Anti-cancer vaccine composition comprising an adjuvant consisting of a peptide or lipopeptide derived from a tumor-associated antigen and an immunoactive substance, and use thereof

Abstract

The present invention relates to an anti-cancer vaccine composition comprising a peptide derived from a tumor-associated antigen (TAA) and an adjuvant consisting of a lipopeptide and an immunoactive substance; and its use. Specifically, the peptide derived from the tumor-associated antigen specifically binds to human leukocyte antigen (HLA), and the combination of peptides having the above characteristics is mixed with an adjuvant in an optimal ratio to prepare a vaccine composition, which is used to prevent or treat cancer.

Description

[Technical field]

[0001] 1. Field of the Invention The present invention relates to an anti-cancer vaccine composition comprising a peptide and a lipopeptide derived from a tumor-associated antigen (TAA) and an adjuvant consisting of an immunoactive substance, and the use thereof. Specifically, the present invention relates to an anti-cancer vaccine consisting of a peptide, a lipopeptide and a poly(I:C) adjuvant that specifically binds to a human leukocyte antigen (HLA), and to the use of a pharmaceutical composition comprising the anti-cancer vaccine as an active ingredient as a preventive or therapeutic agent against cancer. [Background technology]

[0002] 2. Description of Related Art Cancer is characterized by uncontrolled cell proliferation, the ability to invade nearby tissues, and the ability to metastasize to other distant tissues, compared with normal cells.In modern times, with the development of science and medical technology, many diseases have become treatable, and incurable diseases have almost disappeared.However, unlike other diseases, cancer requires very complicated and strict treatment, and the treatment is not completely effective.

[0003] Anticancer treatments are performed by physically removing cancer tissue, suppressing the expression of genes required for cancer cell survival, such as anti-apoptotic molecules, telomerase, growth factor receptor genes, and signaling molecules, or inducing apoptosis via irradiation or administration of anticancer drugs (Knudson AG, Nature reviews. Cancer 1(2): 157-162, 2001).

[0004] However, the problem with this treatment is that it affects not only cancer cells but also normal cells, causing side effects. Therefore, the need for anti-cancer vaccines as an alternative method of treating cancer has emerged.

[0005] Anti-cancer vaccines are vaccines made to attack cancer cells by memorizing antigens specific to cancer cells in the human immune system, and there are various types of antigens and antigen delivery methods (e.g., peptides, DNA, RNA, dendritic cells). Unlike traditional prophylactic vaccines that prevent external viral or bacterial infections, vaccines for cancer treatment should induce immune responses against self-antigens rather than non-self-antigens. Furthermore, vaccines should be effective when administered after cancer formation and exert immune effects to suppress cancer, and should present as many different types of cancer antigens as possible, since the expression rate of each cancer antigen is different even in patients with the same cancer type.

[0006] In fact, many types of tumor-associated antigens are immune self antigens and immune tolerance antigens, so it is difficult to induce specific immune response.These limitations can be overcome by inducing immune response to antigens that are commonly expressed in various cancer types or antigens that are highly expressed in one cancer type.In recent years, antigens that may be common to various cancer types and antigens that are highly expressed in one cancer type have been reported.

[0007] Tumor-associated antigens (TAA), targets of anti-cancer vaccines, are classified as follows: A) Cancer-Testis Antigens: Antigens belonging to this group are not expressed in normal tissues other than testicular spermatocytes / spermatogonia, but are highly expressed in cancer tissues, and therefore were initially called Cancer-Testis (CT) antigens. As testicular cells do not express class I or II HLA molecules, these antigens are not recognized by T cells on normal cells and can therefore be considered immunologically tumor-specific. Well-known examples of CT antigens include MAGE members and NY-ESO-1. B) Differentiation Antigens: These TAAs are proteins expressed during differentiation of cells of a particular tissue and are antigens shared between tumors and the normal cells from which they originate. A representative example of a differentiation antigen is Melan-A / MART-1, found in melanomas and normal melanocytes. This melanocyte lineage-associated protein is involved in the biosynthesis of melanin and, although not tumor-specific, is widely used in cancer immunotherapy. Other examples include tyrosinase for melanoma or prostate-specific antigen (PSA) for prostate cancer. C) Overexpressed TAAs: These are TAAs that generally show low expression levels in normal tissues and are widely expressed in different types of tumors. Many peptide epitopes that can be processed and presented by antigen-presenting cells (APCs) in normal tissues may be below the threshold level of T cell recognition and therefore do not induce an immune response in normal cells, but their overexpression in tumor cells can initiate an anti-cancer response by breaking previously established tolerance. Representative examples of TAAs in this group include Her-2 / neu, survivin, telomerase, or WT-1 (Wilms' tumor 1). D) Tumor-specific antigens: These unique TAAs are caused by mutations in normal genes (such as β-catenin, CDK4, EGFRvIII, etc.). Some of these molecular alterations are associated with tumor transformation and / or progression. Tumor-specific antigens are generally able to induce a strong immune response without the risk of an autoimmune response against normal tissues. On the other hand, in most cases, tumor-specific antigens are generally not shared among many individual cancers, but are only expressed in a particular cancer.E) TAAs caused by aberrant post-translational modifications: These TAAs are neither specific nor overexpressed, but may arise in proteins that are primarily associated with tumors through active post-translational processes. Examples of this class result from events such as protein splicing during degradation that may or may not be tumor-specific, or, in the case of MUC-1 (mucin 1), from altered glycosylation patterns that result in new epitopes in tumors.

[0008] There are methods that use tumor cell lysate, protein, DNA / RNA, plasmid, and peptides that are T cell epitopes as antigen forms of anti-cancer vaccines.Among them, peptide epitopes are the most effective substances that activate antigen-specific T cells to kill cancer cells or inhibit the proliferation of cancer cells.In addition, peptides themselves have the advantages of being very safe, inexpensive, easy to manufacture, and can be combined with various antigens.

[0009] Anticancer vaccines target peptide epitopes derived from tumor-associated antigens presented by molecules of the major histocompatibility complex (MHC). In humans, MHC molecules are called HLA (human leukocyte antigens). There are two classes of MHC molecules: MHC class I and MHC class II. MHC class I molecules consist of α and β2 microglobulin chains, while MHC class II molecules consist of α and β chains. Their three-dimensional shape forms a binding groove that is used for non-covalent binding to peptides.

[0010] MHC class I molecules are found on most nucleated cells and present peptides resulting from endogenous proteins, defective ribosomal products (DRIPs), and proteolytic cleavage of large peptides. However, peptides derived from endosomal compartments or exogenous sources are also found on MHC class I molecules. This non-classical class I presentation is referred to in the literature as cross-presentation (Brossart and Bevan, Blood, 1997).

[0011] MHC class II molecules are found primarily on professional antigen-presenting cells (APCs), which present peptides of foreign or transmembrane proteins taken up by the antigen-presenting cells. Complexes of peptides and MHC class I molecules are recognized by CD8+ T cells with the appropriate T cell receptor (TCR), whereas complexes of peptides and MHC class II molecules are recognized by CD4+ helper T cells with the appropriate TCR. CD4+ helper T cells play a key role in effectively inducing and sustaining CD8+ cytotoxic T cell responses. Thus, CD4+ T cell epitopes induced by tumor-associated antigens are crucial for the development of drugs that induce antitumor immune responses (Gnjatic et al., PNAS, 2003).

[0012] Helper T cells support the environment at the tumor site through cytotoxic T lymphocyte (CTL)-friendly cytokines and attract effector cells, such as CTLs, natural killer cells, macrophages, and granulocytes (Tay et al., Cancer Gene Therapy, 2020). Helper T cells activated by MHC class II play an important role in regulating CTL effector function in antitumor immunity. Helper T cell epitopes that trigger TH1-type helper T cell responses support the effector function of CD8+ killer T cells, including cytotoxic function against tumor cells that present tumor-associated peptide / MHC complexes on their cell surface. In this way, tumor-associated helper T cell peptide epitopes, alone or in combination with other tumor-associated peptides, can be used as active pharmaceutical ingredients in vaccine compositions to stimulate antitumor immune responses. For example, in mammalian models, such as mice, CD4+ T cells are known to suppress tumors through inhibition of angiogenesis by secretion of interferon gamma (IFN-g), even in the absence of CD8+ T lymphocytes (Beatty and Paterson, Immunologic Research, 2001; Mumberg et al., PNAS, 1999).

[0013] Identification and characterization of tumor-associated antigens that are recognized by CD8+ T cells (ligand: MHC class I molecule + peptide epitope) or CD4+ helper T cells (ligand: MHC class II molecule + peptide epitope) and contribute synergistically to the antitumor effects of both types of CD8- and CD4-dependent responses are important for the development of tumor vaccines. Therefore, in the present invention, peptide epitopes that recognize MHC class I molecules and peptide epitopes that recognize MHC class II molecules were used together.

[0014] Essentially, any peptide capable of binding to an MHC molecule can serve as a T cell epitope. The prerequisite for inducing a T cell response in vitro or in vivo is the presence of T cells bearing the corresponding TCR and the absence of immune tolerance to this specific epitope.

