WO2024225764A1 - Immunodominant epitope peptides of fibroblast activating proteins, and uses thereof - Google Patents

Abstract

The present invention relates to immunodominant epitope peptides of fibroblast activating proteins (FAPs), and uses thereof. The peptides of the present invention have excellent immunodominance and exhibit a strong effect of inducing CD8+ T cells specific for FAPs and FAP-expressing fibroblasts (FAP+ CAFs). The peptides of the present invention can exhibit excellent prevention and treatment effects on FAP-associated diseases such as cancer, fibrosis and NASH through immunity induction on FAPs, and has remarkably lower toxicity than other FAP-expressing CAF-targeted vaccines. Particularly, with respect to cancer, the peptides exhibit excellent anti-tumor effects such as depletion of FAP-expressing CAFs, decreased production of ECM in the TME, and suppressed cancer metastasis. If the peptides of the present invention are administered in combination with other anti-cancer agents, accumulation of the anti-cancer agent in tumor cells is increased and a remarkable improvement in anti-tumor effects is exhibited, and thus the peptides can be effectively used in pan-tumor vaccines.

Description

Immunodominant epitope peptide of fibroblast activation protein and uses thereof

The present invention relates to an immunodominant epitope peptide of fibroblast activation protein and uses thereof, and more specifically, to an MHC I-restricted immunodominant epitope peptide of the fibroblast activation protein and uses thereof for the prevention or treatment of fibroblast activation protein alpha (FAP)-related diseases such as tumors, nonalcoholic steatohepatitis (NASH) or fibrosis, and uses thereof for inhibiting cancer metastasis.

Cancer vaccines or tumor vaccines have recently been spotlighted as a treatment for tumors based on the development of personalized neoantigen prediction and detection technology (Nature Reviews Clinical Oncology 2021, 18 (4), 215-229). In particular, neoantigen-based cancer vaccines induce patient-specific antitumor immunity and have shown good results in various clinical trials (Nature 2018, 555 (7696), 402-402; Nat Med 2021, 27 (3), 515~). However, since neoantigen-based cancer vaccines are personalized vaccines, they require a lot of cost and time from neoantigen prediction for each patient to vaccine manufacturing, which increases the burden on patients. Therefore, the need for the development of a pan-cancer vaccine that can be applied to various types of cancer is emerging.

Meanwhile, the tumor microenvironment (TME) is composed of immune cells, cancer-associated fibroblasts (CAFs), signaling molecules, blood vessels, and the extracellular matrix (ECM), and acts as a powerful barrier that limits the effectiveness of conventional cancer treatments, such as chemotherapy and immunotherapy (Cell Commun Signal 2020, 18 (1), 59). In particular, cancer-associated fibroblasts present in the TME play a pivotal role in forming the tumor stroma by secreting extracellular matrix proteins (ECMs) such as collagen, laminin, and fibronectin (Nat Rev Immunol 2015, 15 (11), 669-682; Nat Rev Cancer 2016, 16 (9), 582-598; Nat Rev Clin Oncol 2021, 18 (12), 792-804). These ECM proteins provide a structural network for tumor growth, mediate signaling and interactions between cells, and form a physical barrier between the tumor and the surrounding environment (Signal Transduct Target Ther 2021, 6 (1), 153). In particular, the dense and rigid ECM network formed in desmoplastic tumors such as pancreatic adenocarcinoma (PDAC) and colorectal cancer has been reported to be a major cause of the poor survival rate of patients with such tumors by strongly limiting the therapeutic effect (Proc Natl Acad Sci U S A 2019, 116 (22), 10674-10680; ACS Nano 2019, 13 (10), 11008-11021). In this regard, numerous attempts have been performed to inhibit or deplete CAFs in the TME. However, these approaches have shown mixed results, including inhibition of tumor growth and promotion of tumor metastasis, which are reported to result from randomly targeting heterogeneous CAFs present in various tumors (Nat Rev Clin Oncol 2021, 18 (12), 792-804; Proc Natl Acad Sci U S A 2019, 116 (22), 10674-10680; Clin Cancer Res 2018, 24 (5), 1190-1201).

Fibroblast Activation Protein alpha (FAP) is a transmembrane protein that is highly expressed in CAFs but at very low levels in normal adult tissues, and has been reported as a targeting marker for CAFs (Science 2010, 330 (6005), 827-830). In this regard, therapeutic approaches that inhibit the function of FAP-expressing CAFs, such as small molecule FAP inhibitors (J Clin Invest 2009, 119 (12), 3613-3625), FAP-specific antibodies and drug conjugates (Clin Cancer Res 2020, 26 (13), 3420-3430), and FAP-targeting CAR-T therapy (Cancer Immunol Res 2014, 2 (2), 154-166), have been developed and demonstrated that they promote specific types of tumor regression in vivo, suggesting the potential of this FAP therapeutic strategy (Front Biosci-Landmrk 2018, 23, 1933-1968). However, other than the short-term therapeutic approaches targeting FAP-expressing CAFs as described above, no vaccine has been reported that can induce long-term FAP-specific T cell responses. Although FAP vaccines based on FAP-encoding DNA (. Clin Cancer Res 2018, 24 (5), 1190-1201) or dendritic cells have been reported, they have insufficient therapeutic efficacy and systemic toxicity, making them difficult to apply in clinical practice (J Exp Med 2013, 210 (6), 1137-1151; J Exp Med 2013, 210 (6), 1125-1135; Cancer Cell 2014, 25 (6), 719-734). Therefore, there is a need to develop a novel FAP-expressing CAF-targeted cancer vaccine that has low toxicity but excellent therapeutic efficacy.

Meanwhile, non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic steatosis and inflammation, and is a serious disease that can progress to fibrosis, cirrhosis, and hepatocellular carcinoma (Hepatology 77, 1335-1347(2023)). FAP is expressed in hepatocyte stellate cells and other fibroblasts in liver tissue (Nat Commun 13 (2022)) and is reported to cleave FGF21, a major regulator of lipids. Dysregulation of FGF21 by FAP activity is reported to contribute to the progression of NASH, but no NASH vaccine targeting FAP has been reported.

Under these background technologies, the inventors of the present invention have made diligent efforts to develop a vaccine that exhibits excellent immune-inducing effects by inducing a strong fibroblast-activating protein (FAP)-specific T cell immune response, and as a result, have derived an immunodominant epitope peptide that exhibits excellent immune-inducing effects against FAP, and have confirmed that the immunodominant epitope peptide can effectively treat FAP-related diseases such as cancer, NASH, etc., thereby completing the present invention.

The above information set forth in this Background section is intended solely to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person skilled in the art to which the present invention pertains.

Summary of the invention

The purpose of the present invention is to provide an immunodominant epitope peptide exhibiting an excellent T cell inducing effect against fibroblast activation protein alpha (FAP) and its use for the prevention or treatment of diseases related to fibroblast activation protein alpha (FAP).

The purpose of the present invention is to provide a preparation that specifically binds to the above peptide and a use thereof.

Another object of the present invention is to provide a nucleic acid encoding the peptide.

Another object of the present invention is to provide a recombinant vector comprising the nucleic acid.

Another object of the present invention is to provide a host cell into which the nucleic acid or recombinant vector has been introduced.

To achieve the above purpose, the present invention provides a peptide which is an MHC I-restricted epitope peptide of fibroblast activation protein (FAP), wherein the peptide is composed of 8 to 10 amino acids.

The present invention also provides a nanoparticle comprising the peptide.

The present invention also provides a vaccine composition for preventing or treating a disease related to fibroblast activation protein alpha (FAP), comprising the peptide and/or nanoparticle as an active ingredient.

The present invention also provides a vaccination, prevention and/or treatment method for the prevention or treatment of a fibroblast activation protein alpha (FAP)-related disease comprising the step of administering to a subject the peptide and/or nanoparticle.

The present invention also provides a use of the peptide and/or nanoparticle for the manufacture of a vaccine composition for the prevention or treatment of a disease associated with fibroblast activating protein alpha (FAP).

The present invention also provides a pharmaceutical composition for preventing or treating a disease associated with fibroblast activating protein alpha (FAP), comprising the peptide and/or lipid nanoparticle.

The present invention also provides a nucleic acid encoding the peptide.

The present invention also provides a recombinant vector containing the nucleic acid.

The present invention also provides a host cell into which the nucleic acid or recombinant vector has been introduced.

The present invention also provides a method for producing a peptide, comprising a step of culturing the host cell.

Figure 1 schematically illustrates the development process of a FAP immunodominant epitope peptide in an embodiment of the present invention.

Figure 2 illustrates the gating method of flow cytometry used in ICS analysis.

Figure 3 shows the upper epitope peptide sequences of FAP predicted by each program (netMHC, PREDEP, BIMAS).

Figure 4 shows the immunogenicity and antitumor efficacy of the peptide candidates screened in vivo. Figure 4(a) summarizes the predicted FAP peptides presented by MHC Class I. Figure 4(b) shows the immunogenicity schedule of the predicted peptides (n=3 mice/group). Figure 4(c) and (d) show the representative flow cytometry results of IFN-γ-secreting CD8+ T cells (c) and the percentage of CD8+ T cells producing IFN-γ (d). The statistical significance between the control group and each experimental group was determined by two-tailed Student's t-test. Figure 4(e) and (f) show representative images of IFN-γ-producing spots (e) and the average number of IFN-γ spot-forming cells (f) determined by ELISpot analysis. The statistical significance between the control group and each experimental group was determined by two-tailed Student's t-test. Figure 4(g) shows the immunization schedule to evaluate the antitumor therapeutic efficacy of the predicted peptides. Nine days after E.G7-OVA tumor inoculation, C57BL/6 mice were immunized twice with each peptide at one-week intervals (n = 4 mice/group). Figure 4(h) shows the mean tumor growth curves of E.G7-OVA cells (left) and tumor volumes on day 25 (right). The statistical significance of the differences between the control group and each experimental group was determined by two-tailed Student's t-test. All data are expressed as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5 shows the ICS analysis results of the six screened peptides individually. (Each panel represents: Control (unadministered); peptide administration; peptide and CFA co-administration) The upper panel of each figure shows the results of no ex-vivo restimulation, and the lower panel shows the results of ex-vivo restimulation with peptides.

Figure 6 shows the results of ELISpot analysis performed in the same manner as the ICS analysis.

Figure 7 shows the ratio of FAP+CAF according to tumor volume.

Figure 8 shows the design and antitumor efficacy of FAPPEP-SLNP nanovaccine in an established E.G7-OVA tumor model. Figure 8(a) shows the schematic diagram of FAPPEP-SLNP nanovaccine and its expected mechanism of action after internalization into APC. Figure 8(b) shows the hydrodynamic diameter of FAPPEP-SLNP measured by DLS. Figure 8(c) shows the TEM image of FAPPEP-SLNP showing the morphology and nanoscale size. Scale bar = 200 nm. Figure 3(d) shows the immunization schedule to evaluate the antitumor efficacy of FAPPEP-SLNP nanovaccine against an established E.G7-OVA tumor model. Nine days after E.G7-OVA tumor inoculation, C57BL/6 mice were injected three times with each vaccine modality at 4-day intervals (n = 5 mice/group). Figure 8(e) shows the average growth curve of E.G7-OVA tumors (left) and tumor volume on day 21 (right). The statistical significance of the differences between the control group and each experimental group was determined by two-tailed Student's t-test. All data are expressed as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 9 schematically illustrates the structure of the DSPE-PEG2000-FAPPAP conjugate, one of the components of SLNP.

Figure 10 shows the results of confirming the synthesis of DSPE-PEG2000-FAPPEP using HPLC (a, b) and MALDI-TOF (c, d).

Figure 11 shows the results of ICS analysis confirming that cysteine (Cys) introduced to conjugate FAPPEP to DSPE-PEG2000-PDP does not affect immunogenicity.

Figure 12 shows the results of ICS analysis performed on the spleen and lymph nodes of mice immunized with FAPPEP1-SLNP, confirming the presence of CD8+ T cells specific for FAPPEP1.

Figure 13 shows the results of a tetramer assay using FAPPEP1-H-2Kb.

Figure 14 shows the results confirming that FAPPEP-SLNP nanovaccine increases antigen-specific T cell immunity without inducing autoimmunity by mediating TME remodeling. Figure 14(a) shows the immunization schedule for tumor growth, mouse survival, tumor tissue, and spleen analysis. Nine days after E.G7-OVA tumor inoculation, C57BL/6 mice were immunized three times with each vaccine method at 4-day intervals. Figure 14(b) shows the results of monitoring tumor growth in immunized mice, showing the average growth curve of E.G7-OVA tumors (left) and the average volume of E.G7-OVA tumors on day 21 (right) (n = 9 mice/group). Statistical significance was calculated by one-way ANOVA with post hoc Tukey’s test. Figure 14(c) shows the survival curve of E.G7-OVA tumor-bearing mice (n = 10 mice/group). The statistical significance of the survival difference between the control group and each experimental group was determined by the log-rank (Mantel-Cox) test. Figure 14(d) shows the representative images of Masson’s trichrome-stained tissues from each group as a result of evaluating collagen deposition in tumor tissues using Masson’s trichrome staining. Blue staining indicates collagen; black staining indicates nuclei; red background indicates cytoplasm and muscle; and black dotted lines indicate collagen deposition. Scale bar = 100 μm. Figure 14(e) shows the percentage of collagen-stained areas in three random fields from sections from each tumor group (n = 3 sections per tumor from 3 mice for each staining). Statistical significance was calculated by one-way ANOVA with post hoc Tukey’s test. Figure 14(f) and (g) show the results of tumor tissue analysis at the cellular level (n = 4 mice/group), showing the percentage of FAP+ CAFs (f) and CD8+ TILs (g) in tumors as determined by flow cytometry. Statistical significance was calculated by one-way ANOVA with post hoc Tukey’s test. Figure 14(h) and (i) show the results of spleen cell analysis to evaluate antigen-specific T cell responses (n = 4 mice/group). The percentages of IFN-γ-secreting CD8+ T cells (h) and CD4+ T cells (i) determined by ICS are shown. Statistical significance was determined by two-tailed Student's t-test. Figure 4(j) shows the results of spleen cell analysis to determine whether autoimmunity was induced after immunization with FAPPEP1-SLNP (n = 4 mice/group). The percentages of IL-17A-secreting CD4+ T cells were determined by ICS. Statistical significance was calculated by one-way ANOVA with post hoc Tukey’s test. All data are expressed as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 15 shows the results of confirming the distribution of blood vessels in a tumor (a and b) and the results of confirming metastasis in the lung (c and d). In Figures 15(a) and (b), blood vessels are shown in brown, and in Figures 15(c) and (d), metastatic cells are shown in red.

Figure 16 shows the analysis of the ratio of FAP+ CAF among the cells forming the tumor by flow cytometry analysis of single cells dissociated from various types of tumors (E.G7-OVA, Panc02, MC38).

Figure 17 shows the results of flow cytometry analysis to confirm whether FAP is expressed in cancer cells other than CAFs in MC38 tumors.

Figure 18 is an experiment to confirm whether FAPPEP1-SLNP monotherapy shows therapeutic efficacy in Panc02 and MC38 tumors, which are connective tissue tumors. Experimental method (a) and tumor growth (b) in the Panc02 model, and Kaplan-Meier survival analysis result (c), and experimental method (d) and tumor growth (e) and Kaplan-Meier survival analysis result (f) in the MC38 model.

Figure 19 shows the results of an ICS assay performed to confirm whether CD8+ T cells (a) and CD4+ T cells (b) within the tumor in the MC38 tumor model have immune activity specific to FAP.

Figure 20 shows the results confirming whether FAPPEP1-SLNP exhibits therapeutic efficacy even in large-sized tumors.

