mRNA vaccine containing an adjuvant capable of kinetic control

Abstract

The present invention relates to an mRNA vaccine containing an adjuvant whose immune activation function is dynamically controlled, more specifically, to an mRNA vaccine containing an adjuvant characterized by its sequential activation function after mRNA is transcribed into protein. The present invention also relates to a core technology for optimizing the time interval between mRNA antigen expression and immune activation to effectively control the opposing antigen expression and antigen immunogenicity. The present invention provides a core technology for significantly increasing the efficacy of mRNA vaccines by simultaneously enhancing antigen expression and immunogenicity through dynamically controlling the action of immune activators to optimize the antigen expression level in mRNA and the action time of the adjuvant.

Description

Technical Field

[0001] The present invention relates to an mRNA vaccine containing an adjuvant whose immune activation function is kinetically controlled, and more particularly, to an mRNA vaccine characterized in that after the mRNA is transcribed into a protein, the activation function of the adjuvant sequentially acts.

Background Art

[0002] An mRNA vaccine is a pharmaceutical product that uses mRNA as an antigen for the prevention and treatment of cancer, infectious diseases, autoimmune diseases, etc. Compared with DNA vaccines, mRNA vaccines have the advantages of being stable and easy to mass-produce, and are expected to be widely used as a platform for anti-cancer vaccines and infectious disease vaccines in pandemic situations (Korean Patent Publication No. 10-2020-0118386). However, the immunostimulatory properties of naked mRNA, which induce the stimulation of adjuvants such as toll-like receptor agonists (TLR agonists), conversely interfere with the translation signal transduction system of mRNA, and it is known to inhibit the expression of a sufficient amount of mRNA antigen to exhibit pharmaceutical activity. In order to solve such problems, attempts are actively underway to produce vaccines using modified mRNA with removed or attenuated immunogenicity. However, in this case, due to the low immunogenicity of mRNA, there are limitations in inducing effective humoral and / or cellular immune responses. Therefore, it is very important to develop a novel mRNA vaccine system that can exhibit the best effects by effectively regulating the antigen expression mechanism and the mechanism showing antigen immunogenicity, which have opposite effects on each other.

[0003] On the one hand, the immune reaction is a series of reactions that activated immune cells have against exogenous and endogenous substances, that is, antigens. When microorganisms such as bacteria and viruses, and foreign substances in the body flow into the living body, immune cells recognize and activate them, secrete factors such as cytokines, and induce an inflammatory reaction. Recently, research on the mechanism at the stage of the innate immune reaction that acts non-specifically in the early stage of infection has been actively carried out. Among them, toll-like receptors are receptors that can recognize pathogens in the early stage of inflammation, and are known to recognize the plasma membrane components and nucleic acid components of pathogens and induce an immune reaction. Using this, active research has been conducted on various toll-like receptor ligands for activating immune cells.

[0004] Toll-like receptor agonists are agonists of Toll-like receptors within endosomes and are known to effectively induce not only humoral immunity but also cellular immunity. However, such multifunctional Toll-like receptor agonists are difficult to disperse in aqueous solutions due to their molecular structure. Moreover, they dissolve only in special organic solvents such as DMSO and methanol and do not dissolve in commonly used organic solvents, thus having limitations in manufacturing immunostimulatory agents in various dosage forms. Therefore, they have been commercialized in cream forms mixed with various surfactants (for example, Aldara cream, etc.). In some studies, in order to overcome such problems, they were manufactured in the form of salts and made soluble in aqueous solutions. However, Toll-like receptor agonists manufactured in the form of salts are absorbed into blood vessels in the body and induce many side effects (for example, cytokine storm, various non-specific hypersensitivity immune reactions, etc.) by inducing a systemic immune response in the blood vessels, so they are not easy to use at present. Also, due to such problems of side effects, in order to be actually used for treatment, a concentration lower than the effective dose has to be administered, which becomes a factor in reducing efficacy. Some pharmaceutical companies have attempted to prevent direct absorption into blood vessels by introducing lipids showing lipophilic properties or by directly chemically bonding to high molecular chains having a huge size in order to overcome such problems. However, Toll-like receptor agonists manufactured by such methods still have the possibility of inducing toxicity by inducing non-specific immune reactions in the body because their active sites are still exposed to the outside.

[0005] Therefore, when a Toll-like receptor agonist with a kinetically controlled activation time point and an mRNA vaccine containing mRNA are developed, after administration, initially the immunogenic effect of the mRNA antigen is suppressed. Instead, the expression of the mRNA antigen is normally induced, and then, sequentially, as the Toll-like receptor agonist with a kinetically controlled activation time point is converted to the activated state, the immunological activation of the mRNA is induced, thereby optimizing the time interval of the mechanism of action, and it is expected to have a very large impact in the next-generation mRNA vaccine market.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The present invention has been made to solve the above-mentioned problems in the prior art, and is a composition for an mRNA vaccine containing an adjuvant with a kinetically controlled activation time point and mRNA as active ingredients. After the mRNA moves to the cytosol and is transcribed, the adjuvant binds to the immunologically active site in the endosome / lysosome and the cytosol to induce immunological activation, thereby increasing the amount of antigen expression while simultaneously enhancing the immunogenicity of the antigen through immunological activation. An object of the present invention is to provide an mRNA vaccine composition and the like.

[0007] However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those of ordinary skill in the art from the following description.

Means for Solving the Problems

[0008] The present invention provides a composition for an mRNA vaccine, which contains an mRNA antigen and an immunostimulatory substance capable of kinetic control as active ingredients, wherein the immunostimulatory substance is a conjugate to which a cleavable linker capable of being cleaved at the active site of the immunostimulatory substance is bound. The mRNA antigen is not limited as long as it is an mRNA antigen known to be used in mRNA vaccines.

[0009] In one specific example of the present invention, after the process of transcription of mRNA into protein is carried out after administration, the cleavable linker bound to the active site of the immunostimulatory substance is sequentially cleaved, and the activation function of the immunostimulatory substance is induced.

[0010] In another specific example of the present invention, the kinetic control is such that a cleavable linker to which a cleavable linker capable of being cleaved at the active site of the immunostimulatory substance is bound maintains an inactive state, and after the transcription of mRNA starts, preferably within 2 to 12 hours, more preferably within 3 to 9 hours, the cleavable linker that blocks the active site is cleaved, and the activity of the immunostimulatory substance appears with a time delay. Further, the kinetic control can act on a molecular scale and / or a macro scale.

[0011] In still other specific examples of the present invention, the cleavable linker preferably contains any one or more bonds selected from the group consisting of disulfide, carbamate, hydrazine, ester, peptide, azide, amide, hydrazone, thioether, phosphodiester, thioketal, and combinations thereof. However, it is not limited thereto as long as the bond can be cleaved by endogenous factors (enzymes, redox potential, GSH, pH, etc.) and / or exogenous factors (redox, pH, temperature, photo / light, magnetism, ultrasonic waves, electrical response, etc.) in the living body.

[0012] In still other specific examples of the present invention, the cleavable linker further contains an alkyl derivative such as ethylene oxide or ethylene glycol at both ends or one end thereof, thereby enhancing the solubility and flexibility of the conjugate in an aqueous solution.

[0013] In still other specific examples of the present invention, the chemical bond at the binding site of the cleavable linker is cleaved by any one or more factors selected from the group consisting of enzymes, pH, redox potential, temperature, ultrasonic waves, magnetism, and light sources.

[0014] In still other specific examples of the present invention, any one or more substances selected from the group consisting of cholesterol, lipid, protein, amino acid, peptide, and oligonucleotide are bound to the end of the cleavable linker, and the substances serve to block the activating portion of the toll-like receptor agonist and can be various substances having hydrophilic or lipophilic groups.

[0015] In still another specific example of the present invention, the immunostimulatory substance may preferably be a toll-like receptor agonist, more preferably any one or more selected from the group consisting of a toll-like receptor 1 agonist, a toll-like receptor 2 agonist, a toll-like receptor 3 agonist, a toll-like receptor 4 agonist, a toll-like receptor 5 agonist, a toll-like receptor 6 agonist, a toll-like receptor 7 or 8 agonist, and a toll-like receptor 9 agonist.

[0016] In still another specific example of the present invention, the mRNA antigen and the immunostimulatory substance capable of kinetic control are loaded in any one or more drug delivery carriers selected from the group consisting of nanoliposomes, nanoemulsions, nanomicelles, hydrogels, scaffolds, solid nanoparticles, and polymeric nanoparticles. The loading may be in a form simply encapsulated, regardless of binding, or may be in a form sandwiched between nanoparticle structures or in a bound form, but is not limited thereto as long as it contains the mRNA antigen and the immunostimulatory substance of the present invention.

[0017] In still another specific example of the present invention, after the mRNA antigen loaded inside the drug delivery carrier is first transmitted to the cytosol, the immunostimulatory substance that acts kinetically interacts with a receptor on the cell surface, in endosomes, or in lysosomes.

[0018] In still other specific examples of the present invention, the drug delivery body may further preferably contain any one or more immunostimulatory substances selected from the group consisting of Toll-like receptor agonists, saponins, antiviral peptides, inflammasome inducers, NOD ligands, cytosolic DNA sensor ligands, STING (stimulator of interferon genes) ligands, outer wall components of pathogenic bacteria, alum, lipids, combinations thereof, and derivatives thereof. There is no limitation as long as the immunostimulatory substance is an immunostimulatory substance used as an adjuvant.

[0019] In still other specific examples of the present invention, the mRNA vaccine composition is characterized by being used for the prevention or treatment of any one or more diseases selected from the group consisting of infectious diseases, cancer, metabolic syndrome, autoimmune diseases, and rare diseases.

