Nanostructure comprising cancer antigen peptides conjugated with deoxycholic acid and use thereof

Abstract

An embodiment of the present invention relates to: a cancer antigen peptide nanostructure comprising an assembly of cancer antigen peptides conjugated with deoxycholic acid; a method for preparing same; and use thereof. The inventors of the present invention confirmed that the nanostructure of the present invention effectively stimulates the maturation of dendritic cells, migrates to lymph nodes, and activates T cells. Also, in a cancer cell model, significant infiltration of cytotoxic T lymphocytes into primary tumors was observed, and an anti-metastatic effect was confirmed. Therefore, the nanostructure according to the present invention can be effectively used as a promising immunotherapy platform that can be applied to various intractable cancers.

Description

Nanostructures comprising cancer antigen peptides linked to deoxycholic acid and uses thereof

[0001] One example of the present invention relates to a cancer antigen peptide nanostructure comprising a cancer antigen peptide assembly linked to deoxycholic acid; a method for preparing the same; and a use thereof.

[0002] Immunotherapy is a strategy that utilizes the body's immune system to target and eliminate malignant cells in the fight against cancer. At the heart of this therapy are dendritic cells (DCs), key antigen-presenting cells that bridge the gap between innate and adaptive immunity. Antigens presented by DCs are recognized by the immune system, and during this process, DCs migrate to lymph nodes and interact with immature T cells. DCs effectively control the immune response by regulating T cell activation.

[0003] Among DC-mediated immunotherapy approaches, cancer vaccines aim to enhance therapeutic efficacy by enhancing immune responses to specific tumor antigens. The first DC-mediated vaccine, Sipuleucel-T, was approved by the U.S. Food and Drug Administration (FDA) in 2010. This vaccine induces tumor-specific immune responses by isolating immature DCs from the patient's peripheral blood, maturing them, loading them with antigens, and then reinfusing them. However, the personalized nature of this vaccine necessitates ex vivo manipulation of patient-derived DCs, posing logistical challenges and the potential for hypersensitivity reactions. Therefore, the development of a more universally applicable and effective strategy is needed.

[0004] Developing effective cancer vaccines requires overcoming several hurdles. First, a novel antigen delivery system is needed that can effectively deliver antigens to DCs and address the problem of inadequate antigen loading. Second, tumor-specific antigens must be accurately recognized and delivered. Finally, immunosuppressive cells must be modulated to promote lymphocyte influx. To achieve this, cancer vaccines must be designed to effectively deliver tumor-associated antigens (TAAs) to DCs, induce DC maturation and migration to lymph nodes, and initiate the activation of immature T cells.

[0005] The present inventors hypothesized that effective delivery of survivin antigen, R848, and SD-208 to dendritic cells would induce dendritic cell maturation and migration to lymph nodes, thereby inducing an effective anti-cancer immune response.

[0006] Accordingly, the present invention aims to provide a cancer antigen peptide nanostructure comprising a cancer antigen peptide assembly linked to deoxycholic acid; and a lipid coating the same.

[0007] In addition, the present invention comprises a step of linking a cancer antigen peptide with deoxycholic acid;

[0008] A step of assembling a cancer antigen peptide linked to the above deoxycholic acid; and

[0009] The purpose of the present invention is to provide a method for manufacturing a cancer antigen peptide nanostructure, comprising a step of coating the above assembly with lipid.

[0010] In addition, the present invention aims to provide a vaccine composition for preventing or treating cancer, comprising the cancer antigen peptide nanostructure.

[0011] In addition, the present invention aims to provide a method for preventing or treating cancer using the vaccine composition.

[0012] In addition, the present invention aims to provide a use of the vaccine composition for preventing or treating cancer.

[0013] To achieve the above purpose,

[0014] The present invention provides a cancer antigen peptide nanostructure comprising a cancer antigen peptide assembly linked to deoxycholic acid; and a lipid coating the same.

[0015] In one embodiment of the present invention, the cancer antigen peptide may be survivin.

[0016] In another embodiment of the present invention, the cancer antigen peptide may be an MHC class I binding epitope of survivin.

[0017] In another embodiment of the present invention, the cancer antigen peptide may be composed of an amino acid sequence represented by SEQ ID NO: 1.

[0018] 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).

[0019] In another embodiment of the present invention, an immunomodulator may be additionally loaded onto the cancer antigen peptide nanostructure.

[0020] In another embodiment of the present invention, the immunomodulator 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.

[0021] In another embodiment of the present invention, the nanostructure may increase the expression of one or more selected from the group consisting of CD80 / CD86, MHC I / II, CCR7, TNF-α, IL-6, and IL-12p70 in dendritic cells.

[0022] In another embodiment of the present invention, the nanostructure comprises CD8 + It may induce T cell infiltration into tumors.

[0023]

[0024] In addition, the present invention comprises a step of linking a cancer antigen peptide with deoxycholic acid;

[0025] A step of assembling a cancer antigen peptide linked to the above deoxycholic acid; and

[0026] A method for producing a cancer antigen peptide nanostructure is provided, comprising a step of coating the above assembly with lipid.

[0027] In one embodiment of the present invention, the cancer antigen peptide may be survivin.

[0028] In another embodiment of the present invention, the cancer antigen peptide may be an MHC class I binding epitope of survivin.

[0029] In another embodiment of the present invention, the cancer antigen peptide may be composed of an amino acid sequence represented by SEQ ID NO: 1.

[0030] 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).

[0031] In another embodiment of the present invention, an immunomodulator may be additionally loaded into the assembly.

[0032] In another embodiment of the present invention, the immunomodulator 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.

[0033]

[0034] In addition, the present invention provides a vaccine composition for preventing or treating cancer, comprising the cancer antigen peptide nanostructure.

[0035] 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.

[0036] In addition, the present invention provides a method for preventing or treating cancer using the vaccine composition.

[0037] In addition, the present invention provides a use of the vaccine composition for preventing or treating cancer.

