Nanoparticle comprising cell-penetrating peptides conjugated with deoxycholic acid and use thereof

Abstract

An embodiment of the present invention relates to: a vaccine nanoparticle comprising an assembly of cell-penetrating peptides (CPPs) conjugated with deoxycholic acid (DOCA); a method for preparing same; and a vaccine composition for preventing or treating cancer, comprising same. The effects of migration, maturation, and antigen presentation through MHC I in dendritic cells, as well as antigen-specific T cell immune responses through the nanoparticle of the present invention were evaluated in vitro and in vivo. An improved effect was also confirmed through repolarization of tumor-associated macrophages. Additionally, the effect of combination therapy with an immune checkpoint inhibitor was confirmed in a tumor-bearing animal model. The nanoparticle of the present invention induces antigen-specific immunity and macrophage repolarization, thereby improving anti-tumor immunotherapy, and thus can be applied as an mRNA cancer vaccine.

Description

Nanoparticles comprising cell penetrating peptides linked to deoxycholic acid and uses thereof

[0001] An example of the present invention relates to a vaccine nanoparticle comprising a cell penetrating peptide (CPP) assembly linked to deoxycholic acid (DOCA); a method for producing the same; and a vaccine composition for preventing or treating cancer comprising the same.

[0002] mRNA-based therapies are considered a powerful tool not only as vaccines but also as treatments for various diseases. Research on mRNA vaccines has surged, particularly since the recent COVID-19 pandemic. mRNA vaccines have begun to be applied as cancer vaccines because of their ability to encode various tumor antigens, their high biosafety, and their rapid production compared to conventional vaccines such as peptide and DNA vaccines. However, free mRNA has difficulty penetrating cells due to its large size and negative charge, leading to degradation and limited transduction efficiency. The instability of free mRNA (or naked mRNA) reduces tumor antigen peptide translation within cells, necessitating the development of an appropriate carrier for effective mRNA delivery. Lipid nanoparticles (LNPs) are currently the most well-known mRNA vaccine delivery system in clinical trials, but they have been associated with adverse effects such as excessive inflammation and systemic off-target effects. Therefore, the development of alternative platforms for safe and effective mRNA delivery is necessary.

[0003] The primary goal of therapeutic cancer vaccines is to deliver antigens to dendritic cells, activating them and thereby inducing an antigen-specific T cell immune response. However, adjuvants are required to enhance vaccine efficacy. Adjuvants that can overcome immunosuppressive tumor-associated macrophages (TAMs) within the tumor microenvironment (TME) interfere with vaccine-induced immune responses. Furthermore, precise delivery of adjuvants to dendritic cells and M2 macrophages is crucial for facilitating drug delivery and minimizing systemic toxicity associated with free drug administration.

[0004] Accordingly, the present invention aims to provide a nanoparticle for a vaccine, comprising:

[0005] (i) Deoxycholic acid (DOCA)-linked cell penetrating peptide (CPP) assembly;

[0006] (ii) a mannose-linked lipid coating the assembly;

[0007] (iii) adjuvant; and

[0008] (iv) mRNA.

[0009]

[0010] In addition, the present invention

[0011] A step of linking a cell penetrating peptide to deoxycholic acid;

[0012] A step of assembling a cell penetrating peptide linked to the above deoxycholic acid;

[0013] A step of coating the above assembly with a mannose-linked lipid; and

[0014] The purpose of the present invention is to provide a method for manufacturing a nanoparticle for a vaccine, comprising a step of loading an adjuvant and mRNA inside an assembly coated with the above lipid.

[0015]

[0016] In addition, the present invention aims to provide a vaccine composition for preventing or treating cancer, comprising the nanoparticles.

[0017] In addition, the present invention aims to provide a method for preventing or treating cancer using the vaccine composition.

[0018] In addition, the present invention aims to provide a use of the vaccine composition for preventing or treating cancer.

[0019] To achieve the above purpose,

[0020] The present invention provides a nanoparticle for a vaccine, comprising:

[0021] (i) Deoxycholic acid (DOCA)-linked cell penetrating peptide (CPP) assembly;

[0022] (ii) a mannose-linked lipid coating the assembly;

[0023] (iii) adjuvant; and

[0024] (iv) mRNA.

[0025] In one embodiment of the present invention, the peptide may be composed of an amino acid sequence of SEQ ID NO: 1.

[0026] In another embodiment of the present invention, the adjuvant may be at least one selected from the group consisting of SD-208, Vactosertib, Galunisertib, LY3200882, Resiquimod, Imiquimod, Gardiqiomod, Motolimod, Alum (Aluminium salts), CpG ODNs, GM-CSF, IL-12, poly(I:C), MPL, AS01, IC31, and CFA01.

[0027] In another embodiment of the present invention, the lipid is selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), Dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), DSPE-PEG, monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0 PA), or It may be at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (18:1 PA).

[0028] In another embodiment of the present invention, the mRNA may encode at least one antigenic peptide or protein comprising a pathogenic antigen, a tumor antigen, an allergen, or an autoimmune autoantigen.

[0029] In another embodiment of the present invention, the mRNA may be composed of the base sequence of SEQ ID NO: 2.

[0030] In another embodiment of the present invention, the nanoparticle may increase the expression of any one or more selected from the group consisting of CD80, CD86, CD40, CCR7, IL-12, TNF-α, and IL-6 of dendritic cells.

[0031] In another embodiment of the present invention, the nanoparticles may induce repolarization of M2 macrophages into M1 macrophages.

[0032] In another embodiment of the present invention, the nanoparticles may reduce the expression of Fizz1 or IL-10 in macrophages.

[0033] In another embodiment of the present invention, the nanoparticles may increase tumor-infiltrating lymphocytes in a tumor.

[0034]

[0035] In addition, the present invention comprises a step of linking a cell penetrating peptide with deoxycholic acid;

[0036] A step of assembling a cell penetrating peptide linked to the above deoxycholic acid;

[0037] A step of coating the above assembly with a mannose-linked lipid; and

[0038] A method for producing a nanoparticle for a vaccine is provided, comprising a step of loading an adjuvant and mRNA inside an assembly coated with the lipid.

[0039] In one embodiment of the present invention, the peptide may be composed of an amino acid sequence of SEQ ID NO: 1.

[0040] In another embodiment of the present invention, the adjuvant may be at least one selected from the group consisting of SD-208, Vactosertib, Galunisertib, LY3200882, Resiquimod, Imiquimod, Gardiqiomod, Motolimod, Alum (Aluminium salts), CpG ODNs, GM-CSF, IL-12, poly(I:C), MPL, AS01, IC31, and CFA01.

[0041] In another embodiment of the present invention, the lipid is selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), Dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), DSPE-PEG, monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0 PA), or It may be at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (18:1 PA).

[0042] In another embodiment of the present invention, the mRNA may encode at least one antigenic peptide or protein comprising a pathogenic antigen, a tumor antigen, an allergen, or an autoimmune autoantigen.

[0043] In another embodiment of the present invention, the mRNA may be composed of the base sequence of SEQ ID NO: 2.

[0044]

[0045] In addition, the present invention provides a vaccine composition for preventing or treating cancer, comprising nanoparticles.

[0046] In one embodiment of the present invention, the cancer may be at least one selected from the group consisting of bladder cancer, brain cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer (gastric cancer), head and neck cancer, testicular cancer, melanoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma, and uterine cancer.

[0047] In another embodiment of the present invention, the vaccine composition may be administered in combination with an immune checkpoint inhibitor.

[0048] In another embodiment of the present invention, the immune checkpoint inhibitor may be at least one selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody.

[0049] In addition, the present invention provides a method for preventing or treating cancer using the vaccine composition.

[0050] In addition, the present invention provides a use of the vaccine composition for preventing or treating cancer.

[0051] The effects of the nanoparticles of the present invention on dendritic cell migration, maturation, and antigen presentation via MHC I, as well as on antigen-specific T cell immune responses, were evaluated in vitro and in vivo. Furthermore, enhanced efficacy was confirmed through repolarization of tumor-associated macrophages. Furthermore, the efficacy of combination therapy with immune checkpoint inhibitors was also confirmed in tumor-bearing animal models. The nanoparticles of the present invention can be applied as mRNA cancer vaccines because they enhance anti-tumor immunotherapy by inducing antigen-specific immunity and macrophage reprogramming.

[0052] It should be understood that the effects of the present invention are not limited to the effects mentioned above, but include all effects that can be inferred from the detailed description of the present invention or the composition of the invention described in the claims.

[0053] Figures 1a and 1b are schematic diagrams of mRNA delivery using adjuvant-loaded mannosylated lipid-coated DOCA-9R nanoparticles (A-mL-DRO). Figure 1a shows the structure of deoxycholic acid (DOCA)-9R (DOCA-KGGRRRRRRRRR) and the preparation method of A-mL-DRO. Figure 1b shows that A-mL-DRO targets dendritic cells and M2 macrophages via the mannose receptor, inducing an anti-tumor immune response.

[0054]

[0055] Figures 2a to 2j show the optimization and characterization results of A-mL-DRO. Figure 2a shows that the optimal lipid-to-DR ratio for the mL-DRO formulation was determined by dynamic light scattering (DLS). Figure 2b shows that the optimal amount of R848 in mL-DR for the A-mL-DR formulation was determined by DLS. Figure 2c shows the results of evaluating the drug loading and encapsulation efficiency using UV / Vis spectrophotometry. Figure 2d shows a gel retardation assay to detect the condensation of OVA mRNA into A-mL-DR at various ratios. Figure 2e shows the Z-average diameter and zeta potential of A-mL-DRO at various weight ratios measured by DLS. Figure 2f shows the results of a gel retardation assay after performing an mRNA degradation protection assay in 10% rat serum at 37°C for 30 minutes. Figure 2g shows an in vitro luciferase assay in BMDCs to determine the final A-mL-DR / mRNA ratio. Figure 2h shows TEM images of DRO, mL-DRO, and A-mL-DRO. Figure 2i shows the cumulative drug release profile measured over 72 hours using a UV / Vis spectrophotometer. Figure 2j shows the results of measuring cell viability using a CCK-8 assay after culturing BMDCs with various concentrations of A-mL-DRO.

[0056]

[0057] Figures 3a to 3c show the extent of in vitro cellular uptake of mL-DRO in BMDCs. Figure 3a shows the drug encapsulation efficiency of R848 according to lipid composition (DLPC, DSPE-PEG2000, DSPE-PEG2000-Mannose). Figure 3b shows the flow cytometry histograms and the relative mean fluorescence intensity (MFI) of Cy5.5 in cultured BMDCs treated with Cy55-loaded DRO, L-DRO, and mL-DRO for 2 h. Figure 3c shows confocal microscopy images of the cellular uptake of DRO, L-DRO, and mL-DRO (Cy5.5, red) in BMDCs stained with anti-CD11c antibody (green).

