mRNA vaccine comprising adjuvant capable of kinetic control

Abstract

To provide an mRNA vaccine composition capable of simultaneously enhancing the immunogenicity of an antigen through immune activation while increasing the expression amount of the antigen.SOLUTION: The present invention relates to an mRNA vaccine comprising an adjuvant whose immune activation function is kinetically controlled, and more particularly, to an mRNA vaccine comprising an adjuvant characterized in that the activation function of the adjuvant sequentially acts after mRNA is transcribed into proteins. The invention relates to a core technology for optimizing a time interval between expression of an mRNA antigen and a time point of immune activation in order to effectively regulate expression of an antigen and immunogenicity of an antigen, which are contradictory to each other. The invention provides a core technology of significantly increasing efficacy of the mRNA vaccine by simultaneously increasing an expression level of the antigen and the immunogenicity by dynamically controlling an action of an immunostimulatory substance in order to optimize the antigen expression level of mRNA and an action time of the adjuvant.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention provides an mRNA vaccine containing an adjuvant whose immune activation function is dynamically controlled. More specifically, after the mRNA is transcribed into a protein, the adjuvant is sequentially administered. This relates to an mRNA vaccine that is characterized by its activation function. [Background technology]

[0002] mRNA vaccines use mRNA as an antigen to treat cancer, infectious diseases, and autoimmune diseases. It is a medicine used for the prevention and treatment of autoimmune diseases. Compared with the A vaccine, it has the advantage of being stable and easy to mass-produce, and is expected to be a promising vaccine for the future. It is widely used as a cancer vaccine and infectious disease vaccine platform in pandemic situations. It is expected to be used in the future (Korean Patent Publication No. 10-2020-0118386). , Toll-like receptor agonist (tol l-like receptor agonist (TLR agonist) Conversely, the immune stimulating properties of such adjuvants are similar to those of mR It interferes with the translation signal transduction pathway of NA, thus preventing its pharmacological activity. It is known that the expression of sufficient amounts of mRNA antigen to show the effect of the antibody is inhibited. To solve these problems, we have developed modified mRNA with reduced or eliminated immunogenicity. There are active ongoing efforts to use it to produce a vaccine. In some cases, the efficacy of mRNA is poor due to its low immunogenicity. have limited ability to induce effective humoral and / or cellular immune responses. The mechanism of antigen expression and immunogenicity of antigens, which act in opposition to each other, By effectively regulating the mechanism that shows immunogenicity, it shows the best effect. Therefore, the development of a novel mRNA vaccine system that can achieve this is of great importance.

[0003] On the other hand, immune responses are caused by activated immune cells reacting to foreign and endogenous substances, i.e., antigens. It is a series of reactions that occur in response to microorganisms, including bacteria and viruses, and foreign substances in the body. When it enters the body, immune cells recognize it, become activated, and divide factors such as cytokines. Recently, the innate immune response stage that acts nonspecifically in the early stages of infection has been Research into the mechanism of inflammation is currently being actively conducted, and among these, Toll-like receptors are thought to play a key role in the early stages of inflammation. It is a receptor that can recognize pathogens, recognizing the plasma membrane components and nucleic acid components of the pathogens. It is known to induce an immune response, and this can be used to activate immune cells. Research into various Toll-like receptor ligands is being actively conducted.

[0004] Toll-like receptor agonists are agonists of Toll-like receptors in endosomes, It is known that it effectively induces not only humoral immunity but also cellular immunity. However, such multifunctional Toll-like receptor agonists are insoluble in aqueous solution due to their molecular structure. It is difficult to disperse in special organic solvents such as DMSO and methanol. It is soluble in most commonly used organic solvents and is therefore suitable for a wide range of immunologically active formulations. Therefore, it is difficult to produce a clear solution containing a mixture of various surfactants. Some are commercially available in cream-based formulations (e.g., Aldara cream). In order to overcome these problems, the research team produced the salt and dissolved it in water. Toll-like receptor agonists were prepared in salt form so that they could be dissolved in water. The drug is absorbed into the bloodstream in the body and induces an immune response in the bloodstream (systemic immune By inducing a cytokine response, many side effects (e.g., cytokine stress) can be prevented. (cytokine storm, various non-specific hypersensitivity immune responses, etc.) Therefore, it is not easy to use. For qualitative therapeutic use, a concentration less than the effective dose is required. Some pharmaceutical companies have been trying to reduce the efficacy of drugs by treating them with excessive amounts of alcohol. To overcome these problems, lipids that exhibit lipophilic properties or large-sized lipids have been introduced. By chemically bonding the drug directly to the polymer chains that support it, it is prevented from being absorbed directly into the bloodstream. However, the toll-like particles produced by such methods have Receptor agonists are non-specifically activated in the body because their active sites are still exposed to the outside. However, it still has the potential to induce toxicity by inducing a targeted immune response.

[0005] Therefore, Toll-like receptor agonists and mRNAs with kinetically controlled activation times If an mRNA vaccine containing the mRNA is developed, the immunogenic effect of the mRNA antigen will be suppressed in the early stages after administration. Instead, the expression of mRNA antigen is normally induced, followed by sequential activation. Dynamically controlled activation time of Toll-like receptor agonists is converted to the activated state As a result, immune activation of mRNA is induced, and the time interval of the mechanism of action (tim e interval), which will have a significant impact on the next-generation mRNA vaccine market. It is expected that this will have a significant ripple effect. Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention has been made to solve the problems in the prior art as described above, A drug containing an adjuvant with a kinetically controlled activation time and mRNA as an active ingredient. A composition for an RNA vaccine, in which mRNA is transported to the cytosol and transcribed, and then Adjuvants bind to sites of immune activation within endosomes / lysosomes and the cytosol. By inducing immune activation, the expression of antigens is increased while suppressing immune activation-mediated To provide an mRNA vaccine composition that can simultaneously enhance the immunogenicity of an antigen, etc. The purpose is to:

[0007] However, the technical problem that the present invention aims to achieve is not limited to the problems mentioned above. Further problems not mentioned above and not mentioned can be solved by the following description, which is a conventional method in the art. Engineers can understand it clearly. [Means for solving the problem]

[0008] The present invention relates to mRNA antigens and kinetically controlled The composition is an mRNA vaccine composition containing an immune activator capable of The immunoactivator is attached to the active site of the immunoactivator. A conjugate having a cleavable linker attached thereto. The present invention provides a composition for an mRNA vaccine. The mRNA antigen is used in an mRNA vaccine. There are no limitations as long as the antigen is an mRNA antigen known to be capable of being expressed.

[0009] In one embodiment of the present invention, the composition for an mRNA vaccine is capable of activating the mRNA after administration. After being transcribed into proteins, they bind to the activation sites of immune activators. The cleavable linker is cleaved to induce the activation function of the immunoactivator. It is characterized by:

[0010] In another embodiment of the invention, the dynamic regulation is achieved by switching to the activation site of the immunoactivator. A cleavable linker is attached to maintain the inactive state, and m After the start of RNA transcription, preferably within 2 to 12 hours, more preferably within 3 to 9 hours. Within hours, the cleavable linker blocking the activation site is cleaved, releasing the immunoactivation site. The activity of the activating substance appears with a time delay. , molecular scale and / or macroscale It can work on an acro scale.

[0011] In yet another embodiment of the present invention, the cleavable linker is preferably a disulfide linker. disulfide, carbamate, hydrazine ydrazine), ester, peptide, azide azide, amide, hydrazone, thioether Thioether, phosphodiester, thio Any one selected from the group consisting of thioketals and combinations thereof. However, it is characterized by the fact that it contains one or more bonds. enzymes, redox potential, GSH, pH, etc.) and / or exogenous factors (redox, pH, Temperature, photo / light, magnetism, ultrasound, electrical response The bond can be broken by a compound such as carboxyl responsive. Not determined.

[0012] In yet another embodiment of the invention, the cleavable linker is at both ends or at one end. One end is ethylene oxide or ethylene glycol. By further incorporating alkyl derivatives such as ethylene glycol , which is characterized by increasing the solubility and flexibility of the conjugate in aqueous solution.

[0013] In yet another embodiment of the invention, the cleavable linker is cleavable by enzymes, pH, oxidation Redox potential, temperature, ultrasound, magnetism, and light source The chemical bond at the binding site is broken by one or more factors selected from the group It is characterized by:

[0014] In yet another embodiment of the invention, the cleavable linker has a terminus containing a cholesterol from the group consisting of: lipids, proteins, amino acids, peptides and oligonucleotides The present invention is characterized in that one or more selected substances are bound to the toll-like It acts to block the active part of the receptor agonist and contains hydrophilic or lipophilic groups. The ion exchange material may be a variety of substances having the ion exchange material.

[0015] In yet another embodiment of the invention, the immunoactivator is preferably a Toll-like receptor agonist. Even if it is a toll-like receptor agonist More preferably, 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 agonist, a toll-like receptor 8 agonist, a toll-like receptor 9 agonist, a toll-like receptor 10 agonist, a toll Toll-like receptor 3 agonist, Toll-like receptor 4 agonist, Toll-like receptor 5 agonist , Toll-like receptor 6 agonists, Toll-like receptor 7 or 8 agonists, and Toll-like The present invention is characterized in that the agonist is one or more selected from the group consisting of receptor 9 agonists.

[0016] In yet another embodiment of the present invention, the mRNA antigen and a dynamically controllable immunoactivator are The functionalizing substances are nanoliposomes, nanoemulsions, nanomicelles, hydrogels, scaffolds, etc. any one or more selected from the group consisting of gold, solid nanoparticles and polymeric nanoparticles The drug delivery system is characterized in that it is loaded with a drug carrier. The loading is related to binding. Regardless, it may simply be encapsulated or sandwiched between nanoparticle structures. The mRNA antigen of the present invention may be in the form of a stimulant or in the form of a conjugate. As long as the form contains a substance, it is not limited to this.

[0017] In yet another embodiment of the present invention, the drug delivery vehicle is an internally loaded After initial delivery of the RNA antigen into the cytosol, kinetically acting immune activators Interaction with receptors on the cell surface, in endosomes, or in lysosomes The present invention is characterized in that:

[0018] In yet another embodiment of the present invention, the drug delivery vehicle preferably comprises a Toll-like receptor agonist. Inflammasome agonists, saponins, antiviral peptides, inflammasome inducers flammasome inducer, NOD ligand, CDS ligand (cytosolic DNA sensor ligand), STI NG (stimulator of interferon genes) ligand, disease The outer wall components of the bacteria, alum, lipids, and combinations of these and derivatives thereof. The immunoactivator may further comprise, as an adjuvant, There are no limitations as to the immunostimulant used.

[0019] In yet another embodiment of the present invention, the mRNA vaccine composition is infectious disease), cancer, metabolic syndrome abolic syndrome), autoimmune disease (autoimmune disease) se) and rare disease The present invention is characterized in that it is used for the prevention or treatment of one or more diseases.

[0020] The present invention also relates to mRNA antigens and kinetically controlled The present invention provides a composition for an mRNA vaccine that contains an immune activator capable of immunization as an active ingredient. an infectious disease, comprising administering to an individual cancer, metabolic syndrome, autoimmunity Autoimmune disease or rare disease The present invention provides a method for preventing or treating

[0021] The present invention also relates to mRNA antigens and kinetically controlled A composition for an mRNA vaccine containing an immune activator capable of immunization as an active ingredient Infectious diseases, cancer, metabolic diseases metabolic syndrome, autoimmune disease For the prevention or treatment of rare diseases to provide.

