Live pathogen-mimicking nanoparticles based on pathogen outer wall components and their manufacturing method

Abstract

The present invention relates to a freeze-dried preparation of a pathogen cell wall skeleton containing the pathogen cell wall skeleton as an active ingredient, various live pathogen-mimicking nanoparticles prepared using the freeze-dried preparation and a Toll-like receptor 7 or 8 agonist that can induce the efficacy of live pathogens, their uses, and methods for preparing them.

Description

[Technical Field]

[0001] The present invention relates to live pathogen-mimicking nanoparticles based on pathogen outer wall components and a manufacturing method thereof. Specifically, the present invention relates to live pathogen-mimicking nanoparticles based on pathogen outer wall components, which are manufactured using a freeze-dried preparation of pathogen outer wall components that can be easily dispersed in an aqueous solution and an immunostimulator that can induce the efficacy of live pathogens, as well as uses of the nanoparticles and a manufacturing method thereof. [Background technology]

[0002] Vaccines are pharmaceuticals that confer acquired immunity in humans and other animals. They are widely used to prevent and treat microbial infections, malignant tumors (cancers), allergies, and other diseases using immunogenic substances such as killed or attenuated pathogens, proteins, and synthetic peptides. Vaccines generally come in live, attenuated, killed, and recombinant varieties. Live and attenuated vaccines require a long production period, resulting in high production costs and safety issues. This has led to active research into recombinant vaccines, which offer superior safety and are easily produced. However, recombinant vaccines have the drawback of lower immunogenicity and reduced therapeutic efficacy compared to existing virus-derived vaccines (Korean Patent Publication No. 10-2014-0041134). To overcome these drawbacks, there has been growing interest in developing alternative drugs using outer cell components of pathogenic bacteria. However, most drug formulations using outer cell components suffer from the following problems: First, most pathogen outer wall components are lipid-soluble, limiting their development into various pharmaceutical formulations. Second, vaccine components using pathogen outer wall components are safer than killed or inactivated vaccines or live attenuated vaccines, but they suffer from a fatal limitation: weak immunogenicity. Therefore, to develop effective immunotherapeutics, there is a pressing need for the development of novel pathogen-based vaccines that can be mass-produced, have high stability and excellent immunogenicity, and can be manufactured into various formulations. Summary of the Invention [Problem to be solved by the invention]

[0003] The present invention has been devised to solve the above-mentioned problems of the prior art, and aims to provide live pathogen-mimicking nanoparticles that can be mass-produced, have high safety and immunogenicity, and can be prepared in various dosage forms, as well as uses thereof and a method for producing the same.

[0004] The live pathogen-mimicking nanoparticles of the present invention, which comprise a pathogen outer wall component and a Toll-like receptor 7 or 8 agonist, first activate cellular immune responses through pathogen recognition based on the pathogen outer wall component, and then are delivered to the inside of immune cells through phagocytosis. After that, Toll-like receptor 7 or 8 agonists, which are associated with the live / death signal of living pathogens, are activated, thereby increasing immunogenicity through their activation of signal transduction and cellular immune responses, thereby providing live pathogen-mimicking nanoparticles with high immunogenicity (Figure 1).

[0005] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0006] The present invention provides live pathogen-mimicking nanoparticles containing a Toll-like receptor 7 or 8 agonist and a pathogen cell wall skeleton-based nanodispersion as active ingredients. The Toll-like receptor 7 or 8 agonist is preferably in an inactive state due to a lipid binding to the activation site. The lipid is preferably a fatty acid, steroid, glyceride, phospholipid, cholesterol, or fat-soluble vitamin. However, since the lipid binds to the activation site of the Toll-like receptor 7 or 8 agonist to inactivate it and prevent the Toll-like receptor 7 or 8 agonist from being absorbed into the bloodstream in the body, any known type of lipid can be used.

[0007] Furthermore, the nano-dispersion based on the pathogenic bacterial outer wall component means a form in which the pathogenic bacterial outer wall component from which lipids and membrane proteins have been removed is dispersed in an aqueous solution. The aqueous solution is a general term for all solutions prepared using water as a solvent, and is preferably a buffer solution, a cationic buffer solution, etc., but is not limited as long as the solvent is water.

[0008] In the present invention, the pathogen outer wall components may vary depending on the species and strain, and refer collectively to insoluble residues obtained by physically disrupting pathogens and then subjecting them to a purification process that includes nuclease, protease, and washing with organic solvents, and are generally composed of biopolymer substances, including, for example, long-chain fatty acids, sugar chains, and peptidoglycans. However, they are not limited thereto as long as they are elements generally found in the outer wall components of pathogens.

[0009] In another embodiment of the present invention, the pathogen outer wall component of the present invention is characterized in that it is freeze-dried and easily dispersible in an aqueous solution.

[0010] In yet another embodiment of the present invention, the pathogenic bacteria is a collective term for disease-causing microorganisms, and is preferably pathogenic bacteria, viruses, fungi, etc., and more preferably BCG (bacille Calmette-Guerin) strains, Mycobacterium bacteria, Nocardia bacteria, Corynebacterium bacteria, Rhodococcus bacteria, Gordona bacteria, coronavirus, etc., but is not limited thereto.

[0011] In yet another embodiment of the present invention, the bond between the Toll-like receptor 7 or 8 agonist and the lipid is cleavable, and the cleavable bond is preferably a carbamate, disulfide, ester, peptide, azide, amide, hydrazone, thioether, phosphodiester, thioketal, or a combination thereof, but is not limited thereto as long as the chemical bond at the binding site is cleaved in response to enzymes and pH in the tumor microenvironment or intracellular endosomes and lysosomes, or in response to specific stimuli such as temperature, redox potential, ROS, ATP, ultrasound, magnetic field, or light. The Toll-like receptor 7 or 8 agonist is characterized in that its function is dynamically restored within four days after the activation site is exposed by the cleavage.The enzyme is not limited as long as it is one of various enzymes present in cells, but is preferably acid phosphatase, acid phyrophosphatase, phosphodiesterase, phosphoprotein phosphatase, phosphatidic acid phosphatase, arylsulfatase, proteases, cathepsins, collagenase, arylamidase, peptidase, acid ribonuclease, acid deoxyribonuclease, lipase, or triglyceride. lipase, Phospholipase, Esterase, Carboxyesterase, Clucocerebrosidase, Galactocerebrosidase, Sphingomyelinase, Glycosidases, alpha-Glucosidase, beta-Glucosidase, beta-Galacto sidase, alpha-Mannosidase, alpha-ucosidase, beta-Xylosidase, alpha-N-Acetylhexosaminidase, beta-N-Acetylhexosaminidase, Sialidase, Lysozyme, Hyaluronidase, beta-Glucuronidas It may be e, etc.

[0012] In yet another embodiment of the present invention, the Toll-like receptor 7 or 8 agonist is preferably an imidazoquinoloine, a hydroxyladenine, a pteridone, an aminopyrimidine, a benzoazepine, a thiaoxoguanosine, or a derivative thereof, but is not limited thereto as long as it is a known Toll-like receptor 7 or 8 agonist.