[0058]

[0059] Figures 4a and 4b show in vitro infection of BMDCs with A-mL-DR. Figure 4a shows CD11c infection efficiency of free EGFP mRNA and EGFP mRNA complexes A-DR, AL-DR, and A-mL-DR in BMDCs after 24 h of treatment. + EGFP + Flow cytometry analysis and statistical analysis results of the cells are shown. Figure 4b shows fluorescence microscopy images of BMDCs captured 24 hours after infection with free EGFP mRNA and A-DR, AL-DR, and A-mL-DR (EGFP: green) bound to EGFP mRNA.

[0060]

[0061] Figures 5a to 5f show the results of in vitro dendritic cell maturation of BMDCs. Figure 5a shows CD11c + CD80 + , Figure 5b shows CD11c + CD86 +Flow cytometry plots and ratios of BMDCs are shown. Figure 5c shows confocal microscopy images after CD80 (red) staining to assess dendritic cell maturation and CD11c (green) staining to image the morphology of BMDCs. Figure 5d shows Western blot images of CD80 and CD86 expression in BMDCs. Figure 5e shows CD11c + After gating the cells, the expression levels of CD40, MHCⅠ, MHCⅡ, and CCR7 in BMDCs were analyzed using flow cytometry. Figure 5f shows the results of measuring the levels of proinflammatory cytokines (IL-12p70, TNF-α, and IL-6) secreted from BMDCs using ELISA.

[0062]

[0063] Figures 6a and 6b show the in vitro cellular uptake of mL-DRO in M2 macrophages. Figure 6a shows flow cytometry histograms and relative MFI values ​​of Cy5.5 in M2 macrophages 4 h after treatment. Figure 6b shows confocal microscopy images of M2 macrophages captured 24 h after treatment with DRO, L-DRO, and mL-DRO (Cy5.5, red) and stained with anti-CD206 antibody (green) for morphological observation.

[0064]

[0065] Figures 7a to 7d show the effect of A-mL-DRO on the repolarization of M2 macrophages to the M1 phenotype in vitro. Figure 7a shows CD11c + CD206 + (M2 macrophage marker) cells, Figure 7b shows CD11c + CD80 +(M1 macrophage marker) Flow cytometry plots and ratios for cells are shown. Figures 7c and 7d show qRT-PCR analysis. Figure 7c shows the mRNA expression levels of anti-inflammatory M2 macrophage markers (FiZZ1 and IL-10), and Figure 7d shows the mRNA expression levels of pro-inflammatory M1 macrophage markers (IL-6 and TNF-α), normalized to GAPDH.

[0066]

[0067] Figures 8a to 8e show the biodistribution of mL-DRO in a B16-OVA melanoma mouse model. Figure 8a is a schematic diagram of the experimental schedule for screening biodistribution in a melanoma mouse model. Figure 8b shows in vivo biodistribution images of subcutaneously injected Cy5.5-loaded DRO, L-DRO, and mL-DRO (OVA mRNA: 0.5 mg / kg). Figure 8c shows the average fluorescence intensity per tissue area of ​​major organs, lymph nodes, and tumors 4 and 24 hours after injection. Figure 8d shows the results of immunofluorescence analysis after 24 hours of administration of Cy5.5-loaded DRO, L-DRO, and mL-DRO to lymph nodes, and Figure 8e shows the results of immunofluorescence analysis after 24 hours of administration of Cy5.5-loaded DRO, L-DRO, and mL-DRO to tumors.

[0068]

[0069] Figures 9a to 9c show in vivo infection in a B16-OVA melanoma mouse model. Figure 9a is a schematic diagram of the experimental schedule for in vivo infection studies using a luciferase assay. Figure 9b shows representative images of bioluminescence distribution in B16-OVA melanoma mice treated with A-mL-DR / Fluc mRNA for 6 and 24 hours. Figure 9c shows the average luciferase intensity in regions of interest (ROIs) across the entire body and in the tumor area.

[0070]

[0071] Figures 10a to 10d illustrate ex vivo antigen expression and antigen-specific killing assays. Figure 10a is a schematic diagram of the experimental schedule for antigen expression and antigen-specific killing assays in dendritic cells. Figure 10b is a schematic diagram of OVA (antigen) expression by MHC class I and CD11c in dendritic cells in lymph nodes. + H-2k b / SIINFEKL + A schematic and bar chart of the flow cytometry analysis of cells are shown. Figure 10c shows the experimental protocol scheme for the ex vivo antigen-specific apoptosis assay. Figure 10d shows a flow cytometry histogram at 24 hours after co-culture.

[0072]

[0073] Figures 11a to 11h show the in vivo therapeutic and immunostimulatory effects of A-mL-DRO in a B16-OVA melanoma mouse model. Figure 11a is a schematic diagram of the experimental schedule for evaluating the therapeutic effect of A-mL-DRO on B16-OVA tumors. Figure 11b shows the average tumor growth curve measured 22 days after tumor inoculation. Figure 11c shows the tumor weight, and Figure 11d shows a representative image of the tumor harvested on day 22. Figure 11e shows the results of ELISA measurements of serum IgG levels in vaccinated mice. Figure 11f shows representative plots and bar graphs of CD80+ CD86+ dendritic cells in lymph nodes analyzed by flow cytometry. Figure 11g shows the results of ELISA measurements of proinflammatory cytokine secretion in lymph nodes. Figure 11h shows immunofluorescence staining of the lymph node, showing CD11c+ (green, dendritic cell marker) cells and CD80+ (red, dendritic cell maturation marker) cells.

[0074]

[0075] Figures 12a to 12f show the tumor immunomodulatory effect of A-mL-DRO in a B16-OVA melanoma mouse model. Figure 12a shows the intratumoral CD3 +CD8 + Figure 12b shows the cytological analysis plot and population of T cells in tumors analyzed by flow cytometry. + CD4 + A bar graph showing the population of T cells. Figure 12c shows intratumoral CD8 + IFN-γ + Figure 12d shows the plot and population analysis of T cells using flow cytometry. Figure 12d shows the secretion of IFN-γ from spleen cells measured by ELISA after restimulation with OVA peptide. Figures 12e and 12f show the results of immunofluorescence staining of F4 / 80 (green, macrophage marker). Figure 12e shows the relative CD206 expression in tumors (red, M2 macrophage marker). + Fluorescence intensity, Figure 12f shows the relative CD80 of CD80 (red, M1 macrophage marker) + Shows fluorescence intensity.

[0076]

[0077] Figures 13a to 13i show the results of combination therapy with A-mL-DRO and anti-PD-1 in a B16-OVA melanoma mouse model. Figure 13a is a schematic diagram of the experimental schedule for the in vivo combination therapy study. Figure 13b shows the tumor volume measurements 22 days after tumor inoculation, and Figure 13c shows the tumor weight measurements. Figure 13d shows the results of ELISA detection of IgG and IgM levels in the serum of vaccinated mice. Figure 13e shows the results of intratumoral CD3 + CD8 + Flow cytometry plots and populations of T cells are shown. Figure 13f shows CD45 + After gating the cells, flow cytometry was used to identify intratumoral CD3 + The results of the analysis of CD4+ T cells are shown in Figure 13g. CD8 cells in tumors analyzed by flow cytometry + IFN-γ +Figure 13h shows the T cell plot and population histogram. Figure 13h shows the results of ELISA measurement of IFN-γ secretion from spleen cells after restimulation with OVA peptide. Figure 13i shows TUNEL staining and relative TUNEL intensity.

[0078]

[0079] Figures 14a to 14f show the immunopreventive effect and toxicity evaluation of A-mL-DRO in a B16-OVA melanoma model. Figure 14a is a schematic diagram of the experimental schedule for evaluating the immunopreventive effect and toxicity of A-mL-DRO against B16-OVA tumors in mice. Figure 14b shows the growth curve of B16-OVA tumors in mice. Figure 14c shows the survival rate of B16-OVA tumor-bearing mice in each group. Figure 14d shows the body weights of each group measured 24 days after vaccination. Figure 14e shows histological images of major organs of each group after H&E staining. Figure 14f shows the results of measuring the levels of serum biochemical markers (ALT, AST, BUN, and creatinine) using an automatic chemical analyzer.

[0080]

[0081] Hereinafter, the present invention will be described in detail.

[0082] We developed a novel mRNA nanoparticle (A-mL-DRO) comprising a 9-mer arginine peptide assembly (DOCA-9R) conjugated to deoxycholic acid (DAC) and coated with mannosylated lipids, loaded with mRNA encoding ovalbumin (OVA), a tumor model antigen, and an adjuvant. The nanoparticles were designed to promote antigen-specific immune responses via dendritic cells and, when combined with TAM reprogramming, to achieve enhanced anti-tumor immunotherapy (Fig. 1).

[0083]

[0084] Accordingly, the present invention provides a nanoparticle for a vaccine, comprising:

[0085] (i) Deoxycholic acid (DOCA)-linked cell penetrating peptide (CPP) assembly;

[0086] (ii) a mannose-linked lipid coating the assembly;

[0087] (iii) adjuvant; and

[0088] (iv) mRNA.

[0089] As used herein, the terms "bile acid" or "deoxycholic acid (DOCA)" refer to secondary bile acids produced by the enzymatic oxidation of cholesterol. These bile acids increase cell membrane fluidity and enhance cell permeability. Furthermore, DOCA possesses high hydrophobicity and can be developed as a gene delivery carrier by binding to positively charged amino acids. The immunomodulatory role of bile acids suggests their potential as vaccines.

[0090] The term "cell penetrating peptide (CPP)" used herein was originally used for the delivery of nucleic acids such as pDNA and siRNA. Positively charged CPPs form complexes with negatively charged nucleic acids through electrical interaction, facilitating cell membrane penetration. Furthermore, CPPs disrupt endocrine vesicle membranes, inducing endocrine vesicle escape. This process, a crucial step in mRNA delivery and closely linked to mRNA translation, has led to the emergence of CPPs as promising tools for mRNA delivery.

[0091] The peptide used herein may include not only the peptide but also derivatives thereof. For example, the peptide of the present invention may exhibit at least 80% homology with the peptide of each corresponding sequence number, preferably at least 90%, more preferably at least 95% homology, and the derivative may include a peptide in which the N-terminus, C-terminus, etc. of the peptide is chemically modified or an amino acid is added, substituted, or deleted, and is not particularly limited thereto.

[0092] As used herein, the term "assembly" refers to a structure formed by self-assembly of peptides linked to deoxycholic acid. This assembly is stably formed through hydrophobic bonds and intermolecular interactions, enhancing the structural stability of the peptide and enabling efficient antigen delivery.

[0093] As used herein, the term "vaccine" means a biological preparation or combination that provides a preventive or therapeutic effect against a specific disease by inducing or enhancing an immune response in a living body.

[0094] As used herein, the term "nanoparticle" refers to a particle ranging from several nanometers to several hundred nanometers in size and designed for in vivo drug delivery, vaccine delivery, or delivery to a specific target. In the present invention, the particle comprises a bile acid-linked cell-penetrating peptide assembly and a mannose-linked lipid-coated structure, and is designed to effectively deliver mRNA and an adjuvant.