[0015] Identification of genes that are overexpressed or selectively expressed in tumor tissues or human tumor cell lines does not provide accurate information on the use of antigens transcribed from these genes in immunotherapy.This is because only individual subsets of epitopes of these antigens are suitable for such a response, and this is because T cells with corresponding TCRs must be present and immune tolerance to this specific epitope must be absent or minimal.It is therefore important to select only peptides that are overexpressed or selectively expressed and that appear in the context of MHC molecules, while minimizing immune tolerance and maximizing function and proliferation of T cells.

[0016] However, because MHC has so many alleles, it is not practical to generate candidate peptides for all alleles and test them directly to select those that bind strongly and stably. Instead, in silico algorithmic programs or databases can be used to effectively predict which peptides can bind strongly to the three-dimensional binding grooves present on various MHC molecules.

[0017] Using the information on peptide motifs involved in the binding of MHC to peptides contained in these databases, and the databases obtained by eluting the peptides bound to each MHC molecule using a mass spectrometer, various peptide epitopes can be predicted and found in a given protein sequence.

[0018] Representative databases include SYFPEITHI, MHCPEP, NetMHCpan-4.1EL, and NetMHCIIpan-4.0EL. However, not all peptides predicted by binding ability can effectively induce T cell responses in vitro or in vivo. Although the binding affinity predicted in computer programs is high, in reality they cannot induce sufficient immunogenicity in vitro or in vivo, and even can induce immune tolerance.

[0019] Therefore, in order for these predicted peptides to be used as anti-cancer vaccines, it is necessary to select the most effective and safe peptides through actual experiments and determine whether they cause effective immunogenicity in vitro or in vivo and whether they do not induce immune tolerance. However, conducting these experiments requires a lot of effort in terms of economy, material, and time. Therefore, in the present invention, we preferentially selected peptides that are known to associate with MHC class I and II molecules in various cancer antigens, can maximize T cell function and proliferation, and have already been shown to be safe in various previous clinical trials.

[0020] Although the binding of TCR and peptides is important for increasing the activation and function of T cells mainly against tumor-associated antigens, the results of previous clinical trials using actual peptides as vaccines did not show the expected effects. The main reason is that peptides alone did not induce sufficient immunogenicity. Effective immunogenicity requires costimulatory signaling by antigen-presenting cells and the help of appropriate cytokines. Otherwise, T cells may become anergic and immune tolerance may be induced. Therefore, adjuvants are essential to induce proper and sufficient T cell activation and thereby induce anti-cancer immune responses.

[0021] In the present invention, an adjuvant consisting of a lipopeptide and an immunoactive substance is used as an anti-cancer vaccine composition. This adjuvant is an excellent inducer of humoral and cellular immune responses, and increases Th1-type immune responses, which are particularly important for anti-cancer responses. Thus, the present inventors have completed the present invention by confirming that the adjuvant consisting of a lipopeptide and an immunoactive substance helps peptides to activate cancer antigen-specific T cells, thereby inducing more effective tumor-associated antigen-specific immune responses. Summary of the Invention [Problem to be solved by the invention]

[0022] An object of the present invention is to provide an anti-cancer vaccine composition comprising a peptide derived from a tumor-associated antigen.

[0023] Another object of the present invention is to provide an anti-cancer vaccine composition comprising a peptide derived from a tumor-associated antigen and an adjuvant consisting of a lipopeptide and an immunoactive substance.

[0024] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, which comprises the above-mentioned anti-cancer vaccine composition as an active ingredient.

[0025] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the above-mentioned anti-cancer vaccine composition in combination with one or more treatments selected from the group consisting of chemotherapy, targeted therapy, immune checkpoint inhibitors, and radiation therapy. [Means for solving the problem]

[0026] In order to achieve the above-mentioned objective, the present invention provides an anti-cancer vaccine composition comprising [a peptide derived from one or more tumor-associated antigens selected from the group consisting of hTERT, MAGE-A3, MUC-1, survivin, and WT-1].

[0027] The present invention also provides an anti-cancer vaccine composition comprising [a peptide derived from one or more tumor-associated antigens selected from the group consisting of hTERT, MAGE-A3, MUC-1, survivin, and WT-1] and [an adjuvant consisting of a lipopeptide and an immunoactive substance].

[0028] [Peptides derived from tumor-associated antigens] include one or more of the following peptides: i) peptide of SEQ ID NO: 1 (SEQ ID NO: 1: ILAKFLHWL), derived from hTERT and of HLA-A02 type (MHC class I); ii) peptide of SEQ ID NO: 2 (SEQ ID NO: 2: VYAETKHFL) derived from hTERT and of the HLA-A24 type (MHC class I); iii) peptide of SEQ ID NO: 3 (SEQ ID NO: 3: EARPALLTSRLRFIPK), derived from hTERT and of HLA-DRB1 type (MHC class II); iv) peptide of SEQ ID NO: 4 (SEQ ID NO: 4: RPGLLGASVLGLDDI), derived from hTERT and of the HLA-DRB1 type (MHC class II); v) peptide of SEQ ID NO: 5 (SEQ ID NO: 5: ELTLGEFLKL) derived from survivin and of HLA-A02 type (MHC class I); vi) peptide of SEQ ID NO: 8 (SEQ ID NO: 8: KVAELVHFL) derived from Mage-A3 and of HLA-A02 type (MHC class I); vii) peptide of SEQ ID NO: 9 (SEQ ID NO: 9: IMPKAGLLI), derived from Mage-A3 and of HLA-A24 type (MHC class I); viii) peptide of SEQ ID NO: 12 (SEQ ID NO: 12: TAPPVHNV), derived from MUC-1 and of HLA-A02 type (MHC class I); ix) A peptide of SEQ ID NO: 15 (SEQ ID NO: 15: RMFPNAPYL) derived from WT-1 and of HLA-A02 type (MHC class I), and x) Peptide of SEQ ID NO: 16 (SEQ ID NO: 16: KRYFKLSHLQMHSRKH), derived from WT-1 and of HLA-DRB1 type (MHC class II).

[0029] The adjuvant is prepared by mixing the lipopeptide and the immunologically active substance.

[0030] The adjuvant comprises a liposome having lipopeptides and immunoactive substances inserted into its surface.

[0031] Liposomes are composed of lipids.

[0032] The immunoactive substance is one or more selected from the group consisting of poly(I:C), QS21, MPLA, CpG, and flagellin.

[0033] The present invention also provides a pharmaceutical composition for preventing or treating cancer, which comprises the above-mentioned anti-cancer vaccine composition as an active ingredient.

[0034] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the above-mentioned anti-cancer vaccine composition in combination with one or more treatments selected from the group consisting of chemotherapy, targeted therapy, immune checkpoint inhibitors, and radiation therapy. Effect of the Invention

[0035] The tumor-associated antigen-derived peptide provided in one aspect of the present invention can effectively induce anti-cancer immunity, and an anti-cancer vaccine composition comprising a tumor-associated antigen-derived peptide and an adjuvant comprising a lipopeptide and an immunoactive substance can effectively induce anti-cancer immunity against various cancers, and the anti-cancer vaccine composition can be used in combination with conventional anti-cancer treatments, such as chemical anti-cancer agents having different mechanisms, immune checkpoint inhibitors, and radiation therapy, to significantly enhance the anti-cancer effect, thereby being useful for preventing and treating cancer. [Brief description of the drawings]