[0022] The present invention also relates to mRNA antigens and kinetically controlled The present invention relates to a composition containing an immune activator capable of inhibiting infectious diseases (INF) as an active ingredient. ectious disease, cancer, metabolic syndrome lic syndrome), autoimmune disease or produce drugs used to prevent or treat rare diseases This provides a use for Effect of the Invention

[0023] The composition for an mRNA vaccine according to the present invention is a composition for an mRNA transported to the cytosol and transcribed. After that, they bind to immune activation sites in endosomes / lysosomes and the cytosol, and then By inducing activation, the expression level of antigens is increased while the antigens are suppressed through immune activation. As a technology that can simultaneously enhance the immunogenicity of various mRNA vaccines, / or can significantly improve therapeutic efficacy, so it can be applied to various mRNA vaccines It is expected that this will be possible. [Brief description of the drawings]

[0024] [Figure 1] Figure 1 is a conceptual diagram of a new idea that mRNA antigens can simultaneously improve antigen expression and immunogenicity of mRNA vaccines by sequentially interacting with immune activation sites in endosomes / lysosomes or cytosol after migration and translation of mRNA antigens into the cytosol at the immature antigen-presenting cell stage. [Diagram 2] FIG. 2 shows the differentiated strategy of the present invention for developing an mRNA vaccine with optimal properties by effectively controlling the expression of opposing antigens and the immunogenicity of the antigens, and illustrates the importance of optimizing the time interval between the expression of the mRNA antigen and the time point of immune activation for this purpose. [Diagram 3] FIG. 3 shows that the critical moiety of a Toll-like receptor agonist, i.e., the active site, can be temporarily inactivated by binding a substance having a hydrophilic or lipophilic group to the active site to block it, and the binding site can be linked with a separable linker to control the dynamic behavior of the toll-like receptor agonist-based adjuvant for controlling dynamic immune function in an mRNA vaccine. [Figure 4] FIG. 4 is a diagram showing the concept of dynamic control by drug delivery vehicles, which are designed so that the mRNA antigen is first delivered to the cytosol, and then the immune activator is sequentially released into the endosomes / lysosomes and cytosol; the drug delivery vehicles can be manufactured using nanoliposomes, nanoemulsions, nanomicelles, hydrogels, scaffolds, solid nanoparticles, polymeric nanoparticles, etc. [Diagram 5] FIG. 5 is a diagram showing the results of confirming the degree of antigen presentation when BMDCs according to one embodiment of the present invention are treated with mRNA and R848. [Figure 6]FIG. 6 is a diagram showing the results of confirming the degree of cell activation by measuring cell surface molecules when BMDCs according to one embodiment of the present invention are treated with mRNA and R848. [Figure 7] FIG. 7 is a diagram showing the results of confirming the secretion of inflammatory cytokines when BMDCs according to one embodiment of the present invention are treated with mRNA and R848. [Figure 8] FIG. 8 is a diagram showing the results of confirming the degree of antigen presentation when BMDCs according to one embodiment of the present invention are treated with mRNA and LPS. [Figure 9] FIG. 9 is a diagram showing the results of confirming the degree of antigen presentation when BMDCs according to one embodiment of the present invention are treated with mRNA and Poly I:C. [Figure 10] FIG. 10 is a diagram showing the results of confirming secretion of type 1 interferon, a cytokine, when BMDCs according to one embodiment of the present invention are treated with mRNA and Poly I:C. [Figure 11] FIG. 11 is a diagram showing the results of confirming the degree of activation when mRNA and Poly I:C were treated in a co-culture of T cells and BMDCs according to one embodiment of the present invention, in terms of the amount of IFN-γ+ T cells, the amount of secreted IFN-γ, and the amount of IL-2. [Figure 12] FIG. 12 is a diagram showing the results of confirming the level of antigen presentation and secretion of type 1 interferon, a cytokine, when BMDCs according to one embodiment of the present invention were treated with mRNA, Poly I:C, and R848. [Figure 13] FIG. 13 is a diagram showing the results of confirming the degree of activation when co-cultured T cells and BMDCs according to one embodiment of the present invention were treated with mRNA, Poly I:C, and R848, in terms of the amount of IFN-γ+ T cells, the amount of secreted IFN-γ, and the amount of IL-2. [Figure 14] FIG. 14 is a diagram showing the results of confirming the degree of antigen presentation and the degree of cell activation when BMDC according to one embodiment of the present invention is treated with mRNA, LPS, and R848. [Figure 15]FIG. 15 is a diagram showing the results of confirming the degree of Th1 differentiation when BMDCs according to one embodiment of the present invention were treated with mRNA, LPS, and R848. [Figure 16] FIG. 16 is a diagram showing the results of confirming the correlation between mRNA and type 1 IFN when BMDCs according to one embodiment of the present invention were treated with eGFP-modified mRNA, LPS, and R848. [Figure 17] FIG. 17 shows the results of confirming the level of antigen presentation when pDC according to one embodiment of the present invention was treated with mRNA and R848, or with mRNA and SKKU-078-liposome. [Figure 18] FIG. 18 is a diagram showing the results of confirming the degree of cell activation when pDC according to one embodiment of the present invention is treated with mRNA, or with mRNA and SKKU-078-liposome. [Figure 19] FIG. 19 shows the results of confirming the phenotype of T cells when mRNA and SKKU-078-liposome were treated in a co-culture of T cells and pDC according to one embodiment of the present invention. [Figure 20] FIG. 20 shows the results of confirming the dual phenotype of T cells and the amount of IFN-γ cytokine secreted when mRNA and SKKU-078-liposome were treated in a co-culture of T cells and pDC according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] [Best Mode for Carrying Out the Invention] The present inventors have optimized the time of expression of the mRNA antigen and the time of immune activation of the adjuvant. We have developed a system that can significantly improve the efficacy of mRNA vaccines through a combined administration method. As a result of intensive research, we found that a cleavable linker was attached to the activation site of an adjuvant, which is an immunoactivating substance. The activity of the drug is temporarily inhibited by linking it to an anchor, and after administration to the body, the drug is activated at the target site. After reaching the tissue or cell, the mRNA antigen begins to be transcribed into protein, and The kinetic control of the time required for the cleavable linker to be cleaved and the adjuvant to become active is achieved. The expression of mRNA antigens can be enhanced by using potential immunoactivators together with the mRNA antigens. For mRNA vaccines with significantly increased efficacy by optimizing the time interval between the time of administration and the time of immune activation The composition was invented.

[0026] As shown in FIG. 1, the mRNA vaccine of the present invention is characterized in that the mRNA antigen is delivered to the cytosol (c ytosol) and are transcribed into endosomes / lysosomes or the cytosol. These systems are systems that interact with immune activation sites within the naked body. The fundamental limitation of naked mRNA is that it cannot be fully synthesized after administration to the body. The immune activation effect that induces stimulation of the leukocyte-like receptor is activated, and the transcription signal of mRNA antigen is generated. The delivery system is disrupted, preventing expression of sufficient amounts of mRNA antigen to act as a vaccine. This system solves the problem of the efficacy of the drug being weakened due to damage to the skin (Figure 2). In order to solve these problems, attempts have been made to use modified mRNA with reduced immunogenicity. However, it was concluded that the low immunogenicity of the antibody was responsible for the normal humoral immunity and / or cellular immunity. However, this product still had the problem of not inducing a cytotoxic immune response and thus weakening its efficacy. In order to solve these problems, the present inventors have Toll-like receptor agonists can be transiently activated by attaching a cleavable linker to the Exists in an inactivated state and is subsequently cleaved within the endosomal / lysosomal or cytosolic environment The linker is cleaved and the activity is restored by binding to the receptor. This is a new concept of mRNA that optimizes the time interval between mRNA antigen expression and immune activation. We provided a vaccine system (Figures 2 and 3).

[0027] As used herein, "mRNA vaccine" refers to a vaccine that A general term for vaccines that use messenger ribonucleic acid (mRNA) containing information as an antigen. When you receive the vaccine, the mRNA in your body produces the protein, which your immune system then uses to It works as a principle to detect the virus, trigger an immune response, and generate neutralizing antibodies. It can be applied to a wide range of diseases, including cancer, autoimmune diseases, metabolic syndrome, and rare diseases, in addition to the above. This tends to be the case.

[0028] As used herein, an "immune modulator" refers to a The term "immune system" refers to any substance that activates, induces, or restores normal immune function. The term "adjuvant" refers to a substance that can be used as an adjuvant. A bunt is a substance used in conjunction with an antigen to enhance the immune response. When used together, they increase antibody production and stimulate humoral and / or cellular immunity. The immune activator can preferably increase the Toll-like receptor Agonist (toll-like receptor agonist), saponin, anti Viral peptides, inflammasome inducers inducer), NOD ligand, CDS ligand olic DNA sensor ligand)、STING(stimulator of interferon genes ligand, emulsion n), alum, incomplete Freund's adjuvant, Freund's adjuvant, or a combination thereof, more preferably a Toll-like receptor agonist. It may also include

[0029] As used herein, a "toll-like receptor agonist" "Toll-like receptor agonist" is a membrane protein involved in innate immunity. Ligands that act directly or indirectly on the body and are produced by endogenous or exogenous ligands. May refer to a component capable of initiating a signaling response via a signaling pathway. As used herein, a toll-like receptor agonist refers to a natural toll-like receptor agonist or may be a synthetic Toll-like receptor agonist, Toll-like receptor 1 agonist, Toll Toll-like receptor 2 agonist, Toll-like receptor 3 agonist, Toll-like receptor 4 agonist, Toll-like receptor 5 agonist, Toll-like receptor 6 agonist, Toll-like receptor 7 or 8 agonist agonists, Toll-like receptor 9 agonists, and the like.

[0030] The Toll-like receptor 1 agonist can induce signaling responses via TLR-1. Examples of ligands include triacylated lipid peptides (LPs); phenol-soluble Sex modulin; Mycobacterium tuberculosis Lipid peptides from Actinium tuberculosis; S-(2,3-bis( Palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys -(S)-Ser-(S)-Lys(4)-OH; Borrelia burgdorferi (Bor Lipid peptides of Relia burgdorferi; Acetyl group of OspA lipid peptide and trihydrochloride (Pam3Cys) lipid peptides that mimic the substituted amino termini. However, the present invention is not limited to this.

[0031] The Toll-like receptor 2 agonist can induce signaling responses via TLR-2. Examples of ligands include peptidoglycan, Zymosan, HSP70, HMGB1, HA, bam3Cys-Li p, but is not limited thereto.

[0032] The Toll-like receptor 3 agonist can induce signaling responses via TLR-3. The term refers to a ligand that is capable of binding to a ligand of a specific ligand. For example, the poly IC series includes Poly(I:C) and Poly(I CLC), Poly(IC12U), ampligen, etc. may be used, but are not limited to these. Not determined.

