Kinetically acting adjuvant ensemble

Abstract

To provide an adjuvant ensemble comprising two or more immune activating substances which activate immune cells and whose action time is kinetically regulated.SOLUTION: The present invention relates to: a kinetically acting adjuvant ensemble of a novel concept capable of maximizing a synergistic immune activation response finally exhibited by adjusting treatment order and a time interval when combining immune activating substances having different signal transduction systems in an immune activation mechanism; and a use thereof. The kinetic action is that a second immune activating substance responds to endogenous factors (enzyme, oxidation-reduction potential, GSH and pH) and exogenous factors (oxidation-reduction, pH, temperature, photo / light, magnetism, ultrasound and electrical response) in immune cells, and a chemical bond of a binding site is cleaved to expose an activated site, thereby kinetically recovering a function, or two or more immune activating substances are sequentially released from a carrier at a certain time interval to induce immune activity.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention provides two or more immune cells that activate immune cells, the duration of which is dynamically regulated. The present invention relates to a novel adjuvant ensemble containing an activating agent, more specifically, after injection, The first immune activator binds to the receptor and induces the primary immune response, and then the second The immune activators bind to the receptors and sequentially induce an immune response. The present invention relates to an adjuvant ensemble designed to be directed against [Background technology]

[0002] The immune response is initiated by activated immune cells responding to foreign and endogenous substances, i.e. antigens. igen), which is a series of reactions that occur against microorganisms, including bacteria and viruses, and living organisms. When a foreign substance enters the body, immune cells recognize it, become activated, and release cytokines and other What factors are secreted to induce an inflammatory response? Recently, congenital Research into the mechanism of immune response is being actively conducted, and among them, Toll-like receptors ( Toll-like receptors (TLRs) are receptors that can recognize pathogens at the early stage of inflammation. It is a receptor that recognizes plasma membrane components and nucleic acid components of pathogens and induces immune responses. It is known that this can be used to identify various Toll-like receptors to activate immune cells. Ligand (Toll Like Receptor ligand; TLR ligan Research on d) is being actively conducted (U.S. Patent Publication No. 2012-0294885).

[0003] 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 the cellulose in water. It is also soluble only in special organic solvents such as DMSO and methanol. It is not soluble in commonly used organic solvents, making it suitable for use as an immune activator in a variety of dosage forms. Therefore, it is difficult to produce a cream-type preparation that contains a variety of surfactants. Some studies have shown that In order to overcome these problems, it is prepared in the form of a salt and dissolved in an aqueous solution. However, the toll-like receptor agonist produced in the form of a salt is not effective in vivo. It is absorbed into the bloodstream and causes a systemic immune response in the bloodstream. By inducing cytokine storms, many side effects (e.g., cytokine storms) can be prevented. okine storm, various non-specific hypersensitivity immune reactions, etc.) In addition, due to the problem of side effects, it is practically impossible to treat the disease. In order to use it, you need to treat it at a concentration less than the effective dose. Some pharmaceutical companies have taken measures to address these issues. To overcome this problem, lipids that exhibit lipophilic properties have been introduced, and polymer chains with large sizes have been used. Attempts have also been made to prevent direct absorption into the bloodstream by direct chemical binding. However, the Toll-like receptor agonists produced by such methods Since the active site of the serovar is still exposed to the outside, it is not able to elicit a non-specific immune response in the body. However, there is still the possibility that induction may induce toxicity.

[0004] Various innate immune inducers, including Toll-like receptor agonists, are mixed. It has been consistently reported that when used in combination with other drugs, the effectiveness of the innate immune response is enhanced. Toll-like receptor ligands have different signal transduction pathways in immune activation mechanisms. When used in combination, the immune cell activation ability is higher than when used alone. It has been reported that the increase is 20-50 fold. However, the signals are different from each other. Innate immune inducers that have a signaling system have different mechanisms, so they are processed differently. The order and / or time interval between treatments significantly affected the effectiveness of the immune activation response. However, several innate immune inducers, namely adjuvants, ant) into the body at regular intervals is not only impractical but also The probability that multiple adjuvants stimulate the same immune cells and increase the immune activation effect is so low that it is practically impossible to predict the occurrence of different types of Administering innate immune inducers (asynchronous treatment) ) is not easy.

[0005] Therefore, multiple adjuvants should be administered simultaneously (synchronous treatment), each adjuvant injected into the body is administered at a fixed time interval. By binding to each receptor, it is possible to determine the time interval for immune activation. By optimizing the adjuvant ensemble, we have developed an adjuvant that can maximize the immune activation effect. If released, its use could significantly improve the effectiveness of vaccines. It is expected that this will have a significant ripple effect on the next-generation vaccine market. 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, Different adjuvants, e.g., Toll-like receptor agonists, saponins, antivirals Sex peptides, inflammasome inducers r), NOD ligand, CDS ligand (cytosolic DNA sensor ligand), STING (stimulator of interferon genes) ligands in a set order and at regular intervals A dynamically acting adjuvant ensemble designed to act in a manner that The purpose of this document is to provide a means for

[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 provides a kinetically acting adjuvant ensemble composition, said composition comprising The vaccine contains two or more adjuvants, the first of which binds to immune cell receptors and The primary adjuvant is to induce the primary immune response by binding to the activating site. The conjugates are sequentially immunized with cleavable linkers. An adjuvant antagonist capable of inducing a secondary immune response by binding to immune cell receptors. A bull composition is provided.

[0009] In one embodiment of the invention, the kinetic effect is at the activation site of the second adjuvant. The cleavable linker is attached to maintain the inactive state, and The activation site is blocked within 12 hours, preferably within 3 to 9 hours. The cleavable linker is cleaved, and the activity of the immunoactivator appears in a time-delayed manner. The adjuvant ensemble composition of the present invention is characterized by comprising two or more immune activators. When administered simultaneously, the substances provide a dynamic ensemble that acts at regular time intervals. By providing the above-mentioned power, the synergistic effect of the immunological response can be maximized. The scientific ensemble is a molecular scale and / or It is capable of acting on a macro scale.