[0036] [Figure 1] 1 is a graph showing the results of confirming the immunogenicity of a single peptide derived from a tumor-associated antigen provided in the present invention using an ELISPOT assay. [Diagram 2] FIG. 1 is a graph showing the results of confirming the immunogenicity of the tumor-associated antigen-derived peptides provided by the present invention when combined with each antigen using an ELISPOT assay. [Diagram 3] 1 is a graph showing the results of confirming the immunogenicity of the tumor-associated antigen-derived peptides provided in the present invention when combined according to the number of peptides using an ELISPOT assay. [Figure 4a] FIG. 13 is a graph showing the results of using a tetramer binding assay to compare the frequency (%) of peptide-specific tetramer+CD8+ T cells in groups treated with CHA-2 (IL-2 20 U / ml) peptide alone (5 μg / ml or 50 μg / ml), the 10 peptide combination (50 μg / ml), and the peptide combination (50 μg / ml), lipopeptide and poly(I:C) adjuvant combination. [Figure 4b] FIG. 4b is a graph showing the results of comparing the number of CHA-2 peptide-specific tetramer+CD8+ T cells across the tetramer binding assay of FIG. 4a. [Figure 4c]FIG. 13 is a graph showing the results of using a tetramer binding assay to compare the frequency (%) of peptide-specific tetramer+CD8+ T cells in groups treated with CHA-8 (IL-2 20 U / ml) peptide alone (5 μg / ml or 50 μg / ml), the 10 peptide combination (50 μg / ml), and the peptide combination (50 μg / ml), lipopeptide and poly(I:C) adjuvant combination. [Figure 4d] FIG. 4c is a graph showing the results of comparing the number of CHA-8 peptide-specific tetramer+CD8+ T cells through the tetramer binding assay. [Figure 4e] FIG. 13 is a graph showing the results of using a tetramer binding assay to compare the frequency (%) of peptide-specific tetramer+CD8+ T cells in groups treated with CHA-9 (IL-2 20 U / ml) peptide alone (5 μg / ml or 50 μg / ml), the 10 peptide combination (50 μg / ml), and the peptide combination (50 μg / ml), lipopeptide and poly(I:C) adjuvant combination. [Figure 4f] FIG. 4f is a graph showing the results of comparing the number of CHA-9 peptide-specific tetramer+CD8+ T cells across the tetramer binding assay. [Figure 4g] FIG. 13 is a graph showing the results of using a tetramer binding assay to compare the frequency (%) of peptide-specific tetramer+CD8+ T cells in groups treated with CHA-15 (IL-2 20 U / ml) peptide alone (5 μg / ml or 50 μg / ml), the 10 peptide combination (50 μg / ml), and the peptide combination (50 μg / ml), lipopeptide and poly(I:C) adjuvant combination. [Figure 4h] FIG. 4g is a graph showing the results of comparing the number of CHA-15 peptide-specific tetramer+CD8+ T cells across the tetramer binding assay. [Figure 5a] 1 is a graph showing the results of confirming the cytotoxic effect of a combination of tumor-associated antigen-derived peptides provided in the present invention in colorectal cancer cell line SW620. [Figure 5b]FIG. 5B is a graph showing the results of FIG. 5a with PBMC from three healthy individuals (donors 8, 9 and 10). [Figure 5c] 1 is a graph showing the results of confirming the cytotoxic effect of a combination of tumor-associated antigen-derived peptides provided in the present invention in the prostate cancer cell line PC-3. [Figure 5d] FIG. 5C is a graph showing the results from FIG. 5C with PBMC from three healthy individuals (donors 8, 9 and 10). [Figure 5e] 1 is a graph showing the results of confirming the cytotoxic effect of a combination of tumor-associated antigen-derived peptides provided in the present invention in the breast cancer cell line MCF-7. [Figure 5f] FIG. 5e is a graph showing the results from FIG. 5e with PBMC from three healthy individuals (donors 8, 9 and 10). [Figure 5g] 1 is a graph showing the results of confirming the cytotoxic effect (CTL activity) of a combination of tumor-associated antigen-derived peptides provided by the present invention in the ovarian cancer cell line OVCAR-3. [Figure 5h] FIG. 5g is a graph showing the results from PBMC of three healthy individuals (donors 8, 9, and 10). [Figure 6] 1 is a graph showing the results of comparing the levels of granzyme B secretion in SW620, PC-3, MCF-7, and OVCAR-3 cell lines by combinations of tumor-associated antigen-derived peptides provided by the present invention. [Figure 7a] This is a graph showing the results of comparing the cytotoxic effects (CTL activity) of survivin + MAGE-A3 peptide (S + M peptide), hTERT + WT-1 peptide (H + W peptide), and 10 peptide combinations on the colon cell line SW620 in donor 8 (#P<0.05 10 peptide combination group vs. non-stimulated group, $P<0.05 10 peptide combination group vs. H + W group, &P<0.05 10 peptide combination group vs. S + M group). [Figure 7b]This is a graph showing the results of comparing the cytotoxic effects (CTL activity) of survivin + MAGE-A3 peptide (S + M peptide), hTERT + WT-1 peptide (H + W peptide), and a combination of 10 peptides on the colon cell line SW620 in donor 9 (#P<0.05 10 peptide combination group vs. unstimulated group, $P<0.05 10 peptide combination group vs. H + W group, &P<0.05 10 peptide combination group vs. S + M group). [Figure 7c] This is a graph showing the results of comparing the cytotoxic effects (CTL activity) of survivin + MAGE-A3 peptide (S + M peptide), hTERT + WT-1 peptide (H + W peptide), and a combination of 10 peptides on the colon cell line SW620 in donor 10 (#P<0.05 10 peptide combination group vs. non-stimulated group, $P<0.05 10 peptide combination group vs. H + W group, &P<0.05 10 peptide combination group vs. S + M group). [Figure 7d] This is a graph showing the results of comparing the levels of granzyme B secretion in the colon cancer cell line SW620 by survivin + MAGE-A3 peptide (S + M peptide), hTERT + WT-1 peptide (H + W peptide), and a combination of 10 types of peptides (n is the number of peptides, donors 8 to 10). [Figure 8a] 1 is a graph showing the results of comparing the number of IFN-γ-producing cells in a mouse model for test vaccines prepared using a combination of a tumor-associated antigen-derived peptide provided in the present invention, a lipopeptide, and a poly(I:C) adjuvant or another TLR adjuvant (from left to right: PBS negative control group, peptide treatment group, peptide + TLR2 ligand treatment group, peptide + TLR3 ligand treatment group, peptide + TLR7 / 8 (imiquimod) ligand treatment group, peptide + TLR9 ligand treatment (CpG) group, peptide + L-pampo treatment group). [Figure 8b] FIG. 8B is a graph showing the results of comparing peptide-specific IFN-γ secretion levels in FIG. 8A. [Figure 9]1 is a graph showing the results of comparing the number of IFN-γ producing cells in a mouse model for test vaccines prepared using a combination of 10 peptides provided in the present invention, 10 peptides and L-pampo, and 10 peptides and Lipo-pam. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present invention will be described in detail below.

[0038] The present invention provides an anti-cancer vaccine composition comprising a peptide derived from a tumor-associated antigen.

[0039] Tumor-associated antigens (TAA) are proteins expressed in cancer or tumor cells. Examples of TAA include novel antigens (neoantigens expressed during tumorigenesis and modified from similar proteins in normal or healthy cells), products of oncogenes and tumor suppressor genes, overexpressed or abnormally expressed cellular proteins (e.g., HER2, MUC-1), antigens produced by oncogenic viruses (e.g., EBV, HPV, HCV, HBV, HTLV), testicular cancer antigens (CTAs, e.g., MAGE family, NY-ESO), and cell type-specific differentiation antigens (e.g., MART-1). TAA sequences can be searched experimentally or through published scientific articles or publicly available databases, such as the Ludwig Institute for Cancer Research database (www.cta.lncc.br / ), Cancer Immunity database (cancerimmunity.org / peptide), and TANTIGEN tumor T cell antigen database (cvc.dfci.harvard.edu / tadb / ).

[0040] On the other hand, tumor-specific antigens (TSAs) are antigens produced by a particular type of tumor that do not appear in normal cells of the tissue in which the tumor originates. TSAs include mass antigens, neoantigens, and endogenous antigens. Tumor-specific peptides are not expressed or are expressed only slightly in normal healthy cells or tissues, but are expressed at a high rate (high frequency) in subjects (cells or tissues) with a particular disease or condition, such as a particular type of cancer. Alternatively, tumor-specific peptides may be expressed at low levels in normal healthy cells, but at high levels in cells affected by a disease (e.g., cancer) or in subjects with a disease or condition.

[0041] [Peptides derived from tumor-associated antigens] include a peptide of SEQ ID NO: 1 (SEQ ID NO: 1: ILAKFLHWL) derived from hTERT and being HLA-A02 type (MHC class I), a peptide of SEQ ID NO: 2 (SEQ ID NO: 2: VYAETKHFL) derived from hTERT and being HLA-A24 type (MHC class I), a peptide of SEQ ID NO: 3 (SEQ ID NO: 3: EARPALLTSRLRFIPK) derived from hTERT and being HLA-DRB1 type (MHC class II), a peptide of SEQ ID NO: 4 (SEQ ID NO: 4: RPGLLGASVLGLDDI) derived from hTERT and being HLA-DRB1 type (MHC class II), a peptide of SEQ ID NO: 5 (SEQ ID NO: 5: ELTLGEFLKL) derived from survivin and being HLA-A02 type (MHC class I), Mag The peptide may be at least one selected from the group consisting of a peptide of SEQ ID NO: 8 (SEQ ID NO: 8: KVAELVHFL) derived from e-A3 and being an HLA-A02 type (MHC class I), a peptide of SEQ ID NO: 9 (SEQ ID NO: 9: IMPKAGLLI) derived from Mage-A3 and being an HLA-A24 type (MHC class I), a peptide of SEQ ID NO: 12 (SEQ ID NO: 12: STAPPVHNV) derived from MUC-1 and being an HLA-A02 type (MHC class I), a peptide of SEQ ID NO: 15 (SEQ ID NO: 15: RMFPNAPYL) derived from WT-1 and being an HLA-A02 type (MHC class I), and a peptide of SEQ ID NO: 16 (SEQ ID NO: 16: KRYFKLSHLQMHSRKH) derived from WT-1 and being an HLA-DRB1 type (MHC class II).

[0042] When treated with PBMCs obtained from healthy subjects, the immunogenicity of each peptide was higher than that of untreated controls, but to different degrees (Figure 1). Based on these results, CHA-6, CHA-10, and CHA-11, which consistently showed the lowest immunogenicity, were excluded. The reason for removing peptides showing low immunogenicity is that these peptides induce immune tolerance when added together. Thus, 10 peptides (SEQ ID NOs: 1-5, 8, 9, 12, 15, and 16) out of the 13 peptides were finally confirmed based on the immunogenicity results (Table 1).