[0095] The term "mannose" used in this specification refers to a type of monosaccharide that exists as a structural unit of polysaccharides, and is a sugar component that enables specific binding and delivery to target cells through binding to mannose receptors, and in the present invention, exists in a form coated on the surface of nanoparticles.

[0096] As used herein, the term "mannose receptor" is a type I transmembrane protein abundantly expressed on antigen-presenting cells (APCs), particularly dendritic cells and M2 macrophages. Mannose can be chemically conjugated to lipids and used as a delivery system to target APCs. Furthermore, mannose receptors are pattern recognition receptors (PRRs), and binding to mannose itself stimulates the immune system, playing a crucial role in vaccines. Mannose receptor-mediated delivery helps deliver antigens and adjuvants, and activates APCs, thereby improving the effectiveness of immunotherapy.

[0097] As used herein, the term "coating" refers to the process of covering the exterior with a thin layer to protect the assembly or improve biocompatibility, and in the present invention refers to covering the nanoparticles with mannose-linked lipids.

[0098]

[0099] In one embodiment of the present invention, the peptide may be composed of an amino acid sequence of SEQ ID NO: 1, and more specifically, may be a 9 mer arginine peptide (9R).

[0100] The term "9-mer arginine peptide (9R)" as used herein is reported to be a positively charged poly-arginine peptide exhibiting unique cell-penetrating ability within CPPs. 9R is composed of D-arginine for higher intracellular efficiency and is designed with a linear structure at the N-terminus for efficient translocation across the cell membrane.

[0101]

[0102] In another embodiment of the present invention, the adjuvant may be at least one selected from the group consisting of SD-208, Vactosertib, Galunisertib, LY3200882, Resiquimod, Imiquimod, Gardiqiomod, Motolimod, Alum (Aluminium salts), CpG ODNs, GM-CSF, IL-12, poly(I:C), MPL, AS01, IC31, and CFA01.

[0103] The term "adjuvant" as used herein refers to a substance that induces an anticancer effect by increasing a non-specific immune response to an antigen. The type of adjuvant is not limited, but examples thereof include inhibitors of TLR7, TLR8, and TGF-β (transforming growth factor-β receptor 1), preferably R848.

[0104] As used herein, the term "resiquimod (R848)" is an FDA-approved TLR 7 / 8 antagonist frequently used as an adjuvant in cancer vaccines. It activates dendritic cells and induces cytokine secretion, promoting a more potent immune response. Furthermore, R848 is a well-known drug for reprogramming TAMs from the anti-inflammatory M2 to the inflammatory M1 phenotype. Therefore, using R848 as an adjuvant in vaccines and simultaneously utilizing it for TAM reprogramming could be a strategy for enhancing the efficacy of cancer immunotherapy.

[0105] Toll-like receptors (TLRs) are pattern recognition receptors that detect pathogens or cellular abnormalities and regulate immune responses. These TLRs play a crucial role in the innate immune system and are known to regulate antigen expression and presentation in immune cells such as dendritic cells (DCs).

[0106] The above R848 can enhance dendritic cell maturation and activation, antigen presentation, and T cell activation by activating TLR7 and TLR8. Accordingly, the above R848 can be used as an adjuvant to enhance the immune response.

[0107] The TLR7 / 8 agonist such as R848 is not limited in type, but may be at least one selected from the group consisting of synthetic compounds, natural products, peptides, antibodies, aptamers, siRNA, shRNA, miRNA, ribozymes, DNAzymes, PNA (peptide nucleic acids), antisense oligonucleotides, and other TLR7 / 8 activators.

[0108] The above TLR7 / 8 agonist enhances the antigen expression and immune regulatory function of DCs by activating the signal transduction pathways of TLR7 and TLR8, which can be usefully utilized in antiviral and anticancer immune responses.

[0109] The above TLR7 / 8 agonist may be at least one selected from the group consisting of R848 (Resiquimod), Imiquimod, CL097, Gardiquimod, and other toll-like receptor 7 / 8 activators. More specifically, it may be R848 (Resiquimod).

[0110] In another embodiment of the present invention, the lipid is selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), Dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), DSPE-PEG, monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0 PA), or It may be at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (18:1 PA).

[0111] In another embodiment of the present invention, the mRNA may encode at least one antigenic peptide or protein comprising a pathogenic antigen, a tumor antigen, an allergen, or an autoimmune autoantigen.

[0112] As used herein, the term "mRNA" refers to messenger ribonucleic acid (messenger RNA) containing genetic information that can be translated within a cell to induce the production of a specific protein, and in the present invention, is loaded onto nanoparticles and acts as an active ingredient of a vaccine.

[0113] In another embodiment of the present invention, the mRNA may be composed of the base sequence of SEQ ID NO: 2.

[0114] In another embodiment of the present invention, the nanoparticle may increase the expression of any one or more selected from the group consisting of CD80, CD86, CD40, CCR7, IL-12, TNF-α, and IL-6 of dendritic cells.

[0115] In another embodiment of the present invention, the nanoparticles may induce repolarization of M2 macrophages into M1 macrophages. Repolarization into M1 macrophages is known to promote anti-tumor immune responses in the tumor microenvironment and have a tumor growth inhibitory effect.

[0116] In another embodiment of the present invention, the nanoparticle may reduce the expression of Fizz1 or IL-10 in macrophages. It is known that reducing the expression of Fizz1 or IL-10 in macrophages promotes the transition from the M2 to the M1 phenotype. Fizz1 and IL-10 are characteristic markers of M2 macrophages, and their reduced expression can attenuate the characteristics of M2 macrophages and induce a transition to the M1 phenotype.

[0117] In another embodiment of the present invention, the nanoparticles may increase intratumoral tumor-infiltrating lymphocytes. It is known that increasing intratumoral tumor-infiltrating lymphocytes enhances the intratumoral activity of immune cells, resulting in tumor removal and growth inhibition.

[0118]

[0119] In addition, the present invention comprises a step of linking a cell penetrating peptide with deoxycholic acid;

[0120] A step of assembling a cell penetrating peptide linked to the above deoxycholic acid;

[0121] A step of coating the above assembly with a mannose-linked lipid; and

[0122] A method for producing a nanoparticle for a vaccine is provided, comprising a step of loading an adjuvant and mRNA inside an assembly coated with the lipid.

[0123] The terms peptide, deoxycholic acid, assembly, lipid, nanoparticle, etc. are as described above.

[0124] As used herein, the term "bearing" means mounted at any location internally.

[0125] In one embodiment of the present invention, the peptide may be composed of an amino acid sequence of SEQ ID NO: 1.

[0126] In another embodiment of the present invention, the adjuvant may be at least one selected from the group consisting of SD-208, Vactosertib, Galunisertib, LY3200882, Resiquimod, Imiquimod, Gardiqiomod, Motolimod, Alum (Aluminium salts), CpG ODNs, GM-CSF, IL-12, poly(I:C), MPL, AS01, IC31, and CFA01.

[0127] In another embodiment of the present invention, the lipid is selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), Dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), DSPE-PEG, monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0 PA), or It may be at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (18:1 PA).

[0128] In another embodiment of the present invention, the mRNA may encode at least one antigenic peptide or protein comprising a pathogenic antigen, a tumor antigen, an allergen, or an autoimmune autoantigen.

[0129] In another embodiment of the present invention, the mRNA may be composed of the base sequence of SEQ ID NO: 2.

[0130]

[0131] In addition, the present invention provides a vaccine composition for preventing or treating cancer, comprising nanoparticles.

[0132] The term "cancer" refers to a class of diseases characterized by the development of abnormal cells that multiply uncontrollably and have the ability to invade and destroy normal body tissues.

[0133] The above term “prevention” may mean any act of suppressing or delaying the onset of cancer in an individual by administering a pharmaceutical composition according to one aspect.

[0134] The above term "treatment" may mean any action that improves or beneficially changes the symptoms of cancer in an individual by administering a pharmaceutical composition according to one aspect.

[0135] In one embodiment of the present invention, the cancer may be at least one selected from the group consisting of bladder cancer, brain cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer (gastric cancer), head and neck cancer, testicular cancer, melanoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma, and uterine cancer.

[0136] The above vaccine composition may be provided as a vaccine composition containing the active ingredient alone or including one or more pharmaceutically acceptable excipients or diluents.

[0137] When the above vaccine composition is formulated, it can be formulated using diluents or excipients such as lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be formulated by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppositories may include witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin. When manufacturing in the form of eye drops, known diluents or excipients may be used.

[0138] In another embodiment of the present invention, the composition may be administered parenterally by one or more routes selected from the group consisting of oral administration, topical application to the skin, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intraarterial injection, intramedullary injection, intracardiac injection, intrathecal injection, transdermal injection, intranasal injection, intraenteric injection, local injection, sublingual injection, or intrathoracic injection.

[0139] The above pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration, and the excretion rate, the duration of treatment, concomitant medications, and other factors well known in the medical field.

[0140] The dosage may vary depending on the patient's condition and weight, the extent of the disease, the form of the drug, the route of administration, and the time of administration, but can be appropriately selected by a person skilled in the art.

[0141] The nanoparticles of the present invention can be administered alone or in combination with other therapeutic agents, and when administered in combination, administration can be sequential or simultaneous. At this time, there is no limitation on the type of other therapeutic agents, but examples thereof include amino acids, vaccines, antiviral agents, gene transfer vectors, immune checkpoint inhibitors, immune enhancers, immunomodulators, interleukin inhibitors, neurotrophic factors, neuroprotective agents, antineoplastic agents, chemotherapeutic agents, polysaccharides, anticoagulants, antibiotics, analgesics, anesthetics, antihistamines, anti-inflammatory agents, viruses, Interferon beta-1a, Natalizumab, Daclizumab, Thalidomide, Glatiramer, Teriflunomide, Ocrelizumab, Ustekinumab, Fingolimod, Siponimod, Ozanimod, Dimethyl fumarate, Ponesimod, Darvadstrocel, etc.

[0142] The nanoparticles of the present invention may further comprise a formulation. For example, the formulation may be a therapeutic, prophylactic, or diagnostic agent. Specifically, the formulation may be selected from the group consisting of peptides, proteins, carbohydrates, nucleic acid molecules, lipids, organic molecules, biologically active inorganic molecules, and combinations thereof. For example, a wide range of drugs may be formulated for delivery using the present microneedle devices and methods.

[0143] As used herein, the term "drug" or "drug formulation" is broadly used to refer to any prophylactic, therapeutic, or diagnostic agent, or other substance suitable for introduction into biological tissue, including pharmaceutical excipients and substances for tattooing, cosmetics, and other uses. A drug may be a biologically active substance. Drug formulations may take various forms, such as liquid solutions, gels, solid particles (e.g., microparticles, nanoparticles), or combinations thereof. Drugs may include small molecules, large (i.e., macro-) molecules, or combinations thereof. Drugs may be selected from suitable proteins, peptides, and fragments thereof, which may be naturally occurring, synthetic, or recombinantly produced.