[0010] In another embodiment of the invention, the cleavable linker is preferably a disulfide. (disulfide), carbamate, hydrazine azine, ester, peptide, azide de), amide, hydrazone, thioether (t hioether, phosphodiester, thioketater any one selected from the group consisting of thioketals and combinations thereof However, endogenous factors (enzymes, redox potential, GSH, pH, etc.) and / or exogenous factors (redox, pH, temperature, photo / light, magnetism, ultrasound, electrical response However, the present invention is not limited thereto, so long as the bond can be cleaved by a bond such as ive).

[0011] 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:

[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 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 second a It serves to block the active portion of the adjuvant and has a hydrophilic or lipophilic group. The material can be a variety of substances.

[0014] In yet another embodiment of the present invention, the second adjuvant is a nanoliposome, a nano Emulsions, nanomicelles, hydrogels, scaffolds, solid nanoparticles and polymers The drug delivery system is loaded with one or more drug delivery agents selected from the group consisting of nanoparticles. The loading is characterized in that it is simply encapsulated regardless of binding. or may be sandwiched between or attached to the nanoparticle structure. However, as long as it is in a form that contains the mRNA antigen of the present invention and an immunoactivating substance, Not limited to:

[0015] In yet another embodiment of the invention, the drug delivery vehicle further comprises a first adjuvant. It is characterized by the fact that

[0016] In yet another embodiment of the invention, the drug delivery vehicle is attached to the surface or endonucleases of immune cells. It is particularly notable that the compound further comprises a ligand that reacts with a receptor present in the endosome or cytosol. It is a sign.

[0017] In yet another embodiment of the present invention, the drug delivery vehicle is a Toll-like receptor agonist, Saponins, antiviral peptides, inflammasome inducers ome inducer, NOD ligand, CDS ligand (cytosolic DNA sensor ligand), STING(stim interferon gene regulator ligand, outer wall component of pathogenic bacteria , alum, lipids, combinations thereof and similar Any one or more immunostimulatory substances selected from the group consisting of derivatives The present invention is characterized by further comprising:

[0018] In yet another embodiment of the invention, the second adjuvant is preferably a Toll-like Receptor agonist (toll-like receptor agonist) More preferably, a toll-like receptor 1 agonist, a toll-like receptor 2 agonist, 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 receptor and / or a combination of these. .

[0019] In yet another embodiment of the invention, the first adjuvant is a Toll-like receptor agonist. Streptococcus esculentus, saponin, antiviral peptide, inflammasome inducer, NOD ligand , CDS ligands, STING ligands, outer wall components of pathogens, alum, lipids, and these Any one or more of the immune activators selected from the group consisting of combinations and analogs thereof It can be a substance.

[0020] In yet another embodiment of the invention, the immune cells are antigen presenting cells (dendrit ic cells, macrophages, natural killer cells (NK cells) , T cells, B cells, regulatory T cells, MDSCs ( myeoloid derived suppressor cells), and M2 The cell is characterized in that it is one or more selected from the group consisting of macrophages.

[0021] In yet another embodiment of the invention, the drug delivery vehicle comprises two or more adjuvants in sequence. It may be an ensemble designed to be released next, Structures with two or more layers, such as somes, micelles, emulsions, self-assembled particles, and polymer nanoparticles Core-shell structure with layered structure ), and is a structure containing a firstly release adjuvant. ase of payload and secondarily released adjuvants release of payload).

[0022] In yet another embodiment of the present invention, the drug delivery vehicle is stimuli responsive. The stimulation may include a blockade of endogenous factors in the cells. (enzymes, redox potential, GSH, pH, intracellular proteins, etc.), extrinsic factors (redox , pH, temperature, photo / light, magnetic, ultrasonic, electrical response esponsive) and various physiological environments / immune factors in the body. The stimulus-responsive block may include two or more kinds of stimulus-responsive blocks. They can respond sequentially to different stimuli, or to different intensities of stimuli. It is also possible to react sequentially.

[0023] The present invention also relates to a method for treating infectious diseases comprising the adjuvant ensemble composition as an active ingredient. Infectious disease, cancer, metabolic syndrome metabolic syndrome), autoimmune disease (metabolic syndrome), autoimmune disease (metabolic syndrome), autoimmune disease A pharmaceutical composition for the prevention or treatment of a rare disease The composition is provided.

[0024] In one embodiment of the invention, the pharmaceutical composition is an antigen, a chemotherapeutic agent, or an immunotherapy. It may further include a checkpoint inhibitor.

[0025] In another embodiment of the invention, the antigen is a protein, a recombinant protein, a glycoprotein. Proteins, genes, peptides, polysaccharides, lipopolysaccharides, polynucleotides, cells, cell lysates ( lysate), bacteria and viruses. It is characterized by:

[0026] In yet another embodiment of the present invention, the pharmaceutical composition is for use in preventing cancer growth, metastasis, recurrence or or suppressing resistance to anti-cancer therapeutic therapies.

[0027] The present invention also provides a composition comprising the adjuvant ensemble composition 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

[0028] The present invention also relates to a composition comprising the adjuvant ensemble composition as an active ingredient. Infectious diseases, cancer, metabolic diseases metabolic syndrome, autoimmune disease For the prevention or treatment of rare diseases to provide.

