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WO2024225764A1 - Peptides épitopes immunodominants de protéines d'activation des fibroblastes et leurs utilisations - Google Patents

Peptides épitopes immunodominants de protéines d'activation des fibroblastes et leurs utilisations Download PDF

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WO2024225764A1
WO2024225764A1 PCT/KR2024/005587 KR2024005587W WO2024225764A1 WO 2024225764 A1 WO2024225764 A1 WO 2024225764A1 KR 2024005587 W KR2024005587 W KR 2024005587W WO 2024225764 A1 WO2024225764 A1 WO 2024225764A1
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fap
peptide
present
hla
tumor
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전상용
신호철
김유진
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Korea Advanced Institute of Science and Technology KAIST
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants

Definitions

  • 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.
  • FAP fibroblast activation protein alpha
  • 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 ⁇ ).
  • 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.
  • the tumor microenvironment 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).
  • 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).
  • ECMs extracellular matrix proteins
  • 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).
  • 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).
  • PDAC pancreatic adenocarcinoma
  • numerous attempts have been performed to inhibit or deplete CAFs in the TME.
  • Fibroblast Activation Protein alpha 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).
  • FAP-expressing CAFs 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).
  • FAP-targeting CAR-T therapy Facer Immunol Res 2014, 2 (2), 154-166
  • non-alcoholic steatohepatitis 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.
  • 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.
  • FAP fibroblast-activating protein
  • 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).
  • FAP fibroblast activation protein alpha
  • 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.
  • 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.
  • FAP fibroblast activation protein
  • 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.
  • FAP fibroblast activation protein alpha
  • 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.
  • 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).
  • FAP fibroblast activating protein alpha
  • 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.
  • FAP fibroblast activating protein alpha
  • 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(c) and (d) show the representative flow cytometry results of IFN- ⁇ -secreting CD8+ T cells (c) and the percentage of CD8+ T cells producing IFN- ⁇ (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- ⁇ -producing spots (e) and the average number of IFN- ⁇ 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.
  • 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 3(d) shows the immunization schedule to evaluate the antitumor efficacy of FAPPEP-SLNP nanovaccine against an established E.G7-OVA tumor model.
  • 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.
  • 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).
  • blood vessels are shown in brown
  • 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.
  • 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.
  • 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 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 25 is a histological analysis of liver tissue showing the results of FAPPEP1 peptide vaccine inhibition of cancer metastasis to the liver.
  • 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.
  • 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).
  • ALT a
  • AST b
  • c total bilirubin
  • 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 30 shows the results of evaluating ballooning hepatocytes in liver tissue using H&E staining.
  • 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.
  • 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.
  • “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.
  • 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, pheny
  • Fibroblast Activation Protein alpha is also known as prolyl endopeptidase FAP.
  • FAP Fibroblast Activation Protein alpha
  • 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.
  • FAP fibroblast activation protein
  • the present invention relates, in one aspect, to an immunodominant epitope peptide of fibroblast activation protein (FAP).
  • FAP fibroblast activation protein
  • immunodominance 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.
  • 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.”
  • the peptide may be characterized as being composed of 7 to 11 amino acids, preferably 8 to 10 amino acids.
  • the peptide may be characterized as being an MHC I-restricted epitope peptide of fibroblast activation protein (FAP).
  • FAP fibroblast activation protein
  • MHC Major histocompatibility complex
  • MHC 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
  • type II MHC induces an immune response by presenting it to B cells or helper T cells through APC.
  • the peptide may be characterized as being a human leukocyte antigen (HLA) restricted epitope.
  • HLA human leukocyte antigen
  • HLA Human leukocyte antigen
  • HLA 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the peptide may be characterized as being an epitope peptide restricted to HLA-A1.
  • 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.
  • the peptide may be characterized as being an epitope peptide restricted to HLA-A2.
  • 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.
  • the peptide may be characterized as being an epitope peptide restricted to HLA-A3.
  • 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.
  • the peptide may be characterized as being an epitope peptide restricted to HLA-A24.
  • 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.
  • the peptide may be characterized as being an epitope peptide restricted to HLA-A26.
  • 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.
  • the peptide may be characterized as being an epitope peptide restricted to HLA-B7.
  • 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.
  • the peptide may be characterized as being an epitope peptide restricted to HLA-B8.
  • 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.
  • the peptide may be characterized as being an epitope peptide restricted to HLA-B27.
  • 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.
  • 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.