[0013] In yet another embodiment of the present invention, the nanoparticles may be in a form in which a nanodispersion based on the outer wall components of pathogenic bacteria forms spherical nanoparticles, and a Toll-like receptor 7 or 8 agonist is bound to the spherical nanoparticles, or may be encapsulated regardless of binding, or may be attached to the surface of the nanoparticles, or may be sandwiched between the nanoparticle structures, or may be bound through electrostatic interactions, but are not limited thereto as long as they are in a form that can be contained in the nanoparticles of the present invention.

[0014] In yet another embodiment of the present invention, the nanoparticles may preferably have a diameter of 20 to 500 nm, more preferably 50 to 300 nm.

[0015] In yet another embodiment of the present invention, the nanoparticles may be in the form of nanoliposomes, nanoemulsions, nanomicelles, polymeric nanoparticles, and the like.

[0016] In yet another embodiment of the present invention, the nanoparticles may further comprise, in addition to the cell outer wall components, substances that activate humoral immunity, substances that activate cellular immunity, immunostimulatory agents, immunoadjuvants, etc., including, but not limited to, toll-like receptor 7 or 8 agonists, toll-like receptor agonists, saponins, antiviral peptides, inflammasome inducers, NOD ligands, CDS (cytosolic DNA sensor ligands), STING (stimulator of interferon genes) ligands, antigens, emulsions, alum, chemotherapeutic agents, immune checkpoint inhibitors, and combinations thereof.

[0017] The present invention also provides an immunoadjuvant composition comprising the nanoparticles as an active ingredient.

[0018] The present invention also provides a vaccine composition comprising the adjuvant composition and an antigen.

[0019] In one embodiment of the present invention, the antigen is preferably a protein, a recombinant protein, a glycoprotein, a gene, a peptide, a polysaccharide, a lipopolysaccharide, a polynucleotide, a cell, a cell lysate, a bacterium, a virus, a cancer antigen, etc., but is not limited thereto as long as it is a commonly known antigen.

[0020] In yet another embodiment of the present invention, the vaccine composition may be used to prevent or treat diseases such as microbial infectious diseases, tuberculosis, and cancer, and the prevention or treatment of cancer is characterized by suppressing cancer growth, metastasis, recurrence, etc., or suppressing resistance to anti-cancer therapeutic therapy, but is not limited thereto as long as it is part of a commonly used cancer treatment method.

[0021] In yet another embodiment of the present invention, the cancer may be, but is not limited to, bladder cancer, breast cancer, colon cancer, rectal cancer, lung cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, ovarian cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, uterine cancer, stomach cancer, bone cancer, blood cancer, etc.

[0022] In yet another embodiment of the present invention, the vaccine composition may further comprise a chemotherapeutic agent or an immune checkpoint inhibitor.

[0023] The present invention also provides a method for preventing and / or treating a disease, which comprises administering to an individual a composition containing the nanoparticles as an active ingredient.

[0024] The present invention also provides a use of a composition containing the nanoparticles as an active ingredient for the prevention and / or treatment of a disease.

[0025] The present invention also provides a use of the nanoparticles for producing a drug for use in the prevention and / or treatment of a disease.

[0026] In one embodiment of the present invention, the disease is preferably an infectious disease, tuberculosis, cancer, etc., but is not limited thereto as long as it is a disease that is known to be preventable or treatable using an existing vaccine, since the inclusion of the nanoparticles of the present invention can significantly improve the immunogenicity of existing vaccines.

[0027] The present invention also provides a method for producing live pathogen-mimetic nanomolecules, including the steps of: (a) disrupting pathogenic bacteria; (b) removing lipids from the disrupted bacteria; (c) removing membrane proteins from the lipid-removed disrupted bacteria; (d) separating outer wall components of the pathogenic bacteria by centrifuging the disrupted bacteria from which membrane proteins have been removed; (e) freeze-drying the separated outer wall components of the pathogenic bacteria to produce a freeze-dried preparation; (f) mixing the freeze-dried preparation, a surfactant, and a positively charged buffer solution; and (g) adding a lipid, a lipid conjugate, or a Toll-like receptor 7 or 8 agonist having a lipid bound to its activation site to the mixture of step (f), and sonicating the mixture to produce nanoparticles.

[0028] In one embodiment of the present invention, the surfactant used in step (f) to stably disperse the lyophilized formulation in the aqueous solution may typically be sodium dodecyl sulfate (SDS), didodecyldimethylammonium bromide (DMAB), Pluronic F68, Pluronic F127, polyvinyl alcohol (PVA), Tween-80, Span-85, oleic acid, or a combination thereof, but is not limited thereto as long as it is a commonly known surfactant.

[0029] In another embodiment of the present invention, the positively charged buffer solution used to stably disperse the lyophilized formulation in the aqueous solution in step (f) typically includes L-lysine (L-Lys) buffer solution, L-arginine (L-Arg) buffer solution, L-histidine (L-His) buffer solution, L-tyrosine (L-Tyr) buffer solution, L-aspartic acid (L-Asp) buffer solution, L-glutamine (L-Glu) buffer solution, and combinations thereof, but is not limited thereto as long as it is a commonly known type of positively charged buffer solution.

[0030] In yet another embodiment of the present invention, the method may further comprise, after step (g), adding any one or more selected from the group consisting of a Toll-like receptor 7 or 8 agonist, a Toll-like receptor agonist, a saponin, an antiviral peptide, an inflammasome inducer, a NOD ligand, a CDS ligand (cytosolic DNA sensor ligand), a STING (stimulator of interferon genes) ligand, an antigen, an emulsion, alum, a chemotherapeutic agent, an immune checkpoint inhibitor, a combination thereof, and a combination thereof, followed by mixing and sonicating. [Effects of the Invention]

[0031] The lyophilized preparation of pathogen cell wall components, i.e., a pathogen cell wall component-based nanodispersion, according to the present invention, contains as an active ingredient a pathogen cell wall skeleton from which lipids and membrane proteins have been removed. The pathogen cell wall components, which generally have strong lipid-solubility, can be easily dispersed in aqueous solutions, and live pathogen-mimicking nanoparticles in various dosage forms can be produced using this. Furthermore, by easily incorporating various substances capable of inducing cellular immunity, the pathogen cell wall components can be used to induce not only humoral immunity but also cellular immunity, overcoming the limitations of the low immunogenicity of existing vaccines. Furthermore, because cells are not used, stability can be enhanced and mass production is possible. Therefore, the live pathogen-mimicking nanoparticles based on pathogen cell wall components of the present invention are expected to be effectively used as vaccines for the prevention or treatment of various diseases, such as infectious diseases, tuberculosis, and cancer. [Brief explanation of the drawings]