Figure 21 shows the killing effect of ECM-rich MC38 tumors after combination treatment with FAPPEP1-SLNP nanovaccine and Dox. (a) Penetration of near-infrared dye cypate into MC38 tumors after FAPPEP1-SLNP immunization. Mice were immunized with FAPPEP1-SLNP nanovaccine and administered near-infrared dye cypate as a proxy for small molecule Dox. Penetration into tumors of different groups was compared using IVIS (n = 5 mice/group). The left panels show representative images of intratumoral dye penetration and relative fluorescence signal intensity of the dye distributed in the tumors acquired by IVIS. The data in the right panels show the ratio of fluorescence intensities of tumors of cyphate-treated mice/cyphate-nontreated mice. Figure 21(b) shows the immunization schedule for combination therapy with FAPPEP1-SLNP nanovaccines and Dox for the established MC38 tumor model. Five days after MC38 tumor inoculation, C57BL/6 mice were immunized three times at 4-day intervals with FAPPEP1-SLNP nanovaccines and treated or not with Dox (n = 7 mice/group). Figure 21(c) shows the MC38 tumor growth in mice (left) and tumor volume on day 17 (right). Statistical significance was calculated by one-way ANOVA with post hoc Tukey’s test. Figure 21(d) and (e) show MC38 tumor tissue analysis at the cellular level (n = 4 mice/group). The percentage of FAP+ CAFs (d) and the percentage of CD8+ TILs (e) in the tumors determined by flow cytometry are shown. Statistical significance was calculated by one-way ANOVA with post hoc Tukey’s test. All data are presented as mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 22 schematically illustrates the mechanism by which FAPPEP1-SLNP nanovaccine targets CAFs to kill tumors.

Figure 23 schematically illustrates the mechanism of a tumor metastasis inhibition model targeting CAF of FAPPEP1 peptide vaccine or FAPPEP1-SLNP nanovaccine (top) and the therapeutic efficacy evaluation model of FAPPEP1 peptide vaccine on cancer metastasis used in the examples of the present invention (bottom).

Figure 24 is a histological analysis of lung tissue showing the results of inhibiting cancer metastasis to the lung by the FAPPEP1 peptide vaccine. Figure 24a is an image of lung tissue stained with H&E. The red arrow indicates the area of cancer metastasis to the lung. Scale bar = 50 nm. Figure 24b shows the percentage of cancer metastasis area in three random fields within the sections of each lung group. (n = 3 for three sections per lung from five mice for each staining). Statistical significance was calculated by one-way ANOVA with post hoc Tukey’s test. All data are expressed as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 25 is a histological analysis of liver tissue showing the results of FAPPEP1 peptide vaccine inhibition of cancer metastasis to the liver. Figure 25a is an image of liver tissue stained with H&E. The red arrow indicates the area of cancer metastasis to the lung. Scale bar = 50 nm. (b) Figure 24b shows the percentage of cancer metastasis area in three random fields within the sections of each liver group (n = 3 for three sections per liver from five mice for each staining). Statistical significance was calculated by one-way ANOVA with post hoc Tukey’s test. All data are expressed as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 26 schematically illustrates the NASH treatment mechanism through targeting FAP expressing cells and preventing FGF21 degradation by FAPPEP1 peptide vaccine or FAPPEP1-SLNP nanovaccine.

Figure 27 shows the results showing the therapeutic efficacy of the FAPPEP1 peptide vaccine for the NASH model. Figure 27 schematically shows the experimental method of the NASH establishment and therapeutic effect confirmation model performed in the example of the present invention. After 4 weeks of MCD/MCS diet therapy, C51BL/6 mice were immunized twice with the FAPPEP1 peptide vaccine at one-week intervals. (n = 5 mice/group). Figure 27b shows the body weight monitoring of the mice (left) and the body weight of the mice at 45 days (right). Figure 27c shows the liver weight of the mice at 45 days. Figure 27d shows the liver-to-body weight ratio at 45 days. Statistical significance was calculated by one-way ANOVA with post hoc Tukey’s test. All data are expressed as the mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001). (Illustration created with BioRender.com.)

Figure 28 shows the results of serum analysis of mice after immunization with FAPPEP1 peptide vaccine in NASH model. Mouse sera were collected on day 45 and analyzed for the levels of damage-related markers as follows: ALT (a), AST (b), total bilirubin (c), and HDL (d). Statistical significance was calculated by one-way ANOVA with post hoc Tukey’s test. All data are expressed as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 29 shows the results of evaluating lipid deposition in liver tissue using Oil red O staining after immunization with FAPPEP1 peptide vaccine. Figure 29 shows the images of Oil red O-stained tissues in each group. Red staining area is collagen; black staining area is nucleus; red background staining is cytoplasm and muscle; white arrows indicate lipid deposition. Scale bar = 50 μm. Figure 29b shows the percentage of lipid-stained area in three random fields in sections from each liver group (n = 3 sections per tumor from 5 mice for each stain). Statistical significance was calculated by one-way ANOVA with post hoc Tukey’s test. All data are expressed as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 30 shows the results of evaluating ballooning hepatocytes in liver tissue using H&E staining. Figure 30a shows a histological image of liver tissue stained with H&E. The blank area represents ballooning adipocytes in the liver tissue. Scale bar = 100 nm. Figure 30b shows the percentage of ballooning adipocyte areas in three random fields within the sections of each liver group (n = 3 for three sections per liver from five mice for each staining). Statistical significance was calculated by one-way ANOVA with post hoc Tukey’s test. All data are expressed as mean ± SEM. (*P < 0.05, **P < 0.01, ***P < 0.001).

Detailed description of the invention and preferred embodiments

Hereinafter, the present invention will be described in detail. However, the following detailed description is provided as an example of the present invention, and the present invention is not limited thereby, and the present invention can be variously modified and applied within the scope of the following claims and equivalents interpreted therefrom.

Unless otherwise indicated, nucleic acids and amino acids are listed in 5' to 3' and N-terminal to C-terminal orientations, respectively, from left to right. Numerical ranges listed within the specification are inclusive of the numbers defining the range and include each integer or any non-integer fraction within the defined range.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of testing the present invention, the preferred materials and methods are described herein.

The amino acid sequence referred to in the present invention is interpreted to include variants or fragments thereof in which amino acid residues are conservatively substituted at specific amino acid residue positions.

As used herein, “conservative substitution” means a modification that includes replacing one or more amino acids with amino acids having similar biochemical properties that do not result in loss of biological or biochemical function of the protein.

A “conservative amino acid substitution” refers to the substitution of an amino acid residue with an amino acid residue having a similar side chain. For example, classes of amino acid residues having similar side chains are well known in the art. These classes include amino acids having basic side chains (e.g., lysine, arginine, histidine), amino acids having acidic side chains (e.g., aspartic acid, glutamic acid), amino acids having uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids having non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids having beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Fibroblast Activation Protein alpha (FAP) is also known as prolyl endopeptidase FAP. Although the expression of FAP is generally reported to be very low in adult tissues, FAP is known to be a major factor involved in the onset and progression of various diseases such as cancer, fibrosis, and NASH. Accordingly, various therapies targeting FAP have been studied and reported, but FAP-targeting therapies that can induce long-term immune responses have rarely been reported, and some reported FAP-targeting vaccine therapies also have systemic toxicity or insufficient efficacy, requiring the development of new FAP-targeting vaccine therapeutics.

In one embodiment of the present invention, 8-mer to 10-mer MHC (Major histocompatibility complex) I-restricted epitope peptides of fibroblast activation protein (FAP) were screened through in silico analysis, and it was confirmed that these epitope peptides can induce the production of excellent FAP-specific T cells and exhibit excellent effects in the treatment of tumors, inhibition of metastasis, and treatment of NASH.

Accordingly, the present invention relates, in one aspect, to an immunodominant epitope peptide of fibroblast activation protein (FAP).

The term “immunodominance” of the present invention refers to an immunological phenomenon in which an immune response is induced only to a small number of antigenic peptides among various antigenic peptides that can be derived from a specific protein or polypeptide (Clinical Immunology. 143 (2): 99-115). “Immunodominance” is clearly observed in both antibody-mediated immunity and cell-mediated immunity. The effect of immunodominance is caused by immunodominance. In general, the difference in immunogenicity of hundreds to thousands of peptide antigens that can be produced from a pathogen represents a dominance hierarchy, and an antigen that stimulates a strong immune response is considered an “immunodominant epitope” or “immunodominant antigen.”

In the present invention, the peptide may be characterized as being composed of 7 to 11 amino acids, preferably 8 to 10 amino acids.

In the present invention, the peptide may be characterized as being an MHC I-restricted epitope peptide of fibroblast activation protein (FAP).

MHC (Major histocompatibility complex) is a complex that presents antigens on the cell surface and induces an immune response to the epitopes presented by MHC. MHC can be largely classified into type I and type II. Type I MHC induces a CD8+ T cell response, while type II MHC induces an immune response by presenting it to B cells or helper T cells through APC.

In the present invention, the peptide may be characterized as being a human leukocyte antigen (HLA) restricted epitope.

Human leukocyte antigen (HLA) is a major gene locus of human MHC and refers to an antigenic site that distinguishes whether the MHC presented by a cell is self or non-self. HLA is classified into various families according to its location within the human chromosome. For example, HLA of MHC I is classified into HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, etc., and HLA of MHC II can be classified into HLA-DP, HLA-DQ, HLA-DR, etc., and each family group can be classified in more detail, but is not limited thereto.

In the present invention, the peptide may be an epitope peptide restricted to one or more types of HLA among HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G, and more preferably, may be an epitope peptide restricted to one or more types of HLA among HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-A26, HLA-B7, HLA-B8, and HLA-B27, but is not limited thereto.

The term "MHC restricted epitope" or "HLA restricted epitope" of the present invention means that the antigen can induce an immune cell response when presented by a specific type of MHC or HLA. The restriction of the epitope peptide can be exclusive (restricted to only one type) or non-exclusive (restricted to more than one type) with respect to other types of MHC or HLA. Such restriction of the epitope peptide not only allows more effective immunization depending on the patient's HLA typing, but also prevents toxicity and side effects due to induction of unintended excessive immune response.

In the present invention, the peptide may be characterized by including an amino acid sequence selected from the group consisting of sequence numbers 1 to 149 of Tables 1 to 5 below.

In the present invention, the peptide may be characterized by being composed of an amino acid sequence selected from the group consisting of sequence numbers 1 to 149 of Tables 1 to 5 below.

In the present invention, the peptide may be characterized as being an epitope peptide restricted to HLA-A1. For example, the epitope peptide restricted to HLA-A1 may include or be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 29.

In the present invention, the peptide may be characterized as being an epitope peptide restricted to HLA-A2. For example, the epitope peptide restricted to HLA-A2 may include or be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, 5, and 30 to 45.

In the present invention, the peptide may be characterized as being an epitope peptide restricted to HLA-A3. For example, the epitope peptide restricted to HLA-A3 may include or be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 46 to 65.

In the present invention, the peptide may be characterized as being an epitope peptide restricted to HLA-A24. For example, the epitope peptide restricted to HLA-A24 may include or be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 66 to 85.

In the present invention, the peptide may be characterized as being an epitope peptide restricted to HLA-A26. For example, the epitope peptide restricted to HLA-A26 may include or be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, 14, 19, 21, 28, 58, and 86 to 97.

In the present invention, the peptide may be characterized as being an epitope peptide restricted to HLA-B7. For example, the epitope peptide restricted to HLA-B7 may include or be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 93, and 98 to 115.

In the present invention, the peptide may be characterized as being an epitope peptide restricted to HLA-B8. For example, the epitope peptide restricted to HLA-B8 may include or be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 91, 93, 99, 100, 103, and 116 to 129.

In the present invention, the peptide may be characterized as being an epitope peptide restricted to HLA-B27. For example, the epitope peptide restricted to HLA-B27 may include or be composed of an amino acid sequence selected from the group consisting of SEQ ID NOs: 130 to 149.

In the present invention, the peptide may preferably comprise or consist of an amino acid sequence of SEQ ID NO: 1, 2, 3, 4 or 5, more preferably SEQ ID NO: 1.

In the present invention, the peptide may be characterized by inducing an immune response specific to fibroblast activation protein (FAP).

In the present invention, the peptide may be characterized by, but is not limited to, stimulating a cytotoxic T cell response or a CD8+ T cell response against free FAP or FAP expressing cells.

In the present invention, the FAP expressing cell may be characterized as being a FAP expressing fibroblast, but is not limited thereto.

In the present invention, the peptide may additionally independently include amino acids at the N'-terminus and/or the C'-terminus, respectively. For example, the peptide may additionally include 1 to 100 amino acids, preferably 1 to 50 amino acids, more preferably 1 to 25 or 1 to 10 amino acids, respectively, at the N'-terminus and/or the C'-terminus, but is not limited thereto, and any amino acid may be additionally included within a range in which the activity of the peptide of the present invention as an immunodominant epitope is substantially maintained. For example, in the present invention, the peptide may additionally include amino acids at the N'-terminus and/or the C'-terminus of the amino acid sequence of SEQ ID NOs: 1 to 149. It is preferable that an amino acid according to a sequence of a known fibroblast activation protein (FAP), preferably a sequence of human fibroblast activation protein, is added, but is not limited thereto.

In the present invention, the peptide includes a fragment in which a part of the N'-terminal and/or C'-terminal sequence of the amino acid sequence is deleted. For example, it may be a fragment in which 1 to 5 amino acids, preferably 1 to 3 amino acid sequences are independently deleted from the N'-terminal and/or C'-terminal of the amino acid sequence of SEQ ID NOs: 1 to 149, but is not limited thereto, and includes a fragment in which any amino acid is deleted within a range in which the activity as an immunodominant epitope of the peptide having the amino acid sequence of SEQ ID NOs: 1 to 9 is substantially maintained.

In the present invention, the peptide may be characterized by being obtained through cleavage of a full-length fibroblast activation protein (FAP) obtained from nature or a fragment thereof, or an artificially synthesized oligopeptide, but is not limited thereto.

The peptide of the present invention can be produced and used in the form of a fusion protein by being fused with various functional peptides known in the art.

In the present invention, the fusion protein may be produced and used in the form of a fusion protein in which one or more peptides of the same sequence of the present invention are fused (homo type) and/or peptides of different sequences are fused (hetero type), but is not limited thereto. For example, it may be a fusion protein in which one or more of the amino acid sequences of SEQ ID NOs: 1 to 149 are fused, but is not limited thereto.

For example, in addition to the peptide of the present invention, it may be fused with another FAP epitope peptide or cancer antigen known in the art, or may be fused with an Fc domain, PEG, hyaluronic acid, albumin, serum binding domain, etc. to improve pharmacokinetic properties or ADME properties, but is not limited thereto. In the present invention, the fusion protein may further include an Fc domain. In the present invention, the Fc domain may include the last two constant region immunoglobulin domains of IgA, IgD and IgG and a flexible hinge N-terminus for these domains. In the case of IgA and IgM, the Fc domain may include a J chain. In the case of IgG, the Fc domain may include immunoglobulin domains Cγ2 and Cγ3 and a hinge between Cγ1 and Cγ2. The boundaries of the Fc domain can vary, but the human IgG heavy chain Fc region is generally defined to include residues C226 or P230 of the carboxyl terminus, where the numbering of amino acids uses the Kabat numbering. The terms “Fc,” “Fc domain,” and “Fc region” of the present invention may be used interchangeably, and the Fc domain may refer to the region in isolation, or as part of an antibody, a fragment thereof, or a fusion protein. Polymorphisms in the Fc domain have been reported at various positions and can be used as the fusion domain of the fusion protein of the present invention without limitation.

In the present invention, the peptide of the present invention can be prepared and used in the form of a fusion protein additionally including a membrane penetrating domain for presentation on the surface of a cell or an artificial membrane.

In the present invention, the peptide of the present invention can be prepared and used in the form of a fusion protein additionally including at least one of a hinge domain, a coiled-coil domain, an immune regulatory domain, and an intracellular signaling domain, but is not limited thereto.

The term “transmembrane domain” of the present invention refers to a protein domain that spans the membrane of a cell. In the present invention, the transmembrane domain preferably has an alpha-helical structure, but is not limited thereto.

The term “hinge domain” of the present invention refers to a series of amino acid sequences that exist between the transmembrane domain and the extracellular domain of a membrane anchored protein.

The term “coiled coil domain” of the present invention refers to a structural motif of a protein in which 2 to 7 alpha helices are coiled like a rope strand. Preferably, the coiled coil domain may be characterized by having 2 or 3 alpha helices coiled.

In addition, the peptide of the present invention can be fused without limitation with various domains known in the art for extending half-life, improving pharmacokinetic properties, etc., and it is preferable that the domain does not reduce, inhibit, or mask the function of the peptide of the present invention as an immunodominant epitope.

The peptide of the present invention can be used as a conjugate by being conjugated to a molecule other than a peptide. Methods for producing protein-conjugates known in the art can be used without limitation, and preferably, it can be conjugated to another drug or adjuvant through chemical conjugation, but is not limited thereto.

In another aspect, the present invention relates to a nucleic acid encoding a peptide or fusion protein of the present invention.

The nucleic acids used herein may be present in cells, a cell lysate, or may be present in a partially purified or substantially pure form. A nucleic acid is "isolated" or "rendered substantially pure" when purified away from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques including alkaline/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. A nucleic acid of the present invention may be, for example, DNA or RNA.