  • the peptide may be characterized by inducing an immune response specific to fibroblast activation protein (FAP).
  • FAP fibroblast activation protein
  • 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.
  • the FAP expressing cell may be characterized as being a FAP expressing fibroblast, but is not limited thereto.
  • the peptide may additionally independently include amino acids at the N'-terminus and/or the C'-terminus, respectively.
  • 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.
  • 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.
  • FAP fibroblast activation protein
  • 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.
  • 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.
  • 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.
  • FAP fibroblast activation protein
  • 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.
  • 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.
  • 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.
  • the fusion protein may further include an Fc domain.
  • 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.
  • the Fc domain may include a J chain.
  • the Fc domain may include immunoglobulin domains C ⁇ 2 and C ⁇ 3 and a hinge between C ⁇ 1 and C ⁇ 2.
  • 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.
  • 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.
  • 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.
  • transmembrane domain refers to a protein domain that spans the membrane of a cell.
  • the transmembrane domain preferably has an alpha-helical structure, but is not limited thereto.
  • honey domain refers to a series of amino acid sequences that exist between the transmembrane domain and the extracellular domain of a membrane anchored protein.
  • coiled coil domain refers to a structural motif of a protein in which 2 to 7 alpha helices are coiled like a rope strand.
  • the coiled coil domain may be characterized by having 2 or 3 alpha helices coiled.
  • 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.
  • the present invention relates to a nucleic acid encoding a peptide or fusion protein of the present invention.
  • 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.
  • the present invention relates to a recombinant vector containing the nucleic acid of the present invention.
  • any vector known in the art can be appropriately selected and used without limitation.
  • a vector containing a T7 series (T7A1, T7A2, T7A3, etc.), lac, lacUV5, temperature-dependent ( ⁇ phoA, phoB, rmB, tac, trc, trp or 1PL promoter)
  • yeast 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.
  • 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.
  • 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).
  • control sequence means a DNA sequence essential for the expression of an operably linked coding sequence in a particular host organism.
  • 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.
  • a control sequence suitable for a prokaryote includes a promoter, optionally an operator sequence, and a ribosome binding site.
  • 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.
  • promoters for high expression the SR ⁇ 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.
  • 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 ⁇ 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).
  • 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.
  • "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.
  • 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.
  • the gene 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ and ⁇ NM989, and other DNA phages, such as M13 and filamentous single-stranded DNA phages.
  • Useful expression vectors for yeast cells are the 2 ⁇ 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.
  • transformation means that DNA is introduced into a host so that the DNA becomes replicable as an extrachromosomal element or by chromosomal integration.
  • transfection means that an expression vector is accepted by a host cell, regardless of whether any coding sequence is actually expressed.
  • 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.
  • 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.
  • 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.
  • 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.
  • the present invention relates to a method for producing a peptide, which comprises a step of culturing the 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.
  • 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.
  • 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.
  • 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.
  • APCs antigen-presenting cells
  • a vaccine platform based on neutrally-charged small lipid nanoparticles 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.
  • the present invention from another aspect, relates to nanoparticles comprising the peptide.
  • 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.
  • the nanoparticle may be characterized by including one or more types of the peptide.
  • 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.
  • 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.
  • 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.
  • 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.
  • VLPs virus-like particles
  • protein nanostructures protein nanostructures
  • polymer nanoparticles polymer nanoparticles
  • lipid nanoparticles lipid nanoparticles
  • inorganic nanoparticles but is not limited thereto.
  • virus-like particle 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.
  • 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.
  • HPMA N-(2-hydroxypropyl)-methacrylamide copolymer
  • SMA polystyrene-maleic anhydride copolymer
  • PEG polyethylene glycol
  • PGA poly-L-glutamic acid
  • the inorganic nanoparticles include, but are not limited to, silica nanoparticles, gold nanoparticles, self-associating nanoparticles, etc.
  • the protein nanostructure refers to a nanoparticle formed by self-assembly of a protein.
  • 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.
  • the nanoparticle is a lipid nanoparticle.
  • 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.
  • a nanodisk a unilamellar vesicle
  • MMV multilamellar vesicle
  • MV multivesicular vesicle
  • LNP liposome
  • LNP lipopolyplex
  • 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.
  • 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.