[0144] In another embodiment of the present invention, the vaccine composition may be administered in combination with an immune checkpoint inhibitor.

[0145] When the nanoparticles of the present invention are administered in combination with an immune checkpoint inhibitor, the nanoparticles may be administered simultaneously or sequentially with the immune checkpoint inhibitor.

[0146] The above immune checkpoint inhibitor refers to a therapeutic agent that induces T cell activation by blocking the binding of a ligand to an immune checkpoint receptor and thereby interfering with an immune response suppression signal, and specifically may be at least one selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody, and more specifically may be an anti-PD-1 antibody.

[0147]

[0148] To facilitate understanding of the present invention, the following examples will be described in more detail. However, these examples are intended only to exemplify the content of the present invention and are not intended to limit the scope of the present invention. These examples are provided to more fully explain the present invention to those with average knowledge in the technical field to which the invention pertains.

[0149]

[0150] [Example]

[0151] Example 1. Experimental materials and methods

[0152] 1.1 Experimental materials

[0153] DOCA-9R (Deoxycholic acid-KGGrrrrrrrrr; DR) was synthesized by Peptron (Daejeon, Korea). 1,2-dialuroyl-sn-glycero-3-phosphocholine (DLPC) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-amino(polyethylene glycol)-2000 (DSPE-PEG) 2000) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(polyethylene glycol)-Mannose (DSPE-PEG 2000-Mannose) was purchased from Biopharma PEG (Watertown, MA, USA). Ovalbumin (OVA) mRNA, Firefly Luciferase (FLuc) mRNA, and Enhanced Green Fluorescent Protein (EGFP) mRNA (each modified with 5-methoxyuridine) were obtained from TriLink Biotechnologies (San Diego, CA, USA). Resiquimod (R848), Cy5.5, Cell Counting Kit-8 (CCK-8) assay kit, RBC lysis buffer, dimethyl sulfoxide (DMSO), and dichloromethane (DCM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI1640 medium, Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and trypsin were purchased from WELGENE (Seoul, Korea). Mouse recombinant GM-CSF, IL-4, DNase I solution, and collagenase / hyaluronidase were purchased from STEMCELL Technologies (Vancouver, Canada). Mouse anti-CD11c, CD11b, CD80, CD86, CD40, CCR7, MHC I, MHC II, CD206, CD45, CD3, CD4, CD8, and IFN-γ antibodies were purchased from BioLegend (San Diego, USA). β-Mercaptoethanol, Hank's Balanced Salt Solution (HBSS), lipofectamine 2000, and cell permeabilization buffer were obtained from Thermofisher Scientific (Waltham, MA, USA). All ELISA kits were purchased from Invitrogen (MA, USA).InVivoMAb anti-mouse PD-1 (αPD-1 Ab) was purchased from Bio X cell (Lebanon, USA).

[0154]

[0155] 1.2 Preparation of mRNA nanoparticles (A-mL-DRO)

[0156] LPC, DSPE-PEG 2000 and DSPE-PEG 2000 -mannose was mixed in a molar ratio of 17:2:1 and stored at room temperature (RT) for 30 minutes in a ventilated hood to evaporate chloroform. The resulting lipid mixture was hydrated in distilled water (4% EtOH) to a concentration of 0.2 mg / mL. DR (1 mg) was dissolved in the lipid solution (1.25 mL). R848 (0.3 mg) was dissolved in DMSO (0.15 mL) and DCM (0.85 mL). The drug solution was slowly added to the DR / lipid solution (1.2 mL), sonicated, and evaporated at RT in a ventilated hood until all organic solvents were removed. The mixture was then centrifuged at 20,000 g for 20 minutes to remove free drug and obtain A-mL-DR. Next, A-mL-DR was resuspended in distilled water at a concentration of 1 mg / mL and incubated with OVA mRNA at a ratio of 1:8 (v / v, OVA mRNA: A-mL-DR) for 30 minutes.

[0157]

[0158] 1.3 Agarose gel retardation and mRNA degradation protection assay

[0159] To determine the optimal ratio, A-mL-DRO and 1 μg of OVA mRNA were incubated at various weight ratios ranging from 0.5 to 10 for 15 min at room temperature (RT). Each complex was mixed with 6X DNA loading dye and loaded onto a 1% (w / v) agarose gel. Electrophoresis was performed at 100 V for 20 min in 0.5X TBE buffer.

[0160] Rat serum at a concentration of 10% in RNase-free water and 1 μg of OVA mRNA, either free or loaded in A-mL-DR at various ratios from 2 to 10, were incubated at 37°C for 30 min. Samples incubated with free OVA mRNA were then evaluated by agarose gel retardation analysis.

[0161] 1.4 Characterization of A-mL-DRO

[0162] The mean zeta size and zeta potential of the nanoparticles were analyzed by dynamic light scattering (DLS) using a Zetasizer-Nano ZS (Malvern) after dilution in water. The purpose of this analysis was to identify optimal ratios of lipid / DR, adjuvant / mL-DR, and A-mL-DR / OVA mRNA.

[0163] The morphology of the nanoparticles was visualized using transmission electron microscopy (TEM). The nanoparticles were diluted to a concentration of 0.1 mg / mL, and a drop of the diluted solution was placed on a 200-mesh copper grid coated with carbon film. After the excess nanoparticle solution evaporated, a drop of 2% uranyl acetate solution was placed on the grid to negatively stain it, which was then removed with filter paper. After the liquid on the grid had completely evaporated, the grid was observed using TEM (NEO ARM, JEOL).

[0164]

[0165] 1.5 Drug loading, encapsulation efficiency and release profile

[0166] The prepared A-mL-DR (0.2 mg) was dissolved in DMSO, and the concentration of R848 was measured at 320 nm using a UV / Vis spectrophotometer (Tecan, Korea). The drug loading and encapsulation efficiency were calculated using the equations below. For cumulative release studies, A-mL-DRO (0.2 mg) was resuspended in PBS containing 5% DMSO, and centrifuged to recover the released medium at each time point. The concentration of R848 was then measured at 320 nm.

[0167] Encapsulation efficiency (%) = {Weight of R848 in self-assembly (mg) / Total weight of R848 added to self-assembly (mg)} x 100

[0168] Drug loading (%) = {Weight of R848 in self-assembly (mg) / Weight of self-assembly (mg)} x 100

[0169]

[0170] 1.6 Cell culture

[0171] Immature BMDCs (bone marrow-derived dendritic cells) were isolated from the bone marrow of 8-week-old female C57BL / 6 mice (Orient Bio, Seongnam, Korea). After dissection of the femur and tibia, the bone marrow was washed with HBSS using a 31-gauge syringe and then washed twice with PBS. The collected bone marrow cells were cultured in RPMI1640 medium (10% FBS, 1% penicillin-streptomycin, 100 U / mL) containing GM-CSF (10 ng / mL), IL-4 (10 ng / mL), and β-mercaptoethanol (55 nM) at 37°C in an incubator with 5% CO2. On day 4, fresh medium and all growth factors were added, and on day 8, differentiated cells were harvested and purified using the EasySep Mouse CD11c Positive Selection Kit (STEMCELL Technologies, Canada) according to the manufacturer's protocol.

[0172]

[0173] Raw264.7 cells (a mouse-derived macrophage cell line) were purchased from ATCC (Virginia, USA) and cultured in DMEM (10% FBS, 1% penicillin-streptomycin, 100 U / mL) supplemented with 10% FBS at 37°C in an atmosphere of 5% CO2. To induce M2 macrophage polarization, Raw264.7 cells were cultured with IL-4 (20 ng / mL) for 24 h.

[0174]

[0175] B16F10 (a mouse melanoma cell line) was purchased from ATCC (Virginia, USA), and B16-OVA (a mouse melanoma cell line expressing OVA) was a gift from Professor J. Jung. These cells were cultured in DMEM (10% FBS, 1% penicillin-streptomycin, 100 U / ml) supplemented with 10% FBS at 37°C and 5% CO2. B16F10 (a mouse melanoma cell line) was purchased from ATCC (Virginia, USA), and B16-OVA (a mouse melanoma cell line expressing OVA) was a gift from Professor J. Jung. These cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (100 U / ml) at 37°C and 5% CO2.

[0176]

[0177] 1.7 In vitro luciferase assay

[0178] The final ratio of mRNA to A-mL-DR was determined using a luciferase assay. BMDCs (2 × 105 cells per well) were treated with 1 μg of FLuc mRNA complexed with A-mL-DR at various ratios in serum-free medium. After 4 h, complete medium was added, and the cells were cultured for an additional 20 h. As a positive control, 1 μg of FLuc mRNA was concentrated using Lipofectamine 2000 according to a standard protocol. After 24 h of treatment, cells were lysed, and luciferase activity was measured using a 96-well plate luminometer. The protein concentration of the samples was determined using a DC assay kit. The final luciferase activity was calculated as the relative luminescence value, which is the luciferase activity divided by the protein concentration.

[0179]

[0180] 1.8 CCK-8 analysis

[0181] BMDCs were seeded into 96-well plates and treated with A-mL-DRO at concentrations ranging from 0 to 100 μg / mL, followed by incubation for 24 h. CCK solution was then added to each well and incubated for 30 min. Cell viability was calculated as a relative value by measuring absorbance at 450 nm using a UV / Vis spectrophotometer (Tecan).

[0182]

[0183] 1.9 In vitro cell uptake and microscopic imaging

[0184] BMDCs (2 × 105 cells per well) and polarized M2 macrophages (105 cells per well) were incubated with Cy5.5-loaded nanoparticles (3 μg / mL) for 2 and 4 h, respectively, and then washed twice with PBS. Cells were then stained with anti-mouse CD11c and CD206 antibodies, respectively, and analyzed using a FACS Calibur (BD Biosciences, USA). Cells were stained with DAPI (Southern Biotech), and images were captured using a confocal microscope (Leica).

[0185]

[0186] BMDCs (2 × 105 cells per well) were incubated with EGFP mRNA-loaded nanoparticles (1 μg of EGFP mRNA per well) for 24 h and then washed twice with PBS. Cells were then stained with anti-mouse CD11c, and transduced cells were evaluated by flow cytometry. Cell images were taken using a fluorescence microscope after DAPI staining.

[0187]

[0188] 1.10 In vitro dendritic cell maturation and cytokine secretion

[0189] BMDCs (2 × 105 cells per well) were incubated with nanoparticles (at a concentration of 3 μg / mL) for 24 h. Cells were then harvested and stained with anti-mouse CD80, CD86, CD40, MHC I, MHC II, and CCR7 antibodies. After washing twice, the expression of maturation markers in BMDCs was quantified by flow cytometry. Cells were also stained with DAPI and images were captured using confocal microscopy.

[0190]

[0191] To measure inflammatory cytokine levels, treated cells were centrifuged and culture supernatants were collected. Secreted inflammatory cytokines (IL-12p70, TNF-α, IL-6) in the supernatants were measured using an ELISA kit according to the manufacturer's instructions.