[0033] The Toll-like receptor 4 agonist can induce a signal transduction response via TLR-4. As an example, Shigella flexneri neri outer membrane protein products, AGP, CRX-527, MPLA, PHAD, 3 It may be, but is not limited to, D-PHAD, GLA, LPS, etc.

[0034] The Toll-like receptor 5 agonist can induce signaling responses through TLR-5. It means a ligand, and an example thereof may be flagellin. However, the present invention is not limited to this.

[0035] The Toll-like receptor 6 agonist can induce signaling responses via TLR-6. An example of a ligand that can be used is diacyl lipopeptide. ptide, lipoteichoic acid, etc. However, the present invention is not limited to this.

[0036] The Toll-like receptor 7 or 8 agonist inhibits signaling via TLR-7 or 8. The term "ligand" refers to a ligand capable of inducing a targeting response, and examples thereof include imidazoquinoline agonists (iQAs). midazoquinoloine-based agonist), hydroxyadenine 8-hydroxyadenine-based agonist, Pteridone-based agonist, amino Pyrimidine agonists (2-aminopyrimidine-based agonists IST, benzoazepine-based agonists onist, thiaoxoguanosine agonist (7-thia-8-oxoguan osine-based agonist), etc., and the imidazoquinoline The compounds are described in WO2018 196823, WO2011 049677, and WO2011 027022, WO2017 102652, WO2019 040491, etc. The present invention includes, but is not limited to, compounds of the above-mentioned type or pharma- ceutically acceptable salts. The hydroxyadenine compounds are disclosed in WO2012 080730 and WO2013 068 438, WO2019 036023, WO2019 035969, WO2019 0 35970, WO2019 035971, WO2019 035968, CN 108 948016, US 2014 8846697, WO2016 023511, WO2 017 133683, WO2017 133686, WO2017 133684, W O2017 133687, WO2017 076346, WO2018 210298 , WO2018 095426, WO2018 068593, WO2018 0781 49, WO2018 041763, etc., or a pharma- ceutical acceptable salt thereof The pteridone compounds include, but are not limited to, the salts thereof. 43301, WO2016 007765, WO2016 044182, WO2017 035230, WO2017 219931, WO2011 057148, CN 1 087 94486, or a pharma- ceutically acceptable salt thereof, The aminopyrimidine compounds are not limited thereto. WO2012066335, WO2012 066336, WO2012 067268 , WO2013 172479, WO2012 136834, WO2014 0535 16, WO2014 053595, US 2018 0215720, WO2012 156498, WO2014 076221, WO2016 141092, WO201 8 045144, WO2015 014815, WO2018 233648, WO2 014 207082, WO2014 056593, WO2018 002319, W O2013 117615, etc., or a pharma- ceutical acceptable salt thereof. The benzoazepine compounds include, but are not limited to, those described in WO2007 02461 2, WO2010 014913, WO2010 054215, WO2011 022 508, WO2011 022509, WO2012 097177, WO2012 0 97173, WO2016 096778, WO2016 142250, WO2017 202704, WO2017 202703, WO2017 216054, WO20 Compounds of the type described in WO2017 046112, WO2017 197624, etc. The thiaoxoguanosine compound includes, but is not limited to, a pharma- ceutically acceptable salt thereof. WO2016 180691, WO2016 055553, WO2016 180 743, WO2016 091698, etc., or pharma- ceutical acceptable salts thereof. In addition, the present invention includes, but is not limited to, salts that can be used for the synthesis of compounds of the present invention. 3, PCT / US2015 / 028264, PCT / US2016 / 020499, WO 2015 023598, PCT / US 2015 / 039776, etc. The compound may comprise a steroid-like receptor 7 or 8 compound or a pharma- ceutical acceptable salt. Imiquimod, Resiquimod, Dactolisib ctolisib, Gardiquimod, Sumanirole manirole, motolimod, vesato limod), loxoribine, SM360320, CL264 , 3M-003, IMDQ, Compound 54, etc., but are not limited to these. Toll-like receptor 7 or 8 is not known and can be easily guessed and used by those skilled in the art. This includes all cases where the user is a non-nist.

[0037] The Toll-like receptor 9 agonist can induce signaling responses via TLR-9. The term "ligand" refers to a ligand that binds to a target molecule, and may be, for example, an immunostimulatory oligonucleotide, The immunostimulatory oligonucleotide may contain one or more CpG motifs. Not limited to:

[0038] As used herein, the term "saponin" refers to an amphiphilic glycoside that is Acts as a surfactant. Examples include QS21, Quil A, QS7, QS17, β -escin, digitonin, etc., but are not limited to these.

[0039] As used herein, the term "antiviral peptide" refers to a peptide that is " is a general term for peptides that exhibit antiviral effects. One example is KLK. It may be, but is not limited to, the above.

[0040] As used herein, the term "inflammasome inducer" refers to a "Danger inducer" is a protein complex that recognizes and activates danger signals in the cytoplasm of eukaryotic cells. Inflammasome is a collective term for substances that induce the inflammasome complex. For example, TDB (trehalose-6,6-dibehenate) may be used, but is not limited to this.

[0041] As used herein, the term "NOD ligand" refers to a Nod-ligand. A general term for ligands that activate the ke receptor. Examples include M-TriLYS and N-glycosyltransferase. Cosylated muramyldipeptide id), but is not limited to this.

[0042] As used herein, the term "CDS ligand (cytosolic DNA sensor "cGAS ligand" is a collective term for ligands that activate the DNA sensor cGAS. An example of the polynomial may be Poly(dA:dT), but is not limited to this.

[0043] As used herein, the term "STING ligand (stimulator of interferon)" refers to a The feron genes ligand (FRI) is a molecule that immune cells use to detect cancer. The term is a collective term for ligands that activate STING, a sensor that activates the immune system. One example is cGAMP. , di-AMP, di-GMP, etc., but are not limited thereto.

[0044] As used herein, "cholesterol" refers to a lipid pid) is a general term for steroid-based organic substances with hydrophobic properties, Sterols are a variety of analogues based on the cholesterol structure, some of which are The compound may include any compound that can be obtained by chemically changing it. Preferably, bile acid (cholesterol) acid, deoxycholic acid, lithocholic acid, chenodeoxycholic acid), vitamin D, steroids Hormones (testosterone, estradiol, cortisol, aldosterone, prednisolone) The above-mentioned co-administrations may include, but are not limited to, the following co-administrations: Lesterol binds toll-like receptor 7 or 8 agonists to the surface and / or structure of nanoparticles with various morphologies. and lipid substances that perform similar functions, such as , natural lipids such as phospholipids, synthetic lipids It can also be substituted by other substances such as toll-like receptor 7 or 8 agonists. It binds to the activation site of toll receptors, rendering them inactive, and also binds toll-like receptors 7 and 8. The purpose of this is to prevent the stimulant from being absorbed into the bloodstream in the body, so any known type of lipid can be used. If so, there are no restrictions.

[0045] As used herein, the term "cleavable linker" refers to a They contain cleavable bonds and are involved in the tumor microenvironment, intracellular endosomes and lysosomes. The physiological environment of the body, such as low pH, enzymes, glutathione, etc.; or external stimuli That is, cutting is induced by specific stimuli such as temperature, redox potential, ultrasound, magnetic field, and near-infrared light. The term "linker" generally refers to linkers in which Disulfides, esters, peptides e) a linker containing an azide bond or a cleavable bond The shape is not limited to this.

[0046] As used herein, "prevention" refers to the administration of a composition according to the present invention. By administering the drug, it is possible to suppress infectious diseases, cancer, metabolic syndrome, autoimmune diseases, rare diseases, etc. This refers to any action that increases or delays the onset of the disease.

[0047] As used herein, "treatment" refers to the administration of a composition according to the present invention. has improved symptoms of infectious diseases, cancer, metabolic syndrome, autoimmune diseases, and rare diseases. "Constitution" means any act which beneficially alters the

[0048] As used herein, "individual" or "subject" refers to a This refers to a subject to which the compositions of the invention can be administered, and there is no limitation to the subject.

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

[0050] As used herein, "cancer" refers to a cancer that develops locally through invasion and metastasis. It is a general term for various blood cancers, malignant solid tumors, etc. that can be expanded more systematically. Specific examples of cancer include colon cancer, adrenal cancer, bone cancer, brain cancer, breast cancer, Bronchial cancer, colon and / or rectal cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, Kidney cancer, laryngeal cancer, liver cancer, lung cancer, nervous tissue cancer, pancreatic cancer, prostate cancer, parathyroid cancer Other examples of cancer include adenocarcinoma, Adenoma, basal cell carcinoma, cervical dysplasia and intraepithelial carcinoma, Ewing sarcoma , squamous cell carcinoma, esophageal cell carcinoma, malignant brain tumor, esophageal cell carcinoma, intestinal ganglioneuroma, hyperplasia Corneal nerve cancer, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, leukemia, lymphoma , malignant carcinoma, malignant melanoma, malignant hypercalcemia, Marfan habitus id habitus cancer, medullary carcinoma, metastatic skin cancer, mucosal neuroma, myelodysplastic syndrome group, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, osteogenic and other sarcomas, ovarian cancer, Pheochromocytoma, polycythemia vera, primary brain tumors, small cell lung cancer, ulcerative and Papillary squamous cell carcinoma, seminoma, soft tissue sarcoma, retinoblastoma, renal cell tumor or renal cell carcinoma ( renal cell carcinoma (RCC), reticulum cell sarcoma, and Wilms' disease Also included are astrocytomas and gastrointestinal stromal tumors. al stromal tumor, GIST), glioma or glioblastoma, hepatocellular carcinoma Hepatocellular carcinoma (HCC), pancreatic endocrine cancer, etc. etc. are included.

[0051] As used herein, the term "metabolic syndrome" refers to 5 factors that increase your risk of heart disease, diabetes, stroke and other health problems (High blood pressure, high blood sugar, hypertriglyceridemia, low high-density lipoprotein cholesterol, Obesity is defined as a condition in which an individual has three or more of the following: Metabolic diseases such as obesity, diabetes, hypertension, hyperlipidemia, heart disease, and gout Metabolic syndrome includes all diseases caused by metabolic syndrome.

[0052] As used herein, the term "autoimmune disease" refers to Systemic lupus erythematosus is a general term for diseases caused by a pathological reaction to self-antigens. lupus erythematosus (SLE), rheumatoid arthritis oid arthritis;RA), multiple sclerosis Systemic autoimmune diseases such as MS, insulin-dependent diabetes mellitus (IDD), IDDM, Graves Grave's disease, allergy, etc. do.

[0053] As used herein, a "rare disease" refers to a disease that affects a small proportion of the population. A general term for all diseases that affect only a small proportion of the population, are generally hereditary, and have The incidence and prevalence is very low, meaning diagnosis is difficult and there is no suitable treatment. The definition varies slightly from country to country, but in Korea, the definition of "rare disease" is based on Article 2 of the Rare Disease Management Act. A disease is one in which the prevalence is less than 20,000, or the prevalence is unknown because it is difficult to diagnose. It is defined as a disease that is determined according to the procedures and standards set by the Ministry of Health and Welfare. The World Health Organization (WHO) states that a disease is classified as HIV-positive if its prevalence is approximately 0.65 to 1 per 1,000 people. Countries with less than 200,000 total cases, and the EU per 10,000 people It specifies cases involving five or fewer people and rare diseases.