[0043] To select the most effective peptide combination to induce immune responses against various tumor-associated antigens from the 10 identified peptides, peptides corresponding to each of the five antigens were first mixed and administered to PBMCs obtained from six healthy subjects, and then the increase or decrease in immunogenicity was analyzed using an ELISPOT assay. As a result, it was confirmed that the immunogenicity of the combination of peptides corresponding to hTERT and WT-1, and the combination of peptides corresponding to MAGE-A3 and survivin was effectively enhanced when each of the five antigens was mixed (Figure 2).

[0044] To investigate whether the immunogenicity increases with the number of peptides combined, peptides were combined under various conditions and administered to PBMCs from three healthy individuals, and the ELISPOT results according to the number of peptides are shown in the figure (Figure 3). As a result of the experiment, it was confirmed that the group treated with the combination of 10 peptides showed higher immunogenicity than the groups treated with other combinations, and in particular, the immunogenicity was enhanced more than the combinations of hTERT + WT-1 and survivin + MAGE-A3 (Figure 3).

[0045] The combination of 10 peptides (CHA-1, CHA-2, CHA-3, CHA-4, CHA-5, CHA-8, CHA-9, CHA-12, CHA-15, and CHA-16) consisted of peptides derived from various cancer antigens (hTERT, survivin, MAGE-A3, MUC1, and WT-1). This combination was able to induce anti-cancer immune responses against various cancer antigens, was able to activate CD8 and CD4 T cells together, which are important for anti-cancer immune responses, and contained peptides against MHC class I and II, which provided the highest immunogenicity. Therefore, the above combination was confirmed to be the most effective peptide epitope combination for therapeutic and prophylactic vaccines against various cancers.

[0046] [Peptides derived from tumor-associated antigens] and combinations thereof recognize MHC class I, MHC class II, or both.

[0047] The present invention also provides an anti-cancer vaccine composition comprising a peptide derived from a tumor-associated antigen and an adjuvant consisting of a lipopeptide and an immunoactive substance.

[0048] Lipopeptides may be composed of a fatty acid and several amino acids bound to a glycerol molecule. The number of fatty acids or amino acids constituting the lipopeptide in the glycerol molecule may be one or more. In this case, the fatty acids and amino acids may be chemically modified. Lipopeptides may be lipoproteins in the form of a part of a molecule or in the form of a whole molecule derived from gram-positive or gram-negative bacteria or mycoplasma.

[0049] The lipopeptide is at least one selected from the group consisting of Pam3Cys-SKKKK, Pam3-CSKKKK, PHC-SKKKK, Ole2PamCys-Pam3Cys-SKKKK, Pam3-CSKKKK, PHC-SKKKK, Ole2PamCys-SKKKK, Pam2Cys-SKKKK, PamCys(Pam)-SKKKK, Ole2Cys-SKKKK, Myr2Cys-SKKKK, PamDhc-SKKKK, Pam-CSKKKK, Dhc-SKKKK and FSL-1 (Pam2CGDPKHPKSF), but is not necessarily limited thereto.

[0050] The immunoactive substance may be any one or more selected from the group consisting of poly(I:C), QS21, monophosphoryl lipid A (MPLA), CpG, and flagellin. Poly(I:C) has been used as a potent inducer of type 1 interferon in in vitro and in vivo studies. In addition, poly(I:C) is known to stably and maturely form dendritic cells, the most potent antigen-presenting cells in mammals (Rous, R. et al., International Immunol, 2004). According to these existing reports, poly(I:C) is a potent IL-12 inducer, and IL-12 is an important immunoactive substance that induces cellular immune responses and induces the formation of IgG2a or IgG2b by promoting the expression of Th1 immune responses.

[0051] Poly(I:C) has a length of 50 to 5,000 bp. Poly(I:C) may be contained in the adjuvant at a concentration of, but is not necessarily limited to, 10 to 150, 10 to 90, 10 to 50, 10 to 30, 30 to 60, 30 to 90, 30 to 150, 30 to 50, 10 to 1500, 10 to 900, 10 to 500, 10 to 300, 30 to 600, 30 to 900, 30 to 1500, or 30 to 500 μg / dose.

[0052] QS21 is a fraction of saponin substances called triterpene glucosides with a molecular weight of 1990.14 Da extracted from the bark of the South American Quercus saponaria Molina tree. QS21, when used with lipids such as MPLA (monophosphoryl lipid A) or cholesterol, is known to induce humoral and cellular immune responses by secreting Th1-type cytokines from antigen-presenting cells such as macrophages and dendritic cells.

[0053] QS21 may be contained in the adjuvant at a concentration of, but is not necessarily limited to, 1 to 150, 1 to 90, 1 to 50, 1 to 30, 3 to 60, 3 to 90, 3 to 150, 3 to 50, 1 to 1500, 1 to 900, 1 to 500, 1 to 300, 3 to 600, 3 to 900, 3 to 1500, or 3 to 500 μg / dose.

[0054] Tetramer binding assays were performed to confirm the proliferation of CD8+ T cells. When experiments were performed with tetramerizable CHA-2, CHA-8, and CHA-9, as well as CHA-15 peptides, the number and frequency (%) of peptide-specific CD8+ T cells were increased in the groups treated with the 10 peptide combinations compared to each peptide alone, and the number and frequency of peptide-specific CD8+ T cells were increased by lipopeptide and poly(I:C) adjuvant (Figure 4).

[0055] These results indicate that the combination of 10 selected peptides can increase CD8+ T cells more effectively than a single peptide that can activate CD8+ T cells, and there is a greater synergistic effect when the combination of 10 peptides is administered with an adjuvant. This is because CD4+ T cells are activated by peptides that can bind to MHC class II (CHA-3, CHA-4, and CHA-16) in the combination of 10 peptides, and the activated CD4+ T cells in turn allow the proliferation of peptide-specific CD8+ T cells. Moreover, it can be seen that the synergistic effect in this proliferation of CD8+ T cells was maximized due to the Th1 response induced by the known lipopeptide and poly(I:C) adjuvant (Figures 4a-4h).

[0056] To determine whether T cells expanded and functionally activated by combinations of peptides derived from various tumor-associated antigens could recognize and kill cancer cells as cytotoxic T cells, CTLs, effector cells, and cancer cells were cultured together, and the frequency of cancer cells killed by CTLs was then analyzed by flow cytometry.

[0057] As a result of the experiment, it was confirmed that the effector cells activated by treating PBMCs of three healthy subjects with the peptide combination killed more cancer cells in colorectal cancer cell line SW620, prostate cancer cell line PC-3, breast cancer cell line MCF-7, or ovarian cancer cell line OVCAR-3 than the effector cells cultured without peptide. These results also showed that it was a cell-specific cytotoxic effect, and the more the number of effector cells, the more cancer cells were killed (Figure 5a-5h).

[0058] Furthermore, to confirm the activity of T cells expanded and functionally activated by combinations of peptides derived from various tumor-associated antigens against various cancer cells, we examined the secretion of granzyme B in the culture medium of effector cells cultured together with cancer cells by ELISA. Granzyme B is an enzyme secreted by CD8 T cells, especially CTLs, and plays an important role in directly killing cancer cells.

[0059] As a result of the experiment, it was confirmed that the granzyme B content was significantly higher in the culture medium in which effector cells activated by treating PBMCs of six healthy subjects with the peptide combination were cultured together with the colon cancer cell line SW620, the prostate cancer cell line PC-3, the breast cancer cell line MCF-7, or the ovarian cancer cell line OVCAR-3 than in the culture medium in which effector cells cultured without peptide were cultured together with cancer cells. This indicated that CTLs recognize cancer cells and secrete granzyme B, which induces cancer cell death (Figure 6).

[0060] These results confirmed that tumor antigen-specific T cells proliferated and functionally activated by the combined use of peptides recognized cancer cells, secreted granzyme B, and were able to directly kill cancer cells as cytotoxic T cells.

[0061] Next, the inventors compared whether the combinations of 10 peptides confirmed to have excellent immunogenicity in Figures 3, 4, and 5 showed higher CTL activity than the combinations of survivin + MAGE-A3 or hTERT + WT-1 peptides confirmed to have excellent immunogenicity in Figure 2.

[0062] The cytotoxicity and secretion of granzyme B were analyzed in the colon cancer cell line SW620. The results showed that the effector cells activated by treating PBMCs from three healthy individuals with the 10 peptide combinations secreted significantly higher granzyme B and were able to kill more cancer cells than the effector cells activated by treating them with the survivin + MAGE-A3 or hTERT + WT-1 peptide combinations (Figures 7a-7d).

[0063] This combination of 10 selected peptides consists of peptides derived from various tumor-associated antigens (hTERT, survivin, MAGE-A3, MUC-1, and WT-1), so it can induce anti-cancer immune responses against various cancer antigens. Also, this combination can activate both CD8 and CD4 T cells, which are important for anti-cancer immune responses, so it can induce a high immune response against cancer cells and kill more cancer cells by activated CTLs.