[0029] The present invention also relates to a method for treating infectious diseases using the adjuvant ensemble composition. ous disease, cancer, metabolic syndrome syndrome, autoimmune disease or rare To produce drugs used to prevent or treat rare diseases Provides uses for. Effect of the Invention

[0030] In the present invention, the novel adjuvant whose duration of action is dynamically adjusted includes Compared to the conventional simultaneous administration of two substances, this not only increases the synergistic effect, but also This minimizes potential toxicity issues of adjuvants. The combination of two or more immune activators selected from the above has been shown to have improved immune enhancement. Due to its effectiveness, it can be used for anti-cancer vaccines and the effective prevention and treatment of various diseases, including infectious diseases. It is expected that this compound can be widely used as a therapeutic agent. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 is a conceptual diagram showing a schematic diagram of an adjuvant ensemble for dynamic immune function control on a macroscale. [Diagram 2] Figure 2 shows an example of an adjuvant (Toll-like receptor 7 / 8 agonist) for dynamic immune function control on a molecular scale. The upper part shows a conceptual diagram of a dynamically acting Toll-like receptor 7 / 8 agonist, and the lower part shows the characteristics of a cleavable linker. [Diagram 3] FIG. 3 is a schematic diagram of the mechanism by which the drug exerts an effect different from that of conventional immune function regulators after acting on autoimmune cells (dendritic cells) that are dynamically controlled by cholesterol-toll-like receptor 7 or 8. [Figure 4] FIG. 4 is a diagram showing the results of confirming the intracellular uptake of nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate according to one embodiment of the present invention in terms of the amount of IL-12. [Diagram 5] FIG. 5 shows the results of RT-PCR confirming whether GILT is expressed in antigen-presenting cells according to one embodiment of the present invention. [Figure 6] FIG. 6 shows the results of confirming whether the chemical bond of a nanoliposome containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate according to one embodiment of the present invention is cleaved by GILT. [Figure 7] FIG. 7 shows the results of confirming immune cell regulation by nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate according to one embodiment of the present invention. [Figure 8] FIG. 8 is a diagram showing the results of confirming the effect of a nanoliposome containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate according to one embodiment of the present invention on the maturation of dendritic cells. [Figure 9] FIG. 9 is a diagram showing the results of confirming the sustained immune response induction effect of a nanoliposome containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate according to one embodiment of the present invention. [Figure 10] FIG. 10 is a diagram showing the results of confirming the effect of nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate according to one embodiment of the present invention on inducing differentiation of CD4+ T cells into Th1 response. [Figure 11] FIG. 11 is a diagram showing the results of confirming the effect of combined administration of a Toll-like receptor 3 agonist and a Toll-like receptor 7 or 8 agonist according to one embodiment of the present invention in terms of the amount of IL-12 secreted. [Figure 12] FIG. 12 is a diagram showing the results of confirming the effect of combined administration of a Toll-like receptor 4 agonist and a Toll-like receptor 7 or 8 agonist according to one embodiment of the present invention in terms of the amount of IL-12 secreted. [Figure 13] FIG. 13 is a diagram showing the immune activation effect of the adjuvant ensemble according to one embodiment of the present invention, as confirmed by the secretion levels of TNF-α and IL-6. [Figure 14]FIG. 14 is a diagram showing the results of confirming the degree of cell maturation caused by the adjuvant ensemble according to one embodiment of the present invention based on the expression level of costimulatory molecules. [Figure 15] FIG. 15 is a diagram showing the results of confirming the effect of inducing a sustained immune response by an adjuvant ensemble according to one embodiment of the present invention. [Figure 16] FIG. 16 shows the results of confirming the degree of stabilization of the adjuvant complex according to one embodiment of the present invention by EMSA. [Figure 17] FIG. 17 is a diagram showing the results of confirming the intracellular stability of the adjuvant complex according to one embodiment of the present invention. [Figure 18] FIG. 18 is a diagram showing the in vivo results of confirming the efficacy of an adjuvant complex according to an embodiment of the present invention in inducing a sustained immune response. [Figure 19] FIG. 19 is a diagram showing the in vivo results of confirming the antitumor effect of the adjuvant complex according to one embodiment of the present invention. [Figure 20] FIG. 20 shows the results of analyzing immune cells collected in tumor tissues and lymph nodes using an adjuvant complex according to one embodiment of the present invention. [Figure 21] FIG. 21 shows the results of analyzing immune cells collected in tumor tissues and lymph nodes using an adjuvant complex according to one embodiment of the present invention. [Figure 22] FIG. 22 shows the results of analyzing immune cells collected in tumor tissues and lymph nodes using an adjuvant complex according to one embodiment of the present invention. [Diagram 23] FIG. 23 is a diagram showing the results of analyzing immune cells collected in tumor tissues and lymph nodes using an adjuvant complex according to one embodiment of the present invention. [Figure 24] FIG. 24 is a diagram showing the results of confirming the immune response induction effect in lymph nodes by the adjuvant complex according to one embodiment of the present invention, based on the cytokine secretion ability. [Diagram 25]FIG. 25 is a diagram showing the results of confirming the immune response induction effect in tumor tissues by the adjuvant complex according to one embodiment of the present invention, based on the cytokine secretion ability. [Figure 26] FIG. 26 is a diagram showing the results of confirming the effect of IL-12 on the immune activation efficacy of an adjuvant complex according to one embodiment of the present invention. [Figure 27] FIG. 27 is a diagram showing the results of confirming the antitumor efficacy against metastatic cancer through local injection of an adjuvant complex according to one embodiment of the present invention. [Figure 28] FIG. 28 is a diagram showing the results of confirming the inhibitory effect on pulmonary metastasis via local injection of an adjuvant complex according to one embodiment of the present invention. [Figure 29] FIG. 29 is a diagram showing the results of confirming the antitumor efficacy against orthotopic tumors via local injection of an adjuvant complex according to one embodiment of the present invention. [Diagram 30] FIG. 30 is a diagram showing the results of confirming the antitumor efficacy through the combination of an adjuvant complex according to one embodiment of the present invention and immune checkpoint inhibitor therapy. [Diagram 31] FIG. 31 is a diagram showing the results of confirming the antitumor efficacy through the combination of an adjuvant complex according to one embodiment of the present invention and chemotherapy. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] [Best Mode for Carrying Out the Invention] The present inventors have demonstrated that when two or more adjuvants are used in combination, the synergy of the adjuvants It was confirmed that the order and time difference are important factors in the drug effect, and When using drugs to induce immune activation, in order to maximize the effect, Kinetic control that can adjust the activation time of the adjuvant As a result of intensive research into adjuvant ensemble systems that can The adjuvant acts primarily, and the second adjuvant binds a cleavable linker at the activation site. The activity of the agonist is temporarily inhibited by the agonist, and after administration to the target tissue or After reaching the cells, i.e., within 2 to 12 hours, the linker is cleaved and the secondary Inventing an adjuvant ensemble composition that allows dynamic control of adjuvant activity did.