[0020] The present invention also provides a method for preventing or treating an infectious disease, cancer, metabolic syndrome, autoimmune disease, or rare disease, which includes administering to an individual an mRNA vaccine composition containing an mRNA antigen and an immunostimulatory substance capable of kinetic control as active ingredients.

[0021] Furthermore, the present invention provides a use for preventing or treating an infectious disease, cancer, metabolic syndrome, autoimmune disease or rare disease of a composition for an mRNA vaccine containing an mRNA antigen and an immunostimulatory substance capable of kinetic control as active ingredients.

[0022] Furthermore, the present invention provides a use for producing a drug used for preventing or treating an infectious disease, cancer, metabolic syndrome, autoimmune disease or rare disease of a composition containing an mRNA antigen and an immunostimulatory substance capable of kinetic control as active ingredients.

Advantages of the Invention

[0023] The composition for an mRNA vaccine according to the present invention can, after the mRNA has moved to the cytosol and been transcribed, bind to immunostimulatory sites in endosomes / lysosomes and the cytosol to induce immunostimulation, thereby increasing the expression level of the antigen while simultaneously enhancing the immunogenicity of the antigen through immunostimulation. As a technology, it can significantly improve the preventive and / or therapeutic effects of various mRNA vaccines, and thus it is expected to be applicable to various mRNA vaccines.

Brief Description of the Drawings

[0024]

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[0025] [Best Mode for Carrying Out the Invention] As a result of intensive research on a system that can significantly improve the efficacy of mRNA vaccines by using a combined administration method in which the expression time of the mRNA antigen and the immunostimulatory time of the adjuvant are optimized, a linker that can be cleaved at the activation site of the adjuvant, an immunostimulatory substance, is linked and maintained in a temporarily inhibited state. After in vivo administration, after reaching the target tissue or cell, the mRNA antigen begins to be transcribed into protein, and within 2 to 12 hours, the cleavable linker is cleaved and the activity of the adjuvant appears. By using an immunostimulatory substance capable of kinetic control together with the mRNA antigen, the inventors have invented a composition for an mRNA vaccine in which the efficacy of optimizing the time interval between the expression time of the mRNA antigen and the immunostimulatory time has been significantly increased.

[0026] As shown in FIG. 1, the mRNA vaccine of the present invention is a system in which, after the mRNA antigen moves to the cytosol and is transcribed, an interaction with an immunostimulatory site in the endosome / lysosome or cytosol occurs. Such a system is a system for solving the problem that, after administration in vivo, which is a fundamental limitation of naked mRNA, the immunostimulatory action that induces stimulation of toll-like receptors is activated, and the transcriptional signal transduction system of the mRNA antigen is disturbed, resulting in a decrease in efficacy because the expression of a sufficient amount of mRNA antigen for acting as a vaccine is inhibited (FIG. 2). Conventionally, in order to solve such problems, there have been attempts to use modified mRNA with weakened immunogenicity, but conclusively, due to the low immunogenicity, it does not induce a normal humoral and / or cellular immune response and still has the problem of reduced efficacy. Therefore, in order to solve such problems, the inventors have linked a cleavable linker to the activation site of the toll-like receptor agonist, so that the toll-like receptor agonist exists in a temporarily inactivated state, and then the cleavable linker is cleaved in the endosome / lysosome or cytosolic environment, and the activity is restored to bind to the receptor, thereby providing a new concept of mRNA vaccine system that optimizes the time interval between the expression of the mRNA antigen and the immunostimulation time (FIGS. 2 and 3).

[0027] As used herein, the term "mRNA vaccine" generally refers to a vaccine that uses messenger ribonucleic acid (mRNA) containing genetic information as an antigen. When the vaccine is inoculated, the mRNA produces proteins in the body, and the human immune system senses this and triggers an immune response, generating neutralizing antibodies. Recently, it has been increasingly applied to diseases in various fields, not only infectious diseases but also cancer, autoimmune diseases, metabolic syndrome, rare diseases, etc.

[0028] As used herein, the term "immune modulator" generally refers to a substance that plays a role in activating, inducing, or restoring the normal immune function of the immune system, and means a substance that can be used as an adjuvant. The adjuvant refers to a substance used together with an antigen to enhance the immune response, and can increase the production of antibodies and enhance humoral immunity and / or cellular immunity when used together with the antigen. The immune modulator preferably includes, but is not limited to, a toll-like receptor agonist, saponin, antiviral peptide, inflammasome inducer, NOD ligand, cytosolic DNA sensor ligand, stimulator of interferon genes (STING) ligand, emulsion, alum, incomplete Freund's adjuvant, Freund's adjuvant, or a combination thereof, and more preferably includes a toll-like receptor agonist.

[0029] As used herein, the term "toll-like receptor agonist" refers to a ligand that directly or indirectly acts on toll-like receptors, which are membrane proteins involved in innate immunity, and can induce a signal transduction reaction via a signal transduction pathway by generating endogenous or exogenous ligands. As used herein, the toll-like receptor agonist may be a natural toll-like receptor agonist or a synthetic toll-like receptor agonist, and may be a toll-like receptor 1 agonist, a toll-like receptor 2 agonist, a toll-like receptor 3 agonist, a toll-like receptor 4 agonist, a toll-like receptor 5 agonist, a toll-like receptor 6 agonist, a toll-like receptor 7 or 8 agonist, a toll-like receptor 9 agonist, etc.

[0030] The toll-like receptor 1 agonist refers to a ligand that can induce a signal transduction reaction via TLR-1. As an example, it may be, but is not limited to, triacylated lipid peptide (LP); phenol-soluble modulin; lipid peptide of Mycobacterium tuberculosis; S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH; lipid peptide of Borrelia burgdorferi; trihydrochloride (Pam3Cys) lipid peptide that mimics the acetylated amino terminus of OspA lipid peptide, etc.

[0031] The toll-like receptor 2 agonist refers to a ligand that can induce a signal transduction reaction via TLR-2. As an example, it may be, but is not limited to, peptidoglycan, zymosan, HSP70, HMGB1, HA, bam3Cys-Lip, etc.

[0032] The toll-like receptor 3 agonist means a ligand capable of inducing a signal transduction reaction via TLR-3. As an example, it may be, but is not limited to, Poly(I:C), Poly(ICLC), Poly(IC12U), ampligen, etc. as poly IC systems.

[0033] The toll-like receptor 4 agonist means a ligand capable of inducing a signal transduction reaction via TLR-4. As an example, it may be, but is not limited to, outer membrane protein products of Shigella flexneri, AGP, CRX-527, MPLA, PHAD, 3D-PHAD, GLA, LPS, etc.

[0034] The toll-like receptor 5 agonist means a ligand capable of inducing a signal transduction reaction via TLR-5. As an example, it may be, but is not limited to, Flagellin, etc.

[0035] The toll-like receptor 6 agonist means a ligand capable of inducing a signal transduction reaction via TLR-6. As an example, it may be, but is not limited to, Diacyl lipopeptide, lipoteichoic acid, etc.

[0036] The toll-like receptor 7 or 8 agonist means a ligand capable of inducing a signal transduction reaction via TLR-7 or 8. As an example, it may be an imidazoquinoline-based agonist, an 8-hydroxyadenine-based agonist, a pteridone-based agonist, a 2-aminopyrimidine-based agonist, a benzoazepine-based agonist, a 7-thia-8-oxoguanosine-based agonist, etc. The imidazoquinoline-based compound includes, but is not limited to, compounds or pharmaceutically acceptable salts of the types described in WO2018 196823, WO2011 049677, WO2011 027022, WO2017 102652, WO2019 040491, etc. Also, the 8-hydroxyadenine-based compound includes, but is not limited to, compounds or pharmaceutically acceptable salts of the types described in WO2012 080730, WO2013 068438, WO2019 036023, WO2019 035969, WO2019 035970, WO2019 035971, WO2019 035968, CN 108948016, US 2014 8846697, WO2016 023511, WO2017 133683, WO2017 133686, WO2017 133684, WO2017 133687, WO2017 076346, WO2018 210298, WO2018 095426, WO2018 068593, WO2018 078149, WO2018 041763, etc. The pteridone-based compound includes, but is not limited to, compounds or pharmaceutically acceptable salts of the types described in US 2010 0143301, WO2016 007765, WO2016 044182, WO2017 035230, WO2017 219931, WO2011 057148, CN 1087 94486, etc.The aminopyrimidine-based compounds include, but are not limited to, compounds of the types described in WO2010 133885, WO2012066335, WO2012 066336, WO2012 067268, WO2013 172479, WO2012 136834, WO2014 053516, WO2014 053595, US 2018 0215720, WO2012 156498, WO2014 076221, WO2016 141092, WO2018 045144, WO2015 014815, WO2018 233648, WO2014 207082, WO2014 056593, WO2018 002319, WO2013 117615, etc., or pharmaceutically acceptable salts thereof. The benzazepine-based compounds include, but are not limited to, compounds of the types described in WO2007 024612, WO2010 014913, WO2010 054215, WO2011 022508, WO2011 022509, WO2012 097177, WO2012 097173, WO2016 096778, WO2016 142250, WO2017 202704, WO2017 202703, WO2017 216054, WO2017 046112, WO2017 197624, etc., or pharmaceutically acceptable salts thereof. The thioxoguanosine-based compounds include, but are not limited to, compounds of the types described in WO2016 180691, WO2016 055553, WO2016 180743, WO2016 091698, etc., or pharmaceutically acceptable salts thereof. Additionally, it may also include toll-like receptor 7 or 8 compounds or pharmaceutically acceptable salts described in PCT / US2009 / 035563, PCT / US2015 / 028264, PCT / US2016 / 020499, WO2015 023598, PCT / US 2015 / 039776, etc.Alternatively, it may be, but is not limited to, imiquimod, resiquimod, dactolisib, gardiquimod, sumanirole, motolimod, vesatolimod, loxoribine, SM360320, CL264, 3M-003, IMDQ, Compound 54, etc., and includes all cases of toll-like receptor 7 or 8 agonists that can be easily inferred and used by those skilled in the art.