[0064] Finally, we compared the cellular immune responses induced by test vaccines prepared using combinations of 10 peptides and lipopeptides and poly(I:C) adjuvants, and test vaccines prepared using combinations of 10 peptides and other TLR adjuvants. There are a total of seven experimental groups: PBS negative control, 10 peptides, combinations of 10 peptides and TLR2 ligands, 10 peptides and TLR3 ligands, 10 peptides and TLR7 / 8 ligands (imiquimod), 10 peptides and TLR9 ligands (CpG), and 10 peptides and L-pampo (lipopeptide and poly(I:C) adjuvant) (Figures 8a-8b). As a result of the experiment, it was confirmed that the test vaccine using lipopeptide and poly(I:C) adjuvant, i.e., L-pampo, significantly increased the number of cells producing IFN-γ compared to the test vaccines using TLR2, TLR3, TLR7 / 8, or TLR9 adjuvants. Furthermore, it was confirmed that each peptide-specific IFN-γ secretion was also increased by the test vaccine using L-pampo compared to the test vaccines using TLR2, TLR3, TLR7 / 8 or TLR9 adjuvants (Figures 8a to 8b).

[0065] Furthermore, in the mouse model shown in Figure 8, we determined whether a test vaccine prepared using a combination of 10 peptides and a Lipo-pam adjuvant consisting of liposomes with lipopeptides inserted on their surface and an immunoactive substance could induce a cellular immune response similar to that of L-pampo. As a result, it was confirmed that the test vaccine using Lipo-pam significantly increased the number of cells producing IFN-γ compared to the control group, similar to the vaccine using L-pampo (Figure 9).

[0066] From the above results, it was confirmed that the anti-cancer vaccine composition provided in one embodiment of the present invention is an anti-cancer vaccine composition that can strongly induce a cellular immune response capable of killing cancer cells, compared to vaccine compositions containing other adjuvants, for example, TLR2, TLR3, TLR7 / 8, or TLR9 adjuvants.

[0067] Meanwhile, in one embodiment of the present invention, the anti-cancer vaccine composition is prepared by mixing the lipopeptide and the immunoactive substance as the adjuvant in a certain ratio.

[0068] The anti-cancer vaccine composition may be an oil emulsion or an aqueous solution formulation.

[0069] The anti-cancer vaccine composition may further comprise buffers, isotonicity agents, preservatives, stabilizers, and solubilizers. Buffers include phosphates, acetates, ammonium phosphates, ammonium carbonates, citrates, and the like.

[0070] In terms of the composition ratio of the entire vaccine composition containing the above-mentioned composition, the lipopeptide is preferably contained in the lipopeptide and poly(I:C) adjuvant at a weight ratio of 0.01 to 0.5% by weight, and most preferably at a weight ratio of 0.01 to 0.1% by weight, and the poly(I:C) is preferably contained at a weight ratio of 0.01 to 0.4% by weight, and most preferably at a weight ratio of 0.01 to 0.08% by weight, but are not necessarily limited thereto.

[0071] Furthermore, sodium chloride is contained as an isotonic agent at a weight ratio of preferably 0.1 to 0.8% by weight, more preferably 0.2 to 0.6% by weight, and most preferably 0.44% by weight according to a preferred embodiment of the present invention.

[0072] Furthermore, it is preferable to use phosphate, acetate, ammonium phosphate, ammonium carbonate, citrate, etc. as a buffer, and according to a preferred embodiment of the present invention, it is more preferable to use phosphate and acetate, but the buffer is not necessarily limited thereto. Phosphate is preferably contained at a weight ratio of 0.01 to 0.4% by weight, more preferably at a weight ratio of 0.01 to 0.2% by weight. According to a preferred embodiment of the present invention, it is most preferably contained at a weight ratio of 0.13% by weight. Furthermore, acetate is preferably contained at a weight ratio of 0.01 to 0.3% by weight, more preferably at a weight ratio of 0.01 to 0.2% by weight. According to a preferred embodiment of the present invention, it is most preferably contained at a weight ratio of 0.08% by weight. Therefore, the vaccine composition of the present invention preferably contains a buffer at a weight ratio of 0.02 to 0.6% by weight, and according to a preferred embodiment of the present invention, it is most preferably contained at a weight ratio of 0.15 to 0.25% by weight.

[0073] On the other hand, in another embodiment of the present invention, the adjuvant is prepared by mixing liposomes having lipopeptides inserted on their surface and an immunoactive substance.

[0074] The lipopeptide can be inserted into the liposomes at a concentration of 20-250, 20-50, 50-250, 150-250, 50-150, 20-2500, 20-500, 50-2500, 150-2500, or 50-1500 μg / dose.

[0075] Liposomes are composed of lipids.

[0076] The lipid can be cationic, anionic, or neutral lipid. For example, the lipid is at least one selected from the group consisting of DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DDA (dimethyldioctadecylammonium), DC-chol (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol), DOPG (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and cholesterol, but is not necessarily limited thereto.

[0077] The lipids can be inserted into the liposomes at a concentration of 15-300, 15-150, 15-90, 15-50, 15-40, 20-30, 15-3000, 15-1500, 15-900, 15-500, 15-400, or 20-300 μg / dose.

[0078] The adjuvant consists of positively charged liposomes in which a positively charged lipopeptide is inserted into the lipid bilayer, and a negatively charged immunoactive substance.

[0079] The anti-cancer vaccine composition can induce both humoral and cellular immune responses.

[0080] The anti-cancer vaccine composition can enhance Th1 immune response. IgG2a or IgG2b promotes Th1 immune response that is effective for anti-virus and anti-cancer immune response, and is produced by cytokines secreted by helper T cell 1 (Th1). Therefore, the anti-cancer vaccine composition of the present invention can also be used as a preventive or therapeutic agent for cancer that progresses from viral infection.

[0081] A humoral immune response can be induced by increasing T follicular helper (Tfh) cells and secreting cytokines by the adjuvants of the present invention.

[0082] A cellular immune response can be induced by increasing the secretion of IFN-γ, TNF-α, and granzyme B.

[0083] The present invention also provides a pharmaceutical composition for preventing or treating cancer, comprising the anti-cancer vaccine composition as an active ingredient.

[0084] The anti-cancer vaccine composition may have the characteristics described above.

[0085] In one embodiment of the present invention, the anti-cancer vaccine composition may comprise an adjuvant and an antigen. The adjuvant may comprise a liposome into which the lipopeptide is inserted, and may further comprise an immunoactive substance.

[0086] The preventive or therapeutic agent of the present invention may contain a pharma- ceutically acceptable carrier, may be formulated for human or veterinary use, and may be administered via various routes. The administration route may be oral, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, transdermal, and intranasal, etc. Preferably, it is formulated and administered as an injection. The injection may be prepared using aqueous solvents, such as physiological saline and Ringer's solution, and non-aqueous solvents, such as vegetable oils, high fatty acid esters (e.g., ethyl oleate, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), and may contain pharmaceutical carriers, such as stabilizers that prevent deterioration (e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers that adjust pH, and preservatives that inhibit microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0087] The pharmaceutical composition for preventing or treating cancer of the present invention can be administered in a pharmacologic effective amount. The term "pharmacologic effective amount" refers to an amount sufficient to show a vaccine effect without causing side effects or severe or excessive immune response, and the exact administration concentration may vary depending on the antigen contained in the vaccine. Furthermore, the prophylactic or therapeutic agent can be easily determined by a person skilled in the art according to factors well known in the medical field, such as the patient's age, weight, health, sex, and sensitivity to drugs, administration route, and administration method. The pharmaceutical composition of the present invention can be administered once or several times.

[0088] The cancer is at least one selected from the group consisting of ovarian cancer, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brain stem tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity / paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, breast tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, abdominal tumor, gastric cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, penile cancer, prostate cancer, cervical cancer, endometrial cancer, uterine sarcoma, vaginal cancer, female external genitalia cancer, and skin cancer, but is not necessarily limited thereto.

[0089] The anti-cancer vaccine composition can be used in combination with one or more treatments selected from the group consisting of chemotherapy, targeted therapy, immune checkpoint inhibitors, and radiation therapy, and exhibits synergistic anti-cancer effects compared to existing treatments.

[0090] Hereinafter, the present invention will be described in detail with reference to the following examples and experimental examples.

[0091] However, the following examples and experimental examples are merely intended to illustrate the present invention, and the contents of the present invention are not limited thereto.

[0092] [Example 1] Discovery of MHC class I and MHC class II antigenic peptide epitopes derived from tumor-associated antigens <1-1> Selection of peptide candidates that induce immunogenicity To select peptide candidates for inducing immunogenicity, peptides derived from known tumor-associated antigens undergoing clinical trials for various cancers were selected as candidates. The selected tumor-associated antigens were hTERT, survivin, MAGE-A3, MUC-1, EGFRvIII, NY-ESO-1, and WT-1 antigens, and 16 peptides derived from them were selected (Table 1).

[0093] [Table 1]

[0094] Among these 16 peptides, three peptides (CHA-7, CHA-13, and CHA-14) were excluded because they all had cysteines in their amino acid residues that could form disulfide bonds and prevent binding to MHC molecules. An in silico algorithm was used to confirm whether the 13 peptides thus selected were peptides with high binding affinity to MHC class I or MHC class II.

[0095] As a result, the 13 selected peptides were determined to be derived from various tumor-associated antigens and to be candidates for inducing immunogenicity by confirming that they specifically bind to HLA-A02 / A24 and HLA-DRB1. The 13 confirmed peptide sequences were synthesized into peptides by requesting a peptide synthesis company (Anygen, Korea).