In another aspect, the present invention relates to a recombinant vector containing the nucleic acid of the present invention.

In the present invention, as long as the recombinant vector can induce protein expression of a nucleic acid encoding the peptide, any vector known in the art can be appropriately selected and used without limitation. For example, when E. coli is used as a host, a vector containing a T7 series (T7A1, T7A2, T7A3, etc.), lac, lacUV5, temperature-dependent (λphoA, phoB, rmB, tac, trc, trp or 1PL promoter) can be used, and when yeast is used as a host, a vector containing an ADH1, AOX1, GAL1, GAL10, PGK or TDH3 promoter can be used, and in the case of Bacillus, a vector containing a P2 promoter can be used. However, this is only a listing of some embodiments, and in addition to the vector containing the promoter, as long as it is suitable for the host as a vector containing a promoter for inducing expression of the peptide according to the present invention, various vectors known in the art can be appropriately selected and used by those skilled in the art.

The term "vector" in the present invention means a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases may be integrated into the genome itself. Since plasmids are currently the most commonly used form of vector, the terms "plasmid" and "vector" are sometimes used interchangeably in the present description. However, the present invention includes other forms of vectors that have equivalent functions as known or become known in the art. Protein expression vectors used in E. coli include the pET series from Novagen (USA); the pBAD series from Invitrogen (USA); the pHCE or pCOLD series from Takara (Japan); the pACE series from Xenofocus (Korea); and the like. In Bacillus subtilis, protein expression can be achieved by inserting the target gene into a specific part of the genome, or by using vectors such as the pHT series from MoBiTech (Germany). Protein expression is also possible in molds and yeast by using genome insertion or self-replicating vectors. Plant protein expression vectors can be used using the T-DNA system of Agrobacterium tumefaciens or Agrobacterium rhizogenes. Typical expression vectors for expression in mammalian cell cultures are based on, for example, pRK5 (EP 307,247), pSV16B (WO 91/08291), and pVL1392 (Pharmingen).

The term "expression control sequence" means a DNA sequence essential for the expression of an operably linked coding sequence in a particular host organism. Such control sequences include a promoter for initiating transcription, an optional operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation. For example, a control sequence suitable for a prokaryote includes a promoter, optionally an operator sequence, and a ribosome binding site. For a eukaryote, this includes a promoter, a polyadenylation signal, and an enhancer. The factor that most influences the amount of gene expression in a plasmid is the promoter. As promoters for high expression, the SRα promoter and a cytomegalovirus-derived promoter are preferably used.

Any of a wide variety of expression control sequences may be used in the vector to express the DNA sequence of the present invention. Examples of useful expression control sequences include, in addition to the promoters described above, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the T3 and T7 promoters, the major operator and promoter region of phage lambda, the regulatory region of the fd encoded protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of the phosphatases, e.g. Pho5, the promoter of the yeast alpha-mating system, and any other sequence of structure and inducibility known to control expression of genes in prokaryotes or eukaryotes or their viruses, and any combination thereof. The T7 RNA polymerase promoter Φ can be usefully used to express proteins in E. coli.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. This can be a gene and regulatory sequence(s) that are linked in such a way that an appropriate molecule (e.g., a transcriptional activating protein) can cause gene expression when bound to the regulatory sequence(s). For example, DNA for a pre-sequence or a secretory leader is operably linked to DNA for a polypeptide if it is expressed as a pre-protein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. In general, "operably linked" means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and are in reading frame. However, an enhancer need not be in contact. The connection of these sequences is carried out by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers according to conventional methods are used.

The term "expression vector" as used herein generally refers to a recombinant carrier into which a fragment of heterologous DNA has been inserted, typically a fragment of double-stranded DNA. Here, heterologous DNA refers to heterologous DNA, which is DNA that is not naturally found in the host cell. Once inside the host cell, the expression vector can replicate independently of the host chromosomal DNA, and multiple copies of the vector and its inserted (heterologous) DNA can be produced.

As is well known in the art, in order to increase the level of expression of a transfected gene in a host cell, the gene must be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host. Preferably, the expression control sequences and the gene are contained in a single expression vector that also contains a bacterial selection marker and a replication origin. If the expression host is a eukaryotic cell, the expression vector may further contain expression markers that are useful in the eukaryotic expression host.

In another aspect, the present invention relates to a nucleic acid encoding a peptide of the present invention; or a host cell into which the recombinant vector has been introduced.

In the present invention, the host cell refers to an expression cell into which a gene or a recombinant vector, etc. has been introduced to produce a protein, etc. The host cell may be used without limitation as long as it is a cell capable of expressing the peptide of the present invention, and is preferably a eukaryotic cell, more preferably a yeast, an insect cell, an animal cell, and most preferably an animal cell. For example, a CHO cell line or a HEK cell line, which are mainly used for the expression of peptides, may be used, but is not limited thereto.

A wide variety of expression host/vector combinations can be utilized to express the peptides of the present invention. Suitable expression vectors for eukaryotic hosts include, for example, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus and retroviruses. Expression vectors for use in bacterial hosts include, for example, bacterial plasmids obtained from E. coli, such as pBluescript, pGEX2T, pUCvector, col E1, pCR1, pBR322, pMB9 and derivatives thereof, plasmids having a wider host range, such as RP4, phage DNA, such as a wide variety of phage lambda derivatives, such as λ and λNM989, and other DNA phages, such as M13 and filamentous single-stranded DNA phages. Useful expression vectors for yeast cells are the 2μ plasmids and derivatives thereof. A useful vector for insect cells is pVL 941.

The above recombinant vector can be introduced into a host cell by a method such as transformation or transfection. The term "transformation" as used in the present specification means that DNA is introduced into a host so that the DNA becomes replicable as an extrachromosomal element or by chromosomal integration. The term "transfection" as used in the present specification means that an expression vector is accepted by a host cell, regardless of whether any coding sequence is actually expressed.

It should be understood that not all vectors and expression control sequences are equally effective in expressing the DNA sequences of the present invention. Likewise, not all hosts are equally effective in the same expression system. However, one skilled in the art can make appropriate selections among the various vectors, expression control sequences, and hosts without undue experimental burden and without departing from the scope of the present invention. For example, in selecting a vector, one should consider the host, since the vector must be replicated in it. The copy number of the vector, the ability to control the copy number, and the expression of other proteins encoded by the vector, such as antibiotic markers, should also be considered. In selecting an expression control sequence, several factors should also be considered, such as the relative strength of the sequences, their controllability, and their compatibility with the DNA sequences of the present invention, particularly with respect to possible secondary structures. The unicellular host should be selected by considering factors such as the selected vector, the toxicity of the product encoded by the DNA sequence of the present invention, secretion characteristics, the ability to accurately fold the protein, culture and fermentation requirements, and the ease of purifying the product encoded by the DNA sequence of the present invention from the host. Within the range of these variables, those skilled in the art can select various vector/expression control sequence/host combinations that can express the DNA sequence of the present invention in fermentation or large-scale animal culture. As a screening method when attempting to clone cDNA by expression cloning, the binding method, the panning method, the film emulsion method, etc. can be applied.

The above gene and recombinant vector can be introduced into a host cell through various methods known in the art. A gene encoding a nucleic acid encoding the peptide of the present invention can be directly introduced into the genome of a host cell and exist as a chromosomal element. It will be obvious to those skilled in the art that inserting the gene into the genomic chromosome of a host cell will have the same effect as introducing a recombinant vector into the host cell.

In another aspect, the present invention relates to a method for producing a peptide, which comprises a step of culturing the host cell.

When a recombinant expression vector capable of expressing the above peptide is introduced into a mammalian host cell, the peptide can be produced by culturing the host cell for a period of time sufficient to express the peptide in the host cell or for a period of time sufficient to secrete the peptide into the culture medium in which the host cell is cultured.

In some cases, the expressed peptide can be obtained by separating from the host cell and purifying it to be homogeneous. The separation or purification of the peptide can be performed by a separation or purification method used for general proteins, for example, chromatography. The chromatography can be, for example, a combination of one or more selected from affinity chromatography, ion exchange chromatography, or hydrophobic chromatography, but is not limited thereto. In addition to the chromatography, filtration, ultrafiltration, salting out, dialysis, etc. can be additionally used in combination.

The peptide of the present invention may be presented as a peptide itself, but is not limited thereto, and may be presented attached to the surface of or included within a delivery vehicle for various purposes such as effective delivery, targeting, and improved pharmacokinetic properties.

For example, peptide-based vaccines have several advantages, including cost-effective manufacturing, relatively easy quality control, and good safety profiles, but they exhibit low immunogenicity, require strong adjuvants, and have limited therapeutic efficacy due to low delivery efficiency to antigen-presenting cells (APCs) in vivo.

To overcome these limitations, the present inventors have developed and patented an antigen carrier based on neutrally charged small lipid nanoparticles (SLNPs) and a vaccine system using the same (Korean Patent No. 10-2425028 Theranostics 2016, 6 (2), 192-203, and Angew Chem Int Ed Engl 2020, 59 (34), 14628-14638).

In one embodiment of the present invention, a vaccine platform based on neutrally-charged small lipid nanoparticles (SLNPs) was used to prepare lipid nanoparticles containing the peptide, and it was confirmed that when immunization was performed with the lipid nanoparticles, the number and activity of FAP-specific CD8+ T cells were significantly increased.

Therefore, the present invention, from another aspect, relates to nanoparticles comprising the peptide.

The term nanoparticle of the present invention is defined as a particle having a nanometer size, and each particle may have different physicochemical, biological, and immunogenic characteristics depending on the shape, size, components, surface characteristics, functional groups, etc.

In the present invention, the nanoparticle may be characterized by including one or more types of the peptide. In the present invention, the nanoparticle may be characterized by including the peptide of the present invention including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more types of sequences. For example, the nanoparticle may include any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 149. The one or more peptides included in the vaccine composition of the present invention may be in various forms, such as an epitope peptide, a fusion protein including the same, a nanoparticle, etc., as described above.

Nanoparticle platforms for delivering peptides are well known in the art. For example, the nanoparticle may encapsulate a peptide and contain it inside, or may present the protein on the surface of the nanoparticle, but is not limited thereto.

In the present invention, for example, the nanoparticle may be selected from the group consisting of virus-like particles (VLPs), protein nanostructures, polymer nanoparticles, lipid nanoparticles, and inorganic nanoparticles, but is not limited thereto.

The term virus-like particle (VLP) of the present invention refers to a nanoparticle in the range of several to several hundred nanometers composed of self-assembled viral envelope or capsid proteins from which infectious elements such as genomes have been removed. Virus-like particles are the first nanoparticles for the in vivo delivery of peptide epitopes and are the delivery form for various clinically tested and commercially approved vaccines.

In the present invention, the polymer nanoparticle may be a polymer-based nanoparticle selected from the group consisting of, for example, HPMA (N-(2-hydroxypropyl)-methacrylamide copolymer), SMA (polystyrene-maleic anhydride copolymer), PEG (polyethylene glycol), and PGA (poly-L-glutamic acid), but is not limited thereto. For another example, the polymer nanoparticle may be, but is not limited to, a block copolymer, a dendrimer, or the like.

In the present invention, the inorganic nanoparticles include, but are not limited to, silica nanoparticles, gold nanoparticles, self-associating nanoparticles, etc.

In the present invention, the protein nanostructure refers to a nanoparticle formed by self-assembly of a protein. In the present invention, the protein nanostructure is known to be a platform based on proteins such as, for example, ferritin, lumazine synrhase (LS), dihydrolipoyl acetyltransferase (E2p) nsp10 (nonstructural protein 10), but is not limited thereto.

In the present invention, it is preferably characterized in that the nanoparticle is a lipid nanoparticle.

In the present invention, the lipid nanoparticle may be selected from the group consisting of a nanodisk, a unilamellar vesicle, a multilamellar vesicle (MLV), a multivesicular vesicle (MV), a liposome, a LNP, an emulsion, and a lipopolyplex (LPP), but is not limited thereto.

In the present invention, the lipid nanoparticle may be characterized by further including at least one selected from the group consisting of a phospholipid, a cationic lipid, and an adjuvant.

In the present invention, for example, the phospholipid may be characterized by having an aliphatic carbon number of 10 to 30, preferably 12 to 25, and most preferably 14 to 22, but is not limited thereto. For more specific examples, the phospholipid may be a DSPE-PEG derivative including 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000] (DSPEPEG1000), a functionalized DSPE-PEG derivative including 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000] (DSPE-PEG2000-PDP) and 1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[maleimide(polyethylene glycol)-2000] (DSPE-PEG2000-Maleimide), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Fluorescently labeled phospholipids including 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DPPE-Rhodamine), 1,2-Didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-Dierucoyl-sn-glycero-3-phosphate (DEPA), glycero-3-phosphocholine (DEPC), 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 2-Dierucoyl-sn-glycero-3-Phospho-rac-(1-glycerol) (DEPG), 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine

(DLOPC), 1,2-Dilauroyl-sn-glycero-3-phosphate (DLPA), 1,2-Dilauroyl-sn-glycero-3-phosphocholine (DLPE), 1,2-Dilauroyl-sn-glycero-3- Phospho-rac-(1-glycerol) (DLPG), 1,2-Dilauroyl-sn-glycero-3-phosphoserine (DLPS), 1,2-Dimyristoyl-sn-glycero-3-phosphate (DMPA), 1,2 -Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Dimyristoyl-snglycero-3-Phospho-rac-(1-glycerol) (DMPG), 1,2-Dimyristoyl-sn-glycero-3-phosphoserine (DMPS), 1,2-Dioleoyl-sn-glycero-3-phosphate (DOPA), 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) , 1,2-Dioleoylsn-glycero-3-Phospho-rac-(1-glycerol) (DOPG), 1,2-Dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-Dipalmitoyl-sn-glycero -3-phosphate (DPPA), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-Dipalmitoyl-sn- glycero-3-Phospho-rac-(1-glycerol) (DPPG), 1,2-Dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), 1,2-Distearoyl-sn-glycero-3-phosphate (DSPA), 1,2-Distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-Distearoyl-sn-glycero-3-Phospho-rac-(1-glycerol) (DSPG), 1,2-Distearoyl-sn-glycero-3-phosphoserine (DSPS), Egg-PC (EPC), Hydrogenated Egg PC (HEPC), Hydrogenated Soy PC (HSPC), 1-Myristoyl-sn-glycero-3-phosphocholine (LYSOPC MYRISTIC), 1-Palmitoyl-sn-glycero-3-phosphocholine (LYSOPC PALMITIC), 1-Stearoyl-sn-glycero-3-phosphocholine (LYSOPC STEARIC), 1-Myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine (Milk Sphingomyelin MPPC), 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-Palmitoyl-2- myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) , 1-Palmitoyl-2-oleoyl-sn-glycero-3-Phospho-rac-(1-glycerol) (POPG), 1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-Stearoyl-2-myristoyl-sn-glycero-3-phosphocholine (SMPC), 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) and 1-Stearoyl-2-palmitoyl-sn-glycero- It may be one or more phospholipids selected from the group consisting of, but is not limited to, 3-phosphocholine (SPPC).

In the present invention, the cationic lipid is Dimethyldioctadecyl-ammoniumbromide (DDAB), Dimethyldioctadecylammonium (DDAB), (N,N-dimethyl-N-([2-sperminecarboxamido]ethyl)-2,3-bis(dioleyloxy)-1 -propaniminium pentahydrochloride) (DOSPA), (N-[1-(2,3-dioleyloxy)propyl]-N,N,Ntrimethylammonium) (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]- N,N,N-trimethylammonium) (DOTAP), 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), N4-Cholesteryl-Spermine (GL67), 1,2-dioleyloxy -3-dimethylaminopropane (DODMA), O-alkyl phosphatidylcholines derivatives containing 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (12:0 EPC), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy) It may be at least one cationic lipid selected from the group consisting of DAP derivatives including propan-1-aminium (DOBAQ) and 1,2-distearoyl-3-dimethylammonium-propane (18:0 DAP), but is not limited thereto. no.

In the present invention, in addition to the various examples described above, various nanoparticles known in the art for presenting or encapsulating peptides can be used without limitation.

In the present invention, the cationic lipid may preferably be a cationic cholesterol derivative, more preferably Monoarginine-cholesterol (MA-Chol).

In the present invention, the adjuvant may be, but is not limited to, an immunostimulatory single-chain or double-chain oligonucleotide, poly(I:C), an immunostimulatory small-molecule compound, or a combination thereof.