[0192]

[0193] 1.11 Western Blot

[0194] BMDCs (2 × 105 cells per well) were incubated with nanoparticles (3 μg / mL) for 24 h, and the protein levels of dendritic cell maturation markers were analyzed. Cells were lysed in RIPA buffer and centrifuged at 14,800 rpm for 30 min. The supernatant was collected, mixed with SDS buffer, and proteins were separated by SDS-PAGE. After electrophoresis, the proteins in the gel were transferred to an Immobilon-P PVDF membrane (Sigma-Aldrich). Immunodetection was performed using anti-mouse CD80, CD86, and GAPDH antibodies and anti-rabbit HRP-conjugated antibody (Cell Signaling Technology, MA, USA).

[0195]

[0196] 1.12 In vitro analysis of mRNA expression levels

[0197] M2-polarized Raw264.7 cells were incubated with nanoparticles (3 μg / mL concentration) for 24 h. Total RNA from each group was isolated using the RNeasy Mini Kit (Qiagen, Germany), and cDNA was synthesized using the iScript cDNA Synthesis Kit (Bio-RAD, CA, USA). The relative expression levels of M1 and M2 markers in macrophages were quantified using qRT-PCR (Applied Biosystems 7500, USA). Mouse GAPDH served as an endogenous control, and each sample was calculated using the ΔΔCt method.

[0198]

[0199] 1.13 Biodistribution

[0200] Cy5.5-loaded nanoparticles were injected subcutaneously into female C57BL / 6 mice bearing B16-OVA tumors. Mice were sacrificed 4 and 24 hours after injection, and the heart, lungs, liver, kidney, spleen, tumor, and lymph nodes were collected. The fluorescence intensity of Cy5.5 in each organ was measured using a VISQUE In Vivo Smart Fluorescence device (Vieworks, Korea).

[0201] To preserve the morphology of the lymph nodes and tumors, tissues were fixed in 10% formalin for 1 hour at room temperature and then resuspended in 30% sucrose solution overnight at 4°C. Samples were then fixed in OCT embedding compound (Sakura Finetek, USA), frozen at -70°C, and sectioned using a cryostat. The sectioned samples were rehydrated and subjected to immunofluorescence analysis using FITC-conjugated anti-mouse CD11c and CD206 antibodies according to standard protocols.

[0202]

[0203] 1.14In vivo bioluminescence imaging

[0204] To detect in vivo transduction, A-mL-DR / FLuc mRNA complexes were injected subcutaneously (0.5 mg / kg FLuc mRNA) into female C57BL / 6 mice bearing B16-OVA tumors. Six and 24 h after injection, the mice were injected intraperitoneally with 150 mg / kg D-Luciferin (Promega, USA), and luminescence was measured using an IVIS Spectrum In Vivo Imaging System (PerkinElmer, MA, USA).

[0205]

[0206] 1.15 Ex vivo infection and antigen-specific killing assays

[0207] Female C57BL / 6 mice, 6–8 weeks old, were injected with A-mL-DRO (OVA mRNA 0.5 mg / kg) at a concentration of 4.5 mg / kg twice a week for 3 weeks. The mice were then sacrificed, and lymph nodes and spleens were harvested. To confirm the expression of OVA in dendritic cells in the lymph nodes, the lymph nodes were processed into single-cell suspensions. The cells were then stained with anti-mouse CD11c and H-2K antibodies. b After staining with SIINFEKL antibody, OVA-expressing dendritic cells were analyzed by flow cytometry.

[0208]

[0209] Spleens from vaccinated mice were treated with spleen cells and incubated in 12-well plates for 24 h. Spleen cells were then co-cultured with CFSE-stained B16F10 (0.1 μM) and B16-OVA (1 μM) at a 2:1:1 ratio (# / #, spleen cells: B16F10: B16-OVA) for 4 and 24 h, respectively. Cells were harvested, stained with anti-mouse CD45 antibody, and antigen-specific dead cells were analyzed by flow cytometry.

[0210] Ratio = {B16F10 group (%) / B16-OVA group (%)}

[0211] OVA-specific cell killing rate (%) = [1 - {non-co-cultured control ratio / experimental group ratio} x 100}]

[0212]

[0213] 1.16 In vivo therapeutic efficacy, combination therapy with immune checkpoint blockade, and preventive efficacy of A-mL-DRO

[0214]

[0215] To evaluate the efficacy of A-mL-DRO treatment and combination therapy with anti-PD-1, 2 x 10 5A subcutaneous melanoma model was established by injecting B16-OVA cells into the left flank. When the tumor volume reached 80-100㎣, 4.5 mg / kg of A-mL-DRO (0.5 mg / kg of OVA mRNA) and 5 mg / kg of anti-PD-1 were administered subcutaneously and intraperitoneally, respectively, twice a week. For the analysis of immunoprophylactic efficacy, 6- to 8-week-old female C57BL / 6 mice were vaccinated twice a week with 4.5 mg / kg of A-mL-DRO (0.5 mg / kg of OVA mRNA). Thereafter, 2 x 10 5 B16-OVA cells were injected subcutaneously into the left flank. Tumor volume (㎣) was calculated by measuring tumor height and width, and animal survival was monitored. Mice were sacrificed when tumor volume exceeded 2,000 ㎣.

[0216]

[0217] Ex vivo toxicity of 1.17 A-mL-DRO

[0218] To investigate the potential toxicity of nanoparticles, 6- to 8-week-old female C57BL / 6 mice were injected twice weekly with 4.5 mg / kg of nanoparticles (0.5 mg / kg of OVA mRNA) for 3 weeks, after which they were sacrificed. Blood was collected from each group, centrifuged at 1,000 g for 30 min to obtain serum, and biochemical markers (ALT, AST, BUN, and Creatinine) were analyzed. In addition, major organs (heart, lungs, liver, kidney, and spleen) were harvested and stained with H&E.

[0219]

[0220] 1.18 Analysis of tumor-infiltrating lymphocytes (TILs)

[0221] Tumors from each group were harvested and processed into single-cell suspensions using collagenase / hyaluronidase and DNase I solutions, and red blood cells were removed using red blood cell lysis buffer. Cells were then fixed with 4% paraformaldehyde (Wako, Japan) and permeabilized using cell permeabilization buffer. For the evaluation of tumor-infiltrating lymphocytes (TILs), cells were stained with anti-mouse CD45, CD3, CD4, CD8, and IFN-γ antibodies and analyzed by flow cytometry.

[0222]

[0223] 1.19 Ex vivo analysis of cytokines

[0224] Lymph nodes harvested from each group were homogenized in cold lysis buffer, centrifuged at 14,800 rpm for 20 min, and the supernatant was collected. Relative cytokine levels of IL-12p70 were measured using an ELISA kit.

[0225]

[0226] The spleens collected from each group were processed into spleen cells and seeded at a density of 10 in 12-well plates. 6 Cells were cultured per well. Afterwards, the spleen cells were stimulated with 2 μg / ml OVA peptide at 37°C for 24 h. To measure the level of IFN-γ secreted from the stimulated spleen cells, the culture supernatant was collected and analyzed using an ELISA kit. All ELISA experiments were performed according to the manufacturer's instructions.

[0227]

[0228] 1.20 Immunofluorescence analysis of tissues

[0229] Lymph nodes and tumor tissues harvested from each group were fixed with 10% formaldehyde, processed into paraffin blocks, and sectioned at 8 μm thickness. After deparaffinization with xylene and rehydration through a series of alcohols, permeabilization was performed with Tris-Bisol-Sterile Sterile (TBST) containing 0.025% Triton X-100 for 5 minutes. The samples were blocked with TSB buffer containing 1% BSA and 10% goat serum for 1 hour to prevent nonspecific binding of antibodies. After removing the blocking solution, the sectioned tissues were stained with fluorescently labeled antibodies for 2 hours at room temperature, and cell nuclei were counterstained with DAPI. The slides were coverslipped using Dako Fluorescence Mounting Medium (DAKO, Denmark), scanned using AxioScan.Z1 (Zeiss, Germany), and analyzed with ZEN 3.6 software.

[0230]

[0231] 1.21 Statistical Analysis

[0232] All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism (version 8.02) for Windows (GraphPad Software) using Student's t-test and one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. A p value less than 0.05 was considered statistically significant (ns = not significant, *p < 0.033, **p < 0.01, ***p < 0.001).

[0233]

[0234] Example 2. Experimental Results

[0235] 2.1 Design, optimization, and physicochemical characterization of A-mL-DRO

[0236] Effective mRNA nanoparticles require antigen mRNA and an adjuvant to be delivered and transcribed into target cells. However, free mRNA is unstable, resulting in low intracellular delivery efficiency, necessitating a delivery system. In the present invention, DOCA-9R (DR) was designed as a delivery system for antigen and mRNA by conjugating deoxycholic acid (DOCA) to the N-terminus of a 9-mer arginine peptide. This structure is amphiphilic and can load hydrophobic drugs through self-assembly. To increase uptake into antigen-presenting cells (APCs) and enhance hydrophobic drug loading, DR is composed of DLPC and DSPE-PEG. 2000 , DSPE-PEG 2000 -Coated with lipid layers of various ratios consisting of -mannose (85: 10: 5 molar ratio). At a lipid ratio of 0.25, mannosylated lipid / DOCA-9R self-assemblies (mL-DR) were formed with a zeta potential of +50.87 ± 0.32 mV and an average size of 178 ± 1.04 nm. This contrasts with the zeta potential of +40.8 ± 0.44 mV and an average size of 239.87 ± 4.20 nm measured for DR alone (Fig. 2a). Resiquimod (R848) was used as a hydrophobic adjuvant to induce antitumor responses. Among the various ratios of mL-DR and adjuvant, the optimal loading amount of 30% was confirmed to be the most effective. At the optimized ratio, drug loading and encapsulation efficiency were maintained without loss while maintaining an average size of 218 ± 3.87 nm and a zeta potential of +45.77 ± 0.97 mV (Fig. 2b, Fig. 2c).

[0237] After optimizing the adjuvant-loaded nanoparticles (A-mL-DR), the concentration and stability of the A-mL-DR / OVA mRNA complex (A-mL-DRO) were evaluated using the agarose gel retardation method. It was confirmed that the model antigen OVA mRNA could effectively aggregate with A-mL-DR at a ratio of 2 or higher (Fig. 2d). To ensure complete complexation of the mRNA complex and protection from enzymatic degradation, the average size and zeta potential were measured at ratios from 1 to 10. When the ratio exceeded 2, the average size decreased and tended to be positively charged, showing a pattern similar to that before mRNA binding. At ratios of 8 and 10, the average size was 246 ± 5.55 nm and 221.33 ± 7.48 nm, and the zeta potential was +35.77 ± 2.15 mV and +41.73 ± 0.12 mV, respectively. In contrast, the complex without mRNA binding had a size of 462.03 ± 43.35 nm and a negative charge of -5.49 ± 0.39 mV (Fig. 2e). To evaluate the in vivo stability of mRNA, an mRNA degradation protection test was performed by incubating the A-mL-DR and OVA mRNA complexes in 10% rat serum. Gel retardation test results showed that the complex loaded with OVA mRNA on A-mL-DR was not degraded, unlike free mRNA, indicating protection of mRNA by the enzyme (Fig. 2f). To determine the final ratio, a luciferase assay using FLuc mRNA was performed in BMDCs, and efficient luminescence was confirmed at a ratio of 8 (Fig. 2g).