[0096] <1-2> Confirmation of immunogenicity of selected peptides and selection of final peptides To confirm the immunogenicity of the 13 peptides selected in Example 1, an interferon gamma (IFNg)-enzyme-linked immuno-SPOT assay (ELISPOT) was performed.

[0097] This experiment is a highly sensitive immunoassay that can detect cytokines secreted from single cells. By attaching the cytokines secreted when cells are activated to a capture antibody, the secreted cytokines can be measured without being affected by proteins or proteolytic enzymes expressed on the cell surface. After the cytokines are attached to the capture antibody, the cells are removed, a detection antibody conjugated to an enzyme that can recognize the attached capture antibody is added, a chromogenic substrate for the enzyme is added, and a chromogenic spot is formed at the position of the cells secreting the cytokine, and the number of spots can be used to determine the number of cells that responded to the stimulating antigen.

[0098] Peripheral blood mononuclear cells (PBMCs) required for the experiments were isolated from human venous blood as follows: Venous blood was diluted with 1x PBS, placed in a SepMate-50 tube, and centrifuged at 800 g for 20 min at room temperature. After centrifugation, plasma and isolated PBMCs were washed and then transferred to each well of a 6-well tissue culture plate at 1x10 6 The PBMCs were added at a concentration of 1000 cells / mL and cultured overnight in an incubator (37°C, 5% CO2). The next day, the PBMC culture medium was collected and centrifuged at 2000 rpm for 15 minutes at room temperature. After centrifugation, the PBMCs were seeded in a 6-well cell culture plate, and the peptides were added thereto and cultured to stimulate the cells.

[0099] At this point, the 13 peptides selected in Example 1 were treated alone or in various peptide combinations, and an untreated group and a ConA group were also prepared for comparative analysis. After culturing in a 6-well cell culture plate, 200 μL of PBMC culture solution was dispensed into each well of the ELISPOT plate and cultured for 24 hours under 37° C. and 5% CO2 conditions. After culturing, the plate was washed five times with 1×PBS and prepared for ELISPOT assay. The detection antibody (R4-642-biotin) was diluted in PBS (0.5% FBS) and added to the plate at 100 μL / well, followed by reaction at room temperature for 2 hours. Next, the plate was washed five times with 1×PBS, and streptavidin-HRP diluted in PBS (0.5% FBS) was added to the plate at 100 μL / well, followed by reaction at room temperature for 1 hour. Next, the plate was washed with 1×PBS, and TMB substrate solution was added to the plate at 100 μL / well. When the spot color was clearly developed, the plate was washed six times with DW to stop the reaction. After removing the water, the plate was wrapped in foil and dried in the shade for one day. ELI-FOIL was attached to the dried ELISPOT plate, and the membrane was stamped, and then the number of spots in each well was read using an ELISPOT reader. The number of spots in the test group was subtracted from the number of spots in the untreated group to create a graph.

[0100] As a result, as shown in FIG. 1, it was confirmed that the immunogenicity was increased in the group treated with all 13 peptides compared to the control group that was not treated with peptides. Among them, CHA-6, CHA-10, and CHA-11, which consistently showed the lowest immunogenicity, were excluded. This is because the peptides may induce immune tolerance when peptides showing low immunogenicity are added. Therefore, of the 13 peptides, 10 peptides (CHA-1 to CHA-5, CHA-8, CHA-9, CHA-12, CHA-15, and CHA-16) were finally confirmed based on the immunogenicity results (Table 1).

[0101] To select the peptide combination that could induce the most effective immune response against various cancer antigens among the 10 identified peptides, peptides corresponding to each of the five antigens were first mixed and applied to PBMCs from six healthy volunteers, and then the increase and decrease in immunogenicity were analyzed using an ELISPOT assay.

[0102] As a result, as shown in Figure 2, it was confirmed that the combination of peptides corresponding to hTERT and WT-1, and the combination of peptides corresponding to MAGE-A3 and survivin enhanced immunogenicity more effectively than the combination of peptides corresponding to five antigens individually. Furthermore, it was confirmed that when other antigen combinations were added to this combination, or when antigens other than these two combinations were combined, the immunogenicity decreased or increased, but did not further increase beyond the value of these two combinations.

[0103] In the experiments described above, since there were six peptides (four from hTERT and two from WT-1) in the combination of MAGE-A3 and survivin, and three peptides (two from MAGE-A3 and one from survivin) in the combination of hTERT and WT-1, we performed experiments to determine whether immunogenicity increased with the number of peptides combined.

[0104] For this purpose, peptides were combined under different conditions as shown in FIG. 3 and treated with PBMCs from three healthy individuals, and the ELISPOT results according to the number of peptides were confirmed.

[0105] As a result, as shown in Figure 3, the group treated with the 10 peptides was more immunogenic than the groups treated with other combinations, especially the groups treated with the hTERT + WT-1 combination and the survivin + MAGE-A3 combination.

[0106] The combination of 10 peptides (CHA-1, CHA-2, CHA-3, CHA-4, CHA-5, CHA-8, CHA-9, CHA-12, CHA-15, and CHA-16) induced anti-cancer immune responses against various cancer antigens because it was composed of peptides derived from various cancer antigens (hTERT, survivin, MAGE-A3, MUC1, and WT-1), which contained peptides against MHC class I and II that could activate CD8 and CD4 T cells together, which are important for anti-cancer immune responses, and provided the most enhanced immunogenicity. Therefore, the above combination of 10 peptides was confirmed to be the most effective peptide epitope combination for therapeutic and prophylactic vaccines against various cancers.

[0107] [Example 2] Preparation of anti-cancer vaccines containing peptides derived from tumor-associated antigens All peptides were synthesized by standard, well-established solid-phase peptide synthesis methods using Fmoc chemistry with appropriate resins from C-terminus to N-terminus (Anygen, Korea). Purification and purity of each peptide was confirmed by mass spectrometry and HPLC analysis. Peptides were obtained as white lyophilized compounds with purity of 95% or higher. All peptides were used at a concentration of 10 mg / ml.

[0108] [Example 3] Preparation of an anti-cancer vaccine containing a peptide derived from a tumor-associated antigen and an adjuvant consisting of a lipopeptide and an immunologically active substance <3-1>L-pampo The anti-cancer vaccine was prepared by mixing the peptide combination selected in Example <1-2> with Pam3Cys-SKKKK and the poly(I:C) adjuvant of the present invention.

[0109] First, a test vaccine containing Pam3Cys-SKKKK and poly(I:C) was prepared to observe the adjuvant effect when Pam3Cys-SKKKK and poly(I:C) were simultaneously used. When preparing the vaccine mixture containing the peptide, a 10X buffer solution (pH 7.0) containing 150 mM NaCl and 10 mM sodium phosphate was used.

[0110] <3-2>Lipo-pam First, DOTAP:DPPC liposomes were prepared. For this purpose, DOTAP and DPPC were dissolved in chloroform, respectively, and the organic solvent was evaporated with nitrogen while rotating the mixture of DOTAP and DPPC until it was uniformly distributed on the wall of the glass container. At this time, a thin film was formed on the wall, and the organic solvent remaining in the formed film was removed by storing it in a vacuum desiccator. The completely dried lipid film was rehydrated by adding distilled water. When a multilamellar vesicle (MLV) suspension solution was prepared, 2X buffer (pH 7.0) containing 300 mM NaCl in 20 mM sodium phosphate was added to it in the same amount as distilled water. The formed MLVs were sonicated for 5 min with 5 cycles of 3 s / 3 s (pulse on / off) to prepare DOTAP:DPPC liposomes in the form of small unilamellar vesicles (SUVs).

[0111] Next, Lipo-pam was prepared in the same manner as the preparation of DOTAP:DPPC liposomes, except that DOTAP, DPPC, and Pam3-CSKKKK were each dissolved using an organic solvent, and then DOTAP and DPPC were mixed and Pam3-CSKKKK was added thereto. The vaccine mixture was prepared by adding an immunologically active substance adjuvant (poly(I:C)) and peptide to Lipo-pam. In this case, a 10x buffer solution (pH 7.0) containing 150 mM NaCl and 10 mM sodium phosphate was used.

[0112] Experimental Example 1: Verification of CD8 T cell-specific immunogenicity induced by peptide combination CD8+ T cells are the most important immune cells in anti-cancer immunity, directly killing cancer cells and preventing their proliferation. Therefore, when there are a large number of CD8+ T cells specific to tumor-associated antigens, more effective anti-cancer immunity can be induced. In this Example 1, tetramer binding assays were performed to confirm the proliferation of CD8+ T cells by administering the peptides presented in Example 1 alone, in combination with peptides, and in combination with adjuvants (lipopeptide and poly(I:C)).

[0113] Tetramer binding assay is an experimental method that can sensitively verify antigen-specific CD8 T cell responses, especially CTL (cytotoxic T lymphocyte) responses, by measuring the number or frequency (%) of CD8+ T cells that bind MHC tetramers. This experiment uses four MHC / peptide complexes, "MHC tetramers," to generate a complex, which is then attached to streptavidin labeled with a fluorophore.