  • 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-d
  • 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
  • DDPC 1,2-Didecanoyl-sn-glycero-3-phosphocholine
  • DEPA 1,2-Dierucoyl-sn-glycero
  • DLOPC 1,2-Dilauroyl-sn-glycero-3-phosphate
  • DLPE 1,2-Dilauroyl-sn-glycero-3-phosphocholine
  • DLPS 1,2-Dilauroyl-sn-glycero-3-phosphoserine
  • DMPA 1,2-Dimyristoyl-sn-glycero-3-phosphate
  • DMPC 1,2-Dimyristoyl-sn-glycero-3-phosphocholine
  • DMPE 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine
  • DMPG 1,2-Dimyristoyl-snglycero-3-Phospho-rac-(1-glycerol)
  • DMPS 1,2-Dimyristoyl-sn-glycero-3-phosphoserine
  • 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-
  • DOSPA
  • the cationic lipid may preferably be a cationic cholesterol derivative, more preferably Monoarginine-cholesterol (MA-Chol).
  • MA-Chol Monoarginine-cholesterol
  • 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.
  • 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.
  • 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.
  • the oligonucleotide may be composed of a natural nucleotide or a non-natural nucleotide, or may be composed of a mixture of these.
  • the oligonucleotide may be characterized by containing one or more phosphorothioate linkages and/or being modified with one or more 2'-O-methyl.
  • the single-chain or double-chain oligonucleotide may be characterized as being a CpG oligonucleotide, a STING activating oligonucleotide, or a combination thereof.
  • STING Stimulator of Interferon Genes
  • 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.
  • C cytosine triphosphate deoxynucleotide
  • G guanine triphosphate deoxynucleotide
  • 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.
  • the CpG oligonucleotide of SEQ ID NO: 151 was used, but is not limited thereto.
  • 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.
  • 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
  • the nanoparticle may include an anionic drug in addition to an adjuvant.
  • the anionic drug may be, but is not limited to, an oligonucleotide, an aptamer, mRNA, siRNA, miRNA, or a combination thereof.
  • 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 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)).
  • activated stromal fibroblasts also called activated cancer-associated fibroblasts (CAFs)
  • 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).
  • NASH nonalcoholic steatohepatitis
  • 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.
  • 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.
  • 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.
  • a vaccination therapy based on the peptide of the present invention exhibited an excellent therapeutic effect on nonalcoholic steatohepatitis (NASH), another FAP-related disease.
  • NASH nonalcoholic steatohepatitis
  • 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.
  • FAP-associated diseases such as fibrosis, arthritis, non-alcoholic steatohepatitis, and tumors/cancer through the induction of a strong immune response to FAP.
  • 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.
  • FAP fibroblast activation protein alpha
  • 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.
  • 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.
  • NASH nonalcoholic steatohepatitis
  • the vaccine composition may be characterized by comprising one or more peptides of 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.
  • 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.
  • 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.
  • the vaccine composition when the subject has MHC of the HLA-A1 type, 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.
  • the vaccine composition when the subject has MHC of the HLA-A2 type, 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.
  • the vaccine composition when the subject has MHC of the HLA-A3 type, 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.
  • the vaccine composition when the subject has MHC of the HLA-A24 type, 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.
  • the vaccine composition when the subject has MHC of the HLA-A26 type, 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.
  • the vaccine composition when the subject has MHC of the HLA-B7 type, 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.
  • the subject when it 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.
  • the subject 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.
  • 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.
  • 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.
  • 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.
  • the nanoparticle may be understood as an example of a carrier for presenting the peptide or nucleic acid.
  • the peptide may be included in the vaccine composition in various forms other than the form of the nanoparticle described above.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the vaccine composition may be characterized by additionally comprising a phospholipid, a cationic lipid, and/or an adjuvant.
  • 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.
  • 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 ⁇ -galactosylceramide), detergents, liposomes, saponins such as
  • 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).
  • Amphigen e. phlei
  • MCWE mycobacterial phlei cell wall extract
  • M-DNA M-DNA
  • M-DNA-M. phlei cell wall complex MCC
  • 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).
  • the anionic emulsifiers
  • the adjuvant may be a natural emulsifier, and the natural emulsifier includes, but is not limited to, acacia, gelatin, lecithin, and cholesterol.
  • 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.
  • FCA Freund's complete adjuvant
  • FIA Freund's incomplete adjuvant
  • immunomodulatory cytokines may also be included in the vaccine composition, for example as adjuvants, to enhance vaccine efficacy.
  • cytokines include interferon alpha (IFN- ⁇ ), interleukin-2 (IL-2), and granulocyte macrophage-colony stimulating factor (GM-CSF) or combinations thereof, preferably but not limited to GM-CSF.