[0037] The toll-like receptor 9 agonist means a ligand capable of inducing a signal transduction reaction via TLR-9. As an example, it may be an immunostimulatory oligonucleotide, etc. The immunostimulatory oligonucleotide may include one or more CpG motifs, but is not limited thereto.

[0038] In this specification, "saponin" is an amphiphilic glycoside and acts as a surfactant. As an example, it may be, but is not limited to, QS21, Quil A, QS7, QS17, β-escin, digitonin, etc.

[0039] In this specification, "antiviral peptide" generally refers to a peptide showing an antiviral effect. As an example, it may be, but is not limited to, KLK (kallikrein), etc.

[0040] In this specification, "inflammasome inducer" generally refers to a substance that induces an inflammasome, which is a protein complex that recognizes and activates danger signals in the cytoplasm of eukaryotic cells. As an example, it may be, but is not limited to, TDB (trehalose-6,6-dibehenate), etc.

[0041] As used herein, the term "NOD ligand" generally refers to ligands that activate Nod-like receptors. By way of example, it may be, but is not limited to, M-TriLYS, N-glycosylated muramyldipeptid, etc.

[0042] As used herein, the term "cytosolic DNA sensor ligand" generally refers to ligands that activate cGAS, a DNA sensor. By way of example, it may be, but is not limited to, Poly(dA:dT), etc.

[0043] As used herein, the term "stimulator of interferon genes ligand" generally refers to ligands that activate STING, a sensor used by immune cells to detect cancer. By way of example, it may be, but is not limited to, cGAMP, di-AMP, di-GMP, etc.

[0044] As used herein, "cholesterol" refers to a type of lipid, which is a generic term for steroid-based organic substances having hydrophobic properties. The cholesterol may include various analogs based on the cholesterol structure and all compounds that can be obtained by chemically modifying a part of cholesterol. Preferably, it may include, but is not limited to, bile acids (cholic acid, deoxycholic acid, lithocholic acid, chenodeoxycholic acid), vitamin D, steroid hormones (testosterone, estradiol, cortisol, aldosterone, prednisolone, prednisone), etc. Further, the cholesterol is a substance that helps a toll-like receptor 7 or 8 agonist to be located on the surface and inside of various forms of nanoparticles, and can be replaced by lipid substances having a similar function, such as natural lipids (natureal lipid) like phospholipids, synthetic lipids, etc. It binds to the activation site of the toll-like receptor 7 or 8 agonist to make it in an inactivated state and prevent the toll-like receptor 7 or 8 agonist from being absorbed into blood vessels in the body. Therefore, there is no limitation as long as it is a known type of lipid.

[0045] As used herein, "cleavable linker" refers to a linker that contains a cleavable bond and can be cleaved by conditions in the body such as low pH, enzymes, glutathione, etc. in the tumor microenvironment, intracellular endosomes and lysosomes; or by external stimuli, namely, specific stimuli such as temperature, redox potential, ultrasonic waves, magnetic fields, near-infrared light, etc. Preferably, it means a linker that contains bonds such as carbamate, disulfide, ester, peptide, azide, etc., or is not limited thereto as long as it is in a cleavable form.

[0046] As used herein, "prevention" means all acts of suppressing or delaying the onset of diseases such as infectious diseases, cancer, metabolic syndrome, autoimmune diseases, and rare diseases by administering the composition according to the present invention.

[0047] As used herein, "treatment" means all acts in which the symptoms of infectious diseases, cancer, metabolic syndrome, autoimmune diseases, rare diseases, etc. are improved or beneficially changed by administering the composition according to the present invention.

[0048] As used herein, "individual" or "subject" refers to a subject to whom the composition of the present invention can be administered, and there is no limitation on the subject.

[0049] As used herein, "infectious disease" generally refers to diseases induced by infection with foreign organisms such as viruses, bacteria, and fungi.

[0050] As used herein, the term "cancer" generically refers to various blood cancers, malignant solid tumors, etc. that can expand locally by invasion and systematically by metastasis. Without being particularly limited thereto, specific examples of cancer include colorectal cancer, adrenal cancer, bone cancer, brain cancer, breast cancer, bronchial cancer, colon cancer and / or rectal cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, nerve tissue cancer, pancreatic cancer, prostate cancer, parathyroid cancer, skin cancer, stomach cancer, thyroid cancer, etc. Other examples of cancer include adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and intraepithelial cancer, Ewing sarcoma, squamous cell carcinoma, salivary gland cell carcinoma, malignant brain tumor, blastoma, intestinal gangliocytoma, hyperplastic corneal nerve cancer, islet cell cancer, Kaposi sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid tumor, malignant melanoma, malignant hypercalcemia, marfanoid habitus cancer, myeloid cancer, metastatic skin cancer, mucosal neuroma, myelodysplastic syndrome, myeloma, fungating polyposis, neuroblastoma, osteosarcoma, osteogenic and other sarcomas, ovarian cancer, chromaffin cell tumor, polycythemia vera, primary brain tumor, small cell lung cancer, ulcerative and papillary squamous cell carcinoma, seminoma, soft tissue sarcoma, retinoblastoma, renal cell tumor or renal cell carcinoma (RCC), reticulum cell sarcoma, and Wilms tumor. Also included are astrocytoma, gastrointestinal stromal tumor (GIST), glioma or glioblastoma, hepatocellular carcinoma (HCC), pancreatic endocrine cancer, etc.

[0051] As used herein, "metabolic syndrome" means that an individual has three or more of five risk factors (hypertension, hyperglycemia, hypertriglyceridemia, low high-density lipoprotein cholesterol, and central obesity) that increase the risk of health problems including heart disease, diabetes, and stroke. By way of example, it includes metabolic diseases such as obesity, diabetes, hypertension, hyperlipidemia, heart disease, and gout, and encompasses all diseases caused by metabolic syndrome.

[0052] As used herein, "autoimmune disease" generally refers to diseases caused by pathological reactions against autoantigens, including systemic autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS), insulin-dependent diabetes mellitus (IDDM), Grave's disease, and allergy.

[0053] As used herein, "rare disease" generally refers to all diseases that affect only a small proportion of the population. Generally, they are genetic, have a very low incidence and prevalence of the disease, are difficult to diagnose, and there is no appropriate treatment method. Although the definition varies slightly from country to country, South Korea defines "rare disease" based on Article 2 of the Rare Disease Management Act as "a disease with a diseased population of 20,000 or less, or a disease that is difficult to diagnose and the diseased population is unknown, and refers to a disease determined according to the procedures and criteria specified by the Ordinance of the Ministry of Health and Welfare." The World Health Organization (WHO) designates a disease as a rare disease when the prevalence is about 0.65 to 1 person or less per 1,000 people in the population. In the United States, it is when the total number of patients is less than 200,000, and in the European Union (EU), it is when there are 5 or fewer people per 10,000.

[0054] As used herein, the term "pharmaceutical composition" or "vaccine composition" is characterized by being in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition or vaccine composition is intended for human use. The pharmaceutical composition or vaccine composition may be formulated, but not limited to, in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories and sterile injection solutions by conventional methods. The pharmaceutical composition or vaccine composition of the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc. for oral administration, and buffers, preservatives, soothing agents, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used for injections, and bases, excipients, lubricants, preservatives, etc. may be used for topical administration. The dosage forms of the pharmaceutical composition or vaccine composition of the present invention can be manufactured in various ways by mixing with pharmaceutically acceptable carriers as described above. For example, for oral administration, it can be manufactured in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in unit-dose ampoules or multiple-dose forms. In addition, it can be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0055] Examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil, etc. may be used. Further, it may further contain fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc.

[0056] The administration routes of the pharmaceutical composition or vaccine composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is desirable. The term "parenteral" as used in the present application includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition or vaccine composition of the present invention can also be administered in the form of suppositories for rectal administration.

[0057] The pharmaceutical composition or vaccine composition of the present invention can vary diversely depending on various factors including the activity of the specific compound used, age, weight, general health, gender, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition or vaccine composition varies depending on the patient's condition, weight, degree of illness, drug form, administration route, and duration, but can be appropriately selected by those skilled in the art and can be administered at 0.0001 - 500 mg / kg or 0.001 - 500 mg / kg per day. Administration can be carried out once a day or divided into several times. The said dosage does not limit the scope of the present invention in any way. The pharmaceutical composition or vaccine composition according to the present invention can be formulated into tablets, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

[0058] In addition, the vaccine composition according to the present invention may further contain a conventionally known "immune antigen enhancer (adjuvant)". The immune antigen enhancer generally refers to any substance that increases the humoral and / or cellular immune response against an antigen, and any known substance in the technical field can be used without limitation. For example, Freund's complete adjuvant or incomplete adjuvant can be further included to increase its immunity. In addition, in the case of the vaccine composition, antigen stimulation can be optionally repeated subsequent to the initial dose as necessary.