[0114] Therefore, in this experiment, the following four tetramers were prepared using peptides against MHC class I types capable of recognizing CD8 T cells (CHA-2 / HLA-A24 tetramer, CHA-8 / HLA-A02 tetramer, CHA-9 / HLA-A24 tetramer, and CHA-15 / HLA-A02 tetramer), namely HLA-A02 and HLA-24.

[0115] Similar to the ELISPOT assay, PBMCs were isolated from human venous blood and dispensed into 96-well cell culture plates (200 mL / well). Aliquots of PBMCs were stimulated by treating with peptides. In this case, each peptide was treated at 5 mg / mL or 50 mg / mL, and the same concentrations were used for the combination of 10 peptides identified in Example <1-2>. At this point, control groups that were not treated with peptide (untreated) or treated with irrelevant peptide (non-specific tetramer) were also prepared for the assay.

[0116] The 96-well cell culture plate was placed in an incubator and cultured at 37°C and 5% CO2 for 24 hours. At the end of the culture, the 96-well plate was removed, the cultured cells were collected in a FACS (fluorescence-activated cell sorter) tube, and centrifuged at 2000 rpm at room temperature for 15 minutes. When the centrifugation was completed, the supernatant was removed, FVS (fixable viability stain) was treated, and the cells were resuspended. After resuspension, the FACS tube was covered with foil and reacted at room temperature for 15 minutes. Each tube was washed with 2 mL of PBS, and then centrifuged at 2000 rpm at room temperature for 5 minutes. When the centrifugation was completed, the FACS tube was removed, the supernatant was removed, and each tube was washed with 1 mL of FACS buffer, placed in a centrifuge, and centrifuged at 2000 rpm at room temperature for 5 minutes. When the centrifugation was completed, the FACS tube was removed, the supernatant was removed, and 100 μL of FACS buffer was added to each tube, and resuspended. After resuspension, human FcR blocking solution and MHC tetramer were added to the FACS tube and mixed thoroughly. The FACS tube was wrapped in foil and reacted at room temperature for 20 minutes. After 20 minutes, anti-human CD3, CD45, and CD8 antibodies were added thereto and mixed thoroughly. The FACS tube was wrapped in foil and reacted in a refrigerator, then the FACS tube was removed from the refrigerator, FACS buffer was added, and centrifuged at room temperature. After completion of centrifugation, the FACS tube was removed, the supernatant was removed, and the cells were resuspended in FACS buffer. Then, a flow cytometer was used to analyze the number of MHC tetramer+CD8+ T cells in the total CD8+ T cells and the frequency (%) of MHC tetramer+CD8+ T cells.

[0117] As a result, as shown in Figures 4a-4h, the frequency (%) and total number of tetramer+CD8+ T cells specific to each peptide in the group treated with the 10 peptide combinations were significantly higher than those in the groups treated with CHA-2, CHA-8, CHA-9, and CHA-15 peptides alone (solid line frame). It was confirmed that when lipopeptides and poly(I:C) adjuvant were mixed with the 10 peptide combinations and treated, the frequency and number of tetramer+CD8+ T cells for each peptide were increased compared to when only the 10 peptide combinations were added (dashed line frame).

[0118] The above results show that the combination of 10 selected peptides can increase CD8+ T cells more effectively than a single peptide that can activate CD8+ T cells, and especially when the combination of 10 peptides and adjuvant was administered together, there was a greater synergistic effect. This is because CD4+ T cells are activated by peptides that can bind to MHC class II (CHA-3, CHA-4, and CHA-16), especially in the combination of 10 peptides, and then more peptide-specific CD8+ T cells can proliferate with the help of the activated CD4+ T cells. Furthermore, it is observed that the synergistic effect in the proliferation of CD8+ T cells is maximized due to the Th1 response induced by the known lipopeptide and poly(I:C) adjuvant.

[0119] Experimental Example 2: Verification of cytotoxicity of CD8+ T cells induced by peptide combination To determine whether T cells proliferated and functionally activated by combinations of peptides derived from various tumor-associated antigens could recognize and kill cancer cells as CTLs (cytotoxic T lymphocytes), effector cells were co-cultured with cancer cells, and the frequency of cancer cells killed by CTLs was analyzed using a flow cytometer.

[0120] First, to prepare effector cells, i.e., CTLs proliferated and activated by peptides, PBMCs were isolated from human venous blood, and then 10 mg / mL of each of the 10 peptides selected in Example <1-2> and 5 ng / mL of hIL-2 were added, followed by culturing. As a control, cells cultured with 5 ng / mL of hIL-2 without peptides were used.

[0121] To determine whether peptide-increased effector cells could specifically recognize and kill target cancer cell MHC, they were co-cultured with cancer cells expressing HLA-A02 or HLA-A24 alone, or both HLA-A02 and HLA-A24. Effector cells and target cells (cancer cells) cultured with or without peptide combination were co-cultured for 16 hours at ratios of 2.5:1, 5:1, and 10:1, respectively. The extent of cancer cell killing by effector cells was then measured using CFSE / 7-AAD (carboxyfluorescein succinimidyl ester / 7-aminoactinomycin D) staining.

[0122] In CFSE / 7-AAD staining, target cancer cells were stained with a substance called CFSE, which turned the cancer cells green. The CFSE-stained target cancer cells were cultured with effector cells for 16 hours and then stained again with a substance called 7-AAD. Since 7-AAD only stains dead cells, which turn red, the target cancer cells that express both CFSE and 7-AAD can be determined as dead cells by the effector cells. After staining, the frequency of CFSE+7-AAD+ cancer cells was measured using a flow cytometer, and the results were displayed in a graph.

[0123] The experiments were performed on colon cancer cell line SW620 expressing both HLA-A02 and HLA-A24, prostate cancer cell line PC-3 expressing only HLA-24, breast cancer cell line MCF-7, and ovarian cancer cell line OVCAR-3 expressing only HLA-02. As a result, it was confirmed that effector cells activated by peptide combination killed more cancer cells than effector cells cultured without peptide, and a similar trend was observed in the three donors. Furthermore, as the number of effector cells increased, more cancer cells were killed, confirming the cell-specific cytotoxic effect (Figure 5a-5h).

[0124] Experimental Example 3: Confirmation of induction of granzyme B secretion by combined use of peptides To determine whether T cells expanded and functionally activated by combinations of peptides derived from various tumor-associated antigens could secrete granzyme B capable of directly killing cancer cells as CTLs, the levels of granzyme B in the medium of effector cells co-cultured with cancer cells were analyzed using ELISA.

[0125] Using the same method as in Example 2 above, effector cells and target cells (cancer cells) cultured with or without peptides were added at a ratio of 2.5:1, 5:1, and 10:1, respectively, and cultured together for 16 hours to obtain culture medium. The obtained culture medium was centrifuged at 2000 rpm for 5 minutes to obtain the supernatant, which was used for Granzyme B assay.

[0126] Granzyme B ELISA was performed using the Human Granzyme B (HRP) (Cat. No. 3486-1H-6) kit from MABTECH with the corresponding protocol. 2 μg / mL of capture mAb MT34B6 was added to a 96-well immunoplate (100 μL / well) and reacted overnight at 4°C. After the reaction was completed, the supernatant was removed and PBST (PBS + 0.05% Tween 20) containing 0.1% BSA was added to the plate at 200 μL / well, then reacted at room temperature for 1 hour. The plate was washed five times with PBST (PBS + 0.05% Tween 20), and then 0-2000 pg / mL of each obtained supernatant or ink kit standard was added to the plate at 100 μL / well, and reacted at room temperature for 2 hours. The plate was washed 5 times with PBST (PBS + 0.05% Tween 20), then 1 μg / mL MT28-biotin was added to the plate at 100 μL / well, then reacted at room temperature for 1 hour. The plate was washed 5 times with PBST (PBS + 0.05% Tween 20), then streptavidin-HRP was added to the plate at 100 μL / well, then reacted at room temperature for 1 hour. The plate was washed 5 times with PBST (PBS + 0.05% Tween 20). TMB substrate was added to the plate at 100 μL / well, then reacted at room temperature for 15 minutes, then 0.2 M sulfuric acid was added to the plate at 50 μL / well to stop the TMB substrate reaction. The OD was measured using an ELISA reader.450 was measured and the amount of granzyme B in the supernatant was analyzed by comparison with a standard.

[0127] As a result, as shown in Figure 6, it was confirmed that the effector cells activated by treating PBMCs derived from six healthy subjects with the peptide combination were co-cultured with colon cancer cell line SW620, prostate cancer cell line PC-3, breast cancer cell line MCF-7, or ovarian cancer cell line OVCAR-3, and the culture medium had a significantly higher level of granzyme B than the culture medium in which the effector cells without peptide were co-cultured with the cancer cells. This demonstrated that CTLs recognize cancer cells, secrete granzyme B, and thereby induce the death of the cancer cells.

[0128] Experimental Example 4: Verification of the ability of cytotoxic T cells to kill cancer cells by combinations of cancer antigens or peptide numbers The CTL activity effects of the combination of 10 peptides confirmed to have excellent immunogenicity in FIG. 3 and the combination of survivin + MAGE-A3 or hTERT + WT-1 peptides shown to have high immunogenicity in FIG. 2 were compared.