  • 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.
  • 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.
  • 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.
  • the composition of the present invention can be formulated into a suitable dosage form using a known technique for clinical administration.
  • the vaccine composition may be administered as a single dose or divided into several doses.
  • 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.
  • pharmaceutically effective amount 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.
  • 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.
  • a booster can be administered to support and/or maintain protective immunity.
  • the vaccine composition can be administered to the patient once, twice or more times per month, several months or several years.
  • administration of the vaccine composition may continue, for example, over the course of several years.
  • 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.
  • the vaccine composition may be administered in a lower dose at the beginning of the vaccine therapy and in a higher dose over time.
  • 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.
  • fibrosis 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.
  • the tumor may be characterized as being a FAP-related tumor.
  • 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.
  • 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
  • the tumor may preferably be characterized as being a desmoplastic tumor.
  • prevention means any act of suppressing or delaying the onset of a desired disease by administering a vaccine composition according to the present invention.
  • treatment 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.
  • 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.
  • 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.
  • the vaccine composition may be formulated as a delayed release vehicle or a depot preparation.
  • a long-acting preparation may be administered by inoculation or implantation (e.g., subcutaneously or intramuscularly) or by injection.
  • 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.
  • a suitable polymeric or hydrophobic material e.g., as an emulsion in an acceptable oil
  • an ion exchange resin e.g., a sparingly soluble derivative
  • 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.
  • 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.
  • 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.
  • the oral composition can be formulated to coat the active agent or protect it from decomposition in the stomach.
  • 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.
  • the vaccine composition may be characterized by inducing a FAP-specific cytotoxic T cell response or a FAP-specific CD8+ T cell response.
  • the vaccine composition may be characterized by inducing an immune response to free FAP or FAP-expressing cells in a subject.
  • the FAP-expressing cells may be characterized by being FAP-expressing fibroblasts.
  • the vaccine composition may be characterized by inducing an anti-tumor response.
  • 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.
  • 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.
  • 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.
  • FAP fibroblast activating protein alpha
  • 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.
  • FAP fibroblast activation protein alpha
  • 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).
  • FAP 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.
  • a vaccine composition containing different peptides can be administered depending on the HLA type of the subject.
  • the vaccine composition when the subject has MHC of the HLA-A1 type, 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.
  • the vaccine composition when the subject possesses MHC of the HLA-A2 type, 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.
  • the vaccine composition when the subject possesses MHC of the HLA-A3 type, 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.
  • the vaccine composition when the subject has MHC of the HLA-A24 type, 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.
  • the vaccine composition when the subject has MHC of the HLA-A26 type, 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.
  • the vaccine composition when the subject has MHC of the HLA-B7 type, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a chemotherapy agent cytotoxic anticancer agent
  • a targeted anticancer agent a targeted anticancer agent
  • an immunotherapy agent a hormonal anticancer agent
  • a cell therapy agent etc., but is not limited thereto.
  • 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.
  • alkylating agents such as cyclophosphamide,
  • 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.
  • antibody therapeutics such as cetuximab, trastuzumab, bevacizumab, rituximab, ibltumomab, alemtuzuma
  • 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.
  • a CTLA-4 antibody such as apilimumab
  • a PD-1 antibody such as pembrolizumab or nivolumab
  • a PD-L1 antibody such as atezolizumab
  • an immune checkpoint inhibitor such as an IDO inhibitor.
  • 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.
  • 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.
  • combined administration 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.
  • 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.
  • 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.
  • 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.
  • the combined administration of the peptide, nucleic acid or nanoparticle and the anticancer agent may be characterized by being administered simultaneously.
  • the combined administration of the peptide, nucleic acid or nanoparticle and the anticancer agent may be characterized by sequential administration.
  • 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.
  • the peptide, nucleic acid or nanoparticle and the anticancer agent when 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.
  • the administration of the peptide, nucleic acid or nanoparticle and the anticancer agent can be performed independently and repeatedly one or more times.
  • 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.
  • 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.
  • the pharmaceutical composition may be administered in a formulation and dosage independent of the formulation of the anticancer agent to be administered in combination.
  • 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.
  • 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.
  • 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine DOPE
  • 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000] DSPE-PEG1000
  • 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000] DSPE-PEG2000-PDP) - Avanti Polar Lipids.
  • CpG oligoDNA modified with phosphorothioate backbone (CpG ODN; 5’-TCC ATG ACG TTC CTG ACG TT-3’, SEQ ID NO: 151) - Genotech and Bioneer.