[0238] The morphology of the DR / OVA mRNA complex (DRO), mL-DR / OVA mRNA complex (mL-DRO), and A-mL-DRO appeared as a spherical core-shell structure as shown in transmission electron microscopy (TEM) images (Fig. 2h). The drug release profile of A-mL-DRO showed a sustained release pattern without an initial release. Approximately half and 83.88 ± 3.83% of the drug were released from A-mL-DRO after 12 and 72 h, respectively (Fig. 2i). To evaluate the cytotoxicity of A-mL-DRO, BMDCs were cultured with A-mL-DRO at concentrations ranging from 0 to 100 μg / mL, and no significant change in viability was observed (Fig. 2j).

[0239]

[0240] 2.2 Evaluation of drug encapsulation, cellular uptake, and infection efficiency of A-mL-DRO

[0241] DOCA-9R was intentionally designed so that DOCA constitutes the hydrophobic tail and the 9-mer arginine peptide acts as the hydrophilic peptide. This allows the amphiphilic DOCA-9R monomers to self-assemble into a structure with a hydrophilic shell and a hydrophobic core, and the hydrophobic adjuvant R848 can be encapsulated within the DR to form an adjuvant-loaded DR (A-DR). However, DR alone is not effective in encapsulating drugs, with a drug encapsulation efficiency of only about 30%. To improve drug encapsulation efficiency, we investigated the effect of lipid coating on drug encapsulation, and confirmed that it was effective both when mannosylated lipids were used and when they were not. DLPC and DSPE-PEG 2000 Lipid-coated DR (L-DR) combining DLPC and DSPE-PEG in a molar ratio of 85:15 showed a drug encapsulation efficiency of 80.32 ± 4.57%. 2000 , DSPE-PEG 2000Lipid-coated DR (mL-DR) containing mannose in a molar ratio of 85:10:5 exhibited an efficiency of 82.41 ± 2.15%. As a result, lipid-coated DR enhanced drug encapsulation efficiency by 2.5-fold compared to uncoated DR, suggesting enhanced immune response regardless of the use of mannosylated lipids (Fig. 3a).

[0242] Efficient delivery of mRNA and adjuvants to dendritic cells (DCs) is a critical step in dendritic cell-based immunotherapy, promoting dendritic cell activation and subsequent antigen cross-presentation. Dendritic cells abundantly express the mannose receptor. Therefore, the use of mannosylated lipids (mL) is a clear mannose receptor-mediated targeting strategy for improving capture and uptake by dendritic cells. To confirm the enhanced cellular uptake using common and mannosylated lipids, Cy5.5-labeled DRO, L-DRO, and mL-DRO were treated with BMDCs and analyzed by flow cytometry. Compared to DRO, L-DRO exhibited 5.5-fold higher cellular uptake, while mL-DRO exhibited 8.25-fold higher uptake. This indicates that lipid coating enhances cellular uptake. Furthermore, mL-DRO exhibited 1.58-fold higher cellular uptake in BMDCs compared to L-DRO, suggesting enhanced uptake by dendritic cells through mannose receptor-mediated targeting (Fig. 3b). Confocal microscopy imaging confirmed similar uptake patterns as observed in flow cytometry data. mL-DRO exhibited greater cytoplasmic localization and distribution within BMDCs compared to DRO and L-DRO (Fig. 3c).

[0243] The instability of free mRNA complicates its transcription into cells, necessitating the use of an appropriate delivery system. Negatively charged mRNA can bind to the positively charged peptide coat of DR through electrical interaction. Based on previous results, lipid-coated DR maintains a positive zeta potential, preserving its ability to bind mRNA. To assess transcription efficiency, EGFP mRNA was used as a reporter gene, and the presence of lipid coating and the use of mannosylated lipids were evaluated. BMDCs were treated with EGFP mRNA complexed with A-DR, AL-DR, and A-mL-DR, and fluorescent protein expression was confirmed by flow cytometry. AL-DR showed slightly increased EGFP expression compared to A-DR, while A-mL-DR showed significantly higher transcription efficiency in BMDCs than A-DR and AL-DR. This indicates that lipid coating and mannose receptor targeting enhanced intracellular transcription into dendritic cells (Figure 4a). Furthermore, the transcription efficiency was evaluated using fluorescence microscopy, confirming similar results to those obtained from flow cytometry. A-mL-DR exhibited enhanced cytoplasmic EGFP expression in BMDCs (Fig. 4b). Collectively, these results demonstrate that A-mL-DRO exhibits enhanced drug encapsulation due to the lipid coating, improved cellular uptake due to mannosylated lipids, and enhanced transcription efficiency in dendritic cells.

[0244]

[0245] 2.3 Confirmation of the effect of A-mL-DRO on in vitro dendritic cell maturation

[0246] Among APCs, dendritic cells (DCs) play a crucial role in initiating and regulating innate and adaptive immune responses. When dendritic cells recognize antigens, they present them on MHC I and MHC II molecules. However, antigen presentation alone is insufficient to induce a robust T cell-mediated immune response. Additional positive signals must be provided by costimulatory molecules and soluble factors. Resiquimod (R848), a TLR7 / 8 agonist, acts as an adjuvant to induce these positive signals by targeting all endogenous dendritic cell subtypes and activating NF-κB signaling. Therefore, upregulation of costimulatory molecules and inflammatory cytokines characterizes dendritic cell maturation, playing a crucial role in antigen presentation and subsequent initiation of immune responses. To assess dendritic cell maturation, BMDCs were cultured with A-mL-DRO, which significantly increased the secretion of costimulatory molecules and cytokines. Among the costimulatory molecules, CD80 and CD86 interact with CD28 on T cells on dendritic cells to induce T cell activation. CD80 levels (Fig. 5a) and CD86 levels (Fig. 5b) were analyzed by flow cytometry, and A-mL-DRO significantly increased compared to the control, A-DRO, and mL-DRO. Furthermore, CD80 expression levels were confirmed by confocal microscopy imaging and showed a pattern similar to the flow cytometry data (Fig. 5c). Furthermore, protein levels of CD80 and CD86, measured by Southern blot analysis, were increased in A-mL-DRO-treated BMDCs (Fig. 5d).

[0247] A-mL-DRO induced increased expression of CD40, which interacts with CD40L on T cells. In addition, A-mL-DRO significantly enhanced the levels of MHC I and MHC II, indicating increased intracellular OVA through OVA mRNA transcription. The chemokine receptor CCR7, which interacts with CCL19 and CCL21 and is required for migration to draining lymph nodes, was analyzed by flow cytometry and was confirmed to be upregulated by A-mL-DRO, suggesting dendritic cell maturation (Fig. 5e). Similarly, A-mL-DRO stimulated the production of high concentrations of the proinflammatory cytokine interleukin-12 (IL-12p70) by dendritic cells, thereby promoting T H 1 cell and CD8 + Helps prepare T cells for T cell activation. Secretion of other inflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), which were increased by A-mL-DRO, was also measured by ELISA (Fig. 5f). All results from the dendritic cell maturation assay demonstrate that A-mL-DRO enhances the ability to provide sufficient positive signals through increased costimulatory molecules and inflammatory cytokines, ultimately promoting subsequent immune responses by cytotoxic T cells.

[0248]

[0249] 2.4 Confirmation of the targeting and repolarizing effects of A-mL-DRO on in vitro M2 macrophages.

[0250] The immunosuppressive properties of the tumor microenvironment (TME) can limit the effectiveness of cancer vaccines by producing immunosuppressive factors, such as immunosuppressive cells. Macrophages within the TME, namely tumor-associated macrophages (TAMs), are divided into two groups: a small number of pro-inflammatory M1 macrophages and a large number of anti-inflammatory M2 macrophages. Studies have shown that eliminating TAMs, one of the immunosuppressive cell types in the TME, can enhance the efficacy of cancer vaccine therapy. Therefore, targeting TAMs may be a promising strategy to synergize cancer vaccines and tumor immunotherapy. Macrophage mannose receptor 1 (CD206) is abundantly expressed in TAMs exhibiting the M2 macrophage phenotype. A-mL-DRO inhibits CD206 + To assess the specificity of targeting M2 macrophages, Raw264.7 cells were stimulated with IL-4 to induce an M2 phenotype and then cultured with Cy5.5-loaded DRO, L-DRO, and mL-DRO. Similar to the cellular uptake results observed in BMDCs, flow cytometry analysis showed that L-DRO exhibited a 1.46-fold increase in cellular uptake efficiency compared to uncoated DRO, indicating the effect of lipid coating. However, mL-DRO, which conjugated mannose to lipids, showed approximately 6.28- and 4.3-fold higher cellular uptake than DRO and L-DRO, respectively (Fig. 6a). Confocal microscopy imaging supported the flow cytometry results, showing that mL-DRO increased cellular uptake in M2 macrophages, highlighting the efficiency of mannose receptor targeting (Fig. 6b).

[0251]

[0252] Macrophage repolarization represents a therapeutic strategy to transform the tumor microenvironment from a tumor-promoting to a tumor-suppressive state. Furthermore, reprogramming TAMs offers more advantages than directly eliminating M2 macrophages in terms of optimizing macrophage utilization and can enhance the phagocytosis of immunosuppressive cells, thereby promoting antitumor immunity. R848, a TLR 7 / 8 agonist, has the ability to convert immunosuppressive cells, such as M2 macrophages, to an M1 phenotype. To evaluate macrophage repolarization from M2 to M1, M2-induced Raw264.7 cells were cultured with A-mL-DRO, which resulted in a significant shift from M2 to M1 phenotype. CD11b + CD206 + The proportion of macrophages, i.e., anti-inflammatory M2 type, was decreased (Fig. 7a), and CD11b + CD80 + The proportion of macrophages, i.e., the pro-inflammatory M1 type, increased in the A-mL-DRO-treated group compared to the other groups (Fig. 7b). A-mL-DRO also significantly downregulated the mRNA levels of anti-inflammatory M2 markers (Fizz1 and IL-10), whereas upregulated the mRNA levels of pro-inflammatory M1 markers (IL-6 and TNF-α) (Fig. 7c, d). However, mL-DRO without R848 showed an insignificant conversion pattern in macrophages. These results suggest that A-mL-DRO efficiently induced the conversion from M2 to M1 phenotype by enhancing cellular uptake through mannose receptor targeting, and that the reprogramming effect of R848 and the improved delivery of R848 contributed to this result.