[0032] [Figure 1] FIG. 2 is a diagram illustrating the mechanism of action of a nano-dispersion based on pathogenic bacterial outer wall components according to an embodiment of the present invention. [Figure 2] This figure shows the degree of stabilization when various types of surfactants and positively charged buffer solutions were used to synthesize BCG-CWS nanodispersions according to one embodiment of the present invention. A indicates non-detection. [Figure 3] FIG. 1 shows the results of confirming the immune cell activation properties of a BCG-CWS nanodispersion according to one embodiment of the present invention using ELISA. [Figure 4] 1 is a diagram showing the results of analyzing CWS-T7 / 8a according to one embodiment of the present invention using a transmission electron microscope and DLS. [Figure 5]1 shows the results of ELISA to confirm the cytokines secreted when mouse immune cells were treated with CWS-T7 / 8a or its components according to one embodiment of the present invention. ND means non-detection. [Figure 6] This figure shows the results of administering CWS-T7 / 8a or its constituent substances according to one embodiment of the present invention to mice, and then confirming the changes in AST and ALT levels in the blood over time using ELISA. [Figure 7] FIG. 1 shows the results of confirming the change in antibody titer using ELISA after CWS-T7 / 8a or its constituent substances according to one embodiment of the present invention were mixed with an antigen and administered to mice. [Figure 8] This figure shows the results of confirming the degree of activation of cellular immunity in the spleen using flow cytometry and ELISA after CWS-T7 / 8a or its constituent substances according to one embodiment of the present invention were mixed with an antigen and administered to mice. [Figure 9] This figure shows the results of confirming the degree of activation of antigen-specific T cells in the blood using flow cytometry after CWS-T7 / 8a or its constituent substances according to one embodiment of the present invention were mixed with an antigen and administered to mice. [Figure 10] This figure shows the results of administering CWS-T7 / 8a or its constituent substances according to one embodiment of the present invention to mice, mixed with coronavirus (SARS-CoV-2) recombinant protein, and then confirming the antibody titer in the blood using ELISA and the degree of cellular immune activation in the spleen using flow cytometry and ELISA. [Figure 11] This figure shows the results of confirming the degree of inhibition of cancer growth and survival rate after CWS-T7 / 8a or its constituent substances according to one embodiment of the present invention were mixed with antigens from cancer cells and administered to mice transplanted with cancer cells. [Figure 12]This figure shows the results of confirming the degree of inhibition of cancer growth and survival rate after CWS-T7 / 8a or its constituent substances according to one embodiment of the present invention were mixed with antigens from cancer cells and administered to mice in which cancer had been induced. DETAILED DESCRIPTION OF THE INVENTION

[0033] The lyophilized preparation of pathogenic bacterial outer wall components of the present invention, which contains as an active ingredient the outer wall component of a pathogenic bacterial from which lipids and membrane proteins have been removed, can be prepared in various dosage forms by easily dispersing the highly lipid-soluble outer wall component of a pathogenic bacterial in an aqueous solution. Furthermore, various substances, such as substances capable of inducing cellular immunity, immune activators, and anticancer chemicals, can be easily incorporated to produce nanoparticles such as nanoliposomes, nanomicelles, solid nanoparticles, and nanoemulsions. Therefore, by using the lyophilized preparation of pathogenic bacterial outer wall components of the present invention, which contains as an active ingredient the outer wall component of a pathogenic bacterial from which lipids and membrane proteins have been removed, various forms of live pathogen-mimicking nanoparticles can be easily prepared, and these are expected to be effectively used as vaccines for the prevention or treatment of various diseases, such as infectious diseases, tuberculosis, and cancer.

[0034] As used herein, the term "Toll-like receptor 7 or 8 agonist-based materials" refers to Toll-like receptor 7 or 8 agonists that may be selected from the group consisting of imidazoquinoloines, 8-hydroxyladenines, pteridones, 2-aminopyrimidines, benzoazepines, and 7-thia-8-oxoguanosines. The imidazoquinoline compounds are disclosed in WO 2018 196823, WO 2011 049677, WO 2011 027022, WO 2017 102652, and WO 2019 These include, but are not limited to, compounds of the type referred to in 040491, etc., or pharmaceutically acceptable salts thereof. The hydroxyadenine compounds are disclosed in WO 2012 080730, WO 2013 068438, WO 2019 036023, WO 2019 035969, WO 2019 035970, WO 2019 035971, WO 2019 035968, CN 108948016, US 2014 8846697, WO 2016 023511, WO 2017 133683, WO 2017 133686, WO 2017 133684, WO 2017 133687, WO 2017 076346, WO 2018 The pteridone compounds include, but are not limited to, compounds of the type described in US 2010 0143301, WO 2016 007765, WO 2016 044182, WO 2017 035230, WO 2017 219931, WO 2011 057148, CN 1087 94486, and the like, or pharmaceutically acceptable salts thereof.The aminopyrimidine compounds are disclosed in WO 2010 133885, WO 2012066335, WO 2012 066336, WO 2012 067268, WO 2013 172479, WO 2012 136834, WO 2014 053516, WO 2014 053595, US 2018 0215720, WO 2012 156498, WO 2014 076221, WO 2016 141092, WO 2018 045144, WO 2015 014815, WO 2018 233648, WO 2014 207082, WO 2014 056593, WO 2018 002319, WO 2013 117615, etc., or pharmaceutically acceptable salts thereof. The benzazepine compounds include, but are not limited to, compounds of the type mentioned in WO 2007 024612, WO 2010 014913, WO 2010 054215, WO 2011 022508, WO 2011 022509, WO 2012 097177, WO 2012 097173, WO 2016 096778, WO 2016 142250, WO 2017 202704, WO 2017 202703, WO 2017 216054, WO 2017 046112, WO 2017 197624, etc., or pharmaceutically acceptable salts thereof. The thiaoxoguanosine compounds include, but are not limited to, compounds of the type mentioned in WO 2016 180691, WO 2016 055553, WO 2016 180743, WO 2016 091698, etc., or pharmaceutically acceptable salts thereof.In addition, the present invention may include, but is not limited to, compounds or pharmaceutically acceptable salts of Toll-like receptor 7 or 8 compounds mentioned in PCT / US2009 / 035563, PCT / US2015 / 028264, PCT / US2016 / 020499, WO 2015 023598, PCT / US2015 / 039776, etc., which can be easily guessed and used by those skilled in the art, such as imiquimod, resiquimod, dactolisib, gardiquimod, sumanirole, motolimod, vesatolimod, loxoribine, SM360320, CL264, 3M-003, IMDQ, Compound This includes all cases of Toll-like receptor 7 or 8 agonists such as 54.

[0035] Various Toll-like receptor 7 or 8 agonists having lipid conjugation at their activation sites may be prepared by the method of Korean Patent Publication No. 10-2020-0097656. More specifically, they may be prepared by conjugating a lipid component such as cholesterol to the amine (NH2) group, which is the activation site of the Toll-like receptor 7 or 8 agonist, through a bond characterized by being at least one selected from the group consisting of carbamate, disulfide, ester, peptide, azide, amide, hydrazone, thioether, phosphodiester, thioketal, and combinations thereof. Representative examples are shown in Scheme 1 (carbamate bond) and Scheme 2 (disulfide bond).