In the art, immunostimulatory single- or double-stranded oligonucleotides are known as useful adjuvants. They often contain a CpG motif (a dinucleotide sequence comprising an unmethylated cytosine linked to a guanosine). Oligonucleotides containing a TpG motif, a palindrome sequence, a plurality of consecutive thymidine nucleotides (e.g., TTTT), a plurality of consecutive cytosine nucleotides (e.g., CCCC) or a poly(dG) sequence are also known adjuvants, as are double-stranded RNAs. Any of these various immunostimulatory oligonucleotides can be used without limitation in conjunction with the present invention.

In the present invention, the oligonucleotide may be characterized by having a length of, for example, 10 to 100 nucleotides, for example, 15 to 50 nucleotides, 20 to 30 nucleotides, or 25 to 28 nucleotides, but is not limited thereto.

In the present invention, the oligonucleotide may be composed of a natural nucleotide or a non-natural nucleotide, or may be composed of a mixture of these. For example, the oligonucleotide may be characterized by containing one or more phosphorothioate linkages and/or being modified with one or more 2'-O-methyl.

In the present invention, the single-chain or double-chain oligonucleotide may be characterized as being a CpG oligonucleotide, a STING activating oligonucleotide, or a combination thereof.

The term "Stimulator of Interferon Genes (STING)" of the present invention refers to stimulator of interferon genes (STING), a molecule that plays a major role in the innate immune response.

The term CpG oligonucleotide (CpG oligodeoxynucleotide, or CpG oligodeoxynucleotide, CpG ODN) of the present invention is a short single-stranded synthetic DNA molecule containing unmethylated cytosine triphosphate deoxynucleotide ("C") and guanine triphosphate deoxynucleotide ("G"), which is known as an immunostimulant. When the CpG is included as a component of the nano vaccine of the present invention, it can function as an adjuvant that enhances the immune response of dendritic cells. In the embodiment of the present invention, the CpG oligonucleotide of SEQ ID NO: 151 was used, but is not limited thereto.

In the present invention, the immunostimulatory small molecule compound is used interchangeably with a small molecule adjuvant, and includes a synthetic small molecule adjuvant and a natural small molecule adjuvant. Examples of the immunostimulatory small molecule compound or small molecule adjuvant include, but are not limited to, monophosphoryl lipid A, Muramyl dipeptide, Bryostatin-1, Mannide monooleate (Montanide ISA 720), Squalene, QS21, Bis-(3',5')-cyclic dimeric guanosine monophosphate, PAM2CSK4, PAM3CSK4, Imiquimod, Resiquimod, Gardiquimod, cl075, cl097, Levamisole, 48/80, Bupivacaine, Isatoribine, Bestatin, Sm360320, and Loxoribine. Small molecule adjuvants are described in Flower DR et al. (Expert Opin Drug Discov. 2012 Sep;7(9):807-17.).

In the present invention, the nanoparticle may include an anionic drug in addition to an adjuvant. For example, the anionic drug may be, but is not limited to, an oligonucleotide, an aptamer, mRNA, siRNA, miRNA, or a combination thereof.

Examples of the preparation of the nanoparticles of the present invention are exemplified in the inventors' published documents Theranostics 2016, 6 (2), 192-203, and Angew Chem Int Ed Engl 2020, 59 (34), 14628-14638, and Korean Patent No. 10-2425028.

Those skilled in the art will readily appreciate that modification, fusion, conjugation or formulation of the peptide may be performed at a level that does not significantly impair the immunogenicity of the peptide, and that the nanoparticles, fusion proteins and conjugates described above are embodiments of the peptides of the present invention. Accordingly, the peptides may include various forms, formulations or modifications well known in the field of peptide therapeutics in addition to the lipid nanoparticles, fusion proteins or conjugates.

The peptide of the present invention is an epitope peptide of FAP having excellent immunodominance, and when the peptide of the present invention is administered, an immune response specific to FAP can be induced.

Fibroblast activation protein alpha (FAP) is a type II transmembrane serine protease exclusively expressed in the pathologic states of various non-neoplastic or neoplastic diseases, including fibrosis, arthritis, and tumors or cancer (Cancer Metastasis Rev. 2020 Sep; 39(3): 783-803). It is reported to be expressed in activated stromal fibroblasts, also called activated cancer-associated fibroblasts (CAFs), in more than 90% of all human carcinomas (Oncogene. 37 (32) (August 2018): 4343-4357; rontiers in Bioscience. 23: 1933-1968 (June 2018)). It has been reported that cancer-associated fibroblasts play an important role in the development, growth, and metastasis of cancer, and in NASH, FAP cleaves FGF21, inducing lipid accumulation and causing various diseases such as hepatic steatosis and nonalcoholic steatohepatitis (NASH).

In one embodiment of the present invention, by using a vaccine platform based on neutrally-charged small lipid nanoparticles (SLNPs), lipid nanoparticles including the peptide were manufactured, and it was confirmed that when immunization is performed with the lipid nanoparticles, tumor growth can be effectively inhibited. In particular, it was confirmed that cancer-associated fibroblasts can be effectively removed from the tumor microenvironment, the formation of ECM can be reduced, and CD8+ T cell responses and antigen-specific CD4+ T cell responses against tumor cells can be induced.

In another embodiment of the present invention, it was confirmed that the vaccination therapy based on the peptide of the present invention can exhibit excellent anticancer effects compared to previously reported FAP-targeting cancer vaccines without causing side effects such as induction of systemic toxicity and autoimmune response by not inducing an increase in Th17 cells related to autoimmunity. Furthermore, in another embodiment of the present invention, it was confirmed that it can prevent and inhibit cancer metastasis as well as inhibit and treat cancer growth.

In another embodiment of the present invention, it was confirmed that a vaccination therapy based on the peptide of the present invention exhibited an excellent therapeutic effect on nonalcoholic steatohepatitis (NASH), another FAP-related disease.

Therefore, the excellent immunodominance of the peptide of the present invention can be usefully used for the prevention or treatment of FAP-associated diseases such as fibrosis, arthritis, non-alcoholic steatohepatitis, and tumors/cancer through the induction of a strong immune response to FAP.

Therefore, the present invention, in another aspect, relates to a vaccine composition for preventing or treating a disease associated with fibroblast activation protein alpha (FAP), comprising the peptide (and/or fusion protein), nucleic acid or nanoparticle.

The term “FAP-related disease” in the present invention is used to collectively refer to diseases in which the expression of FAP is the cause or in which excessive expression of FAP is associated with pathological characteristics. For example, FAP-related diseases include, but are not limited to, fibrosis, arthritis, tumors, nonalcoholic steatohepatitis, hepatic steatosis, arteriosclerosis, and myocardial infarction (Cancer Metastasis Rev. 2020 Sep; 39(3): 783-803).

The present invention relates, in a more specific aspect, to a vaccine composition for preventing or treating cancer or a tumor, comprising the peptide (and/or fusion protein), nucleic acid or nanoparticle.

The present invention relates, in a more specific aspect, to a vaccine composition for preventing or treating metastasis of cancer or tumor, comprising the peptide (and/or fusion protein), nucleic acid or nanoparticle.

The present invention relates, in a more specific aspect, to a vaccine composition for preventing or treating nonalcoholic steatohepatitis (NASH) comprising the peptide (and/or fusion protein), nucleic acid or nanoparticle.

In the present invention, the vaccine composition may be characterized by comprising one or more peptides of the present invention. In the present invention, the vaccine composition may be characterized by comprising a peptide of the present invention comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more kinds of sequences. For example, the vaccine composition may comprise any one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 149. The one or more peptides included in the vaccine composition of the present invention may be in various forms, such as an epitope peptide, a fusion protein including the same, a nanoparticle, etc., as described above.

In the present invention, the vaccine composition may be characterized by containing different peptides or a combination thereof depending on the HLA type of the subject to be administered. The HLA type of the subject to be administered can be performed using a genetic analysis method known in the art.

For example, in the present invention, when the subject has MHC of the HLA-A1 type, the vaccine composition may include one or more peptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 29.

In the present invention, when the subject has MHC of the HLA-A2 type, the vaccine composition may include at least one peptide comprising or consisting of an amino acid sequence selected from the group consisting of sequence numbers 1, 3, 4, 5, and 30 to 45.

In the present invention, when the subject has MHC of the HLA-A3 type, the vaccine composition may include at least one peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 46 to 65.

In the present invention, when the subject has MHC of the HLA-A24 type, the vaccine composition may include at least one peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 66 to 85.

In the present invention, when the subject has MHC of the HLA-A26 type, the vaccine composition may include at least one peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, 14, 19, 21, 28, 58, and 86 to 97 as a restricted epitope peptide.

In the present invention, when the subject has MHC of the HLA-B7 type, the vaccine composition may include at least one peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 93, and 98 to 115.

In the present invention, when the subject has MHC of the HLA-B8 type, it may include at least one peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 91, 93, 99, 100, 103, and 116 to 129.

In the present invention, when the subject possesses MHC of the HLA-B27 type, it may include at least one peptide comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 130 to 149.

The vaccine composition according to the above HLA type is one embodiment, and the vaccine composition of the present invention is not limited thereto, and may include one or more different HLA type-restricted peptides.

The term “vaccine composition” of the present invention means a composition containing a substance capable of inducing an immune response by acting as an antigen or immunogen in vivo or in vitro, and in the present invention, can be used interchangeably with “vaccine” or “immunogenic composition” in the same meaning.

The term “immune response” in the present invention is a broad concept that includes both innate immune responses and adaptive immune responses, for example, complement-mediated immune responses, cell-mediated (T cell) immune responses, and/or antibody-mediated (B cell) responses.

The vaccine composition of the present invention can induce or increase an immune response to FAP or FAP-expressing fibroblasts (CAFs) in a subject of administration, and thereby exhibit excellent preventive or therapeutic effects on FAP-related diseases such as fibrosis, arthritis, tumors, and NASH. More specific examples include, but are not limited to, effects of deficiency of FAP-expressing CAFs in a tumor microenvironment, inhibition of ECM formation, induction of CD8+ T cell and CD4+ T cell responses, inhibition of metastasis, and enhancement of tumor penetration ability of immune cells and anticancer agents.

In the present invention, the vaccine composition may contain the peptide, nucleic acid, or nanoparticle of the present invention as an active ingredient, and when containing the peptide or nucleic acid, various means known in the art for effectively presenting and delivering it may be used. In the present invention, the nanoparticle may be understood as an example of a carrier for presenting the peptide or nucleic acid.

In the present invention, the peptide may be included in the vaccine composition in various forms other than the form of the nanoparticle described above. For example, it may be included in the form of a fusion protein or a conjugate, and for another example, it may be presented by being fixed to the surface of a biological membrane such as an exosome or an artificial membrane such as an oligomembrane, or presented by being encapsulated inside, but is not limited thereto.

In the present invention, as an alternative method of direct delivery of the peptide or nanoparticle, the peptide of the present invention may be delivered in vivo by being included in a vaccine composition in the form of a nucleic acid encoding the peptide of the present invention or a delivery vector including the same, and the delivered nucleic acid or vector may be expressed in vivo to synthesize and present the peptide of the present invention in vivo.

In the present invention, various in vivo protein expression platforms for delivery of the nucleic acid can be used, including but not limited to viral vectors, naked DNA or RNA.

In the present invention, the nucleic acid may be contained in a non-viral vector such as a viral vector or an expression plasmid and may be included in the vaccine composition of the present invention. In the present invention, a separate exogenous promoter may be additionally included for the expression of the nucleic acid, but is not limited thereto. Various vectors, promoters, delivery technologies, etc. that can be used for the in vivo delivery and expression of nucleic acids are well known in the art, and a person skilled in the art can perform this without limitation using a known technology depending on the target and purpose.

In the present invention, the vaccine composition may be characterized by additionally comprising a phospholipid, a cationic lipid, and/or an adjuvant.

The term “adjuvant” of the present invention is a concept that arose when Alexander Glenny discovered that aluminum salt increases immune response, and refers to an auxiliary component added to a vaccine composition or a subject to which a vaccine is administered to induce a stronger immune response. For example, the adjuvant may include an emulsifier, muramyl dipeptide, abridin, an aqueous adjuvant, an oil, and more specifically, an aluminum salt such as aluminum phosphate or aluminum hydroxide, a squalene-containing emulsion such as MF59 or an analog thereof (MF59 like), AS03 or an analog thereof (AS03 like), AF03 or an analog thereof (AF03 like), SE or an analog thereof (SE like), a calcium salt, a dsRNA analogue, a lipopolysaccharide, a Lipid A analogue (MPL-A, GLA, etc.), Flagellin, imidazoquinolines, CpG ODN, mineral oil, an agonist of a Toll-like receptor (TLR), a C-type lectin ligand, Examples include, but are not limited to, CD1d ligands (such as α-galactosylceramide), detergents, liposomes, saponins such as QS21, cytokines, and peptides.

In the present invention, the adjuvant may include, for example, Amphigen, LPS, bacterial cell wall extract, bacterial DNA, CpG sequence, poly(I:C), synthetic oligonucleotides and combinations thereof [see: Schijins et al. (2000) Curr. Opin. Immunol. 12:456], mycobacterial phlei (M. phlei) cell wall extract (MCWE) (U.S. Patent No. 4,744,984), M. phlei DNA (M-DNA) and M-DNA-M. phlei cell wall complex (MCC).

In the present invention, the adjuvant may include, for example, compounds which can be used as an emulsifier, natural and synthetic emulsifiers and anionic, cationic and nonionic compounds, and among the synthetic compounds, the anionic emulsifiers include, for example, calcium, sodium and aluminum salts of lauric acid and oleic acid, calcium, magnesium and aluminum salts of fatty acids, and organic sulfonates, for example, sodium lauryl sulfate, the synthetic cationic agents include, for example, cetyltriethylammonium bromide, and the synthetic nonionic agents include, but are not limited to, glyceryl esters (e.g., glyceryl monostearate), polyethylene glycol esters and ethers, and sorbitan fatty acid esters (e.g., sorbitan monopalmitate) and polyoxyethylene derivatives thereof (e.g., polyoxyethylene sorbitan monopalmitate).

In the present invention, the adjuvant may be a natural emulsifier, and the natural emulsifier includes, but is not limited to, acacia, gelatin, lecithin, and cholesterol.

In the present invention, other suitable adjuvants may be formed, for example, of oil components, for example, single oils, oil mixtures, water-in-oil emulsions or oil-in-water emulsions. The oils may be mineral oils, vegetable oils or animal oils. Mineral oils are liquid hydrocarbons obtained from petroleum jelly by distillation techniques and are also referred to in the art as liquid paraffin, liquid petroleum jelly or white mineral oil. Suitable animal oils include, for example, cod liver oil, flounder oil, herring oil, orange roughy oil and shark liver oil, all of which are commercially available. Suitable vegetable oils include, for example, canola oil, almond oil, cottonseed oil, corn oil, olive oil, peanut oil, safflower oil, sesame oil, soybean oil and the like. Freund's complete adjuvant (FCA, or CFA) and Freund's incomplete adjuvant (FIA) are two common adjuvants commonly used in vaccine formulations and are also suitable for use in the present invention. Both FCA and FIA are water-in-mineral-oil emulsions; however, FCA also contains killed Mycobacterium sp.

In the present invention, immunomodulatory cytokines may also be included in the vaccine composition, for example as adjuvants, to enhance vaccine efficacy. Non-limiting examples of such cytokines include interferon alpha (IFN-α), interleukin-2 (IL-2), and granulocyte macrophage-colony stimulating factor (GM-CSF) or combinations thereof, preferably but not limited to GM-CSF.

For example, in a preferred embodiment of the present invention, immunization is performed by administering the peptide of the present invention using CFA as an adjuvant or by administering CpG ODN as an adjuvant together with nanoparticles comprising the peptide of the present invention, but is not limited thereto.

The vaccine composition of the present invention can be manufactured in a unit dose form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains.

At this time, the formulation can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods. Suitable formulations known in the art can be used as disclosed in the literature (Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA). Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

The vaccine composition of the present invention can be administered orally or parenterally. The route of administration of the composition according to the present invention is not limited to these, but for example, intradermal, intravenous, subcutaneous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, intraperitoneal, enteral, sublingual, oral or topical administration is possible. The dosage of the composition according to the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate or disease severity, and can be easily determined by a person skilled in the art. In addition, the composition of the present invention can be formulated into a suitable dosage form using a known technique for clinical administration.