  • Example 1-2 Animals and cell lines
  • mice 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).
  • KAIST Korea Advanced Institute of Science and Technology
  • 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 ⁇ M 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.
  • 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.
  • Example 1-4 Selection of mouse FAP target peptides and vaccine design
  • mice FAP target epitope peptides were predicted 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:
  • Example 1-5 Evaluation of immunogenicity and antitumor effect
  • mice 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 ⁇ g 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 ⁇ g/ml). IFN- ⁇ -producing CD8+ T cells were quantified by intracellular cytokine staining (ICS) (Fig.
  • ICS intracellular cytokine staining
  • IFN- ⁇ -producing cells were measured by enzyme-linked immunospot (ELISpot) assay.
  • 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.
  • cytokine staining For intracellular cytokine staining, cells were permeabilized with Cytofix/Cytoperm solution (BD Biosciences) and incubated with anti-IFN- ⁇ antibody. Samples were then washed and analyzed by flow cytometry. To measure IFN- ⁇ -producing cells by ELISpot, spleen cells (3 ⁇ 10 5 cells/well) were seeded in 96-well microplates coated with mouse IFN- ⁇ -specific monoclonal antibodies and restimulated with each peptide for 30 h. IFN- ⁇ -producing spots were generated using the mouse IFN- ⁇ 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, France, Germany).
  • mice that were inoculated subcutaneously with 2 ⁇ 10 5 E.G7-OVA cells into the right flank.
  • 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 ).
  • 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 ⁇ mol of SLNP.
  • HEPES-buffered glucose HEPES-buffered glucose
  • FAP PEP -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).
  • GMS Gatan Microscopy Suite
  • Example 1-7 Evaluation of antitumor effect of FAP PEP -SLNP nanovaccine
  • E.G7-OVA treatment model 2 ⁇ 10 5 E.G7-OVA cancer cells were injected into the right flank of the mice.
  • 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 ⁇ mol per mouse) by subcutaneous injection into both footpads at the indicated time points.
  • 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.
  • 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.
  • 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.
  • Tumor tissues were resected, fixed in 10% formalin solution, embedded in paraffin, and then cut into 5- ⁇ m-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).
  • 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 ⁇ m) 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-FAP ⁇ antibodies, and stained with Live/Dead cell dye for 20 min at 4°C. The cells were then washed and subjected to flow cytometry.
  • Spleens were harvested from immunized mice, and splenocytes were isolated.
  • 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.
  • cytokine staining For intracellular cytokine staining, cells were permeabilized using Cytofix/Cytoperm solution and then incubated with anti-IL-17A antibody.
  • spleen cells For analysis of antigen-specific splenic T cells, spleen cells were restimulated with FAPPEP or OVAPEP (10 ⁇ g/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- ⁇ antibody. All samples were analyzed by flow cytometry.
  • 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.
  • 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.
  • 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 ⁇ g/head) and CpG-ODN (10 ⁇ g/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- ⁇ m-thick slices.
  • 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
  • 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.
  • MCD methionine/choline deficient
  • MCS methionine/choline sufficient
  • AST aspartate transaminase
  • ALT alanine transferase
  • HDL total bilirubin
  • liver tissues were excised, fixed in 10% formalin solution, embedded in paraffin, and cut into 5- ⁇ m-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 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).
  • Fig. 3 The immunodominant peptide epitopes of FAP were predicted using three epitope peptide prediction programs, netMHC3.0, BIMAS, and PREDEP (Fig. 3).
  • Fig. 4a the identified epitope peptides.
  • 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).
  • CFA complete Freund's adjuvant
  • 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- ⁇ (IFN- ⁇ ) 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- ⁇ -secreting CD8+ T cells than did immunization with the other three peptides (Figs.
  • 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).
  • mice 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
  • FAP peptide-labeled 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).
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
  • DSPE-PEG1000 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000]
  • MA-Chol monoarginine-cholesterol
  • 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).
  • FAP PEP1 -SLNP is the most effective nanovaccine for treating tumors containing FAP+ CAFs in the TME.
  • 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.).
  • MBL Inc. Quant H-2 Kb Tetramer Kit-PE' product
  • 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).
  • 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).
  • 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.
  • 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).
  • FAP PEP1 -SLNP could act on other desmoplastic tumors.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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).