[0038] The inventors of the present invention confirmed that the nanostructure of the present invention effectively stimulates dendritic cell maturation, lymph node migration, and T cell activation. Furthermore, in a cancer cell model, we observed a significant influx of cytotoxic T lymphocytes into the primary tumor, confirming an anti-metastatic effect. Therefore, the nanostructure of the present invention can be usefully utilized as a promising immunotherapy platform applicable to various intractable cancers.

[0039] 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.

[0040] Figures 1a to 1e illustrate the manufacturing method and mechanism of action of DA-L-DSA. Figure 1a illustrates the synthetic procedure of a lipid-coated deoxycholic acid-survivin assembly (DA-L-DSA) loaded with dual adjuvants. Figure 1b shows that subcutaneously injected DA-L-DSA is effectively delivered to dendritic cells, inducing their maturation and migration to lymph nodes, where it activates naïve T cells and induces antigen-specific killing effects. Figure 1c shows the chemical structure of the DS conjugate. Figure 1d shows the results of measuring the CMC of the DS conjugate. Figure 1e shows the hydrodynamic size and zeta potential of L-DSA.

[0041] Figures 2a to 2e show the physicochemical properties of DA-L-DSA. Figure 2a shows the encapsulation efficiency and drug loading degree of SD-208. Figure 2b shows the encapsulation efficiency and drug loading degree of R848. Figure 2c shows the improvement of drug encapsulation efficiency through lipid coating. Figure 2d shows the morphology of DSA, L-DSA, and DA-L-DSA observed using a transmission electron microscope. Figure 2e shows the results of measuring the drug release profile of DA-L-DSA using a UV-vis spectrophotometer.

[0042] Figures 3a to 3d show in vitro evaluations of bone marrow-derived dendritic cell (BMDC) maturation using DA-L-DSA. Figure 3a shows the cellular uptake efficiency of DSA and L-DSA into BMDCs. Figure 3b shows the results of a Western blot analysis for BMDC activation. Figure 3c shows the results of a flow cytometry analysis for BMDC activation. Figure 3d shows the results of an ELISA analysis.

[0043] Figures 4a to 4d show the intracellular and intercellular delivery results of in vivo subcutaneous injection of L-DSA. Figures 4a and 4b show the results 24 hours after injection of Cy5.5-labeled L-DSA. Figure 4a shows a fluorescence image of an ex vivo organ, and Figure 4b shows the quantitative results of the fluorescence signal. Figure 4c is an immunofluorescence image showing the distribution of dendritic cells in an inguinal lymph node. Figure 4d shows the distribution of dendritic cells in the lymph node confirmed by flow cytometry.

[0044] Figures 5a to 5k show the antitumor immune response induced by DA-L-DSA in a survivin-expressing melanoma model. Figure 5a schematically shows the treatment schedule and the average tumor growth profile to verify the antitumor efficacy of DA-L-DSA. Figure 5b shows the growth profile of individual tumors (n=5). Figures 5c to 5d show the results of flow cytometry analysis of dendritic cell maturation in inguinal lymph nodes. Figure 5e shows the results of ELISA quantification of IL-12p70 in lymph nodes. Figures 5f to 5h show the results of CD3 + Tumor-infiltrating lymphocytes and CD8 + Flow cytometric analysis results for cytotoxic T cell recruitment in primary tumors are shown. Figures 5i to 5k show flow cytometric analysis results for regulatory T cells in primary tumors.

[0045] Figures 6a to 6d demonstrate the antitumor and antimetastatic efficacy of DA-L-DSA in a spontaneous metastatic breast cancer model. Figure 6a presents a schematic of the treatment schedule to verify the antitumor and antimetastatic efficacy of DA-L-DSA. Figure 6b shows the average tumor growth profile and the growth profile of individual tumors. Figure 6c shows lung images and H&E staining images. Figure 6d presents the results comparing the number of metastatic nodules.

[0046] Figures 7a to 7g demonstrate the synergistic antitumor efficacy of DA-L-DSA and immune checkpoint inhibitors. Figure 7a depicts a schematic of the treatment schedule to confirm the synergistic effect of DA-L-DSA and conventional immune checkpoint inhibitors, along with the average tumor growth profiles. Figure 7b shows the growth profiles of each tumor (n=5). Figure 7c depicts intratumoral granzyme B. + Figure 7d shows the results of quantifying granzyme B+ cells. Figure 7e shows the flow cytometry images of primary tumors to confirm T cell activation in the tumor microenvironment. Figure 7f shows the TUNEL assay images. Figure 7g shows the TUNEL + Shows the results of quantifying cells.

[0047] Hereinafter, the present invention will be described in detail.

[0048] The present invention provides a cancer antigen peptide nanostructure comprising a cancer antigen peptide assembly linked to deoxycholic acid; and a lipid coating the same.

[0049] As used herein, the term "assembly" refers to a structure formed by self-assembly of cancer antigen peptides linked to deoxycholic acid. The assembly is stably formed through hydrophobic bonds and intermolecular interactions, enhancing the structural stability of the cancer antigen peptides and enabling efficient antigen delivery.

[0050] The term "nanostructure" as used herein refers to a nano-scale structure manufactured using biocompatible materials, which can be utilized for drug delivery, vaccine delivery, and the development of immunotherapeutic agents. The inventors of the present invention constructed a peptide containing an MHC class I binding epitope sequence derived from a tumor-associated antigen called survivin, and linked this peptide to deoxycholic acid to produce a single monomer.

[0051] As used herein, the term "coating" means covering the outer surface of an object with a thin film.

[0052] As used herein, the term "deoxycholic acid" refers to a hydrophobic molecule that can regulate the function of dendritic cells through the TGR5-cAMP-PKA pathway. It is linked to a peptide to increase the binding affinity of the nanostructure to the lipid membrane, while simultaneously contributing to maintaining a stable structure. Activation of TGR5 by deoxycholic acid suppresses excessive dendritic cell activation, thereby reducing excessive inflammatory responses. This suggests that deoxycholic acid may help regulate immune responses, and in particular, may be useful in preventing excessive inflammation, such as cytokine release syndrome, during cancer treatment by making dendritic cells more tolerant.