In the present invention, the vaccine composition may be administered as a single dose or divided into several doses. In the case of divided administration, it is preferable that the doses of the peptide, nucleic acid, nanoparticle and/or adjuvant of the present invention are equally distributed, but this is not limited thereto.

The optimal dosage of the vaccine composition of the present invention can be determined by standard studies involving observation of an appropriate immune response in a subject. After the initial immunization, the subject may be administered one or more booster immunizations at appropriate intervals.

The vaccine composition of the present invention can be administered in a pharmaceutically effective amount. The term “pharmaceutically effective amount” as used herein means an amount sufficient to induce or increase an immune response but not causing side effects or serious or excessive immune responses, and an appropriate dosage may be variously determined by factors such as the formulation method, administration method, patient’s age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity. Various general considerations for determining a “pharmaceutically effective amount” are known to those skilled in the art and are described in, for example, the literature [Gilman et al., eds., Goodman And Gilman's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press, 1990] and [Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Co., Easton, Pa., 1990].

In the present invention, the vaccine composition can be administered to a patient on any suitable schedule to induce and/or support a cytotoxic T lymphocyte response to induce and/or support protective immunity for the prevention or treatment of cancer. For example, after the vaccine composition is administered to the patient as a primary prophylactic administration, a booster can be administered to support and/or maintain protective immunity. In some aspects, the vaccine composition can be administered to the patient once, twice or more times per month, several months or several years.

In the present invention, administration of the vaccine composition may continue, for example, over the course of several years. Typically, the vaccine schedule includes, but is not limited to, more frequent administrations at the beginning of the vaccine regimen, and less frequent administrations (e.g., boosters) for a period of time to maintain protective immunity.

In the present invention, the vaccine composition may be administered in a lower dose at the beginning of the vaccine therapy and in a higher dose over time. In addition, the vaccine may be administered in a higher dose at the beginning of the vaccine therapy and in a lower dose over time, but is not limited thereto.

The term “fibrosis” of the present invention refers to pathological wound healing in which connective tissue is abnormally produced and replaces normal parenchymal tissue. Fibrosis can disrupt or block the normal structure and function of an organ or tissue by depositing connective tissue, and can cause various physical abnormalities as a result. Fibrosis can have excessive expression of fibroblast activation protein alpha as a cause or pathological symptom, and can include abnormal deposition of connective tissue due to proliferation and activation of fibroblasts.

In the present invention, the tumor may be characterized as being a FAP-related tumor.

In the present invention, the FAP-related tumor is used to mean all tumors characterized by an increase in the concentration of free FAP in tumor tissue or tumor microenvironment, the number of FAP-expressing cells, or the level of FAP expression in cells compared to normal tissue or environment. According to existing reports, in almost all tumors, the number of free FAP and/or FAP-expressing cells in the microenvironment increases compared to normal tissue. Therefore, the vaccination therapy and vaccine composition using the FAP peptide of the present invention can be used as an effective therapeutic agent without limitation on the type of tumor.

The term “tumor” of the present invention includes all neoplasms or hyperplasias that are caused by cells that have escaped from the biological control mechanism and have autonomous and excessive proliferation. The above tumors include, for example, benign, premalignant, and malignant tumors, and more specifically, histiocytoma, glioma, astrocytoma, osteoma, various cancers, for example, liver cancer, thyroid cancer, colon cancer, testicular cancer, myelodysplastic syndrome, glioblastoma, oral cancer, lip cancer, oropharyngeal cancer, leukemia, basal cell carcinoma, ovarian cancer, breast cancer, brain tumor, pituitary adenoma, multiple myeloma, gallbladder cancer, biliary tract cancer, colon cancer, retinoblastoma, melanoma, bladder cancer, peritoneal cancer, parathyroid cancer, adrenal cancer, paranasal sinus cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, non-Hodgkin's lymphoma, tongue cancer, esophageal cancer, small intestine cancer, renal pelvis cancer, kidney cancer, heart cancer, duodenal cancer, lymphoma (Hodgkin's lymphoma, FL, MCL, MZBL, CLL, T-ALL, AML, ALL, etc.), vulvar cancer, These may include, but are not limited to, ureteral cancer, urethral cancer, stomach cancer, gastrointestinal cancer, intestinal cancer, sarcoma, osteosarcoma, psoriasis, cervical cancer, uterine cancer, prostate cancer, rectal cancer, vaginal cancer, pancreatic cancer, skin cancer, and laryngeal cancer.

In the present invention, the tumor may preferably be characterized as being a desmoplastic tumor.

The term “prevention” as used in the present invention means any act of suppressing or delaying the onset of a desired disease by administering a vaccine composition according to the present invention.

The term "treatment" as used in the present invention means any action by which the symptoms of a target disease are improved or beneficially changed by administration of a vaccine composition according to the present invention.

The above vaccine composition may additionally contain suitable carriers, excipients and diluents commonly used in vaccine compositions.

Carriers, excipients and diluents that may be included in the above vaccine composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate and mineral oil. When formulating the above composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants and surfactants.

The vaccine composition according to the present invention can be formulated and used in various forms according to conventional methods. Suitable formulations include, but are not limited to, oral formulations such as tablets, pills, powders, granules, sugar-coated tablets, hard or soft capsules, solutions, suspensions or emulsions, injections, aerosols, external preparations, suppositories, and sterile injection solutions.

The vaccine composition according to the present invention can be prepared into a suitable formulation using a pharmaceutically inactive organic or inorganic carrier. That is, when the formulation is a tablet, a coated tablet, a sugar-coated tablet, or a hard capsule, it can contain lactose, sucrose, starch or a derivative thereof, talc, calcium carbonate, gelatin, stearic acid, or a salt thereof. In addition, when the formulation is a soft capsule, it can contain vegetable oil, wax, fat, semi-solid, and liquid polyols. In addition, when the formulation is in the form of a solution or syrup, it can contain water, polyol, glycerol, and vegetable oil, etc.

In the present invention, the vaccine composition may be formulated as a delayed release vehicle or a depot preparation. Such a long-acting preparation may be administered by inoculation or implantation (e.g., subcutaneously or intramuscularly) or by injection. Thus, for example, the vaccine composition may be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or with an ion exchange resin, or as a sparingly soluble derivative, e.g., a sparingly soluble salt. Liposomes and emulsions are well known examples of delivery vehicles suitable for use as carriers.

The vaccine composition according to the present invention may further include, in addition to the carrier described above, a preservative, a stabilizer, a wetting agent, an emulsifier, a solubilizer, a sweetener, a colorant, an osmotic pressure regulator, an antioxidant, etc.

The vaccine composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, the "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dosage level can be determined according to the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the excretion rate, the treatment period, the concurrently used drugs, and other factors well known in the medical field. The vaccine composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects by considering all of the above factors, and this can be easily determined by those skilled in the art.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. The pharmaceutical composition can be administered orally or parenterally. In the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. When administered orally, since proteins or peptides are digested, the oral composition can be formulated to coat the active agent or protect it from decomposition in the stomach. In addition, the composition can be administered by any device that allows the active agent to travel to the target cell.

The method of administration of the vaccine composition according to the present invention can be easily selected according to the formulation, and can be administered orally or parenterally. The dosage may vary depending on the patient's age, sex, weight, degree of disease, and route of administration.

In the present invention, the vaccine composition may be characterized by inducing a FAP-specific cytotoxic T cell response or a FAP-specific CD8+ T cell response. In the present invention, the vaccine composition may be characterized by inducing an immune response to free FAP or FAP-expressing cells in a subject. In the present invention, the FAP-expressing cells may be characterized by being FAP-expressing fibroblasts.

In the present invention, the vaccine composition may be characterized by inducing an anti-tumor response.

In the present invention, the anti-tumor response may include, but is not limited to, induction of a tumor or tumor microenvironment-specific immune response, reduction in the number of tumor cells, reduction in tumor size, death of tumor cells, inhibition of tumor metastasis, etc.

In the present invention, the vaccine composition can induce inhibition of lipid accumulation in the liver, inhibition of hepatic steatosis, inhibition of hepatic inflammation, inhibition of hepatic fibrosis, and reduction of the proportion of swollen cells in the liver, but is not limited thereto.

In another aspect, the present invention relates to the use of the peptide, nucleic acid or nanoparticle of the present invention for the manufacture of a pharmaceutical composition for the prevention or treatment of a fibroblast activating protein alpha (FAP)-related disease.

In another aspect, the present invention relates to a method for preventing or treating a disease associated with fibroblast activation protein alpha (FAP), comprising administering to a subject a peptide, nucleic acid or nanoparticle of the present invention, or administering to a subject a vaccine composition of the present invention.

The preventive or therapeutic method of the present invention includes a vaccination method, a vaccination method, or an immunization method for preventing or treating a disease associated with fibroblast activation protein alpha (FAP).

In the present invention, in the method for preventing or treating a disease related to fibroblast activation protein alpha, a step of analyzing the HLA type of the subject may be additionally included prior to the step of administering to the subject the vaccine composition of the present invention comprising the peptide, nucleic acid, nanoparticle or any one or more of these of the present invention.

In the present invention, a vaccine composition containing different peptides can be administered depending on the HLA type of the subject.

For example, in the present invention, when the subject has MHC of the HLA-A1 type, the vaccine composition may be administered with one or more peptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 to 29.

In the present invention, when the subject possesses MHC of the HLA-A2 type, the vaccine composition may be administered with one or more peptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, 5, and 30 to 45.

In the present invention, when the subject possesses MHC of the HLA-A3 type, the vaccine composition may be administered with one or more peptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 46 to 65.

In the present invention, when the subject has MHC of the HLA-A24 type, the vaccine composition may be administered with one or more peptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 66 to 85.

In the present invention, when the subject has MHC of the HLA-A26 type, the vaccine composition may be administered with one or more peptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12, 13, 14, 19, 21, 28, 58, and 86 to 97 as the restricted epitope peptide.

In the present invention, when the subject has MHC of the HLA-B7 type, the vaccine composition may be administered with one or more peptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 93, and 98 to 115.

In the present invention, when the subject possesses MHC of the HLA-B8 type, one or more peptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 91, 93, 99, 100, 103, and 116 to 129 may be administered.

In the present invention, when the subject possesses MHC of the HLA-B27 type, one or more peptides comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 130 to 149 may be administered.

Administration of peptide or vaccine compositions according to the above HLA type is one embodiment and is not limited thereto, and one or more different HLA type restricted peptides may be administered regardless of the HLA type of the subject or without an analysis step for the HLA type.

Therefore, the present invention relates to a pharmaceutical composition for the prevention or treatment of tumors for co-administration with an anticancer agent comprising the peptide, nucleic acid or nanoparticle of the present invention from another aspect.

In another aspect, the present invention provides a use of the peptide, nucleic acid or nanoparticle in combination with an anticancer agent for the prevention or treatment of tumors.

In another aspect, the present invention provides a method for preventing or treating a tumor, comprising a step of co-administering the peptide, nucleic acid or nanoparticle; and an anticancer agent to a subject.

The present invention relates from another aspect to the use of the peptide, nucleic acid or nanoparticle of the present invention for the preparation of a pharmaceutical composition for the prevention or treatment of tumors in combination with an anticancer agent.

The peptides, nucleic acids and/or nanoparticles of the present invention inhibit the formation of ECM, which acts as a physical barrier to anticancer therapy, by reducing or depleting FAP-expressing CAFs in the tumor microenvironment, and consequently enhance the infiltration or penetration of anticancer agents and immune cells into the tumor, resulting in a more remarkable anti-tumor effect than when each drug is administered as a monotherapy. Therefore, the anticancer agent to be co-administered with the pharmaceutical composition of the present invention can be selected without limitation from various anticancer therapeutic agents or anticancer agents reported in the art.

In the present invention, the anticancer agent may be, for example, a chemotherapy agent (cytotoxic anticancer agent), a targeted anticancer agent, an immunotherapy agent, a hormonal anticancer agent, a cell therapy agent, etc., but is not limited thereto.

In the present invention, the chemical anticancer agent is also called a cytotoxic anticancer agent, and is an anticancer agent that directly attacks cancer cells and exhibits an anticancer effect, and includes, but is not limited to, alkylating agents such as cyclophosphamide, ifosfamide, bendamustine, delphalan, and cisplatin; antimetabolites such as capecitadine, cytarabine, doxifluridine, fluorouracil, clofarabine, fludarabine, and decitamine; DNA rotator cuff inhibitors such as doxorubicin, daunorubicin, idarubicin, etoposide, and topotecan; microtubule inhibitors such as cabazitaxel, docetaxel, vincristine, and the like; and other chemical anticancer agents such as mitomycin C, bleomycin, and hydroxyurea.

In the present invention, the targeted anticancer agent is an anticancer agent containing as a main component a substance that targets and binds to or interacts with a specific antigen expressed by a cancer or an antigen related to a cancer, and examples thereof include, but are not limited to, antibody therapeutics such as cetuximab, trastuzumab, bevacizumab, rituximab, ibltumomab, alemtuzumab, brentuximab, and elotuzumab; and signal transduction inhibitors such as erlotinib, gefitinib, vandetanib, afatinib, rapanitib, axitinib, pazopanib, sunitinib, and sorafenib.

In the present invention, the immunotherapy anticancer agent includes, but is not limited to, a CTLA-4 antibody such as apilimumab, a PD-1 antibody such as pembrolizumab or nivolumab, a PD-L1 antibody such as atezolizumab, and an immune checkpoint inhibitor such as an IDO inhibitor.

In the present invention, the hormonal anticancer agent includes, but is not limited to, androgen suppressants such as bicalutamide and enzalutamide, and female hormone suppressants such as tamoxifen, anastrozole and letrozole.

In the present invention, the cell therapy agent means a therapy agent containing living immune cells as an active ingredient, and includes, but is not limited to, cytotoxic T cell therapy agents, CAR-T cell therapy agents, and CAR-NK cell therapy agents.

The term "combined administration" of the present invention means administering two or more types of effective ingredients simultaneously or sequentially, or administering them simultaneously, sequentially, or at specific intervals so that an improved effect can be achieved compared to the effect expected when each effective ingredient is administered independently due to the action of the two or more types of effective ingredients.

In the present invention, the combined administration may be characterized by administering the peptide, nucleic acid or nanoparticle of the present invention in combination with one or more anticancer agents, and may also be performed in parallel with other anticancer therapies.

In the present invention, each of the effective ingredients to be co-administered may be characterized by being administered through an independent route. Each effective ingredient may be administered independently by a person skilled in the art with a suitable administration method and dosage. For example, as in one embodiment of the present invention, it may be characterized by preferably administering peptides, nucleic acids, or nanoparticles intradermally, and administering anticancer agents through intravenous injection, but is not limited thereto.

In the present invention, the combined administration of the peptide, nucleic acid or nanoparticle and the anticancer agent may be characterized by being administered simultaneously.

In the present invention, the combined administration of the peptide, nucleic acid or nanoparticle and the anticancer agent may be characterized by sequential administration. For example, the anticancer agent may be administered after the administration of the peptide, nucleic acid or nanoparticle, or the peptide, nucleic acid or lipid nanoparticle may be administered after the administration of the anticancer agent.

In the present invention, when the peptide, nucleic acid or nanoparticle and the anticancer agent are administered sequentially, it can be characterized in that they are administered at a certain time interval, and for non-limiting examples, they can be sequentially administered at 1 minute intervals, 5 minutes intervals, 10 minutes intervals, 20 minutes intervals, 30 minutes intervals, 1 hour intervals, 1 day intervals, several days intervals, or 1 week to several weeks intervals, but are not limited thereto, and can be sequentially administered at an appropriate interval by a person skilled in the art.

In the present invention, the administration of the peptide, nucleic acid or nanoparticle and the anticancer agent can be performed independently and repeatedly one or more times. In the present invention, when the peptide, nucleic acid or nanoparticle and the anticancer agent are repeatedly administered, each administration interval can be easily adjusted independently by a person skilled in the art according to the condition of the subject and the level of the desired effect. For example, the peptide, nucleic acid or nanoparticle and the anticancer agent can be independently administered at 1 hour intervals, 6 hours intervals, 8 hours intervals, 12 hours intervals, 1 day intervals, 2 days intervals, 1 week intervals, 2 weeks intervals, or 1 month intervals, but is not limited thereto.

In the present invention, the pharmaceutical composition may be administered in a formulation and dosage independent of the formulation of the anticancer agent to be administered in combination. Preferably, the pharmaceutical composition comprising the peptide, nucleic acid or nanoparticle and the pharmaceutical composition comprising the anticancer agent may each be administered in an effective dosage, but is not limited thereto.