[0129] The experiment was carried out in the same manner as in Experimental Examples 2 and 3, and SW620, a colon cancer cell line, was used as the cancer cells.

[0130] As a result, as shown in Figures 7a to 7d, it was confirmed that effector cells activated by treating PBMCs derived from three healthy individuals with a combination of 10 peptides secreted much higher levels of granzyme B and were able to kill more cancer cells than effector cells activated by treatment with the combination of survivin + MAGE-A3 or hTERT + WT-1 peptides.

[0131] These results suggest that the combination of 10 peptides, which consists of peptides derived from various cancer antigens (hTERT, survivin, MAGE-A3, MUC-1, and WT-1), can induce anti-cancer immune responses against various cancer antigens and can simultaneously activate CD8 and CD4 T cells, which are important for anti-cancer immune responses, resulting in a high induction of immune responses against cancer cells and enabling the killing of more cancer cells by CTLs activated through the induced immune response.

[0132] Experimental Example 5: Evaluation of the efficacy of test vaccines formulated with peptide combinations and lipopeptides plus immunoactive substance adjuvants or other TLR adjuvants in inducing cellular immune responses in mouse models The levels of cellular immune response induction of test vaccines prepared using combinations of the 10 peptides with a lipopeptide plus immunostimulatory substance adjuvant (L-pampo) and test vaccines prepared using combinations of the 10 peptides with other TLR adjuvants were compared in a mouse model.

[0133] Test vaccines were prepared by mixing 50μg of each of the 10 peptides, and then including lipopeptide (Pam3Cys-SKKKK) + immunoactive substance (poly(I:C)) adjuvant or other TLR adjuvants (TLR2 ligand lipopeptide, TLR3 ligand poly(I:C), TLR7 / 8 ligand imiquimod, TLR9 ligand CpG) in the mixture. Each test vaccine was subcutaneously injected into C57BL / 6 mice (100μL / mouse) three times at weekly intervals. One week after the three immunizations, spleens were removed from the mice, and total splenocytes were isolated and analyzed for immunogenicity using ELISPOT assay.

[0134] As a result, as shown in Figures 8a and 8b, when the test vaccine (L-pampo) prepared using Pam3Cys-SKKKK and poly(I:C) adjuvants was administered, the number of cells producing IFN-γ was significantly increased, and the secretion of IFN-γ specific to each peptide was also increased, compared to when the test vaccine was administered using TLR2, TLR3, TLR7 / 8 or TLR9 adjuvants.

[0135] The above results indicate that the anti-cancer vaccine composition provided in one embodiment of the present invention can induce a stronger cellular immune response capable of killing cancer cells compared to a vaccine composition comprising a TLR2, TLR3, TLR7 / 8, or TLR9 adjuvant.

[0136] Experimental Example 6: Evaluation of the efficacy in inducing cellular immune responses of test vaccines prepared using a combination of peptides derived from tumor-associated antigens provided in the present invention and lipopeptide + immunoactive substance adjuvant, or Lipo-pam adjuvant consisting of liposomes with lipopeptides inserted therein and immunoactive substances. The level of induction of cellular immune responses was compared in a mouse model for a test vaccine prepared using a combination of 10 peptides with a lipopeptide plus immunoactive substance adjuvant (Lipo-pam) and a test vaccine prepared using a combination of 10 peptides with a Lipopeptide-loaded liposome and an immunoactive substance adjuvant (Lipo-pam).

[0137] Test vaccines were prepared by mixing 50μg of each of the 10 peptides, and then including in the mixture either a lipopeptide (Pam3Cys-SKKKK) + immunoactive substance (poly(I:C)) adjuvant, or a Lipo-pam adjuvant consisting of liposomes with lipopeptides inserted on their surface and an immunoactive substance (poly(I:C)). Each test vaccine was subcutaneously injected into C57BL / 6 mice (100μL / mouse) three times at weekly intervals. One week after the three immunizations, the spleens were removed from the mice, and total splenocytes were isolated and analyzed for immunogenicity using the ELISPOT method.

[0138] As a result, as shown in Figure 9, when a test vaccine prepared using a Lipo-pam adjuvant consisting of liposomes having lipopeptide (Pam3Cys-SKKKK) inserted on the surface and an immunoactive substance (poly(I:C)) was administered, the number of cells producing IFN-γ was significantly increased compared to when a test vaccine prepared using Pam3Cys-SKKKK and poly(I:C) adjuvant (Lipo-pam) was administered.

[0139] The above results indicate that the anti-cancer vaccine composition provided in one embodiment of the present invention, which is composed of a lipopeptide and an immunoactive substance adjuvant, or Lipo-pam, a liposome having a lipopeptide inserted on its surface and an immunoactive substance, is an anti-cancer vaccine composition that can strongly induce a cellular immune response that can kill cancer cells.

Claims

1. An anti-cancer vaccine composition comprising a peptide derived from a tumor-associated antigen (TAA), An anti-cancer vaccine composition, wherein the peptide derived from a tumor-associated antigen is a peptide set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, or SEQ ID NO:

16.

2. An anti-cancer vaccine composition comprising a peptide derived from a tumor-associated antigen (TAA) and an adjuvant consisting of a lipopeptide and an immunoactive substance, An anti-cancer vaccine composition, wherein the peptide derived from a tumor-associated antigen is a peptide set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, or SEQ ID NO:

16.

3. 3. The anti-cancer vaccine composition according to claim 1, wherein the tumor-associated antigen is at least one selected from the group consisting of hTERT, MAGE-A3, MUC-1, survivin, and WT-1.

4. 3. The anti-cancer vaccine composition of claim 2, wherein the adjuvant comprises a liposome having a lipopeptide inserted into its surface.

5. The anti-cancer vaccine composition of claim 2, wherein the lipopeptide is at least one selected from the group consisting of Pam3Cys-SKKKK, Pam3-CSKKKK, PHC-SKKKK, Ole2PamCys-Pam3Cys-SKKKK, Pam3-CSKKKK, PHC-SKKKK, Ole2PamCys-SKKKK, Pam2Cys-SKKKK, PamCys(Pam)-SKKKK, Ole2Cys-SKKKK, Myr2Cys-SKKKK, PamDhc-SKKKK, Pam-CSKKKK, Dhc-SKKKK, and FSL-1 (Pam2CGDPKHPKSF).

6. The anti-cancer vaccine composition of claim 4, wherein the liposome is composed of lipids.

7. 7. The anti-cancer vaccine composition according to claim 6, wherein the lipid is at least one selected from the group consisting of DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DDA (dimethyldioctadecylammonium), DC-chol (3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol), DOPG (1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and cholesterol.

8. 3. The anti-cancer vaccine composition of claim 2, wherein the adjuvant is composed of a positively charged liposome in which a positively charged lipopeptide is inserted into the lipid bilayer of the liposome, and a negatively charged immunoactive substance.

9. The anti-cancer vaccine composition according to claim 2, wherein the immunoactive substance is at least one selected from the group consisting of poly(I:C), QS21, MPLA (monophosphoryl lipid A), CpG, and flagellin.

10. The anti-cancer vaccine composition according to claim 9, wherein the poly(I:C) has a length of 50 to 5,000 bp.

11. The anti-cancer vaccine composition according to claim 9, wherein the lipopeptide and poly(I:C) are mixed in a composition ratio of 1.25:1 to 2:1, the lipopeptide is 0.01% by weight to 0.1% by weight, and the poly(I:C) is 0.01% by weight to 0.08% by weight.

12. 3. The anti-cancer vaccine composition according to claim 1 or 2, wherein the composition is an aqueous liquid formulation.

13. 3. A pharmaceutical composition for preventing or treating cancer, comprising the anti-cancer vaccine composition according to claim 1 or 2 as an active ingredient.

14. The pharmaceutical composition for preventing or treating cancer according to claim 13, wherein the cancer is at least one selected from the group consisting of ovarian cancer, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, intracranial tumor, ependymoma, brain stem tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity / paranasal sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, breast tumor, small cell lung cancer, non-small cell lung cancer, thymic cancer, mediastinal tumor, esophageal cancer, breast cancer, abdominal tumor, gastric cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer, colon cancer, anal cancer, bladder cancer, kidney cancer, penile cancer, prostate cancer, cervical cancer, endometrial cancer, uterine sarcoma, vaginal cancer, female external genitalia cancer, and skin cancer.

15. The pharmaceutical composition for preventing or treating cancer according to claim 13, wherein the anti-cancer vaccine composition is used in combination with one or more treatments selected from the group consisting of chemotherapy, targeted therapy, immune checkpoint inhibitors, and radiation therapy.

16. 1. Use of a peptide derived from a tumor-associated antigen (TAA) for use in the preparation of a medicament for the prevention, amelioration or treatment of cancer, comprising: The use, wherein the peptide derived from a tumor-associated antigen is a peptide as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, and SEQ ID NO:

16.

17. Use of a peptide derived from a tumor-associated antigen (TAA) and an adjuvant consisting of a lipopeptide and an immunoactive substance for use in the preparation of a medicament for the prevention, amelioration or treatment of cancer, comprising: The use, wherein the peptide derived from a tumor-associated antigen is a peptide as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:12, SEQ ID NO:15, and SEQ ID NO:16.

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