[0053] As used herein, the term "cancer antigen peptide" refers to a specific peptide sequence among the amino acid sequences of a tumor-specific or tumor-associated antigen that can induce an immune response. The peptide induces an immune response by binding to MHC class I or MHC class II molecules and presenting the antigen to T cells from antigen-presenting cells (APCs).

[0054] Cancer antigen peptides can be derived from tumor-specific antigens (TSAs), which are generally overexpressed in cancer cells or created by mutations, or tumor-associated antigens (TAAs), which are also present in normal tissues but whose expression levels are increased in cancer cells.

[0055] The above cancer antigen peptide is presented by CD8 MHC class I + It can induce cytotoxic T lymphocyte (CTL) responses by T cells or play an immune-assisting role through CD4+ T cells presented by MHC class II.

[0056] In one embodiment of the present invention, the cancer antigen peptide may be survivin.

[0057] In another embodiment of the present invention, the cancer antigen peptide may be an MHC class I binding epitope of survivin.

[0058] As used herein, the term "survivin" refers to a member of the anti-apoptotic protein family, also known as baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5). Survivin expression is involved in regulating functions such as survival, growth, division, and angiogenesis. It is overexpressed in various cancers, including melanoma and triple-negative breast cancer, but is rarely expressed in normal cells.

[0059] As used herein, the term "epitope," also known as an antigenic determinant, refers to a single region of an antigen to which an antibody or T-cell receptor (TCR) specifically binds. Epitopes are key elements of the immune response and are determined by a specific amino acid sequence of an antigenic protein or peptide. Epitopes serve as targets for antibodies or T cells in the immune response, and thus can be utilized in the development of vaccines, antibody therapeutics, and immunotherapies. Epitopes for tumor antigens, in particular, are crucial for inducing cancer-specific immune responses.

[0060] Epitopes can be derived from tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). Tumor-specific antigens are antigens that exist only on cancer cells, while tumor-associated antigens are antigens that are expressed on normal cells, but are overexpressed or abnormally expressed in cancer cells. Epitopes are not limited in type but can be divided into B-cell epitopes and T-cell epitopes. B-cell epitopes are three-dimensional structural regions of an antigen that can be directly recognized and bound by antibodies, while T-cell epitopes are peptides presented by MHC molecules after the antigen is degraded and recognized by the T-cell receptor (TCR).

[0061] Herein, the inventors of the present invention have identified a survivin peptide epitope as a TAA-derived peptide capable of attaching to MHC class I of dendritic cells. (66-74) We designed a cancer therapeutic vaccine that provides anti-cancer properties. We also designed a method to deliver R848 (resiquimod) and SD-208 as adjuvants together with antigen to promote DC maturation and enhance antigen presentation.

[0062] In another embodiment of the present invention, the cancer antigen peptide may be composed of an amino acid sequence represented by SEQ ID NO: 1.

[0063] 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.

[0064] As used herein, the term "lipid" refers to a membrane-forming component used to coat cancer antigen peptide assemblies or to impart structural stability to nanostructures. These lipids are amphiphilic molecules possessing both hydrophilic (head) and hydrophobic (tail) portions, and their physicochemical properties aid in the self-assembly and stabilization of nanostructures.

[0065] 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).

[0066] In another embodiment of the present invention, an immunomodulator may be additionally loaded onto the cancer antigen peptide nanostructure.

[0067] As used in the specification, the term "bearing" means mounted in any internal location.

[0068] The term "immunomodulator" as used herein refers to a substance that affects the biological interaction between immune cells and cancer cells, and is a concept that includes immunosuppressants and immunopotentiators.

[0069] In another embodiment of the present invention, the immunomodulator 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.

[0070] According to one embodiment of the present invention, there is no limitation on the type of the immunosuppressant, and for example, it may be an immune checkpoint inhibitor that inhibits immune checkpoint proteins such as PD1, PDL-1, PD-L2, CTLA-4, LAG-3, BTLA, B7H3, B7H4, 4-1BB (CD137), TIM3, KIR, etc.

[0071] According to one embodiment of the present invention, there is no limitation on the type of the immunostimulant, but it may be, for example, an immunostimulant such as monophosphoryl lipid A (MPLA), aluminum salt (alum), or CpG oligodeoxynucleotide (ODN).

[0072] As used herein, the term "adjuvant" 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 or SD-208.

[0073] As used herein, the term "R848 (Resiquimod)" refers to a compound with immunomodulatory and antiviral properties that interacts with Toll-like receptor 7 (TLR7) and Toll-like receptor 8 (TLR8). These Toll-like receptors are pattern recognition receptors that detect pathogens or abnormalities in the external environment and regulate the immune response. In DCs, R848 activates TLR7 and TLR8, enhancing antigen expression and presentation.

[0074] 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).

[0075] 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 immunomodulatory agent that enhances the immune response.

[0076] 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.

[0077] 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.

[0078] 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).

[0079] As used herein, the term "SD-208" is a kinase inhibitor targeting the TGF-β type 1 receptor (TGF-βR1), which blocks the downstream mechanisms that occur when TGF-β is delivered to cells. Previous studies have reported that TGF-β inhibits dendritic cell maturation, antigen presentation, and T cell activation.

[0080] The above TGF-β (transforming growth factor-β) refers to the family of TGF-β1, TGF-β2, and TGF-β3, which are pleiotropic regulatory factors for immune responses, inflammatory responses, cell growth, and cell differentiation. The above TGF-β is a representative immunosuppressive factor for tumor sites and is known to induce epithelial-mesenchymal transition (EMT) in cancer cells.

[0081] The TGF-β inhibitor is not limited in type as long as it inhibits the activity of the TGF-β protein or the expression of the protein or mRNA, but may be at least one selected from the group consisting of compounds, natural products, antibodies, aptamers, siRNA, shRNA, miRNA, ribozymes, DNAzymes, PNA (peptide nucleic acids), antisense oligonucleotides, and peptides that specifically bind to the TGF-β.