[0059] Hereinafter, preferred examples are presented to assist in the understanding of the present invention. However, the following examples are provided only to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

Example

[0060] [Example 1: Synthesis of a conjugate of a toll-like receptor 7 or 8 agonist and cholesterol] Various toll-like receptor 7 or 8 agonists conjugated with cholesterol (imidazoquinoline-based agonist, 8-hydroxyadenine-based agonist, pteridone-based agonist, 2-aminopyrimidine-based agonist, benzoazepine-based agonist, 7-thia-8-oxoguanosine-based agonist, etc.) were produced by the chemical reactions of the following Reaction Formula 1 or 2. More specifically, the amine group (NH 2) Cholesterol (Sigma-Aldrich) was conjugated to the active site of temporarily inactivated Toll-like receptor 7 or 8 agonist via a cleavable carbamate, disulfide, ester, peptide, or azide bond at the [site] to produce a Toll-like receptor 7 / 8 agonist-Cholesterol conjugate.

[0061] [Chemical formula]

[0062] Said R is a side chain containing an aliphatic group or an aromatic group, and may contain -NH-, -CO-, -CONH-, -CSNH-, -COO-, -CSO-, -SO 2 NH-, -SO 2 -, -SO-, -O-, etc.

[0063] [Chemical formula]

[0064] Said R is a side chain containing an aliphatic group or an aromatic group, and may contain -NH-, -CO-, -CONH-, -CSNH-, -COO-, -CSO-, -SO 2 NH-, -SO 2 -, -SO-, -O-, etc.

[0065] [Example 2: Synthesis of 2-methyl-1-(3-nitroquinolin-4-ylamino)propan-2-ol] 2-Methyl-1-(3-nitroquinolin-4-ylamino)propan-2-ol (Compound 2) was synthesized using the method of the following Reaction Formula 3. More specifically, 1-Amino-2-methylpropan-2-ol (14 g) and tetraethylamine (9.6 g) were added to dichloromethane (450 ml) to which Compound 1 (30 g) was added at 10 - 20°C, and the mixture was stirred for 2 hours to produce a mixture. Next, after evaporating the solvent under vacuum to concentrate the mixture, it was resuspended using methyl tert-butyl ether (150 ml). The resuspended mixture was separated using a filter and then concentrated under reduced pressure to obtain Compound 2 (32 g, 85.2%, yellow solid). The structure of the obtained Compound 2 was 1 verified using 1H NMR. 1 1H NMR (400 MHz, DMSO-d6): δ 9.91 (brs, 1H), 9.18 (s, 1H)), 8.46 (d, J = 8.0 Hz, 1H), 7.83 - 7.92 (m, 2H), 7.56 - 7.60 (m, 1H), 5.15 (s, 1H), 3.86 (d, J = 4.8 Hz, 2H), 1.15 (s, 6H).

[0066] [Chemical Formula]

[0067] [Example 3: Synthesis of 1-(3-Aminoquinolin-4-ylamino)-2-methylpropan-2-ol] 1-(3-Aminoquinolin-4-ylamino)-2-methylpropan-2-ol (Compound 3) was synthesized using the method of the following Reaction Scheme 4. More specifically, Compound 2 (32 g), methanol (500 ml), and Pd / C catalyst (3.2 g) were mixed in a reactor at 10 - 20 °C, and then the mixture was degassed and flushed three times with hydrogen. After the hydrogen was vaporized to maintain 1 atm, the mixture was stirred at room temperature for 5 hours. Then, the mixture was resuspended with methyl tert-butyl ether (100 ml). The resuspended mixture was separated using a filter and then concentrated under low pressure to obtain Compound 3 (27 g, 95.4%, yellow solid). The structure of the obtained Compound 3 was 1 verified using 1H NMR. 1 1H NMR (400 MHz, DMSO-d6): δ 8.37 (s, 1H), 7.99 - 8.01 (m, 1H), 7.72 - 7.74 (m, 1H), 7.32 - 7.39 (m, 2H), 5.04 (s, 2H), 4.77 (brs, 1H), 4.67 - 4.70 (m, 1H), 4.12 (brs, 2H), 1.15 (s, 6H).

[0068]

Chemical Structure

[0069] [Example 4: Synthesis of 1-(2-Ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol] 1-(2-Ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol (Compound 4) was synthesized using the method of the following Reaction Formula 5. More specifically, after adding Compound 3 (27 g) and 2-ethoxyacetic acid (30 ml) to a reactor at 10 - 20 °C, the mixture was stirred at 120 - 130 °C for 5 hours. Then, the mixture was cooled to 20 - 25 °C, and saturated sodium carbonate (150 ml) was added. Next, the reaction product was extracted using a mixed solution of dichloromethane and methanol (10 / 1, v / v). The extracted organic solution layer was washed with brine, and then water was removed using sodium sulfate (10 g). Next, the organic solution layer with water removed was filtered using a filter and then concentrated under low pressure to obtain Compound 4 (30 g, 85.8%, yellow gel). The structure of the obtained Compound 4 was 1 verified using 1H NMR. 1 1H NMR (DMSO-d6 400 MHz): δ 9.18 (s, 1H)), 8.63 (d, J = 8.0 Hz, 1H), 8.13 (dd, J = 1.6, 8.0 Hz, 1H), 7.63 - 7.71 (m, 2H), 4.91 (s, 2H), 4.78 (brs, 2H), 3.54 (q, J = 6.8 Hz, 2H), 1.10 - 1.18 (m, 9H).

[0070] [Chemical Formula]

[0071] [Example 5: Synthesis of 2-(Ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinoline 5-oxide] 2-(Ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinoline 5-oxide (Compound 5) was synthesized using the method of the following Reaction Formula 6. More specifically, after adding Compound 4 (30 g), dichloromethane (350 ml), and metachloroperbenzoic acid (26 g) to a reactor at 10 - 20°C, the mixture was stirred at room temperature for 4 hours. Next, after adding a saturated sodium carbonate solution (150 ml) and a sodium sulfate solution (150 ml) to the stirred mixture, the reaction product was extracted using a mixed solution of dichloromethane and methanol (10 / 1, v / v). The moisture in the extracted organic solution layer was removed using sodium sulfate (30 g), filtered using a filter, and then concentrated under low pressure. Thereafter, the concentrated reaction product was resuspended using ethyl acetate (50 ml), separated using a filter, and then dried under low pressure to obtain Compound 5 (30 g, 94.9%, yellow solid). The structure of the obtained Compound 5 was 1 verified using 1H NMR. 1 1H NMR (DMSO-d6 400 MHz): δ 9.04 (s, 1H), 8.79 (d, J = 8.4 Hz, 1H), 8.71 (d, J = 8.4 Hz, 1H), 7.77 - 7.80 (m, 2H), 4.93 (s, 2H), 4.73 (brs, 2H), 3.54 (q, J = 6.8 Hz, 2H), 1.12 - 1.18 (m, 9H).

[0072] [Chemical Formula]

[0073] [Example 6: Synthesis of 1-(4-amino-2-ethoxymethyl)-1H-imidazo[4.5-c]quinolin-1-yl)-2-methylpropan-2-ol] 1-(4-Amino-2-ethoxymethyl)-1H-imidazo[4.5-c]quinolin-1-yl)-2-methylpropan-2-ol (Compound 6) was synthesized using the method of the following Reaction Formula 7. More specifically, to a reactor at 10 - 20 °C were added Compound 5 (30 g), DCM (600 ml), 4-methylbenzene-1-sulfonyl chloride (18.2 g), and aqueous ammonia (NH 3 ·H 2 O, 180 ml), and the mixture was stirred at room temperature for 16 hours. Next, distilled water was added to the stirred mixture, and then the mixture was separated using a mixed solution of dichloromethane and methanol (10 / 1, v / v). The separated organic solution layer was washed with brine and then dried over anhydrous sodium sulfate (50 g) to remove water. The organic solution layer with water removed was filtered using a filter, concentrated under low pressure, and the concentrated reaction product was resuspended for 30 minutes using a mixed solution of methyl tert-butyl ether and methanol (15 / 1, v / v). Then, after separation using a filter, it was dried under low pressure to obtain Compound 6 (18 g, 60%, yellow solid). The structure of the obtained Compound 6 was verified using 1 1H NMR. 1 1H NMR (DMSO-d6 400 MHz): δ 8.27 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.40 (t, J = 7.2 Hz, 1H), 7.21 (t, J = 7.2 Hz, 1H), 6.57 (brs, 2H), 4.89 (s, 2H), 4.68 (brs, 2H), 3.52 (q, J = 6.8 Hz, 2H), 1.11 - 1.17 (m, 9H).

[0074]

Chemical Formula

[0075] [Example 7: Synthesis of 10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo[a]phenanthren-3-yl 2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl carbamate] Using the method of Reaction Scheme 8 below, 10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo[a]phenanthren-3-yl 2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl carbamate (Compound 8) was synthesized. First, Compound 7 (TCI, 50 g, Cholesterol chloroformate) was purified using column chromatography packed with 250 g of silica gel (0% - 20% ethyl acetate in n-hexane) to obtain pure Compound 7 (30 g). Next, Compound 6 (15 g) and dichloromethane (198.9 g) were added to a reactor at 10 - 20 °C, and then pure Compound 7 (30 g) and tetraethylamine (9.6 g) were added in sequence, followed by stirring at 20 - 25 °C for 16 hours. After adding water to the stirred mixture, dichloromethane was added to extract the reactants. The extracted organic solution layer was washed with brine and then dehydrated using anhydrous sodium sulfate (195 g). The dehydrated organic solution layer was filtered using a filter and then concentrated under low pressure. Next, the concentrated reactant was resuspended using a mixed solution of methyl tert-butyl ether and methanol (10 / 1, v / v). Then, after separation using a filter, it was dried under low pressure to obtain Compound 8 (10.2 g, 55.1%, white solid). The structure of the obtained Compound 8 was 1 verified using 1H NMR. 1 Through the 1H NMR results, it was confirmed that a conjugate in which resiquimod (R848) and cholesterol were linked by a carbamate bond was produced. 1 1H NMR (CDCl3 400 MHz): δ 8.13 - 8.19 (m, 2H), 7.59 - 7.63 (m, 1H), 7.46 - 7.50 (m, 1H), 5.42 - 5.43 (m, 1H), 4.92 (brs, 2H), 4.72 - 4.80 (m, 3H), 3.68 (q, J = 6.8 Hz, 2H), 3.24 (s, 1H), 2.51 - 2.59 (m, 1H), 2.36 - 2.47 (m, 1H), 1.96 - 2.11 (m, 3H), 1.81 - 1.95 (m, 2H), 1.45 - 1.75 (m, 9H), 1.02 - 1.35 (m, 27H), 0.94 (d, J = 6.4 Hz, 3H), 0.89 (d, J = 6.4 Hz, 6H), 0.71 (s, 3H).