[0253]

[0254] 2.5 In vivo delivery of A-mL-DRO via mannose-mediated targeting of dendritic cells and M2 macrophages

[0255] A-mL-DRO was confirmed to effectively target dendritic cells and M2 macrophages. These cells comprise the majority of TAMs. As demonstrated in the in vitro experiments, A-mL-DRO exhibited enhanced cellular uptake through lipid coating and active targeting via mannose. To determine whether similar patterns were observed in animal models, experiments were conducted using a C57BL / 6 mouse model implanted with B16-OVA melanoma. Cy5.5-loaded DRO, L-DRO, and mL-DRO were injected subcutaneously into tumor-implanted mouse models. Major organs, lymph nodes, and tumors were harvested, and mL-DRO accumulation was confirmed in lymph nodes where dendritic cells migrate and in tumors with a high proportion of TAMs (Fig. 8a). Four hours after injection, mL-DRO was localized at high concentrations in lymph nodes, and the intranodal signal persisted for up to 24 hours, while the other groups showed a decrease in signal over time. In the tumor, mL-DRO showed some localization at 4 h after injection, and the localization increased over time through systemic circulation after 24 h. However, the other groups did not show significant accumulation in the tumor area (Fig. 8b). The fluorescence intensities of Cy5.5-loaded DRO, L-DRO, and mL-DRO in major organs, lymph nodes, and tumors were then quantified per tissue area and compared among the groups. Similar to the in vitro results, the localization patterns showed that L-DRO and mL-DRO penetrated the tissues more effectively than DRO, which was attributed to the lipid coating. In addition, mL-DRO bound to mannose receptors on dendritic cells and M2 macrophages, and was more effectively transduced into tissues, especially in lymph nodes and tumors, with the signal being well maintained for up to 24 h (Fig. 8c). Frozen section images showed high signals of mL-DRO in lymph nodes and tumors 24 h after injection. mL-DRO was expressed on dendritic cells (CD11c) in lymph nodes. + ) and M2 macrophages (CD206) in tumors +) and localized (Fig. 8d, 8e). This suggests that mL-DRO can effectively move to lymph nodes and tumors through internalization by dendritic cells and uptake by M2 macrophages.

[0256]

[0257] 2.6 Evaluation of in vivo infection efficiency of A-mL-DRO for inducing antigen-specific T cell responses

[0258] Because of additional barriers to in vivo mRNA delivery, not all mRNAs from carriers that demonstrate transgenic activity in vitro can be successfully translated in animal models. In a previous in vitro study, A-mL-DR conjugated with FLuc and EGFP mRNA demonstrated transgenic capacity in BMDCs. To determine whether this could be extended to an animal model, A-mL-DR conjugated with FLuc mRNA was injected subcutaneously into B16-OVA tumor-bearing mice and screened using IVIS at 6 and 24 hours post-injection (Fig. 9a). Images in Fig. 9b show that A-mL-DR conjugated with FLuc mRNA exhibited marked bioluminescence at 6 hours, with a decrease in signal at 24 hours. FLuc expression was quantified in the total area and tumor, and compared to untreated controls and A-mL-DR without FLuc mRNA. A-mL-DR coupled with FLuc mRNA showed significantly higher FLuc expression in both the total and tumor regions at 6 hours post-injection (Fig. 9c). Therefore, we confirmed that mRNA coupled with A-mL-DR was effectively delivered and translated, including into the tumor region, in an animal model. When administered subcutaneously, FLuc expression was detected around the injection site, peaking at 6 hours and gradually decreasing thereafter. Previous drug delivery experiments investigating drug delivery within mL-DRO demonstrated that the signal in the tumor was higher at 24 hours than at 6 hours, but this decrease in luminescence intensity may be explained by the properties of FLuc mRNA.

[0259] Due to the heterogeneity of the TME, TILs contain not only tumor-specific T cells but also T cells expressing non-tumor-related epitopes. However, since tumor antigen-specific T cells play a crucial role in killing cancer cells, increasing the distribution of these cells is essential for effective cancer immunotherapy. To generate tumor antigen-specific T cells, the T cell receptor (TCR) must sufficiently interact with and be activated by tumor antigens expressed via MHC on dendritic cells (DCs). In cancer vaccine development, presenting tumor-specific antigens (TSAs) to APCs, particularly dendritic cells, is crucial for generating tumor antigen-specific T cells and inducing antigen-specific T cell-mediated immune responses. To assess immune responses in mice, OVA mRNA was used as a tumor antigen against B16-OVA melanoma. To investigate the translation of OVA into an animal model and the killing capacity of OVA-specific T cells, passively immunized C57BL / 6 mice were inoculated with A-mL-DRO (Fig. 10a). After harvesting the lymph nodes and preparing them into a single cell suspension, H-2K b / OVA antigen expression on dendritic cells was measured via MHC I staining with SIINFEKL antibody and flow cytometry. A-mL-DRO showed a significant increase in OVA-presenting dendritic cells, indicating a high translation efficiency of OVA mRNA in mice (Fig. 10b). Furthermore, the increased number of OVA-presenting dendritic cells suggests that it may influence the priming of OVA-specific T cells, thereby inducing antigen-specific immunity.

[0260] After confirming the expression of mRNA-encoded antigens in dendritic cells, we conducted antigen-specific killing experiments. Spleen cells were obtained from vaccinated mice and co-cultured with CFSE-stained melanoma cells (B16-OVA and B16F10) for 4 and 24 hours to evaluate the killing capacity of antigen-specific T cells (Fig. 10c). After co-culture, the number of B16-OVA cells was significantly reduced in the A-mL-DRO group, indicating potent antigen-specific cancer cell killing capacity. These results suggest that A-mL-DRO delivers antigens to dendritic cells to induce translation, which in turn delivers the antigens to lymph nodes, inducing a dendritic cell-mediated immune response and promoting T cell activation to recognize and eliminate cancer cells expressing specific antigens. Based on these results, A-mL-DRO demonstrates the potential of a therapeutic mRNA cancer vaccine in antigen-specific anticancer immunotherapy.

[0261]

[0262] 2.7 Confirmation of the therapeutic effect of A-mL-DRO in the B16-OVA melanoma mouse model

[0263] The goal of therapeutic cancer vaccines is to suppress tumor growth by stimulating the adaptive immune system through antigen delivery to dendritic cells (DCs). This can be achieved by ensuring optimal dendritic cell maturation, maintaining cytotoxic T lymphocyte (CTL) and helper T cell responses, and immunomodulating the tumor-like environment (TME). Based on the targeting and activation effects demonstrated in vitro, a mouse model bearing B16-OVA melanoma was established to evaluate the therapeutic vaccine efficacy of A-mL-DRO (Figure 11a). Mice were vaccinated four times with PBS, A-mL-DRO, mL-DRO, and A-mL-DRO, and then sacrificed to evaluate immune responses and antitumor effects. Mice vaccinated with A-mL-DRO exhibited significantly greater tumor growth inhibition compared to mice vaccinated with other formulations. Specifically, mice vaccinated with A-mL-DRO on day 22 had an average tumor volume of 219.52 ㎣, whereas mice vaccinated with PBS, A-DRO, and mL-DRO recorded 1621.27, 750.56, and 643.03 ㎣, respectively (Fig. 11b). Furthermore, measurements of tumors harvested on day 22 showed a significant reduction in both size (Fig. 11c) and weight (Fig. 11d) in mice vaccinated with A-mL-DRO.

[0264] To evaluate the efficacy of immunization, the degree of antibody response and the maturation of dendritic cells in the lymph nodes were measured. Generally, vaccines induce immune responses, leading to the production of immunoglobulin G (IgG), which plays a role in suppressing tumor growth. Serum IgG levels were measured by ELISA, and the highest concentrations were observed in mice vaccinated with A-mL-DRO, indicating that A-mL-DRO induced the strongest humoral immune response (Fig. 11e). To evaluate the maturation of dendritic cells in the lymph nodes, the expression of the co-helper molecules CD80 and CD86 was measured using flow cytometry. In mice vaccinated with A-mL-DRO, CD11c was significantly higher. + CD45+ CD80 among cells + CD86 + The ratio of cells was approximately 4.88-fold, 2.68-fold, and 2.74-fold higher in mice vaccinated with PBS, A-DRO, and mL-DRO, respectively (Fig. 11f). In addition, when the secretion of IL-12p70, an inflammatory cytokine secreted by dendritic cells in lymph nodes, was measured by ELISA, a higher level was observed in mice vaccinated with A-mL-DRO than in the other groups (Fig. 11g). CD80 in lymph nodes + CD11c + Immunofluorescence images of cells and CD80 + The intensity of fluorescence supported the flow cytometric analysis results for dendritic cell maturation (Fig. 11h). The increased IgG levels and enhanced dendritic cell maturation in lymph nodes suggest that A-mL-DRO can induce a potent immune response and induce an adaptive T cell response against tumors.

[0265] To further evaluate the antitumor effect of A-mL-DRO, we analyzed the immune cell composition from tumors and spleens of each vaccinated mouse. First, tumor-infiltrating lymphocytes (TILs) were examined by flow cytometry to identify CD8 cells infiltrating within the tumor. + and CD4 + The presence of T cells was quantified. For this purpose, CD3, a protein that forms a complex with the T cell receptor (TCR), was used. + The signal was gated and used as a marker of T cells. CD8 in T cell-mediated immunity + CTLs are the most potent effector cells that kill tumor cells. Mice vaccinated with A-mL-DRO had significantly higher tumor-infiltrating CD45 cells compared to the other groups. + CD3 + CD8 + The proportion of T cells was the highest (Fig. 12a). In addition, CD4 + Helper T cells play a major role in controlling tumor growth, and CD8 +It acts together with T cells and provides auxiliary signals that activate them. In mice vaccinated with A-mL-DRO, intratumoral CD45 + CD3 + CD4 + T cell infiltration also significantly increased (Fig. 12b). Activated CD8 + T cells eliminate tumors by secreting interferon gamma (INF-γ), a cytokine that acts directly on tumor cells. Mice vaccinated with A-mL-DRO showed a higher level of CD8 cells secreting INF-γ. + T cells were present in relatively greater numbers (Fig. 12c). To confirm the epitope-specific response to antigen peptide stimulation, the levels of INF-γ secreted from spleen cells were measured by ELISA after restimulation with OVA peptide. The mean levels of INF-γ in mice vaccinated with A-mL-DRO were increased by 2.55-fold, 1.54-fold, and 1.39-fold, respectively, compared to mice vaccinated with PBS, A-DRO, and mL-DRO (Fig. 12d). This indicates that an enhanced OVA-specific T cell response was successfully induced in mice bearing B16-OVA tumors vaccinated with A-mL-DRO. Furthermore, the increased infiltration of effector T cells within the tumors supports the expectation that the efficacy will be enhanced when combined with immune checkpoint blockade, particularly anti-PD-1.