[0082] The above TGF-β inhibitor may be a TGF-β receptor inhibitor. The TGF-β receptor inhibitor specifically binds to the TGF-β receptor and inhibits the TGF-β signaling pathway through the receptor.

[0083] The above TGF-β receptor inhibitor is not limited in type as long as it inhibits the activity of the TGF-β receptor protein or the expression of the protein or mRNA, but may be at least one selected from the group consisting of compounds, natural products, antibodies, aptamers, siRNA, shRNA, miRNA, ribozymes, DNAzymes, PNA (peptide nucleic acids), antisense oligonucleotides, and peptides that specifically bind to the TGF-β receptor.

[0084] The above TGF-β receptor inhibitor may be a TGF-β type 1 receptor (TGF-βR1) inhibitor (e.g., a TGF-β type 1 receptor kinase inhibitor), specifically, SD-208 (2-(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-yl-amine), SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), LY2109761 (4-(2-((4-(2-(pyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl)quinolin-7-yl)oxy)ethyl)morpholine), It may be at least one selected from the group consisting of IN-1130 (3-[[5-(6-methylpyridin-2-yl)-4-quinoxalin-6-yl-1H-imidazol-2-yl]methyl]benzamide), LY2157299 (Galunisertib, 4-[2-(6-methylpyridin-2-yl)-5,6-dihydro-4H-pyrrolo[1,2-b]pyrazol-3-yl]quinoline-6-carboxamide), TEW-7197 (2-fluoro-N-[[5-(6-methylpyridin-2-yl)-4-([1,2,4]triazolo[1,5-a]pyridin-6-yl)-1H-imidazol-2-yl]methyl]aniline), and Ki26894, and more specifically, it may be SD-208.

[0085] In another embodiment of the present invention, the nanostructure may increase the expression of one or more selected from the group consisting of CD80 / CD86, MHC I / II, CCR7, TNF-α, IL-6, and IL-12p70 in dendritic cells.

[0086] In another embodiment of the present invention, the nanostructure comprises CD8 + It may induce T cell infiltration into tumors.

[0087]

[0088] In addition, the present invention comprises a step of linking a cancer antigen peptide with deoxycholic acid;

[0089] A step of assembling a cancer antigen peptide linked to the above deoxycholic acid; and

[0090] A method for producing a cancer antigen peptide nanostructure is provided, comprising a step of coating the above assembly with lipid.

[0091] The above terms cancer antigen peptide, deoxycholic acid, assembly, lipid, nanostructure, etc. are as described above.

[0092] In one embodiment of the present invention, the cancer antigen peptide may be survivin.

[0093] In another embodiment of the present invention, the cancer antigen peptide may be an MHC class I binding epitope of survivin.

[0094] In another embodiment of the present invention, the cancer antigen peptide may be composed of an amino acid sequence represented by SEQ ID NO: 1.

[0095] 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).

[0096] In another embodiment of the present invention, an immunomodulator may be additionally loaded into the assembly.

[0097] In another embodiment of the present invention, the immunomodulator 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.

[0098]

[0099] In addition, the present invention provides a vaccine composition for preventing or treating cancer, comprising the cancer antigen peptide nanostructure.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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.

[0104] The above pharmaceutical composition may be provided as a pharmaceutical composition containing the active ingredient alone or including one or more pharmaceutically acceptable excipients or diluents.

[0105] When the above pharmaceutical composition is formulated, it can be prepared 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 prepared 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] The nanostructure of the present invention can be administered alone or in combination with other therapeutic agents, and in the case of combination administration, administration can be sequential or simultaneous. At this time, there is no limitation on the type of other therapeutic agents, but for example, 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. can be used.

[0110] The nanostructure of the present invention may further comprise a formulation along with the immunomodulator. 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 device and method.

[0111] 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.

[0112]

[0113]

[0114] 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.

[0115]

[0116] 1. Experimental materials and experimental methods

[0117] 1.1 Experimental materials

[0118] Deoxycholic acid (DCA)-survivin(66-74) (DS, DCA-Gly-Trp-Glu-Pro-Asp-Asp-Asn-Pro-Ile) was synthesized by GL Biochem Ltd. (Shanghai, China). 1,2-Dipalmitoyl- sn -glycero-3-phosphocholine (DPPC) and 1,2-distearoyl- sn -glycero-3-phosphoethanolamine-N-[amino(polyethyleneglycol)-2000] (DSPE-PEG2000) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). R848, SD-208, pyrene, Cy5.5, and CCK-8 assay kits were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), and RPMI1640 medium were purchased from Welzen (Seoul, South Korea). Mouse recombinant GM-CSF, IL-4, DNase I solution, and collagenase / hyaluronidase were purchased from STEMCELL Technologies (Vancouver, Canada). Anti-mouse CD80, CD40, CD86, CCR7, MHC I, MHC II, CD3, CD8, and IFN-γ antibodies were purchased from Biolegend (San Diego, CA, USA). Anti-mouse CD45 antibody was purchased from BD Biosciences (Franklin Lakes, NJ, USA). β-Mercaptoethanol, Hanks' balanced salt solution (HBSS), and mouse ELISA kits (IL-12p70, IL-6, and TNF-α) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The DeadEnd™ fluorometric TUNEL system was obtained from Promega (Madison, WI, USA).

[0119]

[0120] 1.2 Determination of the critical micelle concentration (CMC) of deoxycholic acid (DCA)-survivin(66-74)(DS)

[0121] To determine the CMC (Critical Micelle Concentration) of DS, various concentrations of DS (0–500 μg mL -1 ) in pyrene solution (25 ng mL -1 ) were incubated for 1 hour. The fluorescence intensity of each solution (excitation at 330 nm) was measured using a plate reader. The critical micelle concentration (CMC) of DS was determined using the ratio between the fluorescence intensities measured at 373 nm and 384 nm.