[0076] [Chemical formula]

[0077] [Example 8: Synthesis of bis(2,5-dioxopyrrolidin-1-yl) 2,2'-disulfanediylbis(ethane-2,1-diyl) dicarbonate] Bis(2,5-dioxopyrrolidin-1-yl) 2,2'-disulfanediylbis(ethane-2,1-diyl) dicarbonate (Compound 9) was synthesized using the method of the following Reaction Scheme 9. First, Compound 7 (TCI, 70 g) was purified using column chromatography (0% - 20% ethyl acetate in n-hexane) packed with 350 g of silica gel to obtain pure Compound 7 (40 g). Next, pure Compound 7 (40 g) and dichloromethane (100 ml) were added to a reactor at 10 - 15 °C, and then a dichloromethane (250 ml) solution containing bis(2-hydroxyethyl) disulfide and pyridine (21 g) were added in sequence. After the mixture was stirred at room temperature for 2 hours, distilled water (200 ml) was added. Then, dichloromethane (150 ml) was added three times to extract the reactants. The extracted organic solution layer was washed with brine and then dried over anhydrous sodium sulfate (20 g). The dried reactant was filtered using a filter and then concentrated under reduced pressure. Subsequently, Compound 9 (20 g, 39.6%, yellow gel) was obtained using column chromatography (silica gel, 300 g, 10% - 30% ethyl acetate in n-hexane). The structure of the obtained Compound 9 was 1 verified using 1H NMR. 1 1H NMR (CDCl 3 3, 400 MHz): δ 5.41 - 5.42 (m, 1H), 4.46 - 4.56 (m, 1H), 4.41 (t, J = 6.8 Hz, 2H), 3.91 (t, J = 6.0 Hz, 2H), 2.98 (t, J = 6.8 Hz, 2H), 2.91 (t, J = 6.0 Hz, 2H), 2.35 - 2.47 (m, 2H), 1.79 - 2.08 (m, 6H), 1.43 - 1.73 (m, 7H), 1.25 - 1.42 (m, 5H), 1.06 - 1.22 (m, 7H), 0.97 - 1.03 (m, 5H), 0.93 (d, J = 6.4 Hz, 3H), 0.88 (dd, J = 1.6, 6.8 Hz, 6H), 0.69 (s, 3H).

[0078]

Chemical Structure

[0079] [Example 9: Synthesis of Compound 10] Compound 10 was synthesized using the method of Reaction Scheme 10 below. More specifically, after adding Compound 9 (20 g) and dichloromethane (200 ml) to a reactor at 10 - 15 °C, bis(2,5-dioxopyrrolidin-1-yl) carbonate (18 g) and tetraethylamine (10.7 g) were added in sequence. The mixture was stirred at room temperature for 3 hours, then distilled water (300 ml) was added, and then dichloromethane (150 ml) was added three times to extract the reaction product. The extracted organic solution layer was washed with brine and then the water was removed using anhydrous sodium sulfate (20 g). The reaction product with the water removed was filtered using a filter, and then the filtrate was concentrated under low pressure. Then, column chromatography (silica gel, 200 g, 5% - 20% ethyl acetate in n-hexane) was used to obtain Compound 10 (18 g, 72%, yellow gel). The structure of the obtained Compound 10 was 1 verified using 1H NMR. 1 1H NMR (CDCl 3 400 MHz): δ 5.39 - 5.40 (m, 1H), 4.57 (t, J = 6.8 Hz, 2H), 4.43 - 4.52 (m, 1H), 4.37 (t, J = 6.4 Hz, 2H), 2.96 - 3.03 (m, 4H), 2.84 (s, 4H), 2.34 - 2.44 (m, 2H), 1.78 - 2.04 (m, 5H), 1.42 - 1.73 (m, 7H), 1.22 - 1.40 (m, 5H), 1.06 - 1.20 (m, 7H), 0.95 - 1.04 (m, 5H), 0.91 (d, J = 6.0 Hz, 3H), 0.86 (d, J = 6.4 Hz, 6H), 0.67 (s, 3H).

[0080] [Chemical formula]

[0081] [Example 10: Synthesis of 2-((2-((10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo[a]phenanthren-3-yloxy)carbonyloxy)ethyl)disulfanyl)ethyl 2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-ylcarbamate] Using the method of Reaction Scheme 11 below, 2-((2-((10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo[a]phenanthren-3-yloxy)carbonyloxy)ethyl)disulfanyl)ethyl 2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-ylcarbamate (Compound 11) was synthesized. More specifically, after adding Compound 6 (15 g) and dichloromethane (198.9 g) to a reactor at 10 - 20 °C, Compound 10 (40.5 g) and tetraethylamine (9.6 g) were added in sequence. The mixture was stirred at 20 - 25 °C for 16 hours, and then distilled water (225 ml) was added. Then, dichloromethane (99.45 g) was added 5 times to extract the reaction product. The extracted organic solution layer was washed with brine and then dried over anhydrous sodium sulfate (195 g). Next, the dried reaction product was filtered using a filter, and the filtrate was concentrated under reduced pressure. Thereafter, Compound 11 (10.8 g, 37.4%, white solid) was obtained using column chromatography (silica gel, 100 g, 10% - 50% ethyl acetate in n-hexane). The structure of the obtained Compound 11 was 1 verified using 1H NMR. 1 Through the 1H NMR results, it was confirmed that a conjugate in which R848 and cholesterol were cross-linked by a disulfide bond was produced. 1 1H NMR (CDCl 3400 MHz): δ 8.15 - 8.17 (m, 2H), 7.60 - 7.64 (m, 1H), 7.47 - 7.51 (m, 1H), 5.39 - 5.40 (m, 1H), 4.93 (s, 2H), 4.81 (s, 2H), 4.56 (t, J = 6.4 Hz, 2H), 4.45 - 4.54 (m, 1H), 4.41 (t, J = 6.4 Hz, 2H), 3.68 (q, J = 6.8 Hz, 2H), 3.13 (s, 1H), 3.09 (t, J = 6.4 Hz, 2H), 3.01 (t, J = 6.4 Hz, 2H), 2.34 - 2.47 (m, 2H), 1.92 - 2.06 (m, 3H), 1.79 - 1.90 (m, 2H), 1.23 - 1.72 (m, 21H), 1.06 - 1.21 (m, 7H), 0.96 - 1.05 (m, 5H), 0.93 (d, J = 6.4 Hz, 3H), 0.88 (dd, J = 1.6, 6.4 Hz, 6H), 0.69 (s, 3H).

[0082] [Chemical formula]

[0083] [Example 11: Production of Nanoparticles Containing a Conjugate of a Cholesterol-Toll-Like Receptor 7 or 8 Agonist] Since the conjugate of the cholesterol-Toll-like receptor 7 or 8 agonist contains cholesterol, it can be easily produced in various nanoparticle forms, thereby maximizing the interaction with immune cells.

[0084] (11.1. Production of Nanoliposomes Containing Resiquimod Conjugated with Cholesterol) To produce anionic nanoliposomes containing resiquimod conjugated with cholesterol, 4 mg of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine, Avanti), 1.2 mg of resiquimod conjugated with cholesterol, and 1 mg of DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-(1’-rac-glycerol), Avanti) were added to 1 ml of chloroform, dissolved to produce a mixture, the mixture was evaporated using a rotary evaporator to produce a thin film form, 2 ml of phosphate buffer solution was added to the thin film, stirred at 45 °C for 30 minutes, and homogenized using a tip sonicator (amplitude: 20%, 2 minutes) to produce anionic nanoliposomes containing resiquimod conjugated with cholesterol. To produce cationic nanoliposomes containing resiquimod conjugated with cholesterol, 4 mg of DOPC, 1.2 mg of resiquimod conjugated with cholesterol, and 2 mg of dimethyldioctadecylammonium bromide (DDAB) were added to 1 ml of chloroform, dissolved to produce a mixture, the mixture was evaporated using a rotary evaporator to produce a thin film form, 2 ml of phosphate buffer solution was added to the thin film, stirred at 45 °C for 30 minutes, and homogenized using a tip sonicator (amplitude: 20%, 2 minutes) to produce cationic nanoliposomes containing resiquimod conjugated with cholesterol.

[0085] (11.2. Production of Nanoemulsion Containing Resiquimod Conjugated with Cholesterol) To produce a nanoemulsion containing resiquimod conjugated with cholesterol, 1 mg of DOPC, 240 μg of cholesterol (Sigma-Aldrich), and 240 μg of resiquimod conjugated with cholesterol were added to 1 ml of chloroform, and then dissolved to produce a mixture. Next, after transferring the mixture into a round-bottom flask, chloroform was completely evaporated using a rotary evaporator to produce a thin-film form. Next, Squalene (5% v / v), Tween 80 (0.5% v / v), and Span 85 (0.5% v / v) were added to 2 ml of phosphate buffer solution and dissolved, and then the solution was added onto the lipid film and dispersed for 1 minute using a tip sonicator and stirred for about 2 hours using a tube revolve to produce a nanoemulsion containing resiquimod conjugated with cholesterol, which was then stored in a 4°C refrigerator until use.