[0266] TAMs are abundant in tumor immunosuppressive M2 macrophages and act as one of the extrinsic resistance mechanisms to vaccine therapy. Therefore, TAM repolarization is important for enhancing antitumor immunity in cooperation with therapeutic cancer vaccines. Previous drug distribution experiments have shown that A-mL-DRO targets M2 macrophages and accumulates in tumors. Immunofluorescence images of tumors from each vaccinated mouse showed the relative repolarization tendency of TAMs. This was observed as a color combination (orange) of the fluorescence of the macrophage marker F480 (green) and the M2 macrophage marker CD206 (red) or the M1 macrophage marker CD80 (red). In mice vaccinated with A-mL-DRO, F480 + CD206 + and CD206 + A significant decrease in fluorescence intensity was observed, indicating that the expression of M2 macrophages was reduced compared to other groups (Fig. 12e). In addition, F480 + CD80 + and CD80 + The fluorescence intensity was increased in mice vaccinated with A-mL-DRO, indicating that the expression of M1 macrophages was increased compared to the other groups (Fig. 12f). When comparing A-mL-DRO and mL-DRO, the main difference was the presence of R848. Mice vaccinated with A-mL-DRO had higher CD206 expression compared to mice vaccinated with mL-DRO. + Fluorescence intensity decreased by 1.29 times, and CD80 +The fluorescence intensity increased by 1.42-fold, indicating that the presence of R848 enhanced the repolarization effect on TAMs. A-mL-DRO, which can simultaneously target dendritic cells and M2 macrophages, utilizes R848 not only as a vaccine adjuvant but also to repolarize TAMs from M2 to M1. Taken together, these results suggest that the therapeutic vaccine efficacy of A-mL-DRO can overcome obstacles to vaccine therapy through TAM repolarization, ultimately enhancing anti-tumor immunotherapy.

[0267]

[0268] 2.8 Confirmation of enhanced efficacy of A-mL-DRO through immune checkpoint blockade in combination therapy

[0269] The combination of therapeutic cancer vaccines and immune checkpoint blockade (ICB) has emerged as a promising strategy for enhancing anti-tumor immune responses. In tumor-intrinsic resistance to therapeutic cancer vaccines, programmed cell death ligand-1 (PD-L1) expressed on cancer cells acts as an immune checkpoint ligand and interacts with programmed cell death-1 (PD-1) expressed on T cells. This interaction interferes with the ability of effector T cells to eliminate cancer cells. In this study, we observed increased PD-1 expression in tumor-infiltrating CD8+ T cells after vaccination, but combined treatment with anti-PD-1 enhanced anti-tumor immunity. Previous results on the therapeutic efficacy of A-mL-DRO observed an increase in tumor-infiltrating effector T cells, suggesting the potential of combining anti-PD-1 with ICB to enhance T cell function. To verify the efficacy of combination therapy, mice bearing B16-OVA melanoma were vaccinated four times with A-mL-DRO and anti-PD-1, respectively, via subcutaneous and intraperitoneal injections (Fig. 13a). Mice vaccinated simultaneously with A-mL-DRO and anti-PD-1 exhibited slower tumor growth compared to mice vaccinated only with anti-PD-1 or A-mL-DRO (Fig. 13b). Furthermore, on day 22, the average tumor weight of the combination-vaccinated mice was only 0.037 g, compared to 0.375 g and 0.224 g in mice vaccinated only with anti-PD-1 or A-mL-DRO (Fig. 13c).

[0270] Among immunoglobulin classes, IgM acts as the first antibody to antigen, while IgG plays an important role in antigen defense. Therefore, the increased serum IgM and IgG levels in combination-vaccinated mice indicate an enhanced humoral immune response to B16-OVA melanoma (Fig. 13d). Combination-vaccinated mice had a higher CD45 infiltrating tumors compared to mice vaccinated with other formulations. + CD3 + CD8 + The proportion of T cells increased, suggesting an increase in effector CTLs (Fig. 13e). In addition, CD45 + CD3 + CD4 + The proportion of helper T cells identified as T cells was also higher in the combination-vaccinated mice (Fig. 13f). Furthermore, among all vaccination groups, the combination-vaccinated mice had a higher proportion of CD8 cells secreting INF-γ. + The proportion of T cells was the highest (Fig. 13g). After restimulation with OVA peptide, the amount of INF-γ secreted from spleen cells was higher in the combined vaccinated mice than in the other groups, indicating an epitope-specific response to the OVA antigen (Fig. 13h). Studies have shown that PD-1 blockade increases tumor-infiltrating T cells by increasing INF-γ levels, and CD8 + It has been reported that CTLs enhance the tumor cell killing ability. TUNEL staining images and TUNEL intensity showed increased cell death in mice co-vaccinated with A-mL-DRO and anti-PD-1. This result suggests that increased tumor-infiltrating CD8 + This suggests that CTLs exhibit an activated function that induces apoptosis of cancer cells (Fig. 13i). In summary, the combined treatment of A-mL-DRO and anti-PD-1 demonstrated effective results by increasing the activation of tumor-infiltrating CTLs and enhancing apoptosis of cancer cells.

[0271]

[0272] 2.9 Immunopreventive effect of A-mL-DRO on B16-OVA melanoma mouse model and safety evaluation of A-mL-DRO

[0273] To investigate the preventive effect of A-mL-DRO against B16-OVA melanoma, mice were vaccinated twice a week for 3 weeks. One week after the last vaccination, mice were inoculated with B16-OVA cells (Fig. 14a). The tumor volumes of each vaccinated mouse were monitored over time after tumor inoculation. Mice vaccinated with A-mL-DRO showed a significant advantage in suppressing tumor growth compared to mice vaccinated with PBS, while mice vaccinated with PBS showed faster tumor growth. Specifically, on day 25, the average tumor volume of mice vaccinated with A-mL-DRO was only 127.19 ㎣, whereas mice vaccinated with PBS, A-DRO, and mL-DRO showed higher average tumor volumes of 1844.28 ㎣, 487.27 ㎣, and 617.50 ㎣, respectively (Fig. 14b). Mice vaccinated with A-mL-DRO showed more effective inhibition of tumor growth compared to other formulations, resulting in prolonged survival (Fig. 14c). Therefore, these results indicate that A-mL-DRO is an mRNA cancer vaccine with excellent preventive effects that delay tumor progression.

[0274] In toxicity assessment, body weight changes are commonly used as an important indirect indicator. To assess the toxicity of A-mL-DRO, the body weights of all vaccinated mice were monitored throughout the vaccination process. No significant changes in body weight were observed after A-mL-DRO vaccination compared to the control mice vaccinated with PBS (Figure 14d). In addition, some vaccinated mice from each group were sacrificed to assess systemic toxicity. Major organs (heart, lungs, liver, kidney, and spleen) and serum were collected before B16-OVA cell inoculation. Histological analysis using H&E staining of major organs revealed no significant pathological changes in any vaccinated mice compared to the PBS control group (Figure 14e). Alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are markers of liver toxicity, and renal function indices represented by blood urea nitrogen (BUN) and creatinine, were measured as biochemical toxicity markers in serum. No significant differences in serum biochemical marker levels were observed between all vaccinated mice and the PBS control group, indicating the biosafety of A-mL-DRO (Fig. 14f). In summary, these results suggest that A-mL-DRO exhibits enhanced immunoprotective effects against tumors without biotoxicity.

[0275]

[0276] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

Claims

1. Nanoparticles for vaccines, comprising: (i) Deoxycholic acid (DOCA)-linked cell penetrating peptide (CPP) assembly; (ii) a mannose-linked lipid coating the assembly; (iii) adjuvant; and (iv) mRNA.

2. A nanoparticle according to claim 1, characterized in that the peptide consists of an amino acid sequence of sequence number 1.

3. A nanoparticle according to claim 1, characterized in that the adjuvant is at least one selected from the group consisting of SD-208, Vactosertib, Galunisertib, LY3200882, Resiquimod, Imiquimod, Gardiqiomod, Motolimod, Alum (Aluminium salts), CpG ODNs, GM-CSF, IL-12, poly(I:C), MPL, AS01, IC31, and CFA01.

4. In claim 1, the lipid is lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), Dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), DSPE-PEG, monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0 PA), or A nanoparticle characterized by comprising at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (18:1 PA).

5. A nanoparticle according to claim 1, characterized in that the mRNA encodes at least one antigenic peptide or protein comprising a pathogenic antigen, a tumor antigen, an allergen, or an autoimmune autoantigen.

6. A nanoparticle according to claim 5, characterized in that the mRNA consists of a base sequence of sequence number 2.

7. A nanoparticle according to claim 1, characterized in that the nanoparticle increases the expression of at least one selected from the group consisting of CD80, CD86, CD40, CCR7, IL-12, TNF-α, and IL-6 of dendritic cells.

8. A nanoparticle according to claim 1, characterized in that the nanoparticle induces repolarization of M2 macrophages into M1 macrophages.

9. A nanoparticle according to claim 1, characterized in that the nanoparticle reduces the expression of Fizz1 or IL-10 in macrophages.

10. A nanoparticle according to claim 1, characterized in that the nanoparticle increases tumor-infiltrating lymphocytes in the tumor.

11. A step of linking a cell-penetrating peptide with deoxycholic acid; A step of assembling a cell penetrating peptide linked to the above deoxycholic acid; A step of coating the above assembly with a mannose-linked lipid; and A method for producing a nanoparticle for a vaccine, comprising the step of loading an adjuvant and mRNA inside an assembly coated with the lipid.

12. A manufacturing method according to claim 11, characterized in that the peptide consists of an amino acid sequence of sequence number 1.

13. A manufacturing method according to claim 11, characterized in that the adjuvant is at least one selected from the group consisting of SD-208, Vactosertib, Galunisertib, LY3200882, Resiquimod, Imiquimod, Gardiqiomod, Motolimod, Alum (Aluminium salts), CpG ODNs, GM-CSF, IL-12, poly(I:C), MPL, AS01, IC31, and CFA01.

14. In claim 11, the lipid is lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoyl-phosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), Dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), DSPE-PEG, monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dielaidoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, 1,2-dimyristoyl-sn-glycero-3-phosphate (14:0 PA), 1,2-distearoyl-sn-glycero-3-phosphate (18:0 PA), or A manufacturing method characterized in that at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (18:1 PA).

15. A method of manufacturing according to claim 11, wherein the mRNA encodes at least one antigenic peptide or protein comprising a pathogenic antigen, a tumor antigen, an allergen, or an autoimmune autoantigen.

16. A manufacturing method according to claim 15, characterized in that the mRNA consists of a base sequence of sequence number 2.

17. A vaccine composition for preventing or treating cancer, comprising the nanoparticle of any one of claims 1 to 10.

18. A vaccine composition according to claim 17, characterized in that the cancer is at least one selected from the group consisting of bladder cancer, brain cancer, breast cancer, cervical cancer, ovarian cancer, colon cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, nasopharyngeal cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer (gastric cancer), head and neck cancer, testicular cancer, melanoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma, and uterine cancer.

19. In claim 17, the vaccine composition is for co-administration with an immune checkpoint inhibitor.

20. A vaccine composition according to claim 19, wherein the immune checkpoint inhibitor is at least one selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody.

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