[0033] As shown in FIG. 1 and FIG. 2, the adjuvant ensemble composition of the present invention comprises a molecular Molecular scale and macro scale e) can be regulated in both directions and by the incorporation of a cleavable phosphoryl group into the activation site of the second adjuvant. Carriers are bound and remain inactive for 2 to 12 hours. Preferably, the cleavable cleavable nucleotide that has blocked the activation site is produced within 3 to 9 hours. This is a system in which the linker is cleaved and the activity of the immune activator appears with a time delay. Thus, the synergistic effect of the immunological response can be maximized.

[0034] In addition, as shown in FIG. 3, the adjuvant ensemble composition of the present invention inhibits dendritic cells. The immune system is not exhausted, i.e., does not induce an immune response, but is in a state of success. By maintaining the cells in a maturing state, i.e. in a state where they can induce an immune response, for a long period of time, They can induce sustained immune responses and encounter naive T cells in lymph nodes. In addition, in the process of presenting antigens, CD40-CD40L functions to induce strong immune activation. This allows for effective induction of Th1 immune response-inducing cytokines such as IL-12. do.

[0035] As used herein, the term "adjuvant" refers to an immunostimulatory agent. It is a stimulant for the immune system, stimulating normal immune function and The term "adjuvant" refers to substances that act to induce or restore immune responses. It means a substance used together with an antigen to improve the It can increase antibody production and increase humoral and / or cellular immunity. The immune activator is preferably a toll-like receptor agonist. like receptor agonist), saponin, antiviral peptide, Inflammasome inducer, NOD linker NOD ligand, CDS ligand (cytosolic DNA sequence) nsor ligand), STING (stimulator of interfe ron genes ligand, emulsion, alum ), incomplete Freund's adjuvant, Freund's adjuvant, or a combination thereof or, more preferably, a Toll-like receptor agonist.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] The Toll-like receptor 4 agonist can induce signaling responses via TLR-4. Ligands are used as an example for Shigella flexneri. eri) outer membrane protein products, AGP, CRX-527, MPLA, PHAD, 3D The agonist may be, but is not limited to, -PHAD, GLA, LPS, etc.

[0041] The Toll-like receptor 5 agonist can induce signaling responses via TLR-5. An example of such a ligand is flagellin. Good, but not limited to this.

[0042] 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.

[0043] 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. are 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.

[0044] 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:

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] As used herein, the term "cleavable linker" refers to a It contains a cleavable bond and is expressed 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 As long as the linker has a structure, it is not limited thereto. An example of a cleavable linker is a linker that can be cleaved by an enzyme. Possible linker groups include tobacco etch virus protease (TE V), trypsin, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase Ze1, matrix metalloproteinase sequence, phosphodiesterase ester, phospholipid, ester, β-galactose ) and the linker group that can be cleaved by nucleophilic / base agents is dialkyldialco Xysilane, cyanoethyl group, sulfone, ethylene glycolyl disulfide Cinate (ethylene glycolyl disuccinate), 2-N- 2-N-acyl nitrobenzenesulfonamide sulfonamide, a-thiophenyl ester er), unsaturated vinyl sulfide e) Sulfonamide after activation tion), malonic aldehyde (MDA)-indole derivative, levulinoyl ester (levulinoyl ester), hydrazone, acyl acylhydrazone, alkyl thioester The linker group that can be cleaved by reducing agents is disulfide. The linker groups that can be cleaved by oxidizing agents include amide bridges and azo compounds. Vicinal diols, selenium compounds, etc., organometallic or metallic Cutting with a catalyst (organometallic or metal catalyst) Possible linker groups include disulfide bridges, azo compounds, etc. Also electrophilic / The acid-cleavable linker group is a para-methoxybenzyl derivative (Param ethoxybenzyl derivative), tert-butyl carbamate Analogue (tert-butylcarbamate analogue), dialkylsilane Dialkyl or Dialkoxy Silane Silane, orthoester, acetal ), aconityl, hydrazone, b-thiopeptide b-thiopropionate, phosphoramidate oramidate, imine, trityl, vinyl amine vinyl ether, polyketal, alkyl 2-( Diphenylphosphino)benzoate derivatives (alkyl 2-(diphenylphosphino)benzoate derivatives These include osphino and benzoate derivatives, which are activated by light irradiation. The cleavable linker group is a 2-nitrobenzyl derivative. yl derivatives, phenacyl esters ), 8-quinolinylbenzenesulfonic acid sulfonate, coumarin, phosphate triester hotriester, bis-arylhydrazone ne), bimane bithiopropionic acid derivatives onic acid derivatives).

[0053] As used herein, "co-administration" refers to administration of a Toll-like receptor 7 or 8 agonist and a cholesterol-lowering agent. Teratol conjugates and antigens, immune checkpoint inhibitors, immune antigen boosters, immune activators It is administered together with various substances such as cancer drugs and chemical anticancer drugs, and there are no restrictions on the type and form. There is no limit.