[0036] [Reaction Scheme 1] TIFF0007811014000001.tif185170

[0037] The R is a branch containing an aliphatic or aromatic group and may include -NH-, -CO-, -CONH-, -CSNH-, -COO-, -CSO-, -SO2NH-, -SO2-, -SO-, -O-, and the like.

[0038] [Reaction Scheme 2] TIFF0007811014000002.tif179170

[0039] The R is a branch containing an aliphatic or aromatic group and may include -NH-, -CO-, -CONH-, -CSNH-, -COO-, -CSO-, -SO2NH-, -SO2-, -SO-, -O-, and the like.

[0040] As used herein, the term "Toll-like receptor 7 or 8 agonist-based materials" refers to Toll-like receptor 7 or 8 agonists, including natural ligands such as single-stranded ribonucleic acids (ssRNAs), guanosine (G)-rich, uridine (U)-rich, and adenosine (A)-rich oligonucleotides, small-interfering RNA (siRNA), and microRNA (miRNA).

[0041] As used herein, the term "toll-like receptor agonist" refers to a substance capable of inducing a signal transduction response via TLR-1, and examples thereof include one or more substances selected from the group consisting of tri-acylated lipopeptides (LPs), phenol-soluble modulin, Mycobacterium tuberculosis lipopeptides, S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH, Borrelia burgdorferi-derived lipopeptides, trihydrochloride (Pam3Cys) lipopeptides mimicking the acetylated amino terminus of OspA lipopeptides, and combinations thereof. Alternatively, the agonist may be a TLR-2 agonist such as Pam3Cys-Lip, or a TLR-3 agonist such as Poly(I:C), Poly(ICLC), Poly(IC12U), or ampligen. Alternatively, the agonist may be a TLR-4 agonist such as a Shigella flexineri outer membrane protein product, AGP, CRX-527, MPLA, PHAD, 3D-PHAD, or GLA, or a TLR-5 agonist such as flagellin or a flagellin fragment. Alternatively, the agonist may be a TLR-9 agonist such as an immunostimulatory oligonucleotide, but is not limited to these, and may include any Toll-like receptor agonist that can be easily predicted and used by those skilled in the art.

[0042] As used herein, "saponin" refers to an amphipathic glycoside found in various plant species, and examples thereof include, but are not limited to, QS21, Quil A, QS7, QS17, β-esquin, digitonin, and combinations thereof.

[0043] As used herein, the term "antiviral peptide" refers to a peptide that can inhibit viral proliferation, and may be, for example, KLK, but is not limited to this.

[0044] As used herein, the term "inflammasome inducer" may be, but is not limited to, TDB (trehalose-6, 6-dibehenate), NLRC4, Nalp3, etc.

[0045] As used herein, the term "NOD ligand" may include, but is not limited to, M-TriLYS (NOD2 agonist-synthetic muramyldipeptide), NOD2 agonist (N-glycolylated muramyldipeptide), mDAP, MDP, etc.

[0046] As used herein, the term "CDS ligand" may be, but is not limited to, Poly(dA:dT).

[0047] As used herein, the term "STING ligand" may include, but is not limited to, cGAMP, di-AMP, di-GMP, etc.

[0048] As used herein, "lipid" may refer to, but is not limited to, fatty acids, steroids, glycerides, phospholipids, cholesterol, fat-soluble vitamins, etc. Furthermore, "lipid conjugate" collectively refers to all compounds bound to the lipids.

[0049] Furthermore, the additional substances that may be included may be respective complex immunomodulatory substances, such as, but not limited to, CL401 (a TLR-2 and TLR-7 agonist) and CL429 (a TLR-2 and NOD2 agonist).

[0050] As used herein, the term "chemo-anticancer agent" refers to any compound known to those skilled in the art for use in cancer treatment, and is not limited thereto. Examples thereof include Paclitaxel, Docetaxel, 5-Flurouracil, Alendronate, Doxorubicin, Simvastatin, Hydrazinocurcumin, Amphotericin B, Ciprofloxacin, Rifabutin, Rifampicin, Efavirenz, Cisplatin, Theophyline, Pseudomonas exotoxin A, Zoledronic acid, Trabectedin, Siltuximab, Dasatinib, Sunitinib, Apatinib, and 5,6-Dimethylxanthenone-4-acetic acid. acid, Silibinin, PF-04136309, Trabectedin, Carlumab, BLZ945, PLX3397, Emactuzumab, AMG-820, IMC-CS4, GW3580, PLX6134, N-acetyl-l-cystein, Vitamin C, bortezomib, aspirin, salicylates, Indolecarboxamide derivatives, quinazoline analogues, Thalidomide, prostaglandin metabolites, 2ME2, 17-AAG, Camptothecin, Topotecan, Pleurotin, 1-methylpropyl, 2-imidazolyl disulphide, Tadalafil, Sildenafil, L-AME, Nitroaspirin, Celecoxib, NOHA, Bardoxolone methyl, D,L-1-methyl-tryptophan, Gemcitabine, Axitinib, Sorafenib, Cucurbitacin B, JSI-124, Anti-IL-17 antibodies, Anti-glycan antibodies, Anti-VEGF antibodies, Bevacizumab, Antracycline, Tasquinimod, Imatinib, cyclophosphamide, etc., but are not limited to these.

[0051] In the specification, the term "immune checkpoint inhibitor" refers to a cancer treatment method that activates the immune function of the body's immune cells to fight cancer cells, and examples of such inhibitors include, but are not limited to, anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-KIR, anti-LAG3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, and anti-TIGIT.

[0052] As used herein, the term "antigen" refers to any substance that induces an immune response in the body. Preferably, the term refers to pathogens (e.g., bacteria, viruses), chemicals, pollen, cancer cells, shrimp, or their partial peptides or proteins, more preferably cancer antigen peptides. However, the term is not limited thereto as long as the substance is capable of inducing an immune response in the body. The antigen may be, for example, a protein, recombinant protein, glycoprotein, gene, peptide, polysaccharide, lipopolysaccharide, polynucleotide, cell, cell lysate, bacteria, virus, or the like, more preferably a cancer antigen peptide. The protein may be, for example, an antibody, antibody fragment, structural protein, regulatory protein, transcription factor, toxic protein, hormone, hormone analog, enzyme, enzyme fragment, transport protein, receptor, receptor fragment, host defense inducer, storage protein, movement protein, exploitative protein, reporter protein, or the like. However, the term is not limited thereto as long as the substance acts as an antigen in the body to induce an immune response.

[0053] As used herein, the term "vaccine" refers to a biological preparation containing an antigen that induces an immune response in the body, and is an immunogen that induces immunity in the body by being injected or orally administered to humans or animals for the prevention or treatment of diseases such as cancer, tuberculosis, and infectious diseases. The animal may be a human or a non-human animal, and the non-human animal may include, but is not limited to, pigs, cows, horses, dogs, goats, sheep, etc.

[0054] As used herein, "prevention" refers to any action of suppressing or delaying the onset of diseases such as infectious diseases, tuberculosis, and cancer by administering the composition according to the present invention.