[0054] As used herein, the term "pharmaceutical composition" refers to a tion" or "vaccine composition" is in the form of a capsule, tablet, granule, injection, ointment, powder or drink. The pharmaceutical composition or vaccine composition is characterized in that it is intended for humans. The pharmaceutical or vaccine composition may include, but is not limited to, It is usually administered in the form of powder, granules, capsules, tablets, aqueous suspensions, and other oral dosage forms, external dosage forms, etc. The pharmaceutical composition of the present invention may be used in the form of a pharmaceutical preparation, a suppository, or a sterile injection solution. The product or vaccine composition may include a pharma- ceutically acceptable carrier. The carriers used for oral administration include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, Stabilizers, suspending agents, dyes, flavorings, etc. may be used. In the case of injections, buffers It can be used by mixing with preservatives, soothing agents, solubilizers, isotonicity agents, stabilizers, etc. In the case of topical administration, bases, excipients, lubricants, preservatives, etc. may be used. The pharmaceutical composition or vaccine composition of the present invention may be in the form of a pharma- ceutically acceptable drug, as described above. For example, for oral administration, it can be prepared in tablets, tro capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, it can be manufactured in a unit dose ampoule or a multiple dose form. In addition, it can be manufactured in the form of a solution, suspension, tablet, capsule, sustained release formulation, etc. It can be formulated.

[0055] On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, and the like. Lose, sucrose, sorbitol, mannitol, xylitol, erythritol, malate Lutitol, starch, gum acacia, alginate, gelatin, calcium phosphate , calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl chloride Nylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc , magnesium stearate or mineral oil may also be used. They may further contain a flocculant, a lubricant, a moisturizer, a flavoring, an emulsifier, a preservative, and the like.

[0056] The administration route of the pharmaceutical composition or vaccine composition according to the present invention is not limited to these. However, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, This includes intraperitoneal, intranasal, intestinal, topical, sublingual or rectal administration. Oral or parenteral administration is preferred. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, Includes intra-articular, intrasynovial, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical or vaccine compositions of the invention may also be administered in the form of suppositories for rectal administration. It can be given.

[0057] The pharmaceutical or vaccine compositions of the present invention may be used in accordance with the activity, age, and other characteristics of the particular compound used. Body weight, general health, sex, diet, time of administration, route of administration, excretion rate, drug combination and prophylaxis The dosage may vary depending on a variety of factors, including the severity of the particular disease being treated. The dosage of the pharmaceutical or vaccine composition depends on the patient's condition, weight, degree of illness, drug form, etc. Although it depends on the form, route of administration and duration, those skilled in the art can appropriately select it. May be administered at 0.0001-500mg / kg or 0.001-500mg / kg The dosage can be once a day or divided into several doses per day. The dosage amounts mentioned above are not intended to limit the scope of the present invention in any way. The pharmaceutical or vaccine composition may be in the form of a pill, dragee, capsule, liquid, gel, syrup, spatula, or the like. It can be formulated into a slurry or suspension.

[0058] In addition, the vaccine composition according to the present invention may further contain a conventionally known "immune antigen adjuvant". The immune antigen adjuvant generally refers to any substance that increases humoral and / or cellular immune responses to an antigen, and any substance known in the art may be used without limitation. For example, Freund's complete or incomplete adjuvant may be further included to increase the immunity. In addition, in the case of the vaccine composition, repeated antigen stimulation may be optionally performed following the initial dose, if necessary. In specific embodiments, the present invention may include the following aspects. [Section 1] A composition for an mRNA vaccine comprising an mRNA antigen and an immunoactivator that can be kinetically controlled as active ingredients, characterized in that the immunoactivator is a conjugate in which a cleavable linker is bound to the active site of the immunoactivator. [Section 2] Item 2. The composition for an mRNA vaccine according to item 1, characterized in that, after administration, the process of transcribing mRNA into a protein is carried out, and then the cleavable linker bound to the activation site of the immune activator is cleaved, thereby inducing the activation function of the immune activator. [Section 3] The dynamic control comprises: The immunoactivator is characterized in that a cleavable linker is bound to the activation site of the immunoactivator to maintain the inactive state, and the cleavable linker that has been blocking the activation site is cleaved within 2 to 12 hours after the start of mRNA transcription, resulting in the activity of the immunoactivator being displayed with a time delay. Item 1. A composition for an mRNA vaccine according to item 1. [Section 4] Item 2. The composition for an mRNA vaccine according to Item 1, wherein the cleavable linker contains one or more bonds selected from the group consisting of disulfide, carbamate, hydrazine, ester, peptide, azide, amide, hydrazone, thioether, phosphodiester, thioketal, and combinations thereof. [Section 5] Item 2. The composition for an mRNA vaccine according to Item 1, wherein the cleavable linker further comprises ethylene oxide or ethylene glycol at both or one of its ends. [Section 6] Item 2. The composition for an mRNA vaccine according to Item 1, wherein the cleavable linker is characterized in that the chemical bond at the binding site is cleaved by any one or more factors selected from the group consisting of an enzyme, pH, redox potential, temperature, ultrasound, magnetism, and a light source. [Section 7] Item 2. The composition for an mRNA vaccine according to Item 1, characterized in that 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. [Section 8] Item 2. The composition for an mRNA vaccine according to Item 1, wherein the immunoactivator is a toll-like receptor agonist. [Section 9] Item 9. The mRNA vaccine composition according to Item 8, wherein the Toll-like receptor agonist is one or more selected from the group consisting of Toll-like receptor 1 agonists, Toll-like receptor 2 agonists, Toll-like receptor 3 agonists, Toll-like receptor 4 agonists, Toll-like receptor 5 agonists, Toll-like receptor 6 agonists, Toll-like receptor 7 or 8 agonists, and Toll-like receptor 9 agonists. [Section 10] Item 2. The composition for an mRNA vaccine according to item 1, characterized in that the mRNA antigen and the dynamically controllable immune activator are loaded into one or more drug delivery vehicles selected from the group consisting of nanoliposomes, nanoemulsions, nanomicelles, hydrogels, scaffolds, solid nanoparticles and polymeric nanoparticles. [Section 11] Item 11. The composition for an mRNA vaccine according to item 10, wherein the drug delivery vehicle is characterized in that the internally loaded mRNA antigen is first delivered to the cytosol, and then the dynamically acting immune activator interacts with a receptor on the cell surface, in an endosome or in a lysosome. [Section 12] Item 11. The composition for an mRNA vaccine according to item 10, wherein the drug delivery vehicle further comprises one or more immune activators selected from the group consisting of toll-like receptor agonists, saponins, antiviral peptides, inflammasome inducers, NOD ligands, CDS ligands (cytosolic DNA sensor ligands), STING (stimulator of interferon genes) ligands, outer wall components of pathogenic bacteria, alum, lipids, combinations thereof, and derivatives thereof. [Section 13] Item 2. The composition for an mRNA vaccine according to Item 1, characterized in that the composition for an mRNA vaccine is 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. [Section 14] A method for preventing or treating an infectious disease, cancer, metabolic syndrome, autoimmune disease, or rare disease, comprising the step of administering to an individual an mRNA vaccine composition comprising an mRNA antigen and a kinetically controllable immune activator as active ingredients. [Section 15] Use of an mRNA vaccine composition comprising an mRNA antigen and a kinetically controllable immune activator as active ingredients for the prevention or treatment of infectious diseases, cancer, metabolic syndrome, autoimmune diseases, or rare diseases. [Section 16] Use of a composition comprising an mRNA antigen and a kinetically controllable immune activator as active ingredients for the production of a drug for the prevention or treatment of an infectious disease, cancer, metabolic syndrome, autoimmune disease, or rare disease.

[0059] In the following, preferred embodiments are presented to aid in understanding the present invention. The following examples are provided to enable the present invention to be more readily understood. However, the contents of the present invention are not limited thereto. EXAMPLES

[0060] [Example 1: Synthesis of Toll-like receptor 7 or 8 agonist-cholesterol conjugates] Diverse Toll-like receptors 7 and 8 with cholesterol conjugation Agonists (imidazoquinoloine-b ased agonist), hydroxyadenine agonist (8-hydroxyadenine agonist denine-based agonist, pteridone agonist one-based agonist, aminopyrimidine agonist (2-aminopyrimidine agonist opyrimidine-based agonist, benzazepine agonist (benzoazepine-based agonist), thiaoxoguanosine Agonists (7-thia-8-oxoguanosine-based agonists t) and the like) were prepared by the chemical reaction of the following reaction scheme 1 or 2. Can be cleaved at the amine group (NH2), which is the active site of agonists for receptors 7 and 8 Carbamates, disulfides, Ester, peptide, or azide bonds Cholesterol (Sigma-Aldrich) was bound via a bond to the activated site. Transiently inactivated Toll-like receptor 7 or 8 agonists conjugated with cholesterol ( Toll-like receptor 7 / 8 agonist-Cholester ol conjugate).

[0061] [ka]

[0062] The R is a side chain containing an aliphatic group or an aromatic group, and is -NH-, -CO-, -CON H-, -CSNH-, -COO-, -CSO-, -SO2NH-, -SO2-, -SO- , -O-, etc.

[0063] [ka]

[0064] The R is a side chain containing an aliphatic group or an aromatic group, and is -NH-, -CO-, -CON H-, -CSNH-, -COO-, -CSO-, -SO2NH-, -SO2-, -SO- , -O-, etc.

[0065] [Example 2: 2-Methyl-1-(3-nitroquinolin-4-ylamino)propane-2 -Synthesis of ol] 2-Methyl-1-(3-nitroquinolin-4-ylamino) ) propan-2-ol (compound 2) was synthesized. More specifically, compound 2 was synthesized at 10 to 20°C. In dichloromethane (450 ml) containing 1 (30 g), 1-amino-2-methylpropane Pan-2-ol (14 g) and tetraethylamine (9.6 g) were added and stirred for 2 hours. The mixture was then concentrated by evaporating the solvent under vacuum. The mixture was then resuspended in methyl tert-butyl ether (150 ml). The mixture was separated using a filter and then concentrated under low pressure to give compound 2 (32 g, 8 The obtained compound 2 has the following structure: 1 1H NMR I testified. 1 H NMR (400MHz, DMSO-d6): δ9.91 (brs, 1H), 9. 18(s, 1H)), 8.46(d, J=8.0Hz, 1H), 7.83-7.92(m , 2H), 7.56-7.60(m, 1H), 5.15(s, 1H), 3.86(d, J =4.8Hz, 2H), 1.15(s, 6H).