  • Example 8 Confirmation of the efficacy of FAP PEP1 peptide vaccine in treating nonalcoholic steatohepatitis (NASH)
  • Nonalcoholic steatohepatitis 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.
  • FGF21 fibroblast growth factor 21
  • Example 8-1 Confirmation of NASH therapeutic effect of FAP PEP1 peptide vaccine
  • 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).
  • Example 8-2 Confirmation of the liver damage alleviation effect of FAP PEP1 peptide vaccine
  • mice were 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).
  • 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
  • tissue analysis was performed using Oil Red O staining and H&E staining.
  • 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).
  • 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.
  • 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.
  • 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.
  • the FAP PEP1 verified has a sequence identical to the peptide epitope candidate predicted from human HLA-A2.
  • 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.
  • 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.
  • an adjuvant such as CpG or poly(I:C)
  • 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).
  • FAP fibroblast activation protein
  • FAP+ CAF FAP-expressing fibroblasts
  • 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.
  • FAP-related diseases such as cancer, fibrosis, NASH, etc.
  • FAP-expressing fibroblasts FAP-expressing fibroblasts
  • anti-tumor effects such as depletion of FAP-expressing CAF, reduction of ECM production in the TME, and inhibition of cancer metastasis.
  • the peptide of the present invention 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.

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Abstract

La présente invention concerne des peptides épitopes immunodominants de protéines d'activation des fibroblastes (FAP), et leurs utilisations. Les peptides selon la présente invention ont une excellente immunodominance et présentent un effet puissant d'induction de lymphocytes T CD8+ spécifiques pour des FAP et des fibroblastes exprimant FAP (FAP + CAF). Les peptides selon la présente invention peuvent présenter d'excellents effets de prévention et de traitement sur des maladies associées à FAP telles que le cancer, la fibrose et la NASH par induction d'immunité sur des FAP, et ont une toxicité remarquablement plus faible que d'autres vaccins ciblant CAF exprimant FAP. Les peptides se distinguent particulièrement dans le cadre du traitement contre le cancer par leurs excellents effets anti-tumoraux, tels que la déplétion de CAF exprimant FAP, la réduction de la production d'ECM dans le TME, et l'inhibition de la propagation des métastases cancéreuses. Une administration combinée des peptides selon la présente invention avec d'autres agents anticancéreux, augmente l'accumulation de l'agent anticancéreux dans les cellules tumorales et améliore de manière remarquable les effets antitumoraux, les peptides peuvent ainsi être efficacement utilisés dans des vaccins pan-tumoraux.
PCT/KR2024/005587 2023-04-26 2024-04-25 Peptides épitopes immunodominants de protéines d'activation des fibroblastes et leurs utilisations Pending WO2024225764A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080058272A1 (en) * 2006-08-29 2008-03-06 Juergen Becker Nonamer Peptides for Cancer Treatment
KR20160106192A (ko) * 2010-03-19 2016-09-09 이매틱스 바이오테크놀로지스 게엠베하 위장암 및 위암을 비롯한 여러가지 종양에 대한 신규한 면역 요법
CN105949302A (zh) * 2016-05-27 2016-09-21 郑州大学 Fap来源的抗肿瘤ctl表位肽p639及其应用
KR20180089522A (ko) * 2015-12-22 2018-08-08 이매틱스 바이오테크놀로지스 게엠베하 유방암 및 기타 암에 대한 면역요법에서의 사용을 위한 펩티드 및 펩티드의 조합
KR20210052924A (ko) * 2019-11-01 2021-05-11 한국과학기술원 작은 지질 나노 입자 및 이를 포함하는 암 백신

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080058272A1 (en) * 2006-08-29 2008-03-06 Juergen Becker Nonamer Peptides for Cancer Treatment
KR20160106192A (ko) * 2010-03-19 2016-09-09 이매틱스 바이오테크놀로지스 게엠베하 위장암 및 위암을 비롯한 여러가지 종양에 대한 신규한 면역 요법
KR20180089522A (ko) * 2015-12-22 2018-08-08 이매틱스 바이오테크놀로지스 게엠베하 유방암 및 기타 암에 대한 면역요법에서의 사용을 위한 펩티드 및 펩티드의 조합
CN105949302A (zh) * 2016-05-27 2016-09-21 郑州大学 Fap来源的抗肿瘤ctl表位肽p639及其应用
KR20210052924A (ko) * 2019-11-01 2021-05-11 한국과학기술원 작은 지질 나노 입자 및 이를 포함하는 암 백신

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