cytotoxic T cell immune response.

[0088] <2-2>Evaluation of the anti-cancer effect of peripheral blood mononuclear cell vaccine The present inventors investigated whether a peripheral blood mononuclear cell vaccine loaded with an antigen peptide can induce anti-cancer therapeutic effects. For this purpose, 2 × 10 5 Three days after subcutaneous implantation of 2 × 10 CT26-HER2 cancer cells, 6 Mice were immunized with PBMC / α-GC, PBMC / HER2 pep, or PBMC / α-GC / HER2 pep (FIG. 8a).

[0089] As a result, as shown in Figure 8b, administration of the peripheral blood mononuclear cell vaccine (PBMC / α-GC / HER2 pep) significantly suppressed tumor growth. In contrast, tumor growth was barely inhibited in mice administered PBMC / α-GC or PBMC / HER2 pep. Therefore, it was revealed that peptide loading alone cannot induce sufficient anticancer therapeutic effects.

[0090] Combining the above results with the anti-cancer effect data of Examples 1-3 (Figures 4 to 6), it was confirmed that the peripheral blood mononuclear cell vaccine prepared by loading α-GC and delivering an antigen exhibited a stronger anti-cancer effect than a cell vaccine loaded with α-GC alone or a cell vaccine delivering only an antigen, inhibiting cancer growth and improving survival rates.

[0091] Experimental Example 3: Superior anti-cancer effect of peripheral blood mononuclear cell vaccine compared to other immune cell component vaccines <3-1> Comparison of immune response induction between peripheral blood mononuclear cell vaccine and B cell / monocyte cell vaccine The ability of peripheral blood mononuclear cell vaccines to induce cytotoxic T cell immune responses was compared with that of conventional anticancer cellular vaccines composed of B cells / monocytes. To this end, we produced vaccines by loading α-GC into B cells / monocytes and peripheral blood mononuclear cells obtained from the spleens of C57BL / 6 mice or by delivering the antigen via Adk35GM, and immunized C57BL / 6 mice with target cells (GP100). Seven days later, we injected α-GC into the target cells (GP100). 25-33 Peptide-loaded CFSE high Labeled target cells: Vehicle-treated CFSE low The immunized mice were administered 1:1 (labeled control cells) and an in vivo cytotoxicity assay was performed (Figure 9a). One day later, flow cytometry was used to detect CFSE in mouse splenocytes.high :CFSE low By calculating the ratio of GP100 25-33 Peptide-loaded CFSE high Lysis of target cells was measured (Fig. 9b).

[0092] As a result, as shown in Figure 9c, in both the group administered the immune cell vaccine composed of B cells / monocytes (Bmo / α-GC / Adk35GM) and the group administered the peripheral blood mononuclear cell vaccine (PBMC / α-GC / Adk35GM), more than 80% of target cells were killed by the response of cytotoxic T cells specific to the cancer antigen GP100, and slightly higher cytotoxicity was confirmed in the group administered the peripheral blood mononuclear cell vaccine.

[0093] <3-2> Confirmation of immune response induction ability of cellular components of PBMC vaccine Next, in vivo cytotoxicity assays were performed to determine whether each type of immune cell, including the peripheral blood mononuclear cell vaccine, induced antigen-specific cytotoxic T cell immune responses. Specifically, α-GC was loaded onto T cells, B cells / monocytes, and peripheral blood mononuclear cells obtained from the spleens of C57BL / 6 mice, and the Adk35GM antigen was delivered to produce an immune cell vaccine. C57BL / 6 mice were then immunized with the vaccine, and 7 days after immunization, a cytotoxicity test was performed using the same method as in Example 3-1 (Figure 10a).

[0094] As a result, as shown in Figures 10b and 10c, the cytotoxic responses were highest in the group administered with the peripheral blood mononuclear cell vaccine (PBMC / α-GC / Adk35GM), the group administered with the B cell / monocyte-based immune cell vaccine (Bmo / α-GC / Adk35GM), and the group administered with the T cell-based immune cell vaccine (T / α-GC / Adk35GM), in that order.

[0095] These results suggest that the immune response of tumor antigen GP100-specific cytotoxic T cells is induced not only by B cells / monocytes, which account for approximately 40% of peripheral blood mononuclear cells, but also by T cells, which account for half of those cells. Combined with the results of Experimental Examples <3-1> and <3-2>, it can be seen that a strong cancer antigen-specific cytotoxic T cell immune response can be induced by T cells, B cells, and monocytes, which account for 90% of peripheral blood mononuclear cell vaccines loaded with α-GC and delivered together with antigens by adenovirus vectors.

[0096] <3-3> Comparison of anticancer drug therapeutic effects between peripheral blood mononuclear cell vaccine and B cell / monocyte cell vaccine Finally, we compared the anticancer therapeutic effects of peripheral blood mononuclear cell vaccine and B cell / monocyte vaccine. For this purpose, 3 x 10 5 B16F10 / GP100 / MAGE-A3 cancer cells were subcutaneously implanted into the flanks of C57BL / 6 mice. After 7 days, the mice received 1.5 × 10 6 Mice were immunized with peripheral blood mononuclear cells (PBMC / α-GC, PBMC / α-GC / Adk35GM) or B cells / monocytes (Bmo / α-GC, Bmo / α-GC / Adk35GM) and the changes in tumor size were measured (Figure 11a).