[0066] [ka]

[0067] [Example 3: 1-(3-aminoquinolin-4-ylamino)-2-methylpropane-2 -Synthesis of ol] Using the method of the following reaction scheme 4, 1-(3-aminoquinolin-4-ylamino)-2-methyl More specifically, dipropan-2-ol (compound 3) was synthesized at a reaction temperature of 10 to 20°C. Compound 2 (32 g), methanol (500 ml), and Pd / C catalyst (3.2 g After mixing, the mixture was degassed and flushed three times with hydrogen. The hydrogen was vaporized and the pressure was kept at 1 atm. After that, the mixture was stirred at room temperature for 5 hours. Then, the mixture was diluted with methyl tert-butyl ether ( The resuspended mixture was separated using a filter and then The mixture was concentrated under low pressure to obtain compound 3 (27 g, 95.4%, yellow solid). The structure of compound 3 is: 1 This was verified using H NMR. 1H NMR (400MHz, DMSO-d6): δ8.37(s, 1H)), 7.9 9-8.01(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] [ka]

[0069] [Example 4: 1-(2-ethoxymethyl)-1H-imidazo[4,5-c]quinoline- Synthesis of (1-yl)-2-methylpropan-2-ol Using the method of the following reaction scheme 5, 1-(2-ethoxymethyl)-1H-imidazo[4,5- c]Quinolin-1-yl)-2-methylpropan-2-ol (Compound 4) was synthesized. More specifically, compound 3 (27 g) and 2-ethoxyacetic acid were added to a reactor at 10 to 20° C. (30 ml) was added, and the mixture was stirred at 120 to 130°C for 5 hours. The mixture was cooled to 20-25°C and saturated sodium carbonate (150 ml) was added. The reaction mixture was extracted with a mixture of chloromethane and methanol (10 / 1, v / v). The extracted organic layer was washed with brine and then diluted with sodium sulfate (10 g). The organic solution layer from which the water had been removed was then filtered using a filter. After purification, the mixture was concentrated under low pressure to obtain compound 4 (30 g, 85.8%, yellow gel). The obtained structure of compound 4 is: 1 This was verified using H NMR. 1 H NMR (DMSO_d6 400MHz): δ9.18(s, 1H)), 8.6 3(d, J=8.0Hz, 1H), 8.13(dd, J=1.6, 8.0Hz, 1H), 7.63-7.71(m, 2H), 4.91(s, 2H), 4.78(brs, 2H), 3.54(q, J=6.8Hz, 2H), 1.10-1.18(m, 9H).

[0070] [ka]

[0071] [Example 5: 2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl) Synthesis of -1H-imidazo[4,5-c]quinoline 5-oxide] Using the method of the following reaction scheme 6, 2-(ethoxymethyl)-1-(2-hydroxy-2-methyl) (ethylpropyl)-1H-imidazo[4,5-c]quinoline 5-oxide (compound 5) More specifically, compound 4 (30 g), dichloromethane, and 1,2-dichloromethane were added to a reactor at 10 to 20°C. After adding 20 ml of benzoic acid (350 ml) and m-chloroperoxybenzoic acid (26 g), the mixture was cooled to room temperature. The mixture was stirred at room temperature for 4 hours. Then, saturated sodium carbonate solution (150 ml) and Sodium sulfate solution (150 ml) was added, followed by dichloromethane and methanol (10 / 10). The reaction product was extracted using a mixed solution of 1, v / v). The water in the extracted organic solution layer was removed with sulfuric acid. After removing the sodium (30g), it was filtered and then stored at low pressure. The reaction mixture was then concentrated and resuspended in ethyl acetate (50 ml). The mixture was filtered and dried under low pressure to obtain compound 5 (30 g, 94. The obtained compound 5 has the following structure: 1 1H NMR Ta. 1H NMR (DMSO_d6 400MHz): δ9.04(s, 1H)), 8.7 9(d, J=8.4Hz, 1H), 8.71(d, J=8.4Hz, 1H), 7.77- 7.80(m, 2H), 4.93(s, 2H), 4.73(brs, 2H), 3.54( q, J=6.8Hz, 2H), 1.12-1.18(m, 9H).

[0072] [ka]

[0073] [Example 6: 1-(4-amino-2-ethoxymethyl)-1H-imidazo[4.5-c ]Synthesis of quinolin-1-yl)-2-methylpropan-2-ol] 1-(4-amino-2-ethoxymethyl)-1H-imidazolium chloride was prepared by the method shown in Reaction Scheme 7 below. Zo[4.5-c]quinolin-1-yl)-2-methylpropan-2-ol (compound 6) More specifically, compound 5 (30 g), DCM ( 600 ml), 4-methylbenzene-1-sulfonyl chloride (18.2 g), and Aqueous ammonia (NH3·H2O, 180 ml) was added and the mixture was stirred at room temperature for 16 hours. Next, distilled water was added to the stirred mixture, followed by dichloromethane and methanol (10 / 1, The mixture was separated using a v / v mixture of 100% ethanol and 100% ethanol. The separated organic layer was washed with brine. After washing with e), the water was removed using anhydrous sodium sulfate (50 g). The organic solution layer from which the condensation products have been removed is filtered using a filter and then concentrated under low pressure. The reaction mixture was dissolved in a mixture of methyl tert-butyl ether and methanol (15 / 1, v / v). The mixture was resuspended in a combined solution for 30 minutes. After that, the cells were separated using a filter and then placed under low pressure. The obtained compound 6 was dried at 40° C. for 1 hour to obtain compound 6 (18 g, 60%, yellow solid). The structure is: 1 This was verified using H NMR. 1 H NMR (DMSO_d6 400MHz): δ8.27(d, J=8.0Hz, 1H), 7.59(d, J=7.6Hz, 1H), 7.40(t, J=7.2Hz, 1H ), 7.21(t, J=7.2Hz, 1H), 6.57(brs, 2H), 4.89(s , 2H), 4.68(brs, 2H), 3.52(q, J=6.8Hz, 2H), 1.1 1-1.17(m, 9H).

[0074] [ka]

[0075] [Example 7: 10,13-dimethyl-17-(6-methylheptan-2-yl)-2, 3,4,7,8,9,10,11,12,13,14,15,16,17-Tetradecahy Doro-1H-cyclo[a]phenanthren-3-yl 2-(ethoxymethyl)-1-(2 -Hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinoline-4-iso Synthesis of arylcarbamates] 10,13-dimethyl-17-(6-methylheptane-2) -Il)-2,3,4,7,8,9,10,11,12,13,14,15,16,17 -Tetradecahydro-1H-cyclo[a]phenanthren-3-yl 2-(ethoxymethyl) (2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]ky We synthesized 4-norin-4-yl carbamate (compound 8). First, compound 7 (TCI, 50 g, Cholesterol chloroformate) in 250 g of silica gel Column chromatography packed with 0% to 20% ethyl acetate in n-hexane Using this method, pure compound 7 (30 g) was obtained. Compound 7 was then added to a reactor at 10-20°C. After addition of 6 (15 g) and dichloromethane (198.9 g), the pure compound 7 (30 g) was ) and tetraethylamine (9.6 g) were added in that order, and the mixture was stirred at 20 to 25° C. for 16 hours. Water was added to the stirred mixture, followed by the addition of dichloromethane to extract the reaction. The organic layer was washed with salt water and then extracted with anhydrous sodium sulfate (195 g). The organic solution layer from which the water had been removed was filtered using a filter and then cooled to low pressure. The concentrated reaction mixture was then concentrated in the presence of methyl tert-butyl ether and methacrylic acid. The cells were then resuspended in a mixture of ethanol (10 / 1, v / v) and filtered. After separation, it was dried under low pressure to give compound 8 (10.2 g, 55.1%, white solid). The structure of the obtained compound 8 is: 1 This was verified using H NMR. 1 H NMR The results showed that resiquimod (R848) and cholesterol are It was confirmed that a conjugate linked by a bamate bond was produced. 1 H NMR (CDCl3400MHz): δ8.13-8.19(m, 2H), 7 .59-7.63(m, 1H)), 7.46-7.50(m, 1H), 5.42-5.4 3(m, 1H), 4.92(brs, 2H), 4.72-4.80(m, 3H), 3.6 8(q, J=6.8Hz, 2H), 3.24(s, 1H), 2.51-2.59(m, 1 H), 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, 2 7H), 0.94(d, J=6.4Hz, 3H), 0.89(d, J=6.4Hz, 6H ), 0.71(s, 3H).

[0076] [ka]

[0077] [Example 8: Bis(2,5-dioxopyrrolidin-1-yl)2,2'-disulfane Synthesis of diylbis(ethane-2,1-diyl)dicarbonate Bis(2,5-dioxopyrrolidin-1-yl) 2,2' is synthesized by the method shown in Reaction Scheme 9 below. - Disulfanediylbis(ethane-2,1-diyl)dicarbonate (compound 9) was synthesized. First, compound 7 (TCI, 70 g) was added to a column packed with 350 g of silica gel. The pure compound was obtained by chromatography (0% to 20% ethyl acetate in n-hexane). Compound 7 (40 g) was obtained. Then, pure compound 7 (40 g) was added to the reactor at 10-15 °C. ) and dichloromethane (100 ml), and then bis(2-hydroxyethyl)dichloromethane (100 ml) was added. A solution of sulfide in dichloromethane (250 ml) and pyridine (21 g) was added. The mixture was stirred at room temperature for 2 hours, and then distilled water (200 ml) was added. The reaction mixture was then extracted with three portions of dichloromethane (150 ml). The solution layer was washed with salt water and then dehydrated with anhydrous sodium sulfate (20 g). The reaction mixture from which water had been removed was filtered and then concentrated under low pressure. Then, column chromatography (silica gel, 300 g, 10% to 30% ethyl acetate) was performed. Compound 9 (20 g, 39.6%, yellow gel) was obtained using ethyl acetate (ethyl acetate in n-hexane). The structure of the obtained compound 9 is as follows: 1 This was verified using H NMR. 1 H NMR (CDCl3400MHz): δ5.41-5.42 (m, 1H), 4. 46-4.56(m, 1H), 4.41(t, J=6.8Hz, 2H), 3.91(t, J=6.0Hz, 2H), 2.98(t, J=6.8Hz, 2H), 2.91(t, J= 6.0Hz, 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=6.4Hz , 3H), 0.88(dd,J=1.6,6.8Hz, 6H), 0.69(s, 3H).

[0078] [ka]

[0079] [Example 9: Synthesis of compound 10] Compound 10 was synthesized by the method of the following reaction formula 10. More specifically, Compound 9 (20 g) and dichloromethane (200 ml) were added to the reactor, and then bis( 2,5-dioxopyrrolidin-1-yl)carbonate (18g) and tetraethylamine (10.7 g) was added in sequence. The mixture was stirred at room temperature for 3 hours, and then distilled water (300 ml) was added, and the reaction was then extracted with three portions of dichloromethane (150 ml). The extracted organic solution layer was washed with salt water and then diluted with anhydrous sodium sulfate (20 g). The reaction mixture from which moisture had been removed was filtered using a filter, and then the filtrate was The filtrate was concentrated under low pressure. Then, column chromatography (silica gel, 200 g, 5% to 20% ethyl acetate in n-hexane) to compound 10 (18 g, 72% The obtained structure of compound 10 is: 1 Verified using H NMR . 1 H NMR (CDCl3400MHz): δ5.39-5.40 (m, 1H), 4 .57(t, J=6.8Hz, 2H), 4.43-4.52(m, 1H), 4.37(t , J=6.4Hz, 2H), 2.96-3.03(m, 4H), 2.84(s, 4H), 2.34-2.44(m, 2H), 1.78-2.04(m, 5H), 1.42-1.7 3(m, 7H), 1.22-1.40(m, 5H), 1.06-1.20(m, 7H), 0.95-1.04(m, 5H), 0.91(d, J=6.0Hz, 3H), 0.86( d, J=6.4Hz, 6H), 0.67(s, 3H).