[0054] As used herein, "chemo-cancer agent" refers to any agent used in the treatment of cancer known to those skilled in the art. There are no restrictions as long as the compound is well-known. Examples include Paclitaxel, Doc etaxel, 5-Flurouracil, Alendronate, Doxorub icin, Simvastatin, Hydrazinocurcumin, Ampho Tericin B, Ciprofloxacin, Rifabutin, Rifamp icin, Efavirenz, Cisplatin, Theophyline, Pse udomonas exotoxin A, Zoledronic acid, Trab ectedin, Siltuximab, Dasatinib, Sunitinib, A patinib, 5,6-Dimethylxanthenone-4-acetic acid, Silibinin, PF-04136309, Trabectedin, C arlumab, BLZ945, PLX3397, Emactuzumab, AMG-8 20, IMC-CS4, GW3580, PLX6134, N-acetyl-l-cys tein, vitamin C, bortezomib, aspirin, salicy lates, indolecarboxamide derivatives, quin azoline analogues, thalidomide, prostaglan din metabolites, 2ME2, 17-AAG, Camptothecin , Topotecan, Pleurotin, 1-methylpropyl, 2-im idazolyl disulfide, Tadalafil, Sildenafi l, L-AME, Nitroaspirin, Celecoxib, NOHA, Bard Oxolone methyl, D, L-1-methyl-tryptophan, G emcitabine, axitinib, sorafenib, cucurbitac in B, JSI-124, Anti IL-17 antibodies, Anti- glycan antibodies, Anti-VEGF antibodies, B evacizumab, Antracycline, Tasquinimod, Imat These include, but are not limited to, inib and cyclophosphamide.

[0055] As used herein, the term "immune checkpoint inhibitor" refers to a "NT inhibitor" is a drug that activates the immune function of immune cells in the human body. A general term for cancer treatment methods that fight cancer cells. Examples include anti-PD-1 and anti- PD-L1, anti-CTLA-4, anti-KIR, anti-LAG3, ant i-CD137, anti-OX40, anti-CD276, anti-CD27, a nti-GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L , anti-BTLA, anti-TCR, anti-TIGIT, etc. Not limited.

[0056] As used herein, the term "antigen" refers to anything that causes an immune response in the body. The term generally refers to substances such as pathogens (bacteria, viruses, etc.), chemicals, pollen, Cancer cells, shrimp, etc., or partial peptides or proteins thereof, are more preferred. This is a cancer antigen peptide, but any substance that can cause an immune response in the body can be used. The antigen is preferably a protein, a recombinant protein, a glycoprotein, or a combination thereof. Proteins, genes, peptides, polysaccharides, lipopolysaccharides, polynucleotides, cells, cell lysates ( lysate), bacteria, viruses, etc., and more preferably, cancer antigens. The glycoprotein may be an antibody, a fragment of an antibody, a structural protein, a regulatory protein, or a peptide. proteins, transcription factors, toxic proteins, hormones, hormone analogs, enzymes, enzyme fragments, Transport protein, receptor, receptor fragment, host defense inducer, storage Protein, movement protein, Xploite Exploitive proteins, reporter proteins, etc. However, if the substance acts as an antigen in the body and induces an immune response, , but is not limited to this.

[0057] 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.

[0058] 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

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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 a 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.

[0065] As used herein, the term "pharmaceutical composition" refers to a "Medicine (production)" refers to the form of capsules, tablets, granules, injections, ointments, powders or beverages. The pharmaceutical composition is characterized in that it is intended for humans. The target composition can be prepared by a conventional method, such as, but not limited to, powder, granules, etc. Granules, capsules, tablets, oral dosage forms such as aqueous suspensions, topical preparations, suppositories and sterile injection solutions The pharmaceutical composition of the present invention may be formulated and used in the form of a pharma- ceutical acceptable The pharmaceutical composition may contain a carrier. Pharmaceutically acceptable carriers include binders, lubricants, and Disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavors, etc. may be used. In the case of injections, it is possible to use buffering agents, preservatives, analgesics, solubilizers, isotonicity agents, stabilizing agents, etc. For topical administration, the drug may be used in combination with a base, excipient, lubricant, etc. Preservatives and the like can be used. The dosage form of the pharmaceutical composition of the present invention can be prepared by adding the above-mentioned pharmaceutical agent. It can be prepared in various forms by mixing with a pharma- ceutical acceptable carrier. For example, Tablets, troches, capsules, elixirs, suspensions, syrups In the case of injections, they can be manufactured in the form of unit dose ampoules or Other types of dosage forms include solutions, suspensions, tablets, capsules, and sustained release. It can be formulated as a release preparation.

[0066] 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.

[0067] The administration route of the pharmaceutical composition according to the present invention includes, but is not limited to, oral, Intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal The term "oral" refers to a pharmaceutical composition that is administered orally, topically, sublingually or rectally. Oral or parenteral administration is preferred. The term "parenteral" includes any of the following: subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrathoracic, intravenous, intravenous, intravenous. This includes intraosseous, intradural, intralesional and intracranial injection or infusion techniques. It may also be administered in the form of suppositories for rectal administration.