[0055] As used herein, "treatment" refers to any action in which the symptoms of infectious diseases, tuberculosis, cancer, etc. are ameliorated or beneficially altered by administering a composition according to the present invention.

[0056] As used herein, the term "individual" refers to a subject to which the composition of the present invention can be administered, and there is no limitation on the subject.

[0057] As used herein, the term "cancer" refers collectively to various blood cancers, malignant solid tumors, etc., which can spread locally through invasion and systematically through metastasis. Specific examples of cancer include, but are not limited to, colorectal cancer, adrenal gland cancer, bone cancer, brain cancer, breast cancer, bronchial cancer, colon and / or rectal cancer, gallbladder cancer, gastrointestinal tract cancer, head and neck cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, nervous tissue cancer, pancreatic cancer, prostate cancer, parathyroid cancer, skin cancer, stomach cancer, and thyroid cancer. Other examples of cancer include adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and carcinoma in situ, Ewing's sarcoma, squamous cell carcinoma, liquid gland cell carcinoma, malignant brain tumor, myeloma, intestinal ganglioneuroma, hyperplastic corneal nerve carcinoma, islet cell carcinoma, Kaposi's carcinoma, leiomyoma, leukemia, lymphoma, malignant carcinoma, malignant melanoma, malignant hypercalcemia, Marpanoid habitus carcinoma, medullary carcinoma, metastatic skin cancer, mucosal neuroma, myelodysplastic syndrome, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, osteogenic and other sarcomas, ovarian cancer, pheochromocytoma, polycythemia vera, primary brain tumor, small cell lung cancer, ulcerative and papillary squamous cell carcinoma, staphylococcus aureus, soft tissue sarcoma, retinoblastoma, rhabdomyoblastoma, renal cell tumor or carcinoma, reticulocyte sarcoma, and Wilms' tumor. Also included are astrocytomas, gastrointestinal stromal tumors (GISTs), gliomas or glioblastomas, renal cell carcinomas (RCCs), hepatocellular carcinomas (HCCs), and pancreatic neuroendocrine carcinomas.

[0058] As used herein, the term "vaccine composition" may be in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and may be intended for humans. The vaccine composition may be formulated and used in the form of oral dosage forms such as powder, granule, capsule, tablet, and aqueous suspension, topical preparation, suppository, and sterile injectable solution, without limitation, by conventional methods. The vaccine composition of the present invention may contain a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may be a binder, lubricant, disintegrant, excipient, solubilizer, dispersant, stabilizer, suspending agent, dye, flavoring, etc.; for injection, a buffer, preservative, soothing agent, solubilizer, isotonicity agent, stabilizer, etc. may be mixed. For topical administration, a base, excipient, lubricant, preservative, etc. may be mixed. The vaccine composition of the present invention may be prepared in various dosage forms by mixing with the above-mentioned pharmaceutically acceptable carrier. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and injections may be in the form of unit dose ampoules or multiple doses. Additionally, the formulation may be a solution, suspension, tablet, capsule, sustained release formulation, etc.

[0059] On the other hand, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. Furthermore, the formulation may further contain a filler, an anti-agglomerating agent, a lubricant, a wetting agent, a flavoring, an emulsifier, a preservative, and the like.

[0060] Routes of administration of the vaccine compositions of the present invention include, but are not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, and rectal. Oral or parenteral administration is preferred. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The vaccine compositions of the present invention may also be administered in the form of suppositories for rectal administration.

[0061] The dosage of the vaccine composition of the present invention may vary depending on several factors, including the activity of the specific compound used, age, body weight, general health, sex, sex, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. The dosage of the vaccine composition varies depending on the patient's condition, body weight, severity of disease, drug form, administration route, and duration, but can be appropriately selected by those skilled in the art. It may be administered at a dose of 0.0001 to 500 mg / kg or 0.001 to 500 mg / kg per day. Administration may be once a day or in several divided doses. The dosage is not intended to limit the scope of the present invention in any way. The vaccine composition of the present invention may be formulated as a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.

[0062] The vaccine composition according to the present invention may further contain a known "adjuvant." The adjuvant generally refers to any substance that enhances humoral and / or cellular immune responses to an antigen, and any adjuvant known in the art may be used without limitation. For example, Freund's complete or incomplete adjuvant may be further included to enhance immunogenicity. Furthermore, in the case of the vaccine composition, repeated antigen stimulation may be optionally performed following the initial dose, if necessary.

[0063] Preferred examples are presented below to aid in understanding the present invention. However, the following examples are provided to facilitate understanding of the present invention, and the content of the present invention is not limited to the following examples. [Example]

[0064] Example 1: Preparation of nanodispersion based on pathogenic bacterial outer wall components To confirm the feasibility of producing nanodispersions based on the cell wall skeleton (CWS) of pathogenic bacteria, we first prepared nanodispersions using the Bacille Calmette-Guérin (BCG) strain (central vaccine). Specifically, the strain was dispersed in phosphate buffered saline (PBS) and then disrupted using an ultrasonic disperser (Sonics & Materials). The disrupted cells were then centrifuged at 1,000 x g for 3 minutes. A 1% Triton X-100 solution was added to the disrupted cells, and the cells were sonicated at 90°C for 2 hours using an ultrasonic disperser (Emerson) in a water bath to separate lipids. The supernatant was then removed by centrifugation at 10,000 x g, and the pellet was then added with 1% SDS. The pellet was then sonicated at 90°C for 2 hours using an ultrasonic disperser (Emerson) in a water bath to separate membrane proteins. The supernatant was then removed by centrifugation at 10,000 x g to obtain BCG-CWS (outer wall component of BCG) from which lipids and membrane proteins had been removed. The resulting BCG-CWS was redispersed in ethanol and then flash-frozen using liquid nitrogen. The ethanol was then removed from the frozen cells using a freeze dryer, resulting in a powdered form. The powdered BCG-CWS was then added to a 40 mM L-(+)-lysine solution to a concentration of 1 mg / mL and dispersed using an ultrasonic disperser at 60°C for 24 hours to produce a BCG-CWS nanodispersion. The mechanism of action of the pathogenic bacterial outer wall component-based nanodispersion of the present invention is shown briefly in Figure 1.

[0065] Generally, the outer wall components of BCG are highly hydrophobic and tend to disperse only in organic phases. To confirm whether the BCG-CWS prepared by the method of the present invention can be easily dispersed in various surfactants and positively charged buffer solutions, we added BCG-CWS nanodispersions to various surfactants, such as sodium dodecyl sulfate (SDS), Tween, Pluronic F, polyvinyl alcohol (PVA), span, and oleic acid, and various types of positively charged buffer solutions, and measured the polydispersity index (PDI). The results are shown in Figure 2.

[0066] As shown in Figure 2, the BCG-CWS nanodispersion was confirmed to be easily dispersed and stable in various surfactants and positively charged buffer solutions. Based on these results, it was confirmed that the method of the present invention can be used to prepare nanodispersions based on various pathogenic outer wall components that are easily dispersed in aqueous solutions.