[0080] [ka]

[0081] [Example 10: 2-((2-((10,13-dimethyl-17-(6-methylheptane -2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16, 17-Tetradecahydro-1H-cyclo[a]phenanthren-3-yloxy)carbo 2-(ethoxymethyl)-1-(2-hydroxyethyl)disulfanyl)ethyl (2-methyl-1H-imidazo[4,5-c]quinolin-4-yl carboxylate) Synthesis of Bamate] Using the method of the following reaction scheme 11, 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-yl 2-(ethoxymethyl)-oxy)carbonyloxy)ethyl)disulfanyl)ethyl 1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinoline In more detail, the 4-yl carbamate (compound 11) was synthesized at 10 to 20 °C. Compound 6 (15 g) and dichloromethane (198.9 g) were added to the reactor, and compound 1 was then added. 0 (40.5 g) and tetraethylamine (9.6 g) were added in that order. After stirring at 25°C for 16 hours, distilled water (225 ml) was added. The reaction mixture was extracted by adding ethanol (99.45 g) five times. The extracted organic layer was washed with brine. After washing with sodium sulfate (195 g), the water was removed. The reactant from which the components have been removed is filtered, and the filtrate is concentrated under low pressure. Then, column chromatography (silica gel, 100 g, 10% to 50% ethyl acetate) was performed. Compound 11 (10.8 g, 37.4%, white solid) was obtained using ethyl acetate in n-hexane. The structure of the obtained compound 11 is: 1 This was verified using H NMR. 1 H NMR As a result, a conjugate in which R848 and cholesterol were cross-linked with disulfide was produced. It was confirmed that this was done. 1 H NMR (CDCl3400MHz): δ8.15-8.17(m, 2H), 7 .60-7.64(m, 1H)), 7.47-7.51(m, 1H), 5.39-5.4 0(m, 1H), 4.93(s, 2H), 4.81(s, 2H), 4.56(t, J=6 .4Hz, 2H), 4.45-4.54(m, 1H), 4.41(t, J=6.4Hz, 2H), 3.68(q, J=6.8Hz, 2H), 3.13(s, 1H), 3.09(t , J=6.4Hz, 2H), 3.01(t, J=6.4Hz, 2H), 2.34-2.4 7(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=6.4Hz, 3H), 0.88(dd,J=1. 6,6.4Hz, 6H), 0.69(s, 3H).

[0082] [ka]

[0083] [Example 11: Cholesterol-Toll-like receptor 7 or 8 agonist conjugates Nanoparticle production Cholesterol-Toll-like receptor 7 or 8 agonist conjugates bind cholesterol Since it contains ruthenium, it can be easily produced in various nanoparticle forms, which can improve immunity. This maximizes interaction with immune cells.

[0084] 11.1. Preparation of nanoliposomes containing cholesterol-bound resiquimod Anionic nanoliposomes containing cholesterol-conjugated resiquimod To prepare the posome, 4 mg of DOPC (1,2- dioleoyl-sn-glycero-3-phosphocholine, Ava nti), 1.2 mg of cholesterol-bound resiquimod, and 1 mg of DPPG (1,2-dipalmitoyl-sn-glycero-3-phospho-(1 '-rac-glycerol), Avanti), and dissolve the mixture. The mixture was then subjected to a rotary evaporator to remove the solvent, resulting in a thin film. The morphology was prepared, and 2 ml of phosphate buffer solution was added to the thin film and stirred at 45°C for 30 min. Stir and homogenize using a tip ultrasonicator (amplitude: 20%, 2 min). We prepared anionic nanoliposomes containing cholesterol-conjugated resiquimod via To prepare cationic nanoliposomes containing cholesterol-conjugated resiquimod 4 mg of DOPC and 1.2 mg of cholesterol were bound to 1 ml of chloroform. resiquimod, and 2 mg of dimethyldioctadecylammonium bromide (DDAB; Dimethyldioctadecylammonium bromide) was added. Then, the mixture is dissolved to produce a mixture, and the mixture is evaporated using a rotary evaporator. The solvent was evaporated to produce a thin film, and the thin film was then soaked in 2 ml of phosphate buffer solution. The mixture was stirred at 45°C for 30 minutes and then subjected to a tip ultrasonic crusher (amplitude: 20% cations containing cholesterol-bound resiquimod through a homogenization step using Nanoliposomes were prepared.

[0085] 11.2. Preparation of Nanoemulsions Containing Cholesterol-Conjugated Resiquimod Nanoemulsions containing cholesterol-bound resiquimod To prepare the 100% chloroform solution, 1 mg of DOPC and 240 μg of cholesterol were added to 1 ml of chloroform. Sterol (Sigma-Aldrich), 240 μg cholesterol-bound Siquimod was added and allowed to dissolve to produce a mixture. The mixture was then placed in a round bottom flask. After transferring the mixture to a container, chloroform was completely evaporated using a rotary evaporator. Then, a thin lipid film was prepared. l with Squalene (5% v / v), Tween 80 (0.5% v / v), and S After adding Pan 85 (0.5% v / v) and dissolving, the solution was added onto the lipid membrane. The mixture was dispersed for 1 minute using a Tip sonicator, and then cooled using a rotating device. The mixture was stirred for about 2 hours using a tube revolve to allow the cholesterol to bind. After preparation of the nanoemulsion containing resiquimod, it was stored in a refrigerator at 4°C until use.

[0086] (11.3. Nanomimetic Composed of Cholesterol-Conjugated Resiquimod and Saponin Cell manufacturing) Nanomicelles composed of cholesterol-conjugated resiquimod and saponin To produce the omicelle, phosphatidylcholine:saponin:cholesterol After mixing the lecithin-bound resiquimod in a weight ratio of 5:3:2, the concentration was 14 mg / ml. The lipids were then added to ether and dissolved to prepare an ether solution containing lipids. Saponin was dissolved in 4 ml of distilled water at a concentration of 1.5 mg / ml and then added to 20 ml of gas. After placing the mixture in a glass bottle and sealing it with a rubber stopper, the bottle was stored in a water jacket at 55°C. Next, 1 ml of the lipid-containing ether solution was injected at 0.2 ml / min using a syringe pump. The solution was added to the glass bottle containing saponin at a rate of 100 ml and stirred for 2 hours. The end of the needle was placed below the surface of the aqueous solution containing saponin, and the second needle was placed for ventilation. Then, transfer the glass bottle to room temperature and stir for 3 days to stabilize it. Through the steps, a nanomycete composed of cholesterol-bound resiquimod and saponin was We manufactured the following.

[0087] 11.4. Preparation of polymeric nanoparticles composed of cholesterol-conjugated resiquimod ) The composition ratio of lactide and glycolide is 50:5. Dissolve 60 mg of PLGA polymer (Eudragit) in 1 ml of chloroform solvent. 5 mg of cholesterol-bound resiquimod was added to the solvent and the mixture was then washed with an ultrasonic cleaner (ult rasonic bath, Emerson Model CPX5800H-E) The cholesterol-resiquimod conjugate and the polymer were dissolved in 2.5% PVA water. The solution was dissolved in 10 ml of solution and 200 μl of the solution was added at a time while dispersing the solution in an ultrasonic disperser (Tip sonicator, Sonics&Materials Model VCX 75 The dispersing was performed for 1 minute using a dispersing machine with a power output of 750 watts and a vibration intensity of 1000 Hz. The frequency was 20 kHz and the amplitude was set to 20%. The aqueous solution was heated at 600 rpm for 8 h at room temperature to completely evaporate the dissolved organic solvent. The mixture was stirred for more than 2 h. In order to remove unreacted macromolecules and cholesterol-resiquimod conjugates, To do this, a centrifuge (Centrifuge, Hanil, Combi-514R) was used. The mixture was centrifuged at 12,000 rpm for 12 minutes, the supernatant was removed, and then the mixture was washed with ultrapure water. 10 ml of the mixture was added and dispersed in an ultrasonic disperser for 30 seconds. The above process was repeated three times, and then It was dried using the freeze-drying method and then stored at -20°C.

[0088] [Example 12: mRNA and cholesterol-containing mRNA containing toll-like receptor agonist Confirmation of efficacy of NA vaccine composition] 12.1. Methods for Processing mRNA Vaccine Compositions In order to confirm the efficacy of the mRNA vaccine composition of the present invention, The experiment was carried out using OVA mRNA, which is widely used in the field. leanCap® OVA mRNA (5 moU, L-7210, TriLin k Biotechnologies) and Opti-MEM TM (Reduced Se rum Medium, ThermoFisher Scientific) at 1:49 The mixture was mixed in a volume ratio of 100:1, and Lipofectamine (Lipofectamine) was used as the carrier. mine TM 2000 Transfection Reagent, ThermoF Isher Scientific) and Opti-MEM TM Mixed in a volume ratio of 2:23 After that, the mixtures were mixed in a 1:1 volume ratio and reacted for 5 minutes to obtain the mR An NA composition was prepared. As a toll-like receptor agonist, a toll-like receptor 3 agonist was used. Polyinosinic-polycytidylic acid (poly I:C, Sigma-Aldrich), and Lipo, a Toll-like receptor 4 agonist. Polysaccharide (LPS, Sigma-Aldrich) and Th The drug was resiquimod (Biorbyt), a pol-like receptor 7 or 8 agonist. y I:C is phosphate buffered saline. e) in PBS at a concentration of 0.8 mg / ml. LPS was used in PBS at 0.3 mg / ml. R848 was used in a DMSO (dimethyoxy) After dissolving it in 100 ml of sulfoxide at a concentration of 20 mg / ml, the solution was The cells were dispersed in PBS to a concentration of 20 μg / ml. When treating with IL-1 receptor agonists, Poly I:C was used at 40 μg / ml. The cells were treated with LPS at a concentration of 30ng / ml and R848 at a concentration of 1μg / ml. In this case, the concentrations of Poly I:C 2μg / ml and R848 1μg / ml were used. The LPS and R848 concentrations were 2ng / ml and 1μg / ml, respectively. Treatment was at a concentration of 100 µg / ml.

[0089] 12.2. Bone marrow derived dendritic cells, plasmacytoid dendritic cells, ovalbumin specific TCR bearing (Experimental mouse spleen cell culture) In order to confirm the efficacy of the mRNA vaccine composition of the present invention, bone marrow cells were used as experimental cells. Bone Marrow-derived Dendritic Cells ll;BMDC), plasmacytoid dendritic cells c cell; pDC), and experimental mice bearing ovalbumin-specific TCR (OT -I transgenic mouse (OT-I mouse) T cells were used. BMDCs and pDCs were generated from experimental mice, C57BL / 6 (female The cells were obtained from the bone marrow of a 6-week-old mouse (Orient Biosciences, Tokyo, Japan). The animals were euthanized, and the femurs and tibias were cut and the medium was poured out to extract the internal materials. The extracted material was treated with red blood cell lysis buffer. The pDCs were used after removal of red blood cells by treatment with ELISA (BioLegend). After euthanasia of 1 year-old C57BL / 6 mice, femurs and tibias were cut and culture medium was poured out to separate the internal structures. The extracted material was then treated with Red Blood Cell Lysate. After removing red blood cells with lysis buffer (BioLegend), C isolation kit(Plasmacytoid Dendritic C ell Isolation Kit, mouse, MACS Miltenyi Bi pDCs were isolated using a otec (transplantation endothelial cell culture) and used in the experiments. Spleen cells were isolated using a otec (transplantation endothelial cell culture) and used in the experiments. Spleens were extracted from mice and physically crushed to prepare single cells, then washed with PBS. The cells were then washed and used after removing red blood cells using a red blood cell lysis solution. 2 × 10 6 Thin The cells were cultured at 5% CO2 and 37°C. ng / ml of GM-CSF (Granulocyte-macrophage colo ny-stimulating factor, Recombinant Mouse Complete with GM-CSF Protein (R&D Systems) e RPMI 1640 medium (Gibco) was used. The medium was replaced with new medium at daily intervals, and the cells on the 7th day of culture were used for the experiments.