[0122]

[0123] 1.3 Preparation of lipid-coated DCA-survivin assemblies (DA-L-DSA) containing dual adjuvants

[0124] DSPE-PEG 2000 A mixture of DPPC and lipids was dissolved in chloroform at a molar ratio of 1:3 and evaporated by stirring at room temperature for 30 minutes. Then, the prepared lipid mixture was dissolved in water (4% EtOH, 10 mL) at a concentration of 0.2 mg / mL. -1 Added and stirred gently. DS (1 mg), R848 (0.15 mg) and SD-208 (0.15 mg) were added to dichloromethane (1 mg mL -1 ) was dissolved in water. The premixed adjuvant / DS solution was slowly dropped into the lipid solution (1.25 mL), sonicated, and then evaporated to remove the organic solvent.

[0125]

[0126] 1.4 Physicochemical properties of DA-L-DSA

[0127] To optimize the lipid / DCA-survivin and adjuvant / DCA-survivin assembly (DSA) ratios, the hydrodynamic size, zeta potential, and polydispersity index (PDI) of DA-L-DSA were measured using a Zetasizer-Nano ZS (Malvern Instrument, Worcestershire, UK). The morphological characteristics of DA-L-DSA were visualized by transmission electron microscopy.

[0128]

[0129] 1.5 Drug loading and in vitro release profiling

[0130] The drug loading and encapsulation efficiency of R848 and SD-208 were calculated by measuring the absorbance at 320 nm and 370 nm, respectively, using DA-L-DSA (0.25 mg). 1 mg mL¹ DA-L-DSA was resuspended in PBS (with or without 10% serum), and the released media were collected by centrifugation at 2, 4, 6, 12, 24, 48, 72, and 120 h. The released media and nanoassemblies were lyophilized and resuspended, and R848 and SD-208 were detected at 320 nm and 370 nm, respectively.

[0131]

[0132] 1.6 Cell culture

[0133] To obtain mouse bone marrow-derived dendritic cells (BMDCs), bone marrow cells were collected from the femurs and tibias of 6- to 10-week-old C57BL / 6 mice (Orient Bio, Korea). Bone marrow contents were washed with HBSS. The collected cells were washed twice with HBSS and then cultured in 1% penicillin-streptomycin (100 U mL -1 ), 10% FBS, IL-4 (20 ng mL -1 ), GM-CSF (20 ng mL -1 ), and cultured in RPMI1640 supplemented with β-mercaptoethanol (55 nM) at 37°C for 6 days. On day 6, cells were sorted using a magnetic EasySep mouse CD11c positive selection kit (STEMCELL Technologies, USA).

[0134] B16F10 (murine melanoma cell line) was obtained from ATCC (Virginia, USA). 1% penicillin-streptomycin (100 U mL -1 ), DMEM supplemented with 10% FBS was used for B16F10 cell culture. Cells were cultured in a 5% CO2, 37℃ environment.

[0135]

[0136] 1.7 In vitro cell viability test

[0137] Mouse bone marrow-derived dendritic cells (BMDCs) were seeded in 96-well plates and cultured at 37°C for 24 h. BMDCs were then treated with DA-L-DSA and other nanoassembly groups at concentrations of 0–20 μM for 24 h, followed by incubation with a CCK-8 solution for 30 min. Cell viability relative to the control group was then measured using a UV / Vis spectrophotometer (Tecan) at 450 nm.

[0138]

[0139] 1.8 Cellular uptake and confocal microscopy imaging

[0140] BMDC(2 Х 10 5 Cell mL -1 ) with Cy5.5-loaded nanoassemblies at 1.5 μg mL -1 After incubation for 2 h at a concentration of 100 μg / ml, the cells were analyzed using a FACS Calibur (BD Biosciences, USA). Cells were stained with DAPI (Southern Biotech) and visualized using a confocal microscope (Leica).

[0141]

[0142] 1.9 Measurement of in vitro dendritic cell maturation

[0143] BMDC(2 Х 10 5 Cell mL -1 ) at 1.5 μg / mL -1 The cells were cultured with nanoassemblies at a concentration of 10 μM for 24 h. The cells were then stained with anti-mouse CD80, anti-mouse CD86, anti-mouse CD40, anti-mouse MHCI, anti-mouse MHCII, and anti-mouse CCR7. The cells were washed twice and resuspended in ice-cold PBS (10% FBS), and analyzed using a FACS Calibur (BD Biosciences, USA).

[0144]

[0145] 1.10 Western Blot

[0146] Cells were lysed in RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich) and centrifuged. Whole cell lysates were resuspended in SDS sample buffer and resolved using SDS-PAGE. Proteins were transferred to Immobilon-P PVDF membranes (Sigma-Aldrich) and blocked with 5% bovine serum albumin (BSA) / TBST buffer for 1 h at room temperature. The membranes were incubated overnight with primary antibodies. The primary antibodies used were anti-mouse CD80 (1:1,000, Abcam, Boston, MA), anti-mouse CD86 (1:1,000, Cell Signaling, Massachusetts, MA), anti-mouse, and mouse anti-GAPDH (1:1,000, Abcam). The membranes were incubated with secondary antibodies for 1 h at room temperature. Anti-rabbit HRP-linked antibody (1:2,000, Cell Signaling) was used as the secondary antibody. The membrane was captured using ChemiDoc XRS+ (Bio-Rad, CA, USA).

[0147]

[0148] 1.11 Measurement of cytokine levels in vitro

[0149] BMDC(2 Х 10 5 Cell mL -1 ) with DA-L-DSA and other nano-assembly groups at 1.5 μg mL -1 The cells were cultured at a concentration of 100 μg / ml for 24 h. Then, the culture medium supernatant was obtained from each group and centrifuged at 13,000 rpm for 5 min at 4°C. The protein expression levels of IL-12p70, IL-6, and TNF-α in the supernatant samples were quantified using ELISA kits (Invitrogen, USA).

[0150]

[0151] 1.12 In vivo biodistribution test

[0152] After subcutaneous injection of Cy5.5-loaded nanoassemblies into mice, the liver, spleen, heart, lungs, and inguinal lymph nodes were collected for fluorescence signal analysis 24 hours later. The isolated major organs were analyzed using a FOBI device.