[0067] Example 2: Confirmation of immune cell activation properties of nanodispersions based on pathogen outer wall components To confirm whether the BCG-CWS nanodispersion prepared in the same manner as in Example 1 possesses immune cell activation properties, bone marrow-derived dendritic cells (BMDCs) were treated with the BCG-CWS nanodispersion at concentrations of 1, 5, 10, 25, and 50 μg / mL. After culturing for 7 days, the secretion levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), representative indicators of inflammatory responses, were measured using an ELISA kit. A positive control group was treated with lipopolysaccharide (LPS), which is known to induce inflammatory responses. The results are shown in Figure 3.

[0068] As shown in Figure 3, the secretion of TNF-α and IL-6 increased in bone marrow-derived dendritic cells treated with the BCG-CWS nanodispersion, demonstrating that the nanodispersion based on pathogen outer wall components prepared by the method of the present invention effectively activates immune cells. Furthermore, it was confirmed that the nanodispersion based on pathogen outer wall components prepared by the method of the present invention can be used as live pathogen-mimicking nanoparticles.

[0069] Example 3: Preparation of live pathogen-mimicking nanoparticles using nanodispersions based on pathogen outer wall components 3.1. Synthesis of Toll-like receptor 7 or 8 agonist-cholesterol conjugates 50g (0.5 molar equivalent, AmBeed, USA) of Bis(2,5-dioxopyrrolidin-1-yl)(disulfanediylbis(ethane-2,1-diyl))dicarbonate) and 1L of DCM (dichloromethane, SIGMA) were added to a reactor at room temperature and mixed. 49g (0.55 molar equivalent, AVANTI Polar lipids) of cholesterol and 23g (1.0 molar equivalent) of TEA (trietylamine, SIGMA) were added to the mixed solution and stirred again at room temperature for 3 hours. 1.5L of distilled water was then added. The aqueous layer was extracted from the mixed solution using 500mL of DCM. This extraction process was repeated three times to achieve high purity. The organic layer in the extracted solution was then removed using 1 L of brine and dried under reduced pressure using 100 g of anhydrous Na2SO4 (SIGMA). The dried material was loaded onto a column packed with 300 g of silica gel and extracted using n-hexane with 5–20% EtOAc to prepare the cholesterol compound. To bind cholesterol to a Toll-like receptor 7 / 8 agonist (TLR7 / 8 agonist), 7.0 g of resiquimod, a Toll-like receptor 7 / 8 agonist, 6.5 g of DCM, 0.6 g of the cholesterol compound, and 0.3 g of TEA were added to a reactor at room temperature and stirred for at least 10 hours. After that, distilled water was added to the solution in an amount 20 times the total volume, and the aqueous layer was extracted five times or more using 3.3 g of DCM. Next, the organic layer contained in the solution after extraction was completed was removed using 1 L of brine, and then dried under reduced pressure using 7 g of anhydrous Na2SO4.The dried material was loaded onto a column packed with 100 g of silica gel and extracted with 10-50% EtOAc in n-hexane to synthesize a conjugate of Toll-like receptor 7 or 8 agonist and cholesterol, which was designated T7 / 8a.

[0070] 3.2. Fabrication of Live Pathogen-Mimicking Nanoparticles Containing BCG-CWS Nanodispersion and Toll-Like Receptor 7 or 8 Agonists To prepare live pathogen-mimetic nanomolecules using BCG-CWS nanodispersions and Toll-like receptor 7 or 8 agonists, BCG-CWS nanodispersions prepared in the same manner as in Example 1 and T7 / 8a were dissolved in a 1:1 (w:w) chloroform solution. The solution was transferred to a round-bottom flask and the organic solvent was completely evaporated to a film using a rotary evaporator. Next, 40 mM L-(+)-lysine solution supplemented with 1% Span-85 was added to the flask to a final concentration of 1 mg / mL of BCG-CWS nanodispersions. The mixture was then stirred at 300 rpm at 60°C for 1 hour to completely disperse the BCG-CWS nanodispersions. After stabilization for 1 hour, live pathogen-mimetic nanoparticles containing BCG-CWS nanodispersions and Toll-like receptor 7 or 8 agonists were prepared and designated CWS-T7 / 8a.

[0071] 3.3. Characterization of live pathogen-mimicking nanoparticles The characteristics of the live pathogen-mimicking nanoparticles prepared in the same manner as in Example 3.2 were confirmed using DLS (dynamic light scattering) and electron microscopy, and the results are shown in Figure 4.

[0072] As shown in Figure 4, the live pathogen-mimicking nanoparticles were confirmed to have an average diameter of approximately 120 nm and to be negatively charged.

[0073] 3.4. Confirmation of immune response activation effect of live pathogen-mimicking nanoparticles To confirm whether live pathogen-mimicking nanoparticles prepared using the same method as in Example 3.2 could activate immune responses, bone marrow-derived dendritic cells (BMDs) and bone marrow-derived macrophages (BMDMs) were treated with various concentrations of CWS nanodispersion (CWS-SLM), T7 / 8a, and live pathogen-mimicking nanoparticles (CWS-T7 / 8a). After 7 days of culture, the secretion levels of TNF-α, IL-6, and IL-12, representative indicators of inflammatory responses, were measured using ELISA kits. All experiments were repeated at least three times, and the results are presented as mean ± standard deviation. Statistical significance was confirmed using Student's t-test. * denotes P<0.05, ** denotes P<0.01, *** denotes P<0.001, and **** denotes P<0.0001. A P<0.05 was considered statistically significant. The results are shown in Figure 5.

[0074] As shown in Figure 5, the secretion of TNF-α and IL-6, cytokines involved in inflammatory immune responses, was significantly increased in immune cells, as was the secretion of IL-12 (p70), an important indicator of cellular immune responses. These results confirm that the live pathogen-mimicking nanoparticles of the present invention primarily activate cellular immune responses through pathogen recognition based on the outer wall components of the pathogen, and then are secondarily delivered into immune cells via phagocytosis. The Toll-like receptor 7 or 8 agonists associated with the live / death signal of the delivered live pathogen-mimicking nanoparticles are separated from cholesterol in endolysosomes, activating signal transduction and cellular immune responses, thereby secreting cytokines involved in inflammatory immune responses in immune cells (Figure 1).

[0075] 3.5. Toxicity assessment of live pathogen-mimicking nanoparticles To evaluate the toxicity of the live pathogen-mimicking nanoparticles, the live pathogen-mimicking nanoparticles, the CWS-nanodispersion, resquimod (R848) (free drug), and the CWS-nanodispersion and resquimod (R848) (free drug) were each added to 50 μL of phosphate buffered saline (PBS) at a resquimod concentration of 25 μg and subcutaneously injected into the right flank of C57BL / 6 mice. Blood was then drawn from the mice at timed intervals and centrifuged at 12,000 rpm to separate plasma. The concentrations of aspartate transaminase (AST) and alanine transaminase (ALT) in the separated plasma were measured using ELISA. The results are shown in Figure 6.