[0086] (11.3. Production of Nanomicelles Composed of Resiquimod Conjugated with Cholesterol and Saponin) To produce a nanomicelle composed of resiquimod conjugated with cholesterol and saponin, phosphatidylcholine:saponin:resiquimod conjugated with cholesterol were mixed at a weight ratio of 5:3:2, and then added to ether to a concentration of 14 mg / ml and dissolved to produce an ether solution containing lipids. Next, saponin was dissolved in 4 ml of distilled water at a concentration of 1.5 mg / ml, placed in a 20 ml glass bottle, the glass bottle was stoppered with a rubber stopper, and then stored in a 55°C water jacket. Next, 1 ml of the ether solution containing lipids was added to the glass bottle containing saponin at a rate of 0.2 ml / min using a syringe pump and stirred for 2 hours. At this time, the tip of the syringe needle was positioned below the surface of the aqueous solution containing saponin, and a second needle was inserted into the rubber stopper for ventilation. Next, the glass bottle was transferred to room temperature and stirred for 3 days for stabilization to produce nanomicelles composed of resiquimod conjugated with cholesterol and saponin.

[0087] (11.4. Preparation of Polymer Nanoparticles Composed of Resiquimod Conjugated with Cholesterol) Dissolve 60 mg of PLGA polymer (Eudragit) with a composition ratio of lactide to glycolide of 50:50 in 1 ml of chloroform solvent. Add 5 mg of resiquimod conjugated with cholesterol to the solvent, and dissolve the cholesterol-resiquimod conjugate and the polymer using an ultrasonic bath (Emerson Model CPX5800H-E). Next, while adding 200 μl of the dissolved solution to 10 ml of 2.5% PVA aqueous solution one by one, disperse it for 1 minute using a tip sonicator (Sonics&Materials Model VCX 750). At this time, the output of the disperser was 750 watts, the vibration intensity was 20 kHz, and the amplitude was set to 20%. Next, the aqueous solution prepared to completely evaporate the organic solvent in which PLGA was dissolved was stirred at 600 rpm at room temperature for 8 hours or more. To remove the unreacted polymer and cholesterol-resiquimod conjugate, centrifugation was performed at 12,000 rpm for 12 minutes using a centrifuge (Centrifuge, Hanil, Combi-514R), and after removing the supernatant, 10 ml of ultrapure water was added and dispersed using an ultrasonic disperser for 30 seconds. After repeating the above process 3 times, it was dried using a freeze-drying method and stored at -20°C.

[0088] [Example 12: Confirmation of Efficacy of mRNA Vaccine Composition Containing mRNA and Cholesterol-Toll-Like Receptor Agonist] (12.1. Treatment Method of mRNA Vaccine Composition) To confirm the efficacy of the mRNA vaccine composition of the present invention, experiments were conducted using OVA mRNA, which is widely used in mRNA vaccines. More specifically, first, CleanCap® OVA mRNA (5moU, L-7210, TriLink Biotechnologies) and Opti-MEM TM(Reduced Serum Medium, ThermoFisher Scientific) was mixed at a volume ratio of 1:49. As the transfection reagent, Lipofectamine (Lipofectamine TM 2000 Transfection Reagent, ThermoFisher Scientific) and Opti-MEM TM were mixed at a volume ratio of 2:23, and then the respective mixtures were further mixed at a volume ratio of 1:1 and reacted for 5 minutes to prepare the mRNA composition. As toll-like receptor agonists, polyinosinic-polycytidylic acid (poly I:C, Sigma-Aldrich), which is a toll-like receptor 3 agonist, lipopolysaccharide (LPS, Sigma-Aldrich), which is a toll-like receptor 4 agonist, and resiquimod (Biorbyt), which is a toll-like receptor 7 or 8 agonist, were used. Poly I:C was dispersed and used at a concentration of 0.8 mg / ml in phosphate buffered saline (PBS), LPS was dispersed and used at a concentration of 0.3 μg / ml in PBS, and R848 was first dissolved at a concentration of 20 mg / ml in DMSO (dimethyl sulfoxide), and then the dissolved solution was further dispersed and used at a concentration of 20 μg / ml using PBS. When treating with a single toll-like receptor agonist, poly I:C was treated at a concentration of 40 μg / ml, LPS was treated at a concentration of 30 ng / ml, and R848 was treated at a concentration of 1 μg / ml. When used in combination, it was carried out at a concentration of 2 μg / ml of poly I:C and 1 μg / ml of R848, or at a concentration of 2 ng / ml of LPS and 1 μg / ml of R848. All mRNAs were treated at a concentration of 1 μg / ml.

[0089] (12.2. Bone marrow-derived dendritic cells, plasmacytoid dendritic cells, spleen cell culture of mice bearing ovalbumin-specific TCR) To confirm the efficacy of the composition for the mRNA vaccine of the present invention, as experimental cells, bone marrow-derived dendritic cells (BMDC), plasmacytoid dendritic cells (pDC), and T cells isolated from the spleen of ovalbumin-specific TCR-bearing experimental mice (OT-I transgenic mouse; OT-I mouse) were used. BMDC and pDC were obtained from the bone marrow of C57BL / 6 (female, Orient Bio), which is an experimental mouse, and used. More specifically, 6-week-old mice were euthanized, the femurs and tibias were cut, and then the medium was flushed to extract the internal substances. Next, the extracted substances were treated with red blood cell lysis buffer (BioLegend) to remove red blood cells and then used. For pDC, after euthanizing 6-week-old C57BL / 6, the femurs and tibias were cut, and the medium was flushed to extract the internal substances. Next, the extracted substances were treated with red blood cell lysis buffer (BioLegend) to remove red blood cells, and then only pDC was isolated using a pDC isolation kit (Plasmacytoid Dendritic Cell Isolation Kit, mouse, MACS Miltenyi Biotec) and used in the experiment. Spleen cells were extracted from OT-I experimental mice, physically crushed to make single cells, washed with PBS, and used after removing red blood cells using red blood cell lysis buffer. Next, the obtained cells were dispensed into a 100 mm × 25 mm culture dish (Corning petri dish) at 2 × 10 6 cells, and 5% CO 2, it was cultured under the condition of 37°C. As the culture medium, complete RPMI 1640 medium (Gibco) supplemented with 20 ng / ml of GM-CSF (Granulocyte-macrophage colony-stimulating factor, Recombinant Mouse GM-CSF Protein, R&D Systems) was used. During the culture period, fresh medium was alternated at 3-day intervals, and the cells on the 7th day of culture were used for the experiment.

[0090] (12.3. Co-culture of splenocytes of OT-I mice and pDCs) To confirm the efficacy of the composition for the mRNA vaccine of the present invention, T cells isolated from the spleen of OT-1 mice and pDCs were co-cultured. Splenocytes (1×10 5 cells / 100 μL) and pDCs (1×10 4 cells / 100 μL) obtained by the same method as in Example 12.2 were dispensed into a 96-well plate, and then treated with the composition for the mRNA vaccine prepared by the same method as in Example 12.1. After 48 hours, analysis of cells and cytokines was performed. For the analysis of intracellular cytokines, before collecting the cells, the cells were treated with cell activation cocktail (BioLgend) and protein transport inhibitor (BD GolgiStop TM Protein Transport Inhibitor) to activate the secretion of cytokines in the cell Golgi apparatus, and then the cells were collected and measured. The collected cells were perforated in the cell membrane and inside the cell using a Fixation / Permeabilization kit (BD Cytofix / Cytoperm TM Plus), and after working to allow the antibody to adhere, the amount of IFN-γ secreted intracellularly was measured using IFN-γ antibody (PE Rat Anti-Mouse IFN-γ; PE-IFN-γ, BioLegend).

[0091] (12.4. Co-culture of splenocytes of OT-I mice and BMDCs) To confirm the efficacy of the composition for mRNA vaccine of the present invention, T cells isolated from the spleen of OT-1 mice were co-cultured with BMDCs. In the case of BMDCs, in order to perform in the form of ex vivo DC therapy (cell therapy), before co-culturing with spleen cells, first, the composition for mRNA vaccine prepared by the same method as in Example 12.1 was treated and cultured for 24 hours. Next, after dispensing the BMDCs (1×10 4 cells / 100 μL) cultured for 24 hours and T cells (1×10 5 cells / 100 μL) into a 96-well plate, after 48 hours, analysis of cells and cytokines was performed. The analysis of cytokines was performed by the same method as in Example 12.3. To measure the degree of cell activation of BMDCs, the mean fluorescence intensity (MFI) of cell surface molecules 80 (Cluster of Differentiation 80; CD80), CD86, and CD40 was confirmed by attaching a fluorescent antibody. For the measurement of CD80, PerCP / Cyanine5.5 anti-mouse CD80 Antibody (PerCP / Cy5.5-CD80, BioLegend) was used, for CD86, PE anti-mouse CD86 Antibody (PE-CD86, BioLegend) was used, and for CD40, FITC anti-mouse CD40 Antibody (FITC-CD40, BioLegend) was used. In the case of BMDCs, first, the forward scatter and side scatter (FSC and SSC) were adjusted to separate into single cells, and then the population of BMDCs was separated using an anti-CD11c antibody (APC anti-mouse CD11c Antibody (APC-CD11c, BD Bioscience)). To measure the degree of antigen presentation, an anti-OVA SIINFEKL antibody was used.