[0153]

[0154] 1.13 In vivo cytotoxic T lymphocyte antigen-specific killing assay

[0155] C57BL / 6 mice were administered 20 μg of DS (DCA-survivin) (66-74) The conjugate) was administered subcutaneously, and the experimental groups were organized according to the drug loading (%) of DA-L-DSA, and a total of three vaccinations were administered at 5-day intervals. Seven days after the final vaccination, spleen cells from untreated healthy mice were collected and treated with 1 μg of survival peptide or PBS for approximately 4 hours. Then, the surviving treated spleen cells and PBS-treated spleen cells were labeled with 5 μM and 0.5 μM carboxyfluorescein succinimidyl ester (CFSE), respectively. Then, equal numbers of each spleen cell were mixed and intravenously injected into each immunized mouse. After 18 hours, CFSE was detected in the spleen of each mouse. [높음] and CFSE [low] The percentage of antigen-specific killing ability was detected by flow cytometry and calculated according to the following formula:

[0156]

[0157] Specific killing ability (%) = [ 1- (CFSE [high] / CFSE [low] ) ] Х 100 %

[0158]

[0159] 1.14 In vivo therapeutic effects on mouse melanoma and breast cancer models.

[0160] 2 Х 10 C57BL / 6 mice aged 6 to 10 weeks 5A murine melanoma model was established by subcutaneously inoculating B16F10 cells into the left flank. A spontaneous metastatic breast cancer model was established in 6- to 10-week-old Balb / c mice. Mice were injected with 2 х 10 5 4T1 cells were inoculated subcutaneously into the fourth mammary gland. Nanoassemblies containing 20 μg of survivin were injected subcutaneously into the right flank, and tumor size (mm 3 ) is (length) Х (width) 2 It was calculated as Х 1 / 2. The experiment was stopped on day 16. In the breast cancer model, surgical resection was performed on day 16 to further observe the anti-metastatic effect, and the metastatic lung was isolated and observed on day 30.

[0161] All animal experiments were performed in accordance with protocols approved by the Hanyang University Institutional Animal Care and Use Committee and registered under number 2022-0051A.

[0162]

[0163] 1.15 Analysis of tumor-infiltrating lymphocytes (TILs) in primary tumors

[0164] On day 20, mice from each group were sacrificed to analyze tumor-infiltrating lymphocytes. Tumor tissue was collected and digested with DNase I solution and collagenase / hyaluronidase. The digested tumor tissue was filtered through a 70 μm strainer and centrifuged at 300 g for 10 minutes at room temperature. Red blood cells (RBCs) were lysed, and the cells were washed twice with phosphate-buffered saline (PBS). The cells were then fixed with 4% paraformaldehyde (Wako, Japan) for 15 minutes and permeabilized with cell permeabilization buffer (ThermoFisher Scientific, USA) for 10 minutes at room temperature. The cells were then stained with anti-mouse CD45, anti-mouse CD3, anti-mouse CD8, and anti-mouse IFN-γ antibodies. The cells were washed twice and resuspended in ice-cold PBS supplemented with 10% fetal bovine serum (FBS) for analysis using a FACS Calibur (BD Biosciences, USA). Paraffin sections of the tumor tissue were prepared for immunofluorescence analysis. Paraffin sections were deparaffinized, permeabilized, and stained with anti-mouse CD3 and anti-mouse CD8 antibodies. After washing the sections twice, cell nuclei were stained with DAPI solution. Tumor cells were also stained according to the protocol using the DeadEnd◎ fluorescent TUNEL system to detect apoptosis. Slides were examined under a fluorescence microscope.

[0165]

[0166] 1.16 Statistical Analysis

[0167] All data are expressed as mean ± SD and SEM. Statistical analyses were performed using the Student's t test and one-way ANOVA with Tukey's post hoc test in GraphPad Prism 8 Project software for Windows (GraphPad Software). All p values ​​were considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant).

[0168]

[0169] 2. Results

[0170] 2.1. Manufacturing of DA-L-DSA

[0171] The fabrication process of a nano self-assembly system capable of delivering both a tumor-associated antigen (TAA) and an adjuvant is illustrated in Figure 1a. Deoxycholic acid, composed of hydrophilic molecules and a hydrophobic cyclopentanophenanthrene nucleus, can form micelles in aqueous solutions. Therefore, when a relatively hydrophilic tumor-associated antigen (TAA) peptide binds to deoxycholic acid, it forms a micellar structure, exhibiting amphipathic properties. Furthermore, two types of immunoadjuvants (dual adjuvants; DA) were loaded into the micelle structure to enhance the immune response (Figure 1b).

[0172] Specifically, we generated deoxycholic acid-survivin (DS) by conjugating the survivin peptide epitope (66-74), a TAA that binds to MHC class I of antigen-presenting cells, to deoxycholic acid (Fig. 1c). The amphipathic DS peptide monomers readily self-assembled to form a core-shell peptide assembly (DSA). DA-DSA was prepared by loading the DSA with two adjuvants, R848 and SD-208. R848 activates immune cells through the TLR7 / 8 MyD88-dependent signaling pathway, which induces interferon (IFN)-α and interleukin (IL)-12. SD-208, a transforming growth factor β receptor (TGF-βR) I kinase inhibitor, enhances dendritic cell and tumor-specific cytotoxic T cell (CTL) responses and stimulates antitumor natural killer cells. The surface of this DA-DSA was coated with a DPPC / DSPE-PEG-based lipid membrane to complete DA-L-DSA. DSA was coated with a lipid layer composed of DPPC: DSPE-PEG2000 = 3:1 (L-DSA).