[0076] As shown in Figure 6, in the experimental group treated with resquimod as a free drug, AST and ALT, indicators of hepatotoxicity, increased initially, whereas in the case of CWS-nanodispersion and live pathogen-mimicking nanoparticles, the concentrations of AST and ALT remained almost unchanged.

[0077] From the above results, it was confirmed that the live pathogen-mimicking nanoparticles of the present invention exhibit little toxicity in the body and not only effectively induce inflammatory immune responses in immune cells but also increase cellular immune responses, and therefore can be used as an effective immune activator.

[0078] Example 4: Production and application of vaccines using live pathogen-mimicking nanoparticles 4.1. Vaccine production using live pathogen-mimicking nanoparticles To use the live pathogen-mimicking nanoparticles prepared in the same manner as in Example 3 as a vaccine, 0.2 mg / mL of the antigen protein ovalbumin (OVA) was mixed with the live pathogen-mimicking nanoparticles in a 1:1 volume ratio and then stirred at 300 rpm at 4°C for 30 minutes to allow the live pathogen-mimicking nanoparticles to interact with the antigen. As a control group, liposomes (T7 / 8a liposomes) containing the CWS-nanodispersion, resquimod, and cholesterol-resquimod conjugate were prepared by reacting them with the antigen protein alone or in combination.

[0079] 4.2. Confirmation of antibody generation efficacy of vaccines utilizing live pathogen-mimicking nanoparticles To confirm the antigenicity and efficacy of the vaccine prepared in the same manner as in Example 4.1, C57BL / 6 mice were injected with 100 μL of the vaccine twice, at two-week intervals. One week after the final injection, blood and spleens were extracted from the mice. The extracted blood was centrifuged at 12,000 rpm to separate plasma, and the antibody titer (IgG titer) was measured. The results are shown in Figure 7.

[0080] As shown in Figure 7, it was confirmed that the vaccine produced using the live pathogen-mimicking nanoparticles of the present invention showed higher antibody titers in all indicators compared to the antigen alone or other control groups.

[0081] 4.3. Confirmation of T cell activation efficacy of vaccines utilizing live pathogen-mimicking nanoparticles To confirm the T cell activation efficacy of the vaccine utilizing live pathogen-mimicking nanoparticles, splenocytes were extracted from the spleen using the same method as in Example 4.2, and 1 x 10 6 The cells were seeded into a 6-well plate (Corning) and cultured. 40 μg of OVA was then added to the cultured spleen cells and cultured for 2 days. The supernatant of the cell culture was then obtained, and the IFNγ concentration was measured using ELISA, and the CD4 + T cells and CD8 +T cells were measured, and the results are shown in Figure 8.

[0082] As shown in FIG. 8, the vaccine prepared using the live pathogen-mimicking nanoparticles of the present invention significantly increased IFNγ secretion and CD4 secretion compared to the antigen alone or other control groups. + T cells and CD8 + The highest proportion of T cells was confirmed in all cases.

[0083] 4.4. Confirmation of the antigen-specific T cell generation efficacy of vaccines utilizing live pathogen-mimicking nanoparticles To confirm the efficacy of antigen-specific T cell generation, peripheral blood monocytic cells (PBMCs) were isolated using Percoll from blood extracted using the same method as in Example 4.2. Next, PBMCs were treated with a fluorescence antibody that reacts with SIINFEKLE, a known site of action of OVA, and the results were confirmed using flow cytometry. The results are shown in Figure 9.

[0084] As shown in Figure 9, it was confirmed that the vaccine prepared using the live pathogen-mimicking nanoparticles of the present invention significantly increased the percentage of antigen-specific T cells in the blood compared to the antigen alone or other control groups.

[0085] 4.5. Production and efficacy of a coronavirus vaccine using live pathogen-mimicking nanoparticles To prepare a coronavirus vaccine using live pathogen-mimicking nanoparticles, SARS-CoV-2 recombinant S1 protein (Arigo) was mixed with live pathogen-mimicking nanoparticles at a 1:1 volume ratio and then stirred at 300 rpm for 30 minutes at 4°C to allow the live pathogen-mimicking nanoparticles to interact with the antigen. The prepared vaccine was then injected into C57BL / 6 mice, 100 μL each, twice, at two-week intervals. One week after the final injection, blood and spleens were extracted from the mice, and the efficacy of the vaccine was confirmed using the same method as in Examples 4.2 and 4.3. The results are shown in Figure 10.

[0086] As shown in Figure 10, it was confirmed that the antibody titers were higher in all indicators compared to the control group administered with the antigen alone. In addition, the control group administered with the antigen alone showed similar levels of IFNγ secretion and CD4 + T cells and CD8 + The percentage of T cells is shown, whereas in the case of the coronavirus vaccine utilizing live pathogen-mimicking nanoparticles, the percentage of IFNγ secretion and CD4 + T cells and CD8 + It was confirmed that the proportion of T cells was increased in all cases.

[0087] From the above results, it was confirmed that the vaccine utilizing the live pathogen-mimicking nanoparticles of the present invention can significantly increase the proportion of T cells, which are immune cells, and antibody titers compared to vaccines using only an antigen. Therefore, it was confirmed that the live pathogen-mimicking nanoparticles of the present invention can be used in various existing vaccines to significantly improve the efficacy of the vaccines.

[0088] Example 5: Confirmation of the anti-cancer immunotherapy effect using live pathogen-mimicking nanoparticles To confirm the cancer prevention efficacy of the vaccine utilizing live pathogen-mimicking nanoparticles, the vaccine prepared in the same manner as in Example 4.1 was injected into C57BL / 6 mice twice, 100 μL at a one-week interval. 5 B16-OVA cancer cells from Cells were subcutaneously injected into C57 / BL6 mice to induce tumors, and tumor growth and survival rates were examined. The results are shown in Figure 11.

[0089] As shown in Figure 11, in the experimental group that received the vaccine, it was confirmed that cancer growth was inhibited and the survival rate was significantly increased.

[0090] To confirm the efficacy of the live pathogen-mimicking nanoparticle vaccine in cancer treatment, 5 × 10 5 B16-OVA cancer cells from Cells were injected subcutaneously to induce tumors, and the tumor size was 50 mm. 3When the tumor volume reached 50 mm, 100 μL of the vaccine prepared in the same manner as in Example 4.1 was injected twice at a weekly interval. Then, tumor growth and survival rate were checked. 3 The results are shown in Figure 12.

[0091] As shown in Figure 12, it was confirmed that in the experimental group that received the vaccine, cancer growth was inhibited and the survival rate was significantly increased.

[0092] From the above results, it was confirmed that the vaccine utilizing the live pathogen-mimicking nanoparticles of the present invention exhibits cancer prevention and treatment effects, and therefore, it was confirmed that the vaccine utilizing the live pathogen-mimicking nanoparticles of the present invention can be used as an immunological anticancer agent.