[0068] The pharmaceutical compositions of the present invention may be prepared in a variety of ways, depending on the activity of the particular compound used, age, weight, general health, Gender, diet, time of administration, route of administration, excretion rate, drug combination and specific disease being prevented or treated The dosage of the pharmaceutical composition may vary depending on various factors, including the severity of the disease. varies depending on the patient's condition, weight, degree of illness, drug form, route of administration and duration. Those skilled in the art can select the appropriate dose, which can be 0.0001 to 500 mg / kg or 0. The dose can be 0.01 to 500 mg / kg. It can also be administered once a day. The dosage may be administered in several divided doses. The pharmaceutical or vaccine composition according to the present invention may be in the form of a pill, sugar, or the like. It can be formulated as coated tablets, capsules, liquids, gels, syrups, slurries, and suspensions. In specific embodiments, the present invention may include the following aspects. [Section 1] 1. A kinetically acting adjuvant ensemble composition comprising: The composition comprises two or more adjuvants; The first adjuvant first binds to immune cell receptors to induce the primary immune response, The second adjuvant is a conjugate having a cleavable linker bound to the activation site, which sequentially binds to immune cell receptors to induce a secondary immune response. Adjuvant ensemble compositions. [Section 2] The kinetic action is The second adjuvant is characterized in that a cleavable linker is bound to the activation site of the second adjuvant, and the second adjuvant is maintained in an inactive state, and the cleavable linker that has been blocking the activation site is cleaved within 2 to 12 hours, thereby causing the activity of the immune activator to appear in a delayed manner. Item 1. The adjuvant ensemble composition according to item 1. [Section 3] The cleavable linker comprises any one or more bonds selected from the group consisting of disulfide, carbamate, hydrazine, ester, peptide, azide, amide, hydrazone, thioether, phosphodiester, thioketal, and combinations thereof. The adjuvant ensemble composition according to item 1. [Section 4] Item 2. The adjuvant ensemble composition according to item 1, wherein the cleavable linker further comprises ethylene oxide or ethylene glycol at both or one of its ends. [Section 5] Item 2. The adjuvant ensemble composition 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 enzymes, pH, redox potential, temperature, ultrasound, magnetism, and light source. [Section 6] Item 2. The adjuvant ensemble composition 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 end of the cleavable linker. [Section 7] Item 1, wherein the second adjuvant is 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 8] Item 8. The adjuvant ensemble composition according to item 7, characterized in that the drug delivery vehicle further comprises a first adjuvant. [Section 9] Item 8. The adjuvant ensemble composition according to Item 7, wherein the drug delivery vehicle further comprises a ligand that reacts with a receptor present on the surface of an immune cell or in an endosome or cytosol. [Section 10] The drug delivery system is Item 8. The adjuvant ensemble composition according to Item 7, further comprising any 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 11] Item 2. The adjuvant ensemble composition according to item 1, wherein the second adjuvant is a toll-like receptor agonist. [Section 12] Item 2. The adjuvant ensemble composition according to item 1, wherein the first adjuvant is any 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 1, wherein the immune cells are any one or more selected from the group consisting of antigen-presenting cells (dendritic cells, macrophages), natural killer cells (NK cells), T cells, B cells, regulatory T cells, MDSCs (myeoloid derived suppressor cells), and M2 macrophages. The adjuvant ensemble composition according to item 1. [Section 14] A pharmaceutical composition for preventing or treating an infectious disease, cancer, metabolic syndrome, autoimmune disease, or rare disease, comprising the adjuvant ensemble composition according to claim 1 as an active ingredient. [Section 15] Item 15. The pharmaceutical composition according to item 14, further comprising an antigen, a chemical anticancer agent, or an immune checkpoint inhibitor. [Section 16] Item 16. The pharmaceutical composition according to item 15, wherein the antigen is one or more selected from the group consisting of a protein, a recombinant protein, a glycoprotein, a gene, a peptide, a polysaccharide, a lipopolysaccharide, a polynucleotide, a cell, a cell lysate, a bacterium and a virus. [Section 17] Item 15. The pharmaceutical composition according to item 14, wherein the pharmaceutical composition inhibits cancer proliferation, metastasis, recurrence, or resistance to anti-cancer treatment. [Section 18] Item 1. A method for preventing or treating an infectious disease, cancer, metabolic syndrome, autoimmune disease, or rare disease, comprising administering to an individual a composition comprising the adjuvant ensemble composition according to item 1 as an active ingredient. [Section 19] Item 1. A composition comprising the adjuvant ensemble composition according to item 1 as an active ingredient for the prevention or treatment of an infectious disease, cancer, metabolic syndrome, autoimmune disease, or rare disease. [Section 20] Item 1. Use of the adjuvant ensemble composition according to item 1 for producing a drug for the prevention or treatment of an infectious disease, cancer, metabolic syndrome, autoimmune disease or rare disease.

[0069] In the following, preferred embodiments are presented to aid in understanding the present invention. The following examples are provided so that the present invention may be more readily understood. Therefore, the contents of the present invention are not limited thereto. EXAMPLES

[0070] Example 1: Synthesis of dynamically acting cholesterol-toll-like receptor agonists ] Initially, it exists in an inactive form, but when it enters the body, it becomes activated. After being delivered to target cells such as tumor microenvironment and immune cells, they are exposed to physiological environments (low pH, enzymes, etc.). It is converted into an activated state by the action of vitamin C, glutathione, etc., and shows immune activation effects. The activity can be kinetically controlled. To produce Toll-like receptor agonists (TLR agonists) To this end, we investigated various Toll-like receptor 7 or 8 agonists (imidazoquinoline agonists ( imidazoquinoloine-based agonist), hydroxyadenosine 8-hydroxyadenine-based agonist , pteridone-based agonist, amine 2-aminopyrimidine-based agonists benzoazepine-based agonists gonist), thiaoxoguanosine agonist (7-thia-8-oxogua The active site of the nosine-based agonist te), i.e., a conjugate in which cholesterol is bound to the amine group (NH2) site (co The toll-like receptor (TLR) was prepared by the chemical reaction shown in Reaction Scheme 1 or 2 below. The bond between the 7 or 8 agonist and cholesterol is a cleavable bond. Carbamates and disulfides of e), ester, peptide, or azide e) Conjugated to cholesterol (Sigma-Aldrich) via linkage.

[0071] [ka]

[0072] 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.

[0073] [ka]

[0074] 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.

[0075] [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).

[0076] [ka]

[0077] [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. 1 H 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).