Although specific amino acid sequences and base sequences are described in the present invention, it will be apparent to those skilled in the art that an amino acid sequence substantially identical to an enzyme to be implemented in the present invention and a base sequence encoding the same fall within the scope of the present invention. Substantially identical includes a case where the homology of the amino acid or base sequence is very high, and also means a protein that shares structural characteristics or has the same function as that used in the present invention regardless of the homology of the sequence. A protein in which a part of a sequence other than the sequence constituting the core of the present invention is deleted or a fragment of the base sequence encoding the same may also be included in the present invention, and therefore the present invention includes all amino acid or base sequences that have the same function as that used in the present invention regardless of the length of the fragment.

Example

The present invention will be described in more detail through the following examples. However, the following examples are only intended to concretize the contents of the present invention and the present invention is not limited thereto.

Example 1: Materials and Methods

Example 1-1: Reagents

1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000] (DSPE-PEG1000) and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000] (DSPE-PEG2000-PDP) - Avanti Polar Lipids.

Boc-Arg(Pbf)-OH and Cholesterol - Sigma Aldrich

CpG oligoDNA modified with phosphorothioate backbone (CpG ODN; 5’-TCC ATG ACG TTC CTG ACG TT-3’, SEQ ID NO: 151) - Genotech and Bioneer.

All peptides used were synthesized in Anigene, and all reagents unless otherwise stated were purchased from Sigma Aldrich.

Example 1-2: Animals and cell lines

Female C57BL/6 mice (Orient Bio) were raised in a pathogen-free environment. Animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of the Korea Advanced Institute of Science and Technology (KAIST) (Accreditation number: KA2020-59).

E.G7-OVA cell line (ATCC; Manassas, VA, USA).

E.G7-OVA cell line was cultured in RPMI-1640 medium (Welgene) supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 4.5 g/l glucose, 10 mM HEPES, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, and 0.5 mg/ml G418 (Gibco).

Panc02 cell line was provided by Professor Seok-Jo Kang, KAIST. Panc02 cells were maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin.

The MC38 cell line was provided by Professor Chan-Hyuk Kim of the Korea Advanced Institute of Science and Technology. MC38 cells were maintained in DMEM medium supplemented with 10% FBS and 1% penicillin/streptomycin.

EO771.lmb cell line (ATCC; Manassas, VA, USA).

EO771.lmb cell line was cultured in DMEM medium (Welgene) supplemented with 10% heat-inactivated FBS and 1% penicillin/streptomycin.

All cells were maintained at 37°C in a humidified atmosphere of 5% _CO2 .

Example 1-3: Flow Cytometry

All antibodies were purchased from BioLegend (San Diego, CA, USA), eBioscience (San Diego, CA, USA), or Bioss Antibodies (Woburn, MA, USA).

Antibodies used were as follows: anti-CD16/CD32 (BioLegend; Catalog #101319, clone 2.4G2), anti-CD45 Pacific Blue (BioLegend; Catalog #103125, clone 30-F11), anti-CD45 PerCP/Cy5. 5 (eBioscience; Catalog #45-0451-82, clone 30-F11), anti-CD3e PerCP/Cy5.5 (BioLegend; Catalog #100327, clone 145-2C11), anti-CD3e PE/Cy7 (BioLegend; Catalog # 100219, clone 145-2C11), anti-CD3e FITC (BioLegend; Catalog #100306, clone 145-2C11), anti-CD8a PE (BioLegend; Catalog #100708, clone 53-6.7), anti-CD8a PE/Cy7 (BioLegend; Catalog #100722, clone 53-6.7), anti-CD8a APC (BioLegend; Catalog #100711, clone 53-6.7), anti -CD8a APC/Cy7 (BioLegend; Catalog #100713, clone 53-6.7), anti-CD4 FITC (eBioscience; Catalog # 11-0041-82, clone GK1.5), anti-IFN-γ PE (eBioscience; Catalog # 12-7311-82, clone XMG1.2), anti-IL-17A PE (eBioscience; Catalog # 12-7177-81, clone eBio17B7), and anti-FAPα Alexa Fluor 647 (Bioss Antibodies; Catalog #bs-5758R-A647).

Additional reagents used for flow cytometry analysis were: 7-AAD Viability Stain Solution (BioLegend; Catalog #420403), Ghost Dye Violet 450 (Tonbo Biosciences, San Diego, CA, USA; Catalog #13-0863), and Fixable Viability Dye eFluor 450.

Cells were blocked by incubation with anti-CD16/CD32 antibodies for 10 min at 4°C and immunostained with different antibodies for 20 min at 4°C. Dead cells were excluded by staining with 7AAD viability stain solution, Ghost Dye Violet 450, or Fixable Viability Dye eFluor 450. Flow cytometry was performed and data were acquired using an LSRFortessa flow cytometer (BD Biosciences, San Jose, CA, USA). Analysis was performed using FlowJo software (TreeStar).

Example 1-4: Selection of mouse FAP target peptides and vaccine design

Prediction of mouse FAP target epitope peptides was performed using epitope peptide prediction algorithms including NetMHC3.0, BIMAS, and PREDEP. The mouse FAP full-length sequence used is SEQ ID NO: 152 (Uniprot NO. P97321). All epitope peptides were selected as 9-mer peptide lengths recognized by MHC-I haplotype H-2Kb. The sequences of the selected peptides are shown in Table 1:

[Table 1]

Example 1-5: Evaluation of immunogenicity and antitumor effect

For immunogenicity testing of the selected peptide candidates, 6-week-old female C57BL/6 mice were immunized twice using a homologous prime-boost regimen at 1-week intervals. Mice were immunized by subcutaneous injection of 100 μg of each peptide emulsified in Complete Freund's Adjuvant (CFA; Sigma Aldrich) into both footpads. Mice were sacrificed 10 days after the final immunization. Antigen-specific T cell responses were evaluated by isolating spleen cells from mice and restimulating them ex vivo with each peptide (10 μg/ml). IFN-γ-producing CD8+ T cells were quantified by intracellular cytokine staining (ICS) (Fig. 2 ), and the number of IFN-γ-producing cells was measured by enzyme-linked immunospot (ELISpot) assay. For ICS analysis, spleen cells (3 × 106 cells per tube) were restimulated with each peptide for 1 h. GolgiStop and GolgiPlug (BD Biosciences) were added to each tube to inhibit intracellular transport of cytokines, and the cells were cultured for an additional 5 h, immunostained with anti-CD3e and anti-CD8a antibodies, and then stained with Live/Dead cell dye for 20 min at 4°C. For intracellular cytokine staining, cells were permeabilized with Cytofix/Cytoperm solution (BD Biosciences) and incubated with anti-IFN-γ antibody. Samples were then washed and analyzed by flow cytometry. To measure IFN-γ-producing cells by ELISpot, spleen cells (3 × 10 ⁵ cells/well) were seeded in 96-well microplates coated with mouse IFN-γ-specific monoclonal antibodies and restimulated with each peptide for 30 h. IFN-γ-producing spots were generated using the mouse IFN-γ ELISpot kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol. Blue-black spots at the sites of cytokine localization were counted using an automated ELISpot reader (AID GmbH, Strasbourg, Germany).

The antitumor therapeutic efficacy of the peptides was evaluated in mice that were inoculated subcutaneously with 2 × 10 ⁵ E.G7-OVA cells into the right flank. Nine days after E.G7-OVA cell inoculation, the mice were randomly divided into groups such that the average tumor volumes of each group were approximately equal, and then immunized twice with each peptide at 1-week intervals using the same method as described above. Tumor growth was monitored every other day using digital calipers, and the tumor volumes were calculated as 0.5 × length × width. The mice were euthanized when the average tumor volume reached the ethical endpoint (~1,500 mm ³ ).

Example 1-6: Preparation of FAP _PEP -SLNP nanovaccine

Monoarginine-cholesterol (MA-Chol) and DSPE-PEG2000-FAP _PEP were synthesized based on previously reported methods.

Cysteinylated FAP _PEP (Cys-FAP _PEP ) prepared using two selected peptide candidates, CKLWRYSYTA and CYFRNVDYLL, was conjugated to DSPE-PEG2000-PDP by disulfide exchange reaction. Cys-FAP _PEP and DSPE-PEG2000-PDP were dissolved in dimethyl sulfoxide and mixed at a molar ratio of 1:2. The solution was gently vortexed overnight at room temperature and the reaction was quenched by the addition of acetonitrile.

The DSPE-PEG2000-FAP _PEP conjugate was purified by high-performance liquid chromatography (HPLC; Agilent, Santa Clara, CA, USA) using a C4 column (Nomura Chemical, Japan) and characterized using MALDI-TOF spectroscopy.

FAPPEP-SLNP nanovaccines containing MA-Chol:DOPE:DSPE-PEG1000:DSPE-PEG2000-FAP _PEP (molar ratio, 48.625:48.625:2.25:0.5) were prepared using a thin film formation and rehydration method. After drying all lipid components dissolved in chloroform and methanol, the solvent was removed under vacuum, and the resulting lipid film was rehydrated with HEPES-buffered glucose (HBG) containing CpG ODN. The solution was sonicated for 10 min, magnetically stirred for more than 4 h at room temperature, and extruded more than 11 times using a mini extruder (Avanti Polar Lipids). The loading efficiency was close to 100% using 1.65 nmol of CpG ODN encapsulated in 8 μmol of SLNP.

For the characterization of FAP _PEP -SLNP, the hydrodynamic size was determined by DLS at room temperature using a Zetasizer Nano range system (Malvern, Worcestershire, UK). Both the morphology and size of FAP _PEP -SLNP were characterized by TEM using a Philips TECNAI F20 instrument (Philips Electronic Instrument Corp., Mahwah, NJ, USA) and 1% uranyl acetate solution was used for negative staining. The average size of the nanovaccines was measured using Gatan Microscopy Suite (GMS) software (Gatan, Pleasanton, CA, USA).

Example 1-7: Evaluation of antitumor effect of FAP _PEP -SLNP nanovaccine

To evaluate the antitumor effect of FAP _PEP1 -SLNP nanovaccine, three subcutaneous tumor models were established in 6-week-old female C57BL/6 mice.

For the E.G7-OVA treatment model, 2 × 10 ⁵ E.G7-OVA cancer cells were injected into the right flank of the mice. Nine days after E.G7-OVA cancer cell inoculation, tumor-bearing mice were immunized three times at 4-day intervals with FAP _PEP1 -SLNP or OVA _PEP -SLNP nanovaccines (CpG, 0.4 nmol per mouse; FAPPEP, 5 nmol per mouse; OVAPEP, 5 nmol per mouse; SLNP, 2 μmol per mouse) by subcutaneous injection into both footpads at the indicated time points.

For the Panc02 treatment model, 1× ¹⁰⁶ Panc02 cancer cells were injected into the right flank of the mice. Sixteen days after Panc02 cell inoculation, the tumor-bearing mice were immunized three times with FAP _PEP1 -SLNP at 4-day intervals using the same method as above.

For the MC38 treatment model, 1x10 ⁵ MC38 cancer cells were injected into the right flank of the mice. Five days after MC38 cancer cell inoculation, the tumor-bearing mice were immunized three times with FAP _PEP1 -SLNP at 4-day intervals using the same method as above.

For evaluation of combination therapy, mice were injected intraperitoneally with Dox (10 mg/kg) every other day for a total of four times as indicated. Tumor growth was monitored every other day and mouse survival was monitored. Mice were euthanized when the mean tumor volume reached the ethical endpoint.

Example 1-8: Histological analysis of tumor tissue

Tumor tissues were resected, fixed in 10% formalin solution, embedded in paraffin, and then cut into 5-μm-thick sections. Collagen deposition in tumor tissues was assessed by Weigert's iron hematoxylin, Biebrich's scarlet-acid fuchsin solution, and Masson's trichrome staining with aniline blue. All section slides were imaged using an inverted microscope (Eclipse Ti2; Nikon, Tokyo, Japan). Masson's trichrome-stained tissue sections were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Example 1-9: TIL and CAF analysis

Tumor-infiltrating CD8+ T cells and FAP+ CAFs were analyzed by dissociating tumor tissues into small pieces and then enzymatically and mechanically dissociating single-cell suspensions using a mouse tumor dissociation kit (Miltenyi Biotech, Auburn, CA, USA) and gentleMACS Dissociator (Miltenyi Biotech). The cell suspension was filtered through a cell strainer (70 μm) and washed with Dulbecco’s phosphate-buffered saline (DPBS). Red blood cells (RBCs) were removed by incubation with 2 ml RBC lysis buffer (BioLegend) for 2 min at room temperature with gentle shaking. The dissociated cells were immunostained with anti-CD45, anti-CD3e, anti-CD8a, and anti-FAPα antibodies, and stained with Live/Dead cell dye for 20 min at 4°C. The cells were then washed and subjected to flow cytometry.

Example 1-10: Splenic T cell analysis

Spleens were harvested from immunized mice, and splenocytes were isolated. For analysis of splenic Th17 cells, the isolated cells were stimulated with soluble anti-CD3e (BD Biosciences; Catalog #553057, clone 145-2C11) and anti-CD28 (BD Biosciences; Catalog #553294, clone 37.51) antibodies for 1 h at 37°C, and GolgiStop and GolgiPlug were added to inhibit intracellular transport of cytokines. The cells were incubated for an additional 5 h and then stained with anti-CD3e and anti-CD4 antibodies and Live/Dead cell dye for 20 min at 4°C. For intracellular cytokine staining, cells were permeabilized using Cytofix/Cytoperm solution and then incubated with anti-IL-17A antibody. For analysis of antigen-specific splenic T cells, spleen cells were restimulated with FAPPEP or OVAPEP (10 μg/ml) for 1 h, followed by addition of GolgiStop and GolgiPlug and incubation for an additional 5 h as described above. Cells were then stained with anti-CD3e, anti-CD4 and anti-CD8a antibodies and Live/Dead cell dye for 20 min at 4°C. After surface marker staining, cells were permeabilized and incubated with anti-IFN-γ antibody. All samples were analyzed by flow cytometry.

Example 1-11: Tumor Penetration of Cypate Dye

Tumor penetration was assessed using cypate, a near-infrared dye, as a surrogate for small-molecule chemotherapeutic drugs. Mice were injected with 1 × 10 ⁵ MC38 cancer cells into the right flank and immunized three times at 4-day intervals with FAPPEP1-SLNPs 5 days after MC38 cancer cell inoculation according to the same schedule used in the MC38 tumor treatment study. After the final immunization, when the tumors reached an average size of ~300 mm ³ , cypate dye (2.5 mg kg-1) was injected intravenously via the retro-orbital route. Tumor tissues were harvested 2 h after cypate dye injection, and fluorescence signals were evaluated using an in vivo imaging system (IVIS; PerkinElmer, Waltham, MA, USA).

Example 1-12: Evaluation of the cancer metastasis inhibition effect of FAP _PEP1 peptide vaccine and tissue analysis

The evaluation of the anti-cancer metastasis effect was tested in the form of a peptide vaccine using the FAPpep1 peptide.

To evaluate the metastatic inhibition efficacy of the FAP _PEP1 peptide vaccine, orthotopic tumor models were established in female C57BL/6 mice. 1 × 10 ⁵ EO771.lmb cancer cells were injected into the right abdominal mammary glands (4th pair) of mice. Tumor growth was monitored daily using a digital caliper, and the tumor volume was calculated as (0.5 × length × width ² ). Primary tumors were resected when the average tumor volume reached ~300 mm ³ . Four days after resection of the primary tumors, the mixture of FAPpep ₁ (100 μg/head) and CpG-ODN (10 μg/head) was immunized twice at 1-week intervals via subcutaneous injection into both footpads. The mice were sacrificed and analyzed 1 week after the final vaccination.

Lung and liver tissues of sacrificed mice were excised, fixed in 10% formalin solution, embedded in paraffin, and cut into 5-μm-thick slices. To confirm the metastatic inhibition effect of FAPpep vaccine, the lung and liver tissue samples were stained with H&E, and all section slides were imaged using an inverted microscope (Eclipse Ti2; Nikon, Tokyo, Japan). The stained tissue sections were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Example 1-13: NASH therapeutic effect of FAP _PEP1 peptide vaccine and serum and tissue analysis

To evaluate the therapeutic effect of FAP _PEP1 peptide vaccine on NASH, 6-week-old female C57BL/6 mice were fed a methionine/choline deficient (MCD) diet for 4 weeks, or a methionine/choline sufficient (MCS) diet as a control group to establish a model. Body weight was monitored three times per week. After 4 weeks of MCD/MCS diet, mice were immunized by subcutaneous administration of a mixture of FAPpep1 (100 μg/head) and CpG-ODN (10 μg/head) twice at 1-week intervals into the footpad. The diet was continued during the vaccine administration period. One week after the last immunization, the mice were sacrificed, the livers were removed, and the liver weights were measured.