[0093] Generally, pathogenic bacterial outer wall components have strong hydrophobic properties and are characterized by being dispersed only in organic phases. However, the pathogenic bacterial outer wall component-based nanodispersions prepared by the method of the present invention are easily dispersed in aqueous solutions without the use of lipophilic solvents, and therefore can be easily prepared in various nanoparticle forms, such as nanoliposomes, nanoemulsions, nanomicelles, and polymeric nanoparticles. Furthermore, the above results confirmed that the pathogenic bacterial outer wall component-based nanodispersions prepared by the method of the present invention maintain the immune cell activation efficacy of the pathogen itself, and therefore can be used to prepare various pharmaceutical dosage forms and can be widely applied as immune-activating therapeutic agents for various diseases. Furthermore, it was confirmed that the inclusion of various adjuvants in the pathogenic bacterial outer wall component-based nanodispersions of the present invention can effectively induce not only humoral immunity but also cellular immunity, further improving their immunogenicity.

[0094] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, the above-described embodiments should be understood to be illustrative in all respects and not restrictive. [Industrial Applicability]

[0095] The nanodispersion based on outer wall components of pathogenic bacteria of the present invention is easily dispersible in aqueous solutions, allowing it to be prepared as vaccines in various dosage forms. Furthermore, by mixing it with various antigens and / or adjuvants, its immunogenicity can be significantly increased, allowing it to be used as an immune-activating therapeutic agent for various diseases. Therefore, it is expected to be widely applicable in the field of vaccines used for the prevention or treatment of various diseases.

Claims

1. A live pathogen-mimicking nanoparticle comprising a Toll-like receptor 7 or 8 agonist and a nanodispersion based on a pathogen cell wall skeleton as active ingredients, The Toll-like receptor 7 or 8 agonist is in an inactive state due to a lipid binding to the activation site, The Toll-like receptor 7 or 8 agonist is at least one selected from the group consisting of imidazoquinoline compounds, hydroxyadenine compounds, pteridone compounds, aminopyrimidine compounds, benzazepine compounds, and thiaoxoguanosine compounds; The outer wall of the pathogen is derived from the Bacillus Calmette-Guerin (BCG) strain, A live pathogen-mimicking nanoparticle, characterized in that the binding of the Toll-like receptor 7 or 8 agonist and lipid is in a cleavable form.

2. The live pathogen-mimicking nanoparticles according to claim 1, wherein the pathogen outer wall component is a freeze-dried pathogen outer wall component dispersed in an aqueous solution.

3. 2. The live pathogen-mimicking nanoparticle of claim 1, wherein the detachable bond is at least one selected from the group consisting of carbamate, disulfide, ester, peptide, azide, amide, hydrazone, thioether, phosphodiester, thioketal, and combinations thereof.

4. The live pathogen-mimicking nanoparticle of claim 1, characterized in that the bond between the Toll-like receptor 7 or 8 agonist and lipid reacts to the enzymes and pH of the tumor microenvironment or the intracellular endosomes and lysosomes, cleaving the chemical bond at the binding site and exposing the activation site of the Toll-like receptor 7 or 8 agonist, thereby dynamically restoring function within four days.

5. The live pathogen-mimicking nanoparticles of claim 1, wherein the nanoparticles have a diameter of 20 to 500 nm.

6. The live pathogen-mimicking nanoparticle of claim 1, wherein the nanoparticle is at least one selected from the group consisting of nanoliposomes, nanoemulsions, nanomicelles, and polymeric nanoparticles.

7. 2. The live pathogen-mimicking nanoparticle of claim 1, further comprising at least one selected from the group consisting of a toll-like receptor agonist, a saponin, an antiviral peptide, an inflammasome inducer, a NOD ligand, a cytosolic DNA sensor ligand (CDS ligand), a stimulator of interferon genes (STING) ligand, an antigen, alum, a chemotherapeutic agent, an immune checkpoint inhibitor, and a combination thereof.

8. An adjuvant composition comprising the nanoparticles of claim 1 as an active ingredient.

9. A vaccine composition comprising the immunogen potentiator composition of claim 8 and an antigen as active ingredients.

10. The vaccine composition according to claim 9, wherein the antigen is at least one selected from the group consisting of a protein, a recombinant protein, a glycoprotein, a gene, a peptide, a polysaccharide, a lipopolysaccharide, a polynucleotide, a cell, a cell lysate, a bacterium, and a virus.

11. The vaccine composition according to claim 9, further comprising a chemical anticancer agent or an immune checkpoint inhibitor.

12. The vaccine composition according to claim 9, which is used for the prevention or treatment of cancer.

13. The vaccine composition according to claim 12, wherein the vaccine composition suppresses cancer growth, metastasis, recurrence, or resistance to anti-cancer treatment.

14. (a) disrupting pathogens; (b) removing lipids from the disrupted material; (c) removing membrane proteins from the lipid-depleted lysate; (d) centrifuging the membrane protein-removed lysate to separate the outer wall components of the pathogen; (e) freeze-drying the isolated outer wall components of the pathogen to prepare a freeze-dried preparation; (f) mixing the lyophilized formulation, a surfactant, and a positively charged buffer solution; (g) a method for producing live pathogen-mimicking nanoparticles, comprising the step of adding a lipid, a lipid conjugate, or a Toll-like receptor 7 or 8 agonist having a lipid bound to its activation site to the mixture of step (f), and sonicating the mixture to produce nanoparticles; The Toll-like receptor 7 or 8 agonist is at least one selected from the group consisting of imidazoquinoline compounds, hydroxyadenine compounds, pteridone compounds, aminopyrimidine compounds, benzazepine compounds, and thiaoxoguanosine compounds; The pathogen is a BCG (Bacille Calmette-Guerin) strain, A method for producing live pathogen-mimicking nanoparticles, wherein the binding of the Toll-like receptor 7 or 8 agonist and lipid is in a cleavable form.

15. 15. The method for producing live pathogen-mimicking nanoparticles according to claim 14, wherein after step (g), any one or more selected from the group consisting of a Toll-like receptor 7 or 8 agonist, a Toll-like receptor agonist, a saponin, an antiviral peptide, an inflammasome inducer, a NOD ligand, a cytosolic DNA sensor ligand (CDS ligand), a stimulator of interferon genes (STING) ligand, an antigen, alum, a chemotherapeutic agent, an immune checkpoint inhibitor, and combinations thereof are further added, mixed, and sonicated.

16. 15. The method for producing live pathogen-mimicking nanoparticles according to claim 14, wherein the surfactant in step (f) is at least one selected from the group consisting of sodium dodecyl sulfate (SDS), didodecyldimethylammonium bromide (DMAB), Pluronic F68, Pluronic F127, polyvinyl alcohol (PVA), Tween-80, Span-85, oleic acid, and combinations thereof.

17. 15. The method of claim 14, wherein the positively charged buffer solution in step (f) is at least one selected from the group consisting of an L-lysine (L-Lys) buffer solution, an L-arginine (L-Arg) buffer solution, an L-histidine (L-His) buffer solution, an L-tyrosine (L-Tyr) buffer solution, an L-aspartic acid (L-Asp) buffer solution, an L-glutamine (L-Glu) buffer solution, and combinations thereof.

18. 10. Use of the nanoparticles of claim 1 for the manufacture of a medicament for use in the prevention or treatment of a disease.

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