[0078] [ka]

[0079] [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).

[0080] [ka]

[0081] [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 100 ml of benzoic acid (350 ml) and metachloroperoxybenzoic 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 it with sodium (30g), it was filtered and then stored under 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. 1 H 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).

[0082] [ka]

[0083] [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).

[0084] [ka]

[0085] [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).

[0086] [ka]

[0087] [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. HCl in n-hexane to obtain compound 9 (20 g, 39.6%, yellow gel). 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, 6 H), 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=6.4H z, 3H), 0.88(dd,J=1.6,6.8Hz, 6H), 0.69(s, 3H) .

[0088] [ka]

[0089] [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 filtered 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).

[0090] [ka]

[0091] [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).

[0092] [ka]

[0093] [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.

[0094] (11.1. Nanoparticles containing cholesterol-bound Toll-like receptor 7 or 8 agonists (Production of liposomes) Nanoliposomes containing cholesterol-Toll-like receptor 7 or 8 agonist conjugates ( In order to prepare nanoliposomes, cholesterol was prepared in the same manner as in Example 1. Cholesterol-Toll-like receptor 7 or 8 agonist complex Nanoliposomes were prepared using the chymotrypsin conjugate. More specifically, 0.5 ml of chloroform was added to the 5 mg of DOPC (1,2-dioleoyl-sn- glycero-3-phosphocholine, Avanti), 1.5 mg D DAB (dimethyldioctadecylammonium bromide, Avanti) and 1 mg of cholesterol-resiquimod conjugate were dissolved and mixed. The mixture was evaporated in a rotary evaporator. The chloroform was evaporated using a torr oven for 30 min to prepare a thin film of multiple layers. Then, 2 ml of phosphate buffered saline was added to the mixture. The mixture was then stirred at room temperature for 2 hours using a tip ultrasonic disintegrator (T Homogenization step using ip sonication (amplitude: 20%, 2 min) After obtaining unilamellar liposomes through the above process, a mini-extrusion process was performed to homogenize the nanoliposomes. Stable cholesterol-toll-like receptor 7 was obtained by a mini extruder. Nanoliposomes containing the agonist conjugate were prepared. , Ultraviolet and Visible Spectroscopy (UV-Vis spectrophotomete r) using an internalized cholesterol-toll-like receptor 7 or 8 agonist The amount of was quantified.

[0095] (11.2. Nanoparticles containing cholesterol-bound Toll-like receptor 7 or 8 agonists (Production of emulsion) Nanoemulsions containing cholesterol-Toll-like receptor 7 or 8 agonist conjugates In order to prepare nanoemulsion, collagen was prepared in the same manner as in Example 1. Cholesterol-toll-like receptor 7 or 8 agonist complex The Siquimod conjugate was used to prepare nanoemulsions. More specifically, 1 ml of chloroform was added to 1 mg DOPC, 240 μg cholesterol (Sigma-Aldrich) 100 μg of cholesterol-resiquimod conjugate was added, dissolved, and mixed. The mixture was then transferred into a round-bottom flask and then rotary evaporated. Completely evaporate the chloroform using a rotary evaporator. Then, a thin lipid film was prepared. ml with Squalene (5% v / v), Tween 80 (0.5% v / v), and After adding Span 85 (0.5% v / v) and dissolving it, the solution was applied onto the lipid membrane. The mixture was added, dispersed for 1 minute using a Tip sonicator, and then cooled using a rotating device. Stir for about 2 hours using a tube revolve to mix the cholesterol-tol After preparing the nanoemulsion containing the receptor 7 or 8 agonist conjugate, and stored in a refrigerator at 4°C.

[0096] 11.3. Cholesterol-bound Toll-like receptor 7 or 8 agonists and synapses Preparation of nanomicelles composed of ponin Consisting of cholesterol-Toll-like receptor 7 or 8 agonist conjugate and saponin To prepare the nanomicelle, the same method as in Example 1 was used. One of the cholesterol-Toll-like receptor 7 or 8 agonist conjugates we have constructed is cholesterol. Nanomicelles were prepared using phosphatidylcholine-resiquimod conjugates. Zycorin:saponin:cholesterol-resiquimod conjugate mixed in a weight ratio of 5:3:2 After that, add it to ether to a concentration of 14 mg / ml, dissolve it, and Then, saponin was dissolved in 4 ml of distilled water at a concentration of 1.5 mg / ml. After dissolving it in water, place it in a 20 ml glass bottle, seal the glass bottle with a rubber stopper, and then pour the mixture into a 55 ml flask. The mixture was stored in a water jacket at 10 °C. Next, 1 ml of the lipid-containing ether solution was The solution was added to the glass bottle containing saponin at a rate of 0.2 ml / min using a pump. The solution was stirred for 2 hours. During this time, the tip of the syringe needle was positioned below the surface of the aqueous solution containing saponin. The second needle was inserted into the rubber stopper for ventilation. Next, the glass bottle was transferred to room temperature. After stirring for 3 days to stabilize the mixture, cholesterol-bound resiquimod and Nanomicelles composed of saponin and glycerin were prepared.