Blood was collected immediately before sacrifice of the mice, serum was separated by centrifugation, and the levels of aspartate transaminase (AST), alanine transferase (ALT), total bilirubin, and HDL were measured.

Lung and liver tissues were excised, fixed in 10% formalin solution, embedded in paraffin, and cut into 5-μm-thick slices. The prepared liver tissue samples were stained with H&E and analyzed, or stained with isopropanol and Oil Red O. All section slides were imaged using an inverted microscope (Eclipse Ti2; Nikon, Tokyo, Japan). The stained tissue sections were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Example 1-12: Statistical Analysis

Data are expressed as mean ± standard error of the mean (S.E.M.). Groups were compared using two-tailed Student’s t-test or one-way analysis of variance (ANOVA), as appropriate, using GraphPad Prism 5 (GraphPad Software). Statistical analysis of survival was performed using the log-rank (Mantel-Cox) test. Statistically significant was defined as P < 0.05.

Example 2: Prediction and screening of immunodominant peptide epitopes of FAP.

The immunodominant peptide epitopes of FAP were predicted using three epitope peptide prediction programs, netMHC3.0, BIMAS, and PREDEP (Fig. 3). Among the identified epitope peptides, those with high scores for MHC class I of the H-2Kb haplotype and sharing 8–9 amino acid peptide sequences were selected (Fig. 4a). Ultimately, six of the nine selected peptides were tested and screened for in vivo immunogenicity. C57BL/6 mice were subcutaneously immunized twice at 1-week intervals with a mixture of each peptide and complete Freund's adjuvant (CFA). One week after the last immunization, the mice were sacrificed, and spleen cells were isolated to evaluate the antigen-specific T cell responses to each peptide (Fig. 4b). Isolated splenocytes were restimulated with each peptide, and interferon-γ (IFN-γ) production by the cells was analyzed by intracellular cytokine staining (ICS) and enzyme-linked immunospot (ELISpot) assays. The ICS assay showed that immunization with each of the three peptides (KLWRYSYTA, GLFKCGIAV, and YFRNVDYLL) induced significantly higher frequencies of IFN-γ-secreting CD8+ T cells than did immunization with the other three peptides (Figs. 4c,d and 5a–f). The three ICS-positive peptides were also selected as positive candidates in the ELISpot assay (Figs. 4e,f and 6a–f). Based on the ICS and ELISpot assays, these three peptides (KLWRYSYTA, GLFKCGIAV, and YFRNVDYLL) were ultimately selected for further evaluation of their antitumor efficacy. The murine lymphoma cell line E.G7-OVA is a popular model system to study MHC class I-restricted CD8+ T cell responses, and was used to induce tumor formation in syngeneic mice, and the presence of FAP+ CAFs was confirmed in the generated tumor tissues by flow cytometry (Fig. 7). When E.G7-OVA tumors reached an average volume of ~100 mm3, mice were randomly divided into four groups and immunized twice with a mixture of each peptide and CFA at 1-week intervals (Fig. 4g). Immunization with GLFKCGIAV + CFA was ineffective in suppressing tumor growth, whereas immunization with two other peptide candidates, KLWRYSYTA (designated FAP _PEP1 ) and YFRNVDYLL (designated FAP _PEP2 ), resulted in effective tumor growth inhibition compared to the control (Fig. 4h). These results imply that these two peptides are suitable epitope peptide candidates that enable potent induction of peptide epitope-specific CD8+ T cells and high antitumor efficacy.

Example 3: Synthesis, characterization, and antitumor efficacy evaluation of FAP epitope peptide-presenting nanovaccine

Using the small lipid nanoparticle platform reported in our previous study, we designed a nanovaccine displaying two epitope peptides of FAP (Theranostics 2016, 6 (2), 192-203; Angew Chem Int Ed Engl 2020, 59 (34), 14628-14638; Korean Patent No. 10-2425028).

These FAP peptide-labeled SLNPs (FAPPEP-SLNPs) were composed of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) to promote endosomal escape; 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000] (DSPE-PEG1000), which provides colloidal stability in vivo; and monoarginine-cholesterol (MA-Chol), which enables complexation with CpG adjuvants and provides mechanical stability (Figure 8). Peptide epitope peptides were introduced to the surface of SLNPs via cleavable disulfide bonds using cysteine-modified peptides at the N-terminus (N'-Cys-FAP _PEP1 and N'-Cys-FAP _PEP2 ) and DSPE-PEG2000 (Figures 9 and 10).

We confirmed that the two cysteine-modified peptides above maintained immunogenicity in vivo (Fig. 11a,b). We predict that once FAP _PEP -SLNPs are taken up by APCs, free FAP _PEP is released through cleavage of disulfide bonds in the reducing environment of the cytoplasm and presented by MHC class I on the cell membrane (Fig. 8a). The hydrodynamic size of FAP _PEP -SLNPs prepared using the thin film rehydration method was measured to be ~144 nm by dynamic light scattering (DLS) (Fig. 8b). Transmission electron microscopy (TEM) showed that FAP _PEP -SLNPs had a spherical morphology with an average diameter of ~104 nm (Fig. 8c).

Next, we evaluated the therapeutic efficacy of FAP _PEP1 -SLNP and FAP _PEP2 -SLNP in the E.G7-OVA tumor model. E.G7-OVA tumor-bearing mice were immunized three times with each nanovaccine method at 4-day intervals (Fig. 8d). As expected, the control SLNP without the FAP _PEP epitope peptide on the surface did not inhibit tumor growth (Fig. 8e). Treatment with FAP _PEP1 -SLNP showed significantly greater antitumor efficacy than treatment with FAP _PEP2 -SLNP, and FAP _PEP2 -SLNP showed moderate tumor regression effect. These results imply that FAP _PEP1 -SLNP is the most effective nanovaccine for treating tumors containing FAP+ CAFs in the TME.

To confirm the presence of CD8+ T cells specific for FAP _PEP1 , ICS analysis was performed on the spleen and lymph nodes of mice immunized with FAP _PEP1 -SNLP. As a result, when restimulated with a control peptide having the same amino acid sequence as FAP _PEP1 but with a random sequence, IFN-ν secretion was low, whereas when restimulated with FAP _PEP1 , IFN-ν activity was greatly increased, confirming that CD8+ cells specific for FAP _PEP1 were formed through immunization (Fig. 12).

To further confirm the formation of CD8+ cells specific for FAP _PEP1, the spleen and lymph nodes of mice immunized with FAP _PEP1 -SNLP were extracted, and FAP _PEP1 -H-2Kb tetramer, an antibody that specifically binds to CD8+ cells specific for FAP _PEP1 , was prepared using the 'QuickSwitchTM Quant H-2 Kb Tetramer Kit-PE' product (MBL Inc.) (MBL Inc.). When FAP _PEP1 -H-2Kb tetramer was treated, it was confirmed that the fluorescence reaction indicating that the tetramer was bound was very high in the group immunized with FAP _PEP1 -SLNP (Fig. 13).

Example 4: Confirmation of therapeutic efficacy of FAP _PEP1 -SLNP nanovaccine by CAF removal effect in tumor microenvironment

Next, we investigated the mechanism of action of FAP _PEP1 -SLNP nanovaccine to inhibit tumor growth. For comparison, we used an OVA _{PEP -SLNP cancer cell-targeted nanovaccine (Angew Chem Int Ed Engl 2020, 59 (34), 14628-14638) displaying an epitope peptide (SIINFEKL, SEQ ID NO: 150) that exhibits high antitumor efficacy against E.G7-OVA tumors by inducing OVA (ovalbumin)-specific CD8} + T cell responses and eliminating cancer cells. E.G7-OVA tumor-bearing mice with an average tumor volume of ~100 mm ³ were immunized three times in total with FAP _PEP1 -SLNP or OVA _PEP -SLNP at 4-day intervals (Fig. 14a). Treatment with FAP _PEP1 -SLNP significantly inhibited tumor growth, showing similar or slightly better efficacy compared to OVA _PEP -SLNP nanovaccine targeting cancer cells (Fig. 14b). It also significantly prolonged survival compared to untreated controls (Fig. 14c).

Vaccination with FAP _PEP1 -SLNPs may affect ECM production in tumors because it induces a cytotoxic immune response against FAP+ CAFs in the TME. Masson's trichrome (MT) staining showed that immunization with FAP _PEP1 -SLNPs significantly reduced the collagen-positive area in tumor tissues compared with the untreated control tumor tissues (Fig. 14d, e). However, although OVA _PEP -SLNPs showed similar antitumor efficacy as FAP _PEP1 -SLNPs, the former failed to reduce ECM production, suggesting that the antitumor efficacy of the latter may be the result of the depletion of FAP+ CAFs in the TME. Flow cytometry analysis confirmed that immunization with FAP _PEP1 -SLNPs significantly reduced the proportion of FAP+ CAFs and increased CD8+ tumor-infiltrating lymphocytes (TILs) in tumor tissues (Fig. 14f, g).

Next, we evaluated antigen-specific CD8+ T cell responses in splenocytes isolated from mice immunized with FAP _PEP1 -SLNP or OVA _PEP -SLNP. The frequency of IFN-γ-secreting CD8+ T cells in splenocytes was substantially increased after ex vivo restimulation with FAP _PEP1 or OVA _PEP , indicating successful induction of antigen-specific CD8+ T cell responses (Fig. 14h). Interestingly, although FAP _PEP1 is predicted to be a MHC class I epitope, we observed a significant increase in the frequency of IFN-γ-secreting CD4+ T cells among splenocytes treated with FAP _PEP1 -SLNP. However, OVA _PEP -SLNP nanovaccine failed to generate antigen-specific CD4+ T cell responses (Fig. 14i).

Meanwhile, since FAP is also selectively expressed in bone marrow stromal cells, pancreatic α cells, and uterine stroma, our FAP-targeted nanovaccine may induce autoimmune-related adverse effects or systemic toxicity. To evaluate such adverse effects and toxicity, we analyzed the generation of interleukin-17A (IL-17A)-secreting T helper 17 (Th17) cells after immunization with FAP _PEP1 -SLNP. We found no significant difference in the frequency of Th17 cell generation between FAP _PEP1 -SLNP and OVA _PEP -SLNP immunization (Fig. 14j), indicating that FAP _PEP1 -SLNP does not induce autoimmune responses. In conclusion, FAP _PEP1 -SLNP is a nanovaccine that can induce not only FAP-specific CD8+ T cell responses but also CD4+ T cell responses, indicating that it can exhibit high antitumor efficacy with a low possibility of inducing autoimmune responses (J Exp Med 2013, 210 (6), 1137-1151; J Exp Med 2013, 210 (6), 1125-1135; Cancer Cell 2014, 25 (6), 719-734; Nat Rev Immunol 2018, 18 (10), 635-647; Immunity 2021, 54 (12), 2701-2711).

Next, blood vessels were identified in the tumors of the mice immunized with FAP _PEP1 -SLNP. As a result, it was confirmed that blood vessels in the tumors of the mice immunized with FAP _PEP1 -SLNP were significantly reduced compared to the control group (Fig. 15a and b). This suggests that angiogenesis was reduced due to the reduction of CAFs, based on the fact that CAFs secrete angiogenic factors such as VEGF.

We confirmed tumor metastasis in the lungs of the mice immunized with FAP _PEP1 -SLNP. As a result, we confirmed that tumor metastasis in the mice immunized with FAP _PEP1 -SLNP was significantly reduced compared to the control group (Fig. 15c and d). Based on the fact that CAFs are known to play a major role in forming the pre-metastatic niche, this can be interpreted as an inhibitory effect of metastasis due to the reduction of CAFs.

Example 5: Confirmation of the efficacy of FAP _PEP1 -SLNP nanovaccine against various tumors

To determine whether FAP _PEP1 -SLNP could act on other desmoplastic tumors, we examined whether FAP ⁺ CAFs existed in MC38 and Panc02 tumors, which are known as desmoplastic tumors. After reaching an average of 300 mm ³ , E.G7-OVA, MC38, and Panc02 tumors were excised, dissociated into single cells, and analyzed by flow cytometry. As a result, we confirmed that the ratio of FAP ⁺ CAFs was high in both tumor models, including E.G7-OVA tumors (Fig. 16).

In addition, we confirmed that FAP _PEP1 -SLNP showed a continuous tumor suppression effect upon additional inoculation even after FAP ⁺ CAF were removed in the early stage of the tumor. Therefore, we conducted an experiment to confirm whether cancer cells themselves also express the FAP protein. When the size of MC38 tumors reached 300 mm ³ , the tumors were excised and dissociated into single cells, and the abundance ratio of cancer cells and FAP ⁺ CAF was confirmed through flow cytometry. As a result, it was found that FAP was expressed in the cancer cells themselves in large tumors, and therefore, it was confirmed that 7FAP _PEP1 -SLNP showed a continuous therapeutic efficacy upon additional inoculation even after FAP ⁺ CAF were removed in the early stage (Fig. 17).

Based on these results, we confirmed whether the FAP _PEP1 -SLNP nanovaccine had therapeutic efficacy in Panc02, a pancreatic ductal adenocarcinoma (PDAC) cell line with abundant ECM, and MC38, a colon cancer cell line. In both tumor models, immunization was started when the average tumor size was 100 mm ³ , and a total of three immunizations were performed at 4-day intervals, the same schedule as the previous experiment. As a result, in both the Panc02 tumor model (Fig. 18a-c) and the MC38 tumor model (Fig. 18d-f), it was confirmed that the mice vaccinated with FAP _PEP1 -SLNP showed slower tumor growth and increased survival rate (Fig. 18).

Additionally, to determine whether CD8 T cells and CD4 T cells within the tumor have immune activity against FAP ⁺ cells in the MC38 tumor model, MC38 tumors were extracted, dissociated into single cells, and ICS analysis was performed. As a result, in the case of TILs (tumor infiltrating lymphocytes) of mice vaccinated with FAP _PEP1 -SLNP, a significant increase in the secretion of IFN-γ was confirmed compared to the control group. This confirmed that FAP-specific CD8 ⁺ T cells and CD4 ⁺ T cells produced by FAP _PEP1 -SLNP are present within the tumor (Fig. 19), which means that they effectively act on FAP ⁺ cells within the tumor.

These results (Figs. 16 to 19) imply that the epitope peptide of the present invention and the vaccine comprising it can be used as a pan-tumor vaccine against other connective tissue tumors or FAP expressing tumors.

Example 6: Confirmation of antitumor effect through combination therapy of FAP _PEP1 -SLNP nanovaccine and anticancer drug

The dense and rigid ECM of connective tissue tumors acts as a powerful physical barrier not only to chemotherapeutic agents but also to biological agents such as antibodies and CAR-T cells, reducing the therapeutic efficacy of various anticancer therapies (Signal Transduct Target Ther 2021, 6 (1), 153, Proc Natl Acad Sci USA 2019, 116 (6), 2210-2219;, Nat Nanotechnol 2021, 16 (1), 25-36). We predicted that the ability of FAP _PEP1 -SLNP nanovaccine to deplete CAFs in the TME could reduce ECM density, thereby enhancing the penetration of chemotherapeutic drugs into connective tissue tumors.

To verify this, the murine pancreatic ductal adenocarcinoma cell line Panc02 and the colorectal cancer cell line MC38 were chosen as connective tissue tumor models (PLoS One 2013, 8 (11), e80580; Cancer Res 2018, 78 (5), 1321-1333; Nat Commun 2020, 11 (1), 515). First, we confirmed that immunization with FAP _PEP1 -SLNP nanovaccine significantly slowed tumor growth and prolonged the survival period of immunized mice. Non-immunized mice were used as controls in both Panc02 (Fig. 20a-c) and MC38 tumor models (Fig. 20d-f), confirming that the FAP _PEP1 -SLNP nanovaccine was also effective against these connective tissue tumors.