[0092] (12.5. Confirmation of the Efficacy of the Composition for mRNA Vaccine in BMDCs) (12.5.1. Effects of Single Toll-Like Receptor Agonists) To confirm the effect of the composition for mRNA vaccine containing a single toll-like receptor in BMDCs, first, a toll-like receptor agonist and modified mRNA (5-methoxyuridine modified OVA mRNA, TriLink) were used to treat BMDCs in the same method as in Example 12.1, and the degree of antigen presentation, cell surface molecules, and the amounts of cytokines interleukin-12p70 (IL-12p70) and interferon were measured. The amount of inflammatory cytokines was measured using ELISA. As toll-like receptor agonists, R848, a toll-like receptor 7 or 8 agonist, LPS, a toll-like receptor 4 agonist, and poly I:C, a toll-like receptor 3 agonist, were used, and poly I:C was used at concentrations of 20 μg / ml, 30 μg / ml, and 40 μg / ml separately. Thereafter, all experiments were repeated at least three times, and the results were shown as the mean value ± standard deviation. Statistical significance was confirmed by Student’s t-test, and if P < 0.05, it was determined to be statistically significant. The results using R848 are shown in Figures 5 to 7, the results using LPS are shown in Figure 8, and the results using poly I:C are shown in Figures 9 to 10.

[0093] As shown in Figure 5, compared with the control group (mRNA only) treated with mRNA alone, in the experimental group (0 h) treated with mRNA and R848 simultaneously, although the degree of antigen presentation increased somewhat, there was no significant difference. However, in the experimental group (4 h) treated with mRNA and then with R848 4 hours later and the experimental group (8 h) treated with R848 8 hours later, a significant difference was shown, and it was confirmed that the degree of antigen presentation increased.

[0094] As shown in Figure 6, compared with the control group (mRNA only) treated with mRNA alone, in the experimental group treated with both mRNA and R848, regardless of the difference in treatment time, the cell surface molecules all increased, and thus it was confirmed that the degree of cell activation can be increased by the combined administration of mRNA and a toll-like receptor agonist.

[0095] As shown in Fig. 7, in the case of IL-12p70, it was confirmed that the secretion of IL-12p7 increased in the experimental group treated with a time interval (4 h) compared to the experimental group treated simultaneously with mRNA and R848 (0 h). Also, in the case of the ratio of IL-12p70 to IL-10 (IL-12p70 / IL-10), which is used as an index for inducing differentiation into Th1 (Th1 polarization), it was confirmed that it increased significantly in the experimental groups treated with a time interval (4 h and 8 h) compared to the experimental group treated simultaneously with mRNA and R848 (0 h).

[0096] As shown in Fig. 8, in the case of the experimental group treated simultaneously with mRNA and LPS (0 h), the degree of antigen presentation decreased compared to the control group treated with mRNA alone (mRNA only). However, in the experimental groups treated with mRNA and then LPS with a time interval (4 h and 8 h), it was confirmed that the degree of antigen presentation increased.

[0097] As shown in Fig. 9, in the case of the experimental group treated simultaneously with mRNA and Poly I:C (0 h), it was confirmed that the degree of antigen presentation decreased significantly compared to the control group treated with mRNA alone (mRNA only), regardless of the concentration of Poly I:C. On the other hand, in the experimental group treated with Poly I:C with a time interval (4 h), it was confirmed that the degree of antigen presentation increased significantly regardless of the concentration of Poly I:C (20, 30, and 40).

[0098] Fig. 10 shows the results of comparing the tendency of type 1 interferon (type 1 IFN), an antiviral cytokine, in the case of treating mRNA with 40 μg / ml of poly I:C. In the control group treated with mRNA alone (mRNA), the secretion of type 1 IFN was not confirmed. However, in the case of the experimental group treated simultaneously with mRNA and poly I:C (0 h), it was confirmed that the secretion of type 1 IFN increased significantly. Also, in the case of the experimental group treated with poly I:C with a 4-hour time difference (4 h), it was confirmed that the secretion of type 1 IFN decreased.

[0099] When each dendritic cell group having different antigen-presenting ability, degree of cell activation, and inflammatory cytokine secretion ability was co-cultured with T cells isolated from the spleen of ovalbumin-specific TCR transgenic mice (OT-I mice) to confirm the effect of the mRNA vaccine composition, the degree of activation of OT-1 T cells was confirmed. To confirm the degree of activation of T cells, interferon-γ (Interferon-γ; IFN-γ), a cytokine secreted from activated T cells, was confirmed. More specifically, the ratio of cells carrying IFN-γ inside the cells and the concentration of IFN-γ secreted outside the cells among the total T cells were measured. Also, the concentration of interleukin-2 (Interleukin-2; IL-2), a cytokine secreted from T cells and contributing to the differentiation and increased activity of T cells, was also confirmed. The results are shown in Fig. 11.

[0100] As shown in Fig. 11, compared with the control group (mRNA) in which BMDC treated with mRNA alone and T cells were cultured, in the experimental group (0 h) in which BMDC treated with mRNA and poly I:C simultaneously and T cells were cultured, IFN-γ + It was confirmed that the amounts of T cells, secreted IFN-γ, and IL-2 all decreased significantly. On the other hand, in the case of the experimental group (4 h) in which BMDC treated with mRNA and then poly I:C 4 hours later and T cells were cultured, or the experimental group (8 h) in which BMDC treated with mRNA and then poly I:C 8 hours later and T cells were cultured, IFN-γ + It was confirmed that the amounts of T cells, secreted IFN-γ, and IL-2 all increased significantly. In particular, in the case of IL-2, it was confirmed that the experimental group (4 h) treated at an interval of 4 hours showed the highest secretion amount.

[0101] (12.5.2. Combined administration effect of toll-like receptor 3 agonist and toll-like receptor 7 or 8 agonist) To confirm the effect of a composition for an mRNA vaccine containing a complex toll-like receptor, namely, a toll-like receptor 3 agonist and a toll-like receptor 7 or 8 agonist, in BMDCs, first, poly I:C, R848, and modified mRNA were treated with BMDCs in the same manner as in Example 12.1, and the degree of antigen presentation and the amount of interferon were measured. The results are shown in Fig. 12.

[0102] As shown in Fig. 12, in the experimental group (0 h) where mRNA and the complex toll-like receptor agonist were treated simultaneously, the degree of antigen presentation was significantly reduced compared to the case where mRNA was treated alone. Conversely, in the experimental group (4 h) where mRNA was treated and the complex toll-like receptor agonist was treated 4 hours later, it was confirmed that the degree of antigen presentation was significantly increased. Also, in the control group (mRNA) where mRNA was treated alone, secretion of type 1 IFN was not confirmed, but in the experimental group (0 h) where mRNA and poly I:C were treated simultaneously, it was confirmed that secretion of type 1 IFN was significantly increased. Also, in the experimental group (4 h) where poly I:C was treated with a 4-hour time difference, it was confirmed that secretion of type 1 IFN decreased.

[0103] Also, to confirm the effect of the composition for an mRNA vaccine when BMDCs and T cells were co-cultured, the degree of activation of OT-1 T cells was confirmed. The results are shown in Fig. 13.

[0104] As shown in Fig. 13, compared to the control group (mRNA) where BMDCs treated with mRNA alone and T cells were cultured, the experimental group (0 h) where BMDCs treated with mRNA, Poly I:C, and R848 simultaneously and T cells were cultured had IFN-γ +It was confirmed that the amount of T cells, the amount of secreted IFN-γ, and the amount of IL-2 all showed results similar to those of the control group treated with mRNA alone. On the other hand, in the experimental group (4h) where BMDC and T cells treated with mRNA were cultured with Poly I:C and R848 4 hours after mRNA treatment, or in the experimental group (8h) where BMDC and T cells treated with mRNA were cultured with poly I:C and R848 8 hours after mRNA treatment, IFN-γ + It was confirmed that the amount of T cells, the amount of secreted IFN-γ, and the amount of IL-2 all increased significantly.

[0105] (12.5.3. Combined administration effect of toll-like receptor 4 agonist and toll-like receptor 7 or 8 agonist) To confirm the effect of a composition for an mRNA vaccine containing a combined toll-like receptor, namely, a toll-like receptor 4 agonist and a toll-like receptor 7 or 8 agonist, in BMDC, first, LPS, R848, and modified mRNA were treated on BMDC in the same manner as in Example 12.1, and the degree of antigen presentation and the degree of cell activation were confirmed. The degree of cell activation was confirmed by measuring cell surface molecules. The results are shown in Figure 14.

[0106] As shown in Figure 14, compared with the control group treated with mRNA alone (mRNA only), in the experimental group (0h) treated with mRNA, LPS, and R848 simultaneously, the degree of antigen presentation decreased slightly, but no significant difference was shown. However, in the experimental group (4h) treated with mRNA and then with LPS and R848 4 hours later and the experimental group (8h) treated with mRNA and then with LPS and R848 8 hours later, it was confirmed that the degree of antigen presentation increased significantly and showed a significant difference. Also, in terms of cell surface molecules, compared with the control group treated with mRNA alone (mRNA only), it was confirmed that all cell surface molecules increased in the experimental group treated with mRNA, LPS, and R848 together.

[0107] Also, the ratio of IL-12p70 to IL-10 (IL-12p70 / IL-10), which is used as an index for inducing differentiation into Th1 (Th1 polarization), was confirmed. The results are shown in Figure 15.