[0173] As a result, the amphiphilic DS peptide monomers readily self-assembled to form core-shell peptide assemblies (DSA), which exhibited a critical micelle concentration (CMC) of 133.25 μg mL-1 in PBS (pH 7.4) (Fig. 1d). The hydrodynamic size and zeta potential were measured at various weight ratios of lipid to DS. When the weight ratio of lipid to DS was 25%, the zeta potential was -13.50 ± 2.40 mV and the Z-average size was 209.73 ± 1.04 nm, which were increased compared to DSA at -29.4 ± 1.20 mV and 177 ± 0.96 nm, respectively (Fig. 1e).

[0174]

[0175] The drug loading and encapsulation efficiencies of R848 and SD-208 were measured at various weight ratios of adjuvants to L-DSA. Both adjuvants were loaded into L-DSA and detected at different wavelengths. As a result, it was found that 15% of adjuvant to L-DSA was optimal for maximum loading of both adjuvants (Figs. 2a and 2b). The drug encapsulation efficiency was further improved by coating the DSA surface with lipids. DA-L-DSA showed 1.8- and 3.8-fold higher encapsulation efficiencies than R848 and SD-208, respectively (Fig. 2c). Transmission electron microscopy (TEM) images confirmed the presence of a lipid layer on the surface of L-DSA compared to DSA. In addition, it was observed that the spherical shape was maintained even when the adjuvant was loaded (Fig. 2d).

[0176] The release profiles of R848 and SD-208 were monitored in PBS (pH 7.4) and PBS containing 10% serum, with fresh media replaced at each time point. DA-L-DSA exhibited a slightly faster release in serum-containing media, demonstrating a sustained release pattern of both adjuvants without an initial burst. R848 and SD-208 were released from DA-L-DSA by 57.19 ± 7.57% and 62.52 ± 8.71%, respectively, after 120 h at 37°C in serum-containing media (Fig. 2e).

[0177]

[0178] 2.2. Confirmation of dendritic cell maturation using DA-L-DSA in vitro

[0179] To determine the maximum non-toxic concentration (NOC) for further experiments, the in vitro cytotoxicity of L-DSA and DA-L-DSA was measured using the CCK-8 assay using bone marrow-derived dendritic cells (BMDCs) cultured at various concentrations. Neither L-DSA nor DA-L-DSA exhibited cytotoxicity at concentrations up to 100 μM. Therefore, the treatment concentration was set at 100 μM for in vitro experiments. To evaluate the contribution of the lipid membrane coated on the nanoassemblies to enhancing the delivery efficiency to dendritic cells, BMDCs were treated with DSA and L-DSA encapsulated with Cy5.5. Flow cytometry analysis revealed that the MFI value of L-DSA was 3.03-fold higher than that of DSA (Fig. 3a).

[0180] To determine whether DA-L-DSA can effectively mature dendritic cells and induce T cell activation, BMDCs were treated with DA-L-DSA for 24 hours, and Western blot and immunohistochemical analyses were performed. As a result, CD80 / 86 expression in the DA-L-DSA-treated group was similar to that in the LPS-treated group used as a positive control (Fig. 3b). In addition, flow cytometry analysis confirmed that the expression of CD80, CD86, CD40, MHC I, MHC II, and CCR7 was significantly higher in the DA-L-DSA-treated group compared to the untreated group, the group treated with only the adjuvant, and the L-DSA-treated group (Fig. 3c).

[0181] Additionally, the DA-L-DSA treatment group showed the highest levels of IL-12p70, IL-6, and TNF-α release, which are known to activate naïve T cells, as measured by ELISA, and was almost the same level as the positive control group, the LPS treatment group (Fig. 3d).

[0182]

[0183] 2.3. Confirmation of L-DSA migration into lymph nodes and lymph node-resident dendritic cells in vivo

[0184] To evaluate the ability of DA-L-DSA to promote an effective antitumor immune response, L-DSA and DSA were injected subcutaneously into mice. After 24 hours, the biodistribution of Cy5.5-labeled L-DSA and DSA was investigated in vivo. We hypothesized that the subcutaneously injected nanoassemblies would be partially degraded and some would be delivered to antigen-presenting cells. Among these cells, dendritic cells would be activated and migrate to lymph nodes. Indeed, L-DSA exhibited significantly higher lymph node localization than DSA (Figures 4a and 4b).

[0185] To confirm that the observed signal was due to the migration of dendritic cells delivered with nanoassemblies to the lymph nodes, we evaluated the distribution of dendritic cells using immunofluorescence staining and flow cytometry. As a result, the overlap between the dendritic cell marker CD11c and the fluorescent label Cy5.5 of the nanoassemblies appeared yellow, indicating that the nanoassemblies were localized in the lymph nodes. The L-DSA-treated group showed a significantly brighter signal than the DSA-treated group (Fig. 4c). Similarly, flow cytometry analysis of the lymph nodes showed that the L-DSA-treated group had a significantly higher distribution of dendritic cells with Cy5.5 signals than the DSA-treated or untreated groups (Fig. 4d).

[0186]

[0187] 2.4. Analysis of the antitumor and anti-metastatic efficacy of DA-L-DSA in survivin-expressing cancer.

[0188] To evaluate the efficacy of DA-L-DSA in inducing an antitumor immune response against survivin-expressing cancer, a melanoma allograft mouse model was utilized. DA-L-DSA was administered subcutaneously to mice on days 7 and 10 after tumor inoculation. All groups were sacrificed on day 16. Results showed that the primary tumor size in the DA-L-DSA-treated group was 3.52 times smaller than that in the untreated group (Figures 5a and 5b).