[0090] (12.3. Co-culture of OT-I mouse spleen cells and pDCs) To confirm the efficacy of the mRNA vaccine composition of the present invention, the spleen of OT-1 mice was used. The T cells and pDCs isolated from the spleen cells were co-cultured with the spleen cells obtained in the same manner as in Example 12.2. (1×10 5 cells / 100 μL) and pDCs (1 × 10 4 Cells / 100μL) in 96 wells After dispensing into plates, the mRNA vaccine composition was prepared in the same manner as in Example 12.1. After 48 hours, cell and cytokine analysis was performed. For analysis of the in vitro cell activation assay, cells were incubated with tail(BioLgend) and protein transport inhibit or (BD GolgiStop TM Protein Transport Inhibitor After treatment with β-aminobutyric acid (BITOR) to activate cytokine secretion in the Golgi apparatus, cells were The cells were harvested and measured. The harvested cells were fixed and permeabilized. tion kit (BD Cytofix / Cytoperm TM Plus) After creating holes in the cell membrane and inside the cells so that the antibody can attach, FN-γ antibody(PE Rat Anti-Mouse IFN-γ;PE -IFN-γ, BioLegend) to measure the amount of IFN-γ secreted into cells. did.

[0091] (12.4. Co-culture of OT-I mouse splenocytes and BMDCs) To confirm the efficacy of the mRNA vaccine composition of the present invention, the spleen of OT-1 mice was used. T cells isolated from ex vivo DCs were co-cultured with BMDCs. Co-culturing with spleen cells for use in herapy (cell therapy) First, the mRNA vaccine composition prepared in the same manner as in Example 12.1 was treated. The BMDCs (1 × 10 4 cells / 100 μL) and T cells (1 × 10 5 cells / 100μL) was dispensed into a 96-well plate. Cell and cytokine analysis was then performed after 48 hours. The same procedure as in Example 12.3 was used to measure the degree of cell activation of BMDCs. Cell surface molecule 80(Cluster of Differentiation 80;C D80), CD86, and CD40 mean fluorescence intensity The molecular weight intensity (MFI) was confirmed by attaching a fluorescent antibody. D80 was measured using PerCP / Cyanine5.5 anti-mouse CD80 Antibody (PerCP / Cy5.5-CD80, BioLegend) was used. , CD86 is PE anti-mouse CD86 Antibody(PE-CD 86, BioLegend) and CD40 was detected using FITC anti-mouse C Confirmed using D40 Antibody (FITC-CD40, BioLegend) In the case of BMDCs, first, forward scatter and scattering (S After adjusting FSC and SSC) to separate into single cells, , CD11c Antibody (APC anti-mouse CD11c Antibody(A PC-CD11c (BD Bioscience) to isolate the BMDC population. To measure the level of antigen presentation, anti-OVA SIINFEKL and tibody was used.

[0092] 12.5. Confirmation of efficacy of mRNA vaccine composition in BMDCs 12.5.1. Effects of Single Toll-Like Receptor Agonists To confirm the efficacy of an mRNA vaccine composition containing a single Toll-like receptor in BMDCs, To this end, we first synthesized Toll-like receptor agonists and modified mRNA (5-methoxyuridi ne modified OVA mRNA, TriLink) was used in the same manner as in Example 12.1. The BMDCs were treated with the method and the antigen presentation level, cell surface molecules, and cytokines were examined. Interleukin-12p70 (IL-12p70) and In The amount of inflammatory cytokines was measured using ELISA. Toll-like receptor agonists include R8, which is a Toll-like receptor 7 or 8 agonist. 48, LPS, a Toll-like receptor 4 agonist, and Toll-like receptor 3 agonist Poly I:C was used at 20 μg / ml, 30 μg / ml Concentrations of 100 μg / ml and 40 μg / ml were used separately. All subsequent experiments were performed at a minimum The results were repeated three times and are shown as the mean ± standard deviation. Statistical significance was determined by the Student's The results were confirmed by t-test, and P<0.05 was considered to be statistically significant. The results using R848 are shown in Figures 5 to 7, and the results using LPS are shown in Figure 8. The results when poly I:C was used are shown in FIGS.

[0093] As shown in FIG. 5, compared with the control group treated with mRNA alone (mRNA only), In contrast, in the experimental group treated simultaneously with mRNA and R848 (0 h), the degree of antigen presentation was Although there was a partial increase, the difference was not significant. There was a significant difference between the experimental group treated with R848 (4h) and the experimental group treated with R848 8 hours later (8h). It was confirmed that the level of antigen presentation was increased.

[0094] As shown in FIG. 6, compared with the control group treated with mRNA alone (mRNA only), In contrast, in the experimental group treated with both mRNA and R848, no significant changes were observed regardless of the treatment time. Both of these increase cell surface molecules, which in turn increases the mRNA and the Toll-like receptor agonist. It was confirmed that the degree of cell activation could be increased by co-administration.

[0095] As shown in Figure 7, in the case of IL-12p70, simultaneous treatment with mRNA and R848 The IL-1 expression level in the time-interval treated group (4 h) was significantly higher than that in the control group (0 h). We confirmed that the secretion of 12p7 increased. In addition, it induced Th1 differentiation (Th1 polarization). The ratio of IL-12p70 to IL-10 (IL-12p70 / IL- In the case of 10), the time was also longer than that of the experimental group (0 h) treated simultaneously with mRNA and R848. A significant increase was observed in the experimental groups treated at intervals (4h and 8h). did.

[0096] As shown in Figure 8, in the experimental group (0h) in which mRNA and LPS were simultaneously treated, Compared with the control group treated with mRNA alone (mRNA only), the degree of antigen presentation was The experimental group treated with mRNA and LPS at time intervals (4h and 8h), it was confirmed that the level of antigen presentation increased.

[0097] As shown in FIG. 9, the experimental group (0 h) in which mRNA and Poly I:C were treated simultaneously In the case of the control group treated with mRNA alone (m It was confirmed that the degree of antigen presentation was significantly reduced compared to that of the control (RNA only). In the experimental group treated with Poly I:C at a time interval (4h), The degree of antigen presentation was significantly increased regardless of the concentration of C (20, 30, and 40). confirmed.

[0098] FIG. 10 shows the antiviral site of the mRNA and 40 μg / ml poly I:C. Type 1 Interferon (type 1 interferon, type The results are compared between the control group treated with mRNA alone (mRN P1 and IFN). In A), type 1 IFN secretion was not confirmed, but mRNA and poly I In the experimental group (0h) treated with :C, the secretion of type 1 IFN was significantly increased. In addition, the results showed that the increase in the number of IL-1-positive cells was due to the poly I:C treatment with a 4-hour time lag. In the experimental group (4h), it was confirmed that the secretion of type 1 IFN was decreased.

[0099] Different antigen-presenting abilities, levels of cell activation, and proinflammatory cytokine secretion abilities Each group of dendritic cells carrying ovalbumin-specific TCR transgenic When co-cultured with T cells isolated from the spleens of OT-I mice, the mRNA vaccine To confirm the effect of the composition for cutin, the activation level of OT-1 T cells was examined. To confirm the degree of cell activation, we used cytokines secreted by activated T cells. The researchers confirmed that the IFN-γ (Interferon-γ) is involved in the inflammatory process. The ratio of cells that have IFN-γ inside the cells and those that have IFN-γ outside the cells was The concentration of IFN-γ secreted from T cells was also measured. Interleukin-2, a cytokine that contributes to increased activity The concentrations of IL-2 and IL-3 were also confirmed, and the results are shown in Figure 11.

[0100] As shown in FIG. 11, control T cells were cultured with BMDCs treated with mRNA alone. Compared with the group (mRNA), BMDCs and T treated simultaneously with mRNA and poly I:C The experimental group (0h) in which cells were cultured was treated with IFN-γ + T cells, the amount of IFN-γ secreted, The amounts of IL-1 and IL-2 were all significantly decreased. , and the experimental group (4h) in which T cells were cultured with BMDCs treated with poly I:C 4 hours later. Alternatively, BMDCs and T cells were treated with mRNA and cultured 8 hours later with poly I:C. In the experimental group (8h) where the mice were fed IFN-γ + The amount of T cells and secreted IFN-γ The amount of IL-2 and the amount of IL-1 were all significantly increased. It was confirmed that the experimental group treated with a time interval of 4 hours (4h) showed the highest secretion amount. I acknowledged.

[0101] (12.5.2. Toll-like receptor 3 agonists and Toll-like receptor 7 or 8 agonists (Effect of combined administration of In BMDCs, a composite Toll-like receptor, i.e., Toll-like receptor 3 agonist and To confirm the efficacy of mRNA vaccine compositions containing Toll-like receptor 7 or 8 agonists To do this, first, poly I:C, R848, and modified mRNA were used as in Example 12.1. BMDCs were treated in the same way, and the degree of antigen presentation and interferon levels were measured. is shown in Figure 12.

[0102] As shown in Figure 12, mRNA and a composite Toll-like receptor agonist were simultaneously treated. In the experimental group (0h), the degree of antigen presentation was significantly lower than that in the control group treated with mRNA alone. On the contrary, the expression of the complex Toll-like receptor agonist was significantly decreased after 4 hours of treatment with mRNA. In the experimental group treated with methionine (4h), the level of antigen presentation was significantly increased. In addition, in the control group treated with mRNA alone (mRNA), the expression of type 1 IFN was not observed. Although no secretion was observed, the experimental group (0 h) treated simultaneously with mRNA and poly I:C In the case of , it was confirmed that the secretion of type 1 IFN was significantly increased. In the experimental group treated with poly I:C with a time lag between the two (4h), 1. We confirmed that IFN secretion was reduced.

[0103] In addition, we will confirm the effect of the mRNA vaccine composition when BMDCs and T cells are co-cultured. To investigate the activation level of OT-1 T cells, the results are shown in Figure 13.

[0104] As shown in FIG. 13, control T cells were cultured with BMDCs treated with mRNA alone. Compared with group (mRNA), mRNA, Poly I:C, and R848 were treated simultaneously. In the experimental group (0 h), BMDCs and T cells were cultured, and IFN-γ + T cells, secreted The amount of IFN-γ and IL-2 were similar to that of the control group treated with mRNA alone. On the other hand, we confirmed that the effect of mRNA treatment was observed after 4 hours of treatment with Poly I:C and In the experimental group, BMDCs and T cells were cultured with R848 or mRNA. After 8 hours, BMDCs and T cells treated with poly I:C and R848 were cultured. In the experimental group (8h), IFN-γ + Amount of T cells and amount of secreted IFN-γ , and IL-2 were all significantly increased.