[0108] As shown in Fig. 15, it was confirmed that, compared with the experimental group (0 h) treated simultaneously with mRNA, LPS, and R848, the experimental groups administered with a time lag (4 h, 8 h) significantly increased. Also, to confirm the correlation between mRNA and type 1 IFN, BMDCs were treated with eGFP-modified mRNA, and 12 hours later, the proportion of cells expressing the eGFP protein and the amount of type 1 IFN were measured. The results are shown in Fig. 16.

[0109] As shown in Fig. 16, in the experimental group (0 h) treated simultaneously with mRNA, LPS, and R848, the number of cells expressing GFP decreased compared with the control group (mRNA only) treated with only mRNA, but it was confirmed that the number of cells expressing GFP significantly increased in the experimental groups administered with a time lag (4 h, 8 h). Next, in the case of Type 1 IFN, it was not secreted in the control group treated with only mRNA alone, but was secreted the most in the experimental group (0 h) treated simultaneously with mRNA, LPS, and R848, and it was confirmed that the secretion amount decreased as the processing time interval between mRNA and toll-like receptor increased.

[0110] Through the above results, when the mRNA antigen and the toll-like receptor agonist are administered simultaneously, the expression of mRNA is suppressed, the immunogenic effect of the mRNA antigen is activated, and the expression of Type 1 IFN increases. However, when the mRNA antigen and the toll-like receptor agonist are administered with a time lag, it was confirmed that the expression of the mRNA antigen is induced initially, and the immunogenicity of the mRNA antigen is activated after a time interval.

[0111] (12.6. Confirmation of the efficacy of the mRNA vaccine composition in pDCs) Experiments were conducted using pDCs, which are known to secrete a relatively large amount of type 1 IFN compared to BMDCs. The treatment method was the same as that for BMDCs. Next, as toll-like receptor agonists, R848 and nanoliposomes (SKKU-078-liposome) produced in the same manner as in Example 11.1 using a conjugate in which cholesterol was cross-linked to R848 via a disulfide bond were used. Next, the degree of antigen presentation and the secretion level of interleukin-12p70 were measured. The results are shown in FIGS. 17 and 18.

[0112] As shown in FIG. 17, the control group treated with mRNA alone (mRNA only) showed no significant difference compared to the control group treated with only PBS (PBS), and it was confirmed that there was a partial increase in the experimental group (0 h) treated with mRNA and R848 simultaneously. However, it was confirmed that the degree of antigen presentation was significantly increased in the experimental group (4 h) treated with mRNA and then treated with R848 4 hours later. Also, in the case of the experimental group treated with liposome and mRNA simultaneously (mRNA+SKKU-078-liposome), it was confirmed that the degree of antigen presentation was significantly increased. This was confirmed to be the same result as the experimental group treated with R848 at an interval of 4 hours after mRNA treatment.

[0113] Also, as shown in FIG. 18, it was confirmed that the secretion level of interleukin-12p70 was significantly increased in the experimental group (mRNA+SKKU-078-liposome) treated with liposome and mRNA simultaneously compared to the control group treated with mRNA alone (mRNA only).

[0114] Also, when co-culturing pDCs and T cells of the spleen, in order to confirm the effect of the mRNA vaccine composition, liposomes containing modified mRNA and R848-cholesterol conjugate were treated while co-culturing. Next, the ratio of T cells by phenotype was analyzed 48 hours later. The results are shown in FIGS. 19 and 20.

[0115] As shown in FIGS. 19 and 20, in the case of the control group treated with mRNA alone (mRNA only), the results are similar to or slightly increased compared to the control group treated with PBS (PBS). However, in the case of the experimental group treated with mRNA and liposome (mRNA+SKKU-078-liposome), it was confirmed that the proportions of interferon gamma (IFN-γ) positive T cells, tumor necrosis factor alpha (TNF-α) positive T cells, and double positive T cells were significantly increased. In addition, it was confirmed that the secretion amount of IFN-γ cytokine increased.

[0116] Through the above results, in the production of a composition for an mRNA vaccine containing a toll-like receptor agonist, when the toll-like receptor agonist and mRNA are injected into the body and act simultaneously, while reducing the pharmaceutical activity of the mRNA, after the mRNA is transcribed into protein, when the activation function of the toll-like receptor agonist, i.e., the adjuvant, acts sequentially, it was confirmed that the immunization reaction is optimized and the efficacy of the vaccine can be improved most effectively. That is, it was confirmed that sequential activation of mRNA and the toll-like receptor agonist is important in producing an effective composition for an mRNA vaccine. In addition, the conjugate of the toll-like receptor agonist and cholesterol of the present invention has cholesterol linked to the active site of the toll-like receptor agonist by a cleavable bond, and initially the activity is temporarily inhibited. Then, when the conjugate reaches the target position by the physiological environment, the toll-like receptor agonist and cholesterol are separated and show activity, and it was confirmed that the same effect as that treated with a time difference from mRNA is shown. That is, by using the conjugate of the toll-like receptor agonist-cholesterol of the present invention as an adjuvant, a composition for a vaccine containing the conjugate of the toll-like receptor agonist-cholesterol and mRNA as active ingredients can not only significantly enhance the effect of the composition for an mRNA vaccine by the activation function of the toll-like receptor agonist, which is an adjuvant, acting sequentially after the mRNA is transcribed into protein even in simultaneous administration, but also be widely used in a wide variety of mRNA vaccine fields. It was also confirmed that by further including various adjuvants in the nanoparticles that can be easily produced using the conjugate of the toll-like receptor agonist-cholesterol, the immunostimulatory efficacy of the composition for an mRNA vaccine can be further increased.

[0117] The above description of the present invention is for illustrative purposes, and those with ordinary knowledge in the technical field to which the present invention pertains can understand that it can be easily transformed into other specific forms without changing the technical idea and essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all aspects and not restrictive.

Industrial Applicability

[0118] The mRNA vaccine containing an adjuvant whose immune activation function is kinetically controlled can significantly enhance the effect of the mRNA vaccine composition by the activation of the adjuvant after the induction of mRNA transcription, even in the case of co-administration, or by the activation function of the toll-like receptor agonist, which is the adjuvant, acting sequentially after the mRNA is transcribed into protein. Therefore, it can be widely used in various types of mRNA vaccine fields.

Claims

1. A composition for an mRNA vaccine, comprising an mRNA antigen and a toll-like receptor 7 or 8 agonist capable of kinetic control as an active ingredient, wherein the toll-like receptor 7 or 8 agonist is any one selected from the group consisting of imidazoquinoline agonists, hydroxyadenine agonists, pteridone agonists, aminopyrimidine agonists, benzazepine agonists and thioxoguanosine agonists, and the toll-like receptor 7 or 8 agonist is a conjugate to which a cleavable linker that can be cleaved at the active site of the toll-like receptor 7 or 8 agonist is bound, and after administration of the composition for the mRNA vaccine, after the process of transcription of mRNA into protein is carried out, the cleavable linker bound to the active site of the toll-like receptor 7 or 8 agonist is sequentially cleaved, and the activation function of the toll-like receptor 7 or 8 agonist is induced. A composition for an mRNA vaccine.

2. The kinetic control is such that a cleavable linker to which a cleavable linker that can be cleaved at the active site of the toll-like receptor 7 or 8 agonist is bound and maintains an inactive state, and after the transcription of mRNA starts, the cleavable linker that blocks the active site within 2 to 12 hours is cleaved, and the activity of the immunostimulatory substance appears with a time delay. The composition for an mRNA vaccine according to claim 1.

3. The cleavable linker according to claim 1, characterized in that it contains any one or more bonds selected from the group consisting of disulfide, carbamate, hydrazine, ester, peptide, azide, amide, hydrazone, thioether, phosphodiester, thioketal and combinations thereof.

4. The composition for mRNA vaccine according to claim 1, wherein the cleavable linker further comprises ethylene oxide or ethylene glycol at both ends or one end thereof.

5. The composition for mRNA vaccine according to claim 1, wherein the chemical bond at the binding site of the cleavable linker is cleaved by any one or more factors selected from the group consisting of enzymes, pH, redox potential, temperature, ultrasonic waves, magnetism, and light sources.

6. The composition for mRNA vaccine according to claim 1, wherein any one or more substances selected from the group consisting of cholesterol, lipids, proteins, amino acids, peptides, and oligonucleotides are bound to the ends of the cleavable linker.

7. The composition for mRNA vaccine according to claim 1, wherein the mRNA antigen and the kinetically controllable toll-like receptor 7 or 8 agonist are loaded into any one or more drug delivery carriers selected from the group consisting of nanoliposomes, nanoemulsions, nanomicelles, hydrogels, scaffolds, solid nanoparticles, and polymeric nanoparticles.

8. The composition for mRNA vaccine according to claim 7, wherein after the mRNA antigen loaded inside is first delivered to the cytosol, the kinetically acting toll-like receptor 7 or 8 agonist interacts with the receptor on the cell surface, endosome, or lysosome.

9. The composition for mRNA vaccine according to claim 7, wherein the drug delivery carrier further comprises any one or more immunostimulatory substances selected from the group consisting of toll-like receptor agonists, saponins, antiviral peptides, inflammasome inducers, NOD ligands, CDS ligands, STING ligands, outer wall components of pathogenic bacteria, alum, lipids, combinations thereof, and derivatives thereof.

10. The composition for the mRNA vaccine is used for the prevention or treatment of any one or more diseases selected from the group consisting of infectious disease, cancer, metabolic syndrome, autoimmune disease, and rare disease, and is characterized in that it is used for the prevention or treatment of any one or more diseases selected from the group consisting of infectious disease, cancer, metabolic syndrome, autoimmune disease, and rare disease. The composition for the mRNA vaccine according to claim 1.

11. Use of the composition according to claim 1 for producing an agent for increasing the immune response of the mRNA antigen in the composition according to claim 1.

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