[0097] As shown in Figure 11b, administration of the peripheral blood mononuclear cell vaccine (PBMC / α-GC / Adk35GM) and the B cell / monocyte vaccine (Bmo / α-GC / Adk35GM) inhibited tumor growth. However, the solid tumors in the mice treated with Bmo / α-GC / Adk35GM subsequently regrew, whereas the tumors in the mice treated with PBMC / α-GC / Adk35GM remained suppressed for more than 4 weeks, demonstrating a strong anticancer therapeutic effect.

[0098] In summary, in Experiments <3-1> and <3-2>, there was no difference in the level of cytotoxic T cell induction between the peripheral blood mononuclear cell vaccine and the B cell / monocyte vaccine, but in Experiment <3-3>, it was confirmed that the peripheral blood mononuclear cell vaccine had a much stronger anti-cancer therapeutic effect than the B cell / monocyte vaccine. This suggests that various immune cell populations within peripheral blood mononuclear cells interact complementarily to induce an enhanced anti-cancer treatment response.

[0099] Industrial Applicability The vaccine of the present invention comprising peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen is easy to obtain because it does not require the isolation of specific cells from peripheral blood mononuclear cells, and immunization with peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen not only induces significant levels of natural killer cell and natural killer T cell activation and cytotoxic T lymphocyte responses, but also exhibits synergistic therapeutic effects against malignant tumors, and therefore can be applied not only to cancer prevention but also to cancer treatment through immunotherapy.

Claims

1. Immunotherapeutic and prophylactic vaccines comprising peripheral blood mononuclear cells loaded with a natural killer T cell ligand and an antigen.

2. The immunotherapeutic and prophylactic vaccine of claim 1, wherein the peripheral blood mononuclear cells are obtained by removing red blood cells from peripheral blood.

3. 2. The immunotherapeutic and prophylactic vaccine of claim 1, wherein the peripheral blood mononuclear cells are composed of lymphocytes, consisting of T cells, B cells and natural killer cells, and monocytes.

4. 2. The immunotherapeutic and prophylactic vaccine of claim 1, wherein the natural killer T cell ligand is selected from the group consisting of α-galactosylceramide, α-glucuronosylceramide, phosphatidylinositol tetramannoside, isoglobotrihexosylceramide, ganglioside GD3, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, sulfitide, β-galactosylceramide, lipophosphoglycan, glycoinositol phospholipid, β-anomeric galactosylceramide and α-anomeric galactosylceramide which are analogs of α-galactosylceramide, bacterial lipid antigens, and mutants of α-galactosylceramide.

5. 2. The immunotherapeutic and prophylactic vaccine of claim 1, wherein the antigen is derived from a pathogen, including pathogenic bacteria, viruses, and parasites, or is a cancer antigen.

6. 6. The immunotherapeutic and prophylactic vaccine of claim 5, wherein the antigen from a pathogenic bacterium is selected from the group consisting of Bordetella pertussis antigens (pertussis toxin, filamentous hemagglutinin, and pertactin), tetanus toxoid, diphtheria toxoid, Helicobacter pylori antigens (capsular polysaccharides of serogroups A, B, C, Y, and W-135), Streptococcus pneumoniae antigens (pneumococcal 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.

7. 6. The immunotherapeutic and prophylactic vaccine of claim 5, wherein the virus-derived antigen is selected from the group consisting of influenza virus antigens (hemagglutinin and neuraminidase), human papillomavirus (HPV) antigens (glycoproteins), vesicular stomatitis virus antigens (vesicular stomatitis virus glycoproteins), cytomegalovirus (CMV) antigens, hepatitis virus antigens (hepatitis A (HAV), hepatitis B (HBV), hepatitis C (HCV), hepatitis D (HDV), and hepatitis G (HGV) antigens) (core and surface antigens), 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.

8. 6. The immunotherapeutic and prophylactic vaccine of claim 5, 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), muringlobulin 1 (MUC-1), carcinoembryonic antigen (CEA), p53, alpha-fetoprotein, breast cancer protein expressed by Her-2 / neu, proteinase 3, WT-1, PAP, PSA, PSMA, G250, BAGE, GAGE, NY-ESO-1, MART-1, MCIR, Ig idiotype, CDK4, caspase-8, beta-catenin, CIA, BCR / ABL, EBV LMP2a, HCV, HHV-8, 5T4, and neoplasms derived from tumor-specific mutations.

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

10. 9. The immunotherapeutic and prophylactic vaccine of 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, biliary tract 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 prophylactic vaccine of claim 1, wherein the antigen is introduced and expressed by a recombinant virus.

12. 12. The immunotherapeutic and prophylactic vaccine of claim 11, wherein the recombinant virus is an adenovirus, a retrovirus, a vaccinia virus, a poxvirus, or a Sindbis virus carrying a transgene that expresses an antigen.

13. Steps below: (a) obtaining peripheral blood mononuclear cells; (b) removing red blood cells; and (c) delivering a natural killer T cell ligand and an antigen to peripheral blood mononuclear cells; 10. A method for producing an anti-cancer immunotherapeutic and prophylactic vaccine, comprising:

14. 14. An anti-cancer immunotherapeutic and prophylactic vaccine prepared by the method of claim 13.

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

16. Use of peripheral blood mononuclear cells loaded with a natural killer T cell ligand and a cancer antigen to prevent or treat cancer.

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

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