Next, we investigated whether the FAP+ CAF-targeted nanovaccines could enhance the tumor penetration of small molecule anticancer drugs. Cypate dye was chosen as a model drug to visualize drug penetration in tumors. MC38 colorectal tumor-bearing mice were immunized with FAP _PEP1 -SLNP nanovaccines for a total of three injections at 4-day intervals. After the tumor volume reached ~300 mm ³ , free Cypate was intravenously injected into the immunized mice, and the tumors were harvested for ex vivo fluorescence imaging 2 h later. The accumulation of dye in the tumors was significantly higher in the vaccinated group than in the unvaccinated control group ( Figure 21a ), suggesting that the drug penetration was enhanced due to the decrease in the density of the intratumoral ECM.

Furthermore, we performed combination therapy with FAP _PEP1 -SLNP immunotherapy and doxorubicin (Dox) to determine whether the combination therapy could exhibit the predicted synergistic effects. After MC38-derived colorectal tumors reached a size of ~100 mm ³ , mice were immunized three times with FAP _PEP1 -SLNP at 4-day intervals followed by intraperitoneal injection of Dox every other day for a total of 4 times (Fig. 21b). Although both FAP _PEP1 -SLNP nanovaccine and Dox monotherapy exhibited significant antitumor efficacy, the combination therapy was much more effective in inhibiting tumor growth than either monotherapy (Fig. 21c). In addition, flow cytometry analysis of the treated tumors showed that the proportion of FAP+ CAFs was significantly reduced in both the FAP _PEP1 -SLNP nanovaccine and combination groups, but not in the Dox monotherapy group (Fig. 21d). In addition, although the proportion of CD8+ TILs tended to increase in the combination treatment group, this difference was not statistically significant (Fig. 21e). Taken together, these results suggest that the FAP _PEP1 -SLNP nanovaccine is not only effective in inhibiting the growth of connective tissue tumors, but also substantially enhances its efficacy in combination with chemotherapeutic drugs by increasing drug penetration within the tumor (Figure 22).

Example 7: Confirmation of the cancer metastasis inhibition effect of FAP _PEP1 peptide vaccine

FAP+CAF secrete extracellular vesicles from primary tumors to promote metastasis and protect cancer cells from anoikis and shear stress in the blood circulation by forming clusters with cancer cells. Therefore, the previously used tail vein injection metastasis model is not suitable for confirming the role of FAP+CAF.

For this reason, in this example, to confirm the association of FAP+CAF and the metastasis inhibition effect of the FAP _PEP1 peptide vaccine, an orthotopic breast tumor model was constructed using the EO771.lmb metastatic breast cancer cell line by the method described in Examples 1-13. Since it was confirmed that the ratio of FAP+CAF was high at an average tumor size of 300 mm ³ in Example 5 (Fig. 16), the tumor was resected when the tumor size reached 300 mm ³ .

Four days after resection, the FAP _PEP1 peptide vaccine and CpG ODN were immunized twice by subcutaneous injection into the footpad at one-week intervals to confirm the metastasis-suppressing effect of the FAP _PEP1 peptide vaccine on metastasis (Fig. 23).

Since EO771.lmb tumors are known to metastasize primarily to the lungs and liver, lung and liver tissues were removed one week after the immunization injection and subjected to histological analysis.

H&E staining analysis of lung tissue showed that administration of the FAP _PEP1 peptide vaccine significantly reduced metastasis, whereas the metastatic area increased in the control group (Fig. 24).

In addition, H&E staining of liver tissue showed that metastasis was significantly reduced when the FAP _PEP1 peptide vaccine was administered compared to the control group (Fig. 25).

These results demonstrate that the FAP _PEP1 vaccine of the present invention can effectively prevent cancer metastasis by targeting FAP+CAF in metastasis.

Example 8: Confirmation of the efficacy of FAP _PEP1 peptide vaccine in treating nonalcoholic steatohepatitis (NASH)

Nonalcoholic steatohepatitis (NASH) is a chronic liver disease characterized by hepatic steatosis and inflammation, which may progress to fibrosis, cirrhosis, and liver cancer. FAP cleaves fibroblast growth factor 21 (FGF21), an important regulator of lipids, and dysregulation thereof may contribute to the progression of NASH.

Example 8-1: Confirmation of NASH therapeutic effect of FAP _PEP1 peptide vaccine

To confirm the therapeutic effect of the FAP _PEP1 peptide vaccine of the present invention on nonalcoholic steatohepatitis, a NASH model was established using a methionine-choline deficient diet (MCD) (Nat Rev Gastro Hepat 16, 411-428 (2019)). The establishment of the NASH model was verified through monitoring of body weight and liver weight. Stages 2 and 3 NASH models and a control group model were prepared through 4 weeks of MCD diet and MCS diet therapy, and a mixture of the FAP _PEP1 peptide vaccine and CpG ODN was subcutaneously injected twice into both heel pads of each mouse at 1-week intervals (Fig. 27a). After administration of the FAP _PEP1 peptide vaccine, body weight was significantly recovered (Fig. 27b), and when the livers of the mice were removed one week after the final immunization and their weights were measured, it was confirmed that the liver weight was significantly recovered after administration of the FAP _PEP1 peptide vaccine (Fig. 27c).

In addition, in the MCD diet-induced NASH model immunized with the FAP _PEP1 peptide vaccine, the liver weight-to-body weight ratio was markedly increased (Fig. 27d), confirming that the FAP _PEP1 peptide vaccine also exhibited excellent effects in the treatment of NASH.

Example 8-2: Confirmation of the liver damage alleviation effect of FAP _PEP1 peptide vaccine

To confirm the alleviation of liver damage, serum was collected just before the sacrifice of mice. Mice of the NASH model immunized with the FAP _PEP1 peptide vaccine showed a significant decrease in the levels of AST, ALT, and total bilirubin, which are indicators of liver damage (Figs. 28a to 28c). In addition, the level of HDL was increased (Fig. 28d), confirming that the FAP _PEP1 peptide vaccine had a protective effect against liver damage induced by the MCD diet.

Example 8-3: Liver tissue analysis results after immunization with FAP _PEP1 peptide vaccine

To verify the effect of the FAP _PEP1 peptide vaccine on liver tissue in the NASH model, tissue analysis was performed using Oil Red O staining and H&E staining.

In the NASH model immunized with the FAP _PEP1 peptide vaccine, Oil Red O staining showed a remarkable effect related to hepatic steatosis. Specifically, as shown in Figures 29a and 29b, the livers of mice immunized with the FAP _PEP peptide vaccine showed a marked decrease in the intensity of Oil Red O staining, confirming a significant decrease in intracellular lipid accumulation.

H&E staining revealed balloon-shaped adipocytes in the liver tissue, and while unstained areas representing adipocytes were seen in the liver of the MCD diet-fed control group, a marked reduction in hepatocyte swelling was observed in mice immunized with the FAP _PEP1 peptide vaccine (Figs. 30a and 30b).

As a result, the histological analysis results indicate that the FAP _PEP1 peptide vaccine exhibits an excellent therapeutic effect on hepatic steatosis, which can induce liver tissue recovery in the NASH model. These histological results support the results in the examples, such as body weight, liver weight, and serum analysis, implying an excellent therapeutic effect of the FAP _PEP -SLNP nanovaccine in the treatment of NASH.

Example 9: Human FAP epitope and nanovaccine

The results of Examples 2 to 8 demonstrate that immunotherapy using the predicted FAP epitope can exhibit excellent therapeutic effects not only for the treatment of cancer and inhibition of metastasis, but also for FAP-related fibrotic diseases such as NASH. It has already been well known through many studies that FAP is selectively overexpressed in cancer-associated fibroblasts present in human cancers and myofibroblasts or hepatic stellate cells in fibrotic diseases and that it promotes the growth and metastasis of cancer or mediates fibrotic diseases by the same mechanism as in mouse models. Therefore, it is suggested that the efficacy results of the FAP epitope vaccine shown in mouse cancer models and NASH models can be applied to humans. In addition, the similarity between the FAP protein of mice and the FAP protein of humans is about 94%, so there is a high possibility that the FAP _PEP1 peptide epitope verified in mice has cross-reactivity with a specific HLA type in humans. In this example, the FAP _PEP1 verified has a sequence identical to the peptide epitope candidate predicted from human HLA-A2.

In this example, the epitope of human FAP (SEQ ID NO: 153, Uniprot No. B4DLR2) according to HLA Super type was additionally confirmed. Prediction of FAP target epitope peptides was performed using epitope peptide prediction algorithms including NetMHC3.0, BIMAS, and PREDEP. All epitope peptides were selected as 8-mer to 10-mer peptide lengths recognized by H-2Kb, which is an MHC-I haplotype. HLA Super types were analyzed for HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-A26, HLA-B7, HLA-B8, and HLA-B27. The final derived FAP epitope sequences are shown in Tables 2 to 5 below.

[Table 2]

[Table 3]

[Table 4]

[Table 5]

A clinical trial of a FAP vaccine capable of targeting the FAP protein and eliminating cells expressing FAP can be performed as follows. 1) Analyze the HLA type of each patient, and select one or more FAP epitope peptides corresponding to the individual patient from the peptide sequences described in Tables 1 to 5. 2) The selected peptide sequence candidates can be administered together with an adjuvant (such as CpG or poly(I:C)), or 3) the selected peptide sequence candidates can be manufactured into a nanovaccine including nanoparticles and administered together with an adjuvant. Based on the effects confirmed in the above examples, it can be clearly predicted that the vaccination therapy using the peptide sequences described in Tables 2 to 5 of the present invention will exhibit excellent effects in the prevention and treatment of FAP-related diseases such as cancer, cancer metastasis, NASH, etc.

The peptide of the present invention has excellent immunodominance and exhibits a strong induction effect of CD8+ T cells specific for fibroblast activation protein (FAP) and FAP-expressing fibroblasts (FAP+ CAF). The peptide of the present invention can exhibit excellent preventive and therapeutic effects on FAP-related diseases such as cancer, fibrosis, NASH, etc. through immunity induction against fibroblast activation protein, and has significantly lower toxicity compared to other FAP-expressing CAF targeting vaccines. In particular, in the case of cancer, it exhibits excellent anti-tumor effects such as depletion of FAP-expressing CAF, reduction of ECM production in the TME, and inhibition of cancer metastasis. When the peptide of the present invention is administered in combination with other anticancer agents, it increases the accumulation of anticancer agents in tumor cells and exhibits a remarkable enhancement of the antitumor effect, so that it can be usefully used as a pan-tumor vaccine.

While the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Electronic file attached.

Claims (38)

A peptide comprising an MHC I-restricted epitope peptide of fibroblast activation protein (FAP), said peptide consisting of 8 to 10 amino acids. A peptide characterized in that, in claim 1, it is an epitope peptide restricted to at least one HLA super family selected from the group consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-A26, HLA-B7, HLA-B8 and HLA-B27. A peptide according to claim 1, characterized in that it comprises an amino acid sequence selected from the group consisting of sequence numbers 1 to 149. A peptide characterized in that it consists of an amino acid sequence selected from the group consisting of sequence numbers 1 to 149 in the first paragraph. A nucleic acid encoding the peptide of claim 1. A recombinant vector comprising the nucleic acid of claim 5. A host cell into which the nucleic acid of claim 5 or the recombinant vector of claim 6 has been introduced. A nanoparticle comprising the peptide of claim 1. In claim 8, the nanoparticle is characterized in that the nanoparticle is selected from the group consisting of virus-like particles (VLPs), protein nanostructures, polymer nanoparticles, lipid nanoparticles, and inorganic nanoparticles. A nanoparticle in claim 9, characterized in that the nanoparticle is a lipid nanoparticle. In claim 10, the lipid nanoparticle is a nanoparticle further comprising at least one selected from the group consisting of a phospholipid, a cationic lipid, and an adjuvant. A nanoparticle in claim 11, characterized in that the phospholipid has 14 to 22 aliphatic carbon atoms. The method of claim 11, wherein the cationic lipid is Dimethyldioctadecyl-ammoniumbromide (DDAB), Dimethyldioctadecylammonium (DDAB), (N,N-dimethyl-N-([2-sperminecarboxamido]ethyl)-2,3-bis(dioleyloxy)- 1-propaniminium pentahydrochloride) (DOSPA), (N-[1-(2,3-dioleyloxy)propyl]-N,N,Ntrimethylammonium) (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl] -N,N,N-trimethylammonium) (DOTAP), 3ß-[N- (N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), N4-Cholesteryl-Spermine (GL67), 1,2- dioleyloxy-3-dimethylaminopropane (DODMA), A lipid nanoparticle characterized by comprising at least one selected from the group consisting of O-alkyl phosphatidylcholines derivatives, and dimethylammoniumpropane (DAP) derivatives. A nanoparticle according to claim 11, characterized in that the cationic lipid is a cationic cholesterol derivative. A nanoparticle in claim 11, characterized in that the adjuvant is an immunostimulatory single-chain or double-chain oligonucleotide, an immunostimulatory small-molecule compound, or a combination thereof. A nanoparticle in claim 15, wherein the single-chain or double-chain oligonucleotide is a CpG oligonucleotide, poly(I:C), a STING activating oligonucleotide, or a combination thereof. A nanoparticle in claim 16, characterized in that the CpG oligonucleotide comprises a nucleic acid sequence of SEQ ID NO: 151. A vaccine composition for preventing or treating a disease associated with fibroblast activating protein alpha (FAP), comprising at least one of the peptide of any one of claims 1 to 4, the nucleic acid of claim 5, and the nanoparticle of any one of claims 8 to 17. A vaccine composition according to claim 18, wherein the fibroblast activation protein alpha (FAP)-related disease is selected from the group consisting of fibrosis, arthritis, tumors, cancer metastasis, non-alcoholic steatohepatitis, hepatic steatosis, arteriosclerosis, and myocardial infarction. In claim 19, a vaccine composition characterized in that the tumor has an increased number of FAP expressing cells in the tumor microenvironment compared to normal tissue. A vaccine composition according to claim 19, characterized in that the tumor is a desmoplastic tumor. A vaccine composition characterized in that, in claim 18, it further comprises at least one selected from the group consisting of phospholipids, cationic lipids, and adjuvants. A vaccine composition in claim 22, characterized in that the adjuvant is an immunostimulatory single-chain or double-chain oligonucleotide, poly(I:C), an immunostimulatory low-molecular-weight compound, or a combination thereof. A vaccine composition characterized in that it comprises an MHC I-restricted epitope peptide of at least one type of fibroblast activation protein in claim 18. A vaccine composition characterized in that it comprises at least one kind of epitope peptide restricted to any one or more HLA super families selected from the group consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-A26, HLA-B7, HLA-B8 and HLA-B27 in claim 24. A vaccine composition characterized in that it comprises MHC I-restricted epitope peptides of at least 10 types of fibroblast activation proteins in claim 24. A vaccine composition characterized in that it contains a peptide comprising at least one amino acid sequence selected from the group consisting of sequence numbers 1 to 149 in claim 18. A vaccine composition according to claim 18, characterized in that it comprises at least two different peptides, wherein each of the peptides independently comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 149. A pharmaceutical composition for preventing or treating cancer, characterized in that it comprises at least one of the peptide of any one of claims 1 to 4, the nucleic acid of claim 5, and the nanoparticle of any one of claims 8 to 17, and is administered in combination with an anticancer agent. A method for treating a disease associated with fibroblast activating protein alpha (FAP) in a subject, comprising administering to the subject a vaccine composition of claim 18. A method according to claim 30, characterized in that it further comprises a step of confirming the HLA type of the subject prior to the administering step. A method according to claim 30, wherein the fibroblast activation protein alpha (FAP)-related disease is selected from the group consisting of fibrosis, arthritis, tumors, cancer metastasis, nonalcoholic steatohepatitis, hepatic steatosis, arteriosclerosis, and myocardial infarction. In claim 32, a method characterized in that the tumor has an increased number of FAP expressing cells in the tumor microenvironment compared to normal tissue. A method according to claim 32, wherein the tumor is a connective tissue tumor. A method according to claim 30, wherein the vaccine composition induces an anti-tumor response in a subject. In claim 35, a method characterized in that the anti-tumor response is a decrease in the number of tumor cells, a decrease in tumor size, and/or a decrease in tumor metastasis in the subject. Use of a peptide according to any one of claims 1 to 4, a nucleic acid according to claim 5 and a nanoparticle according to any one of claims 8 to 17 for the prevention or treatment of a disease associated with fibroblast activating protein alpha (FAP). Use of a peptide according to any one of claims 1 to 4, a nucleic acid according to claim 5 and a nanoparticle according to any one of claims 8 to 17 for the manufacture of a vaccine composition for the prevention or treatment of a disease associated with fibroblast activating protein alpha (FAP).

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