[0189] To confirm the anti-tumor immune response by DA-L-DSA, lymph nodes were collected from mice, and flow cytometry and ELISA were performed to determine the degree of maturation and activation of dendritic cells distributed within the lymph nodes. The flow cytometry results showed that the number of activated dendritic cells in the lymph nodes was 4.64 times higher in the dual adjuvant-treated group, 5.79 times higher in the DA-L-DSA-treated group, and 16.84 times higher in the DA-L-DSA-treated group than in the untreated group (Fig. 5c and 5d). In addition, the ELISA results confirmed that IL-12p70, a T cell activation indicator derived from activated dendritic cells, was 1.84 times higher in the DA-L-DSA-treated group than in the untreated group (Fig. 5e). In addition, primary tumors were collected from mice for flow cytometry and immunohistochemical analyses to determine the degree of T cell infiltration and immune regulation. Flow cytometry analysis confirmed immune activation due to an intratumoral increase in tumor-infiltrating lymphocytes (TILs) (Fig. 5g), among which CD8 + T cells infiltrated 4.21 times more than the untreated group (Fig. 5f and 5h). In addition, immunofluorescence staining images of the primary tumors also showed that DA-L-DSA infiltrated intratumoral CD8 + It was demonstrated that DA-L-DSA increased the recruitment of T cells. In addition, the proportion of regulatory T cells (Treg) in tumors of the DA-L-DSA-treated group was 3.33 times lower than that of the untreated group (Fig. 5i and 5j). Overall, CD8 cells in the tumor microenvironment + The ratio of T cells and Tregs was significantly higher in the DA-L-DSA group than in the other groups (Fig. 5k).

[0190]

[0191] To verify the efficacy of DA-L-DSA in metastatic cancer expressing survivin, a metastatic breast cancer model was established by injecting 4T1 cells into the fourth mammary gland of Balb / c mice. After cancer cell inoculation, the cancer therapeutic vaccine DA-L-DSA was administered three times at three-day intervals, starting on day 6. Tumor tissue was surgically removed on day 16, and the lungs were examined on day 30 to assess the extent of metastasis (Fig. 6a).

[0192] Consistent with the results in the murine melanoma model, the primary tumor size in the DA-L-DSA-treated group in the metastatic breast cancer model was 3.60 times smaller than that in the untreated group (Fig. 6b). Furthermore, the anti-metastatic effect of DA-L-DSA treatment was also confirmed through the experiment. While the untreated group had an average of 56.75 metastatic nodules, the DA-L-DSA-treated group had 7.5 and 7.57 times fewer nodules, respectively (Figs. 6c and 6d).

[0193]

[0194] 2.5. Confirmation of antitumor efficacy through combined administration of immune checkpoint inhibitors and DA-L-DSA.

[0195] As DA-L-DSA administration has been shown to increase the influx of cytotoxic T cells and decrease Tregs in various cancers expressing survivin, we further investigated whether DA-L-DSA treatment could improve the response rate of immune checkpoint inhibitors by overcoming the limited response rate of existing immune checkpoint inhibitors. To this end, anti-PD-1 antibodies were administered every 3 days starting on day 4 after tumor inoculation in an allograft mouse melanoma model, followed by two doses of DA-L-DSA (Fig. 7a). As a result, the combination therapy of DA-L-DSA and anti-PD-1 antibodies barely inhibited tumor growth (Fig. 7b). Immunofluorescence staining showed that the DA-L-DSA / anti-PD-1 combination treatment group significantly produced granzyme B, indicating the activation of cytotoxic T cells within the tumor (Figs. 7c and 7d). In the flow cytometry analysis results, the DA-L-DSA / anti-PD-1 combination treatment group showed higher IFN-γ levels than the anti-PD-1 monotherapy group. + CD8 + The number of T cells was induced 1.98 times more in the DA-L-DSA monotherapy group and 1.7 times more in the CD8 group. + IFN-γ secretion by T cells promotes effector responses and enhances antitumor efficacy (Fig. 7e). Tumor cell apoptosis was confirmed through TUNEL analysis of tumor tissues. Apoptosis was induced in both DA-L-DSA and anti-PD-1 treatment groups, and the combination treatment group showed the highest number of apoptotic tumor cells. These results suggest that combination therapy may increase the infiltrated CD8 + This suggests that T cells have antitumor properties (Fig. 7f and Fig. 7g).

[0196]

[0197] 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. A cancer antigen peptide nanostructure comprising a cancer antigen peptide assembly linked to deoxycholic acid; and a lipid coating the same.

2. A cancer antigen peptide nanostructure according to claim 1, characterized in that the cancer antigen peptide is survivin.

3. A cancer antigen peptide nanostructure, wherein the cancer antigen peptide in claim 1 is an MHC class I binding epitope of survivin.

4. A cancer antigen peptide nanostructure, characterized in that the cancer antigen peptide in claim 1 is composed of an amino acid sequence represented by sequence number 1.

5. 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 cancer antigen peptide nanostructure characterized by comprising at least one selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) (18:1 PA).

6. A cancer antigen peptide nanostructure, wherein an immunomodulator is additionally loaded onto the cancer antigen peptide nanostructure in claim 1.

7. A cancer antigen peptide nanostructure, characterized in that in claim 6, the immunomodulator 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.

8. A cancer antigen peptide nanostructure according to claim 1, characterized in that the nanostructure increases the expression of at least one selected from the group consisting of CD80 / CD86, MHC I / II, CCR7, TNF-α, IL-6, and IL-12p70 in dendritic cells.

9. In claim 1, the nanostructure is CD8 + A cancer antigen peptide nanostructure characterized by inducing intratumoral infiltration of T cells.

10. A step of linking a cancer antigen peptide with deoxycholic acid; A step of assembling a cancer antigen peptide linked to the above deoxycholic acid; and A method for producing a cancer antigen peptide nanostructure, comprising the step of coating the above assembly with lipid.

11. A manufacturing method according to claim 10, characterized in that the cancer antigen peptide is survivin.

12. A manufacturing method according to claim 10, wherein the cancer antigen peptide is an MHC class I binding epitope of survivin.

13. A manufacturing method according to claim 10, characterized in that the cancer antigen peptide is composed of an amino acid sequence represented by sequence number 1.

14. In claim 10, 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 manufacturing method characterized in that claim 10 further includes a step of loading an immunomodulator into the assembly.

16. A manufacturing method according to claim 15, characterized in that the immunomodulator 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.

17. A vaccine composition for preventing or treating cancer, comprising a cancer antigen peptide nanostructure according to any one of claims 1 to 9.

18. A vaccine composition for preventing or treating cancer, characterized in that in claim 17, 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.

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