[0105] (12.5.3. Toll-like receptor 4 agonists and Toll-like receptor 7 or 8 agonists (Effect of combined administration of Combined Toll-like receptors in BMDCs, i.e., Toll-like receptor 4 agonist and To confirm the efficacy of mRNA vaccine compositions containing Toll-like receptor 7 or 8 agonists To do this, first, LPS, R848, and modified mRNA were mixed in the same manner as in Example 12.1. The antigen presentation and cell activation were confirmed by treating BMDCs. The surface molecules were measured and confirmed, and the results are shown in Figure 14.

[0106] As shown in FIG. 14, the control group (mRNA only) treated with mRNA alone In comparison, in the experimental group (0 h) treated simultaneously with mRNA, LPS, and R848, Although the antigen presentation level was partially decreased, the difference was not significant. The experimental group was treated with LPS and R848 after 4 h and the experimental group was treated with LPS and R848 after 8 h. In the experimental group treated with (8h), the degree of antigen presentation was significantly increased, demonstrating a significant difference. In addition, the cell surface molecules were also not expressed in the control group treated with mRNA alone (mRNA only ), the experimental group treated with mRNA, LPS and R848 all showed cell surface It was confirmed that the number of surface molecules increased.

[0107] In addition, IL-12p70, which is used as an indicator of Th1 differentiation (Th1 polarization), The IL-10 ratio (IL-12p70 / IL-10) was confirmed. The results are shown in Figure 15. did.

[0108] As shown in FIG. 15, the experimental group ( Compared with 0h, the experimental groups administered at different times (4h, 8h) showed a significant increase. In addition, to confirm the correlation between mRNA and type 1 IFN, Then, eGFP-modified mRNA was treated in BMDCs, and after 12 hours, eGFP protein was expressed. The percentage of cells expressing type 1 IFN and the amount of type 1 IFN were measured, and the results are shown in Figure 16.

[0109] As shown in FIG. 16, the experimental group ( 0h) showed a higher expression of GFP than the control group treated with mRNA only (mRNA only). The number of expressing cells decreased, but in the experimental groups administered with a time lag (4 h, 8 h) We confirmed that the number of cells expressing GFP increased significantly. In the case of N, no secretion was observed in the control group treated with only mRNA, whereas the mRNA A, The highest amount was secreted in the experimental group treated simultaneously with LPS and R848 (0 h). We confirmed that the amount of secretion decreased as the time interval between mRNA and Toll-like receptor treatment increased. Ta.

[0110] Based on the above results, when mRNA antigen and Toll-like receptor agonist were administered simultaneously, In Type 2 encephalopathy, mRNA expression is suppressed and the immunogenic effect of mRNA antigen is activated. 1. IFN expression increases, but there is a time lag between the administration of mRNA antigen and Toll-like receptor agonist. When administered at 100 mg / kg, expression of mRNA antigen is induced initially, and then at intervals, It was possible to confirm that the immunogenicity of the RNA antigen was activated.

[0111] (12.6. Confirmation of efficacy of mRNA vaccine composition in pDC) They are known to secrete relatively large amounts of type 1 IFN compared to BMDCs. The experiment was carried out using pDCs. The treatment method was the same as for BMDCs. As a cholesterol-like receptor agonist, R848 is linked to cholesterol by a disulfide bond. Prepared in the same manner as in Example 11.1 using a cross-linked conjugate. The nanoliposome (SKKU-078-liposome) was used. The presentation level and the amount of interleukin-12p70 secreted were measured. The results are shown in Figure 17 and Shown in Figure 18.

[0112] As shown in FIG. 17, the control group treated with mRNA alone (mRNA only) , no significant difference was observed compared to the control group treated with PBS alone (PBS), and mRNA and R84 In the experimental group (0h) in which 8 was simultaneously treated, a partial increase was confirmed. However, In the experimental group (4h) in which R848 was treated 4 hours after treatment with mRNA, the degree of antigen presentation was It was confirmed that the number of cells increased significantly. In the case of mRNA+SKKU-078-liposome, antigen presentation was significantly increased. This was confirmed by measuring the level of R84 at 4-hour intervals after mRNA treatment. It was confirmed that the results were the same as those of the experimental group treated with 8.

[0113] As shown in FIG. 18, the control group treated with mRNA alone (mRNA only) y), compared with the experimental group treated with liposomes and mRNA simultaneously (mRNA+SKKU- In the case of 078-liposome, the secretion of interleukin-12p70 was significantly increased. It was confirmed that it increased.

[0114] In addition, the effect of the mRNA vaccine composition was enhanced when pDCs were co-cultured with splenic T cells. To confirm this, we co-cultured the modified mRNA with the R848-cholesterol conjugate. The researchers then analyzed the percentage of T cells by phenotype after 48 hours. The results are shown in Figures 19 and 20.

[0115] As shown in Figures 19 and 20, the control group treated with mRNA alone (mRNA In the case of the PBS-treated control group (PBS only), the results were similar to or slightly different from those of the PBS-treated control group (PBS). The experimental group treated with mRNA and liposomes (mRNA+SKKU- In the case of 078-liposome, interferon gamma (interferon gamma; IFN-γ) positive T cells, tumor necrosis factor α (tumor necrosis Percentage of tumor necrosis factor alpha (TNF-α) positive and double positive T cells It was also confirmed that the amount of IFN-γ cytokine secreted increased significantly. It was confirmed that.

[0116] Based on the above results, a composition for an mRNA vaccine containing a Toll-like receptor agonist was prepared. To achieve this, a Toll-like receptor agonist and mRNA are injected into the body and act simultaneously. In some cases, this reduces the pharmaceutical activity of the mRNA while preventing the mRNA from being transcribed into protein. After the toll-like receptor agonist is activated, the activation function of the adjuvant is activated. When combined with other drugs, the immune response is optimized and the efficacy of the vaccine is most effectively improved. In other words, in order to produce an effective mRNA vaccine composition, Therefore, we confirmed that sequential action of mRNA and Toll-like receptor agonists is important. In addition, the toll-like receptor agonist of the present invention and a cholesterol conjugate (co njugate is an active site inhibitor of Toll-like receptor agonists. ) is linked to cholesterol by a cleavable bond. Initially, the activity is temporarily inhibited, and then the conjugate is activated physiologically. When the target location is reached, the toll-like receptor agonist and cholesterol are released. The role of the protein was isolated and showed activity, which was consistent with the time lag treatment with mRNA. It was confirmed that the Toll-like receptor agonist of the present invention has the same effect. The use of a toll-cholesterol conjugate as an adjuvant increased the A vaccine containing a receptor agonist-cholesterol conjugate and mRNA as active ingredients The composition for use may be administered simultaneously or sequentially after the mRNA is transcribed into protein and then administered with an adjuvant. The activation function of toll-like receptor agonists, which are Not only can it significantly enhance the efficacy of the composition for vaccination, but also it can be used for various types of mRNA vaccines. It was confirmed that the toll-like receptor agonist can be widely used in the field of medicine. Various applications have been achieved by using the cholesteryl-cholesterol conjugate to easily prepare nanoparticles. By further including an adjuvant, the immune activation efficacy of the mRNA vaccine composition can be further enhanced. It was confirmed that the ion exchange rate can be further increased.

[0117] The above description of the present invention is given by way of example only and is intended to be illustrative and not restrictive of the principles of the present invention. A person having knowledge of the present invention may create other specific It is understood that the above embodiment can be easily modified in shape. It should be understood that these are illustrative in all respects and not restrictive. [Industrial Applicability]

[0118] mRNA vaccine containing an adjuvant whose immune activation function is dynamically controlled according to the present invention In the case of co-administration, the adjuvant is activated after the transcription of mRNA is induced. After the mRNA is transcribed into protein, the toll-like receptor agonist, which acts as an adjuvant, is produced. The activation function of the agonist enhances the effect of the mRNA vaccine composition. Since it can significantly improve the expression of ribosomal RNA, it can be widely used in various types of mRNA vaccines. do.

Claims

1. A composition for an mRNA vaccine, comprising an mRNA antigen and an immunoactivator capable of being kinetically controlled as active ingredients, wherein the immunoactivator is a conjugate in which a cleavable linker is bound to an active site of the immunoactivator.

2. The composition for an mRNA vaccine according to claim 1, characterized in that, after administration, the process of transcribing mRNA into protein is carried out, and then the cleavable linker bound to the activation site of the immune activator is cleaved, thereby inducing the activation function of the immune activator.

3. The dynamic control comprises: The immunoactivator is characterized in that a cleavable linker is bound to the activation site of the immunoactivator to maintain the inactive state, and the cleavable linker blocking the activation site is cleaved within 2 to 12 hours after the start of mRNA transcription, resulting in the activity of the immunoactivator appearing in a time-delayed manner. The composition for an mRNA vaccine according to claim 1 .

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

5. The composition for an mRNA vaccine according to claim 1, wherein the cleavable linker further comprises ethylene oxide or ethylene glycol at both or one of its ends.

6. The composition for an mRNA vaccine according to claim 1, characterized in that the cleavable linker is characterized in that the chemical bond at the binding site is cleaved by any one or more factors selected from the group consisting of an enzyme, pH, redox potential, temperature, ultrasound, magnetism, and a light source.

7. The composition for an mRNA vaccine described in claim 1, characterized in that one or more substances selected from the group consisting of cholesterol, lipids, proteins, amino acids, peptides and oligonucleotides are bound to the end of the cleavable linker.

8. The composition for an mRNA vaccine according to claim 1, wherein the immunoactivator is a toll-like receptor agonist.

9. The mRNA vaccine composition according to claim 8, characterized in that the Toll-like receptor agonist is one or more selected from the group consisting of Toll-like receptor 1 agonists, Toll-like receptor 2 agonists, Toll-like receptor 3 agonists, Toll-like receptor 4 agonists, Toll-like receptor 5 agonists, Toll-like receptor 6 agonists, Toll-like receptor 7 or 8 agonists, and Toll-like receptor 9 agonists.

10. The composition for an mRNA vaccine according to claim 1, characterized in that the mRNA antigen and the dynamically controllable immune activator are loaded into one or more drug delivery vehicles selected from the group consisting of nanoliposomes, nanoemulsions, nanomicelles, hydrogels, scaffolds, solid nanoparticles and polymeric nanoparticles.

11. The composition for an mRNA vaccine according to claim 10, characterized in that the drug delivery vehicle is characterized in that the internally loaded mRNA antigen is first delivered to the cytosol, and then the dynamically acting immune activator interacts with a receptor on the cell surface, in an endosome or in a lysosome.

12. The composition for an mRNA vaccine according to claim 10, wherein the drug delivery vehicle further comprises one or more immune activators 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 outer wall component of a pathogen, an alum, lipids, a combination thereof, and a derivative thereof.

13. The composition for an mRNA vaccine according to claim 1, which is 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.

14. 13. Use of the composition of claim 1 for producing a drug for increasing the immune response of an mRNA antigen.

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