[0097] (11.4. Cholesterol-bound Toll-like receptor 7 or 8 agonist (Production of Polymer Nanoparticles) Polymeric nanoparticles composed of cholesterol-Toll-like receptor 7 or 8 agonist conjugates To prepare the particles, cholesterol-toll-like receptor was prepared in the same manner as in Example 1. The cholesterol-resiquimod conjugate, one of the 7 or 8 agonist conjugates, was used to We produced molecular nanoparticles. More specifically, we synthesized lactide and glycolide nanoparticles. lycolide) composition ratio of 50:50 PLGA polymer (Eudragit) 60mg was dissolved in 1 ml of chloroform. Next, 5 mg of cholesterol-resiquimod bond was The coalescence was added to a solvent and subjected to ultrasonic cleaning (ultrasonic bath, Emerson Model CPX5800H-E was used to measure the cholesterol-resiquimod conjugate and The molecule was dissolved. Next, the solution was added to 10 ml of 2.5% PVA aqueous solution for 20 minutes. 0 μl at a time while dispersing with an ultrasonic disperser (Tip sonicator, Sonics & Microelectronics, Inc.). The mixture was dispersed for 1 minute using a 300V ferrite core (Model VCX 750). The power of the disperser was 750 watts, the vibration intensity was 20 kHz, and the Amplitude The de was set to 20%. Next, the organic solvent in which the PLGA was dissolved was completely evaporated. The aqueous solution was stirred at 600 rpm for more than 8 hours at room temperature. To remove the esterol-resiquimod conjugate, The mixture was centrifuged at 12,000 rpm for 12 minutes using a centrifuge (Hanil, Combi-514R). After centrifuging and removing the supernatant, 10 ml of ultrapure water was added and the mixture was placed in an ultrasonic disperser for 30 seconds. The above process was repeated three times, and then the mixture was dried using a freeze-drying method. Stored at 20°C.

[0098] [Example 12: γ-interactin in endolysosomes Dynamically acting cholesterol by ferron-induced lysosomal thiol reductase - Confirmation of chemical bond cleavage effects of Toll-like receptor 7 or 8 agonists] 12.1 Dynamically Acting Cholesterol Toll-Like Receptor 7 or 8 Agonists Intracellular delivery of nanoliposomes containing Cholesterol and resiquimod prepared in the same manner as in Example 11.1 were dissolved in water to form a disulfide. Nanoliposomes containing the conjugates linked by disulfide bonds are To confirm whether the antibody migrates into cells via endocytosis, bone marrow derived dendritic cell In particular, we used D, which suppresses intracellular transfer into BMDCs. The cells were treated with ynasore (40 μg) and incubated for 1 hour. Nanoliposomes containing agonist conjugates of L-like receptor 7 or 8 were treated with 4, 8, 12, and The cell culture medium was harvested after 1, 5 and 24 hours, respectively. After centrifugation at 1000 rpm for 3 minutes to separate the cells from the supernatant, The amount of interleukin-12 (IL-12) was measured by enzyme immunoassay. All experiments were then repeated at least three times, and the results were The values ​​are shown as mean ± standard deviation. Statistical significance was determined by Student's t-test. The results were confirmed by FTIR and were considered to be statistically significant if P<0.05. The results are shown in Figure 4. did.

[0099] As shown in Figure 4, the intracellular import inhibitor-treated group had a very low amount of I These results confirmed that cholesterol-toll-like receptors secrete L-12. Nanoliposomes containing agonist conjugates of agonists 7 and 8 were successfully transported into cells and inhibited immune activation. It was confirmed that the activation reaction was observed.

[0100] 12.2. Gamma interferon-induced lysosomal protein expression in BMDCs and macrophages (Checking for the presence or absence of murine thiol reductase expression) γ-Interferon-induced lysosomal thiol reduction in BMDCs and macrophages Gamma-interferon-inducible lysosomal enzyme To confirm whether BMD expresses GILT (Glycine thiol reductase), The experiment was carried out using C and macrophage RAW 264.7 cells. Total ribonucleic acid (RNA) from BMDCs and RAW 264.7 cells was analyzed using Trizol reagent. The isolated RNA was then used as a template to generate complementary deoxyribonucleic acid (cDNA ) were synthesized using the Maxime RT PreMix tool. Real-time polymerase chain reaction (RT-PCR) was performed using the cDNA obtained, and PCR was performed using the The R product was analyzed by agarose gel electrophoresis. The expression level of GILT was confirmed by lysis, and the results are shown in Figure 5.

[0101] As shown in FIG. 5, antigen presenting cells l) We confirmed that GILT is expressed in dendritic cells and macrophages.

[0102] (12.3. Dynamic action of cholesterol by GILT - Toll-like receptor 7 or or 8) Confirmation of chemical bond cleavage of agonist Disulfide chemical conjugation of cholesterol with Toll-like receptor 7 or 8 agonists Disulfide bonds are formed by GILT present in endolysosomes. To confirm whether cleavage occurs, the same method as in Example 11.1 was used. The produced cholesterol and resiquimod form a disulfide bond. 50 μl of nanoliposomes containing the conjugate bound in d) was placed in a 5 ml tube. Cysteine ​​(cyst) dissolved in PBS containing or not containing GILT was added to the PBS. eine) was mixed with nanoliposomes and incubated in a shaking incubator at 37°C. The samples were then flash frozen using liquid nitrogen and stored in a -20°C freezer. After 12 hours, all samples were stored in a vacuum freeze dryer at 10 Pascal and The freeze-dried sample was then subjected to liquid chromatography. The cholesterol-toll-like receptor 7 or The amount of resiquimod (R848) separated from the agonist complex was quantified. The results are shown in Figure 1. Shown in Figure 6.

[0103] As shown in Figure 6, the disulfide bond between cholesterol and R848 is catalyzed by GILT. It was confirmed that R848 was detected in a separated form after cleavage.

[0104] The results show that cholesterol and Toll-like receptor 7 or 8 agonists are disulfide-coupled. Nanoliposomes containing conjugates linked by amide bonds were transported into cells via intracellular import. The bond is then cleaved by GILT present in the endolysosomes within the cell, and cholesterol is released. Confirmation that Toll and Toll-like receptor 7 or 8 agonists exhibit separate immune stimulatory effects I was able to do it.

[0105] Example 13: Dynamically acting cholesterol-toll-like receptor 7 or 8 agonists Confirmation of immune response induction effect by stimuli 13.1. Nanoliposomes Containing Cholesterol Toll-Like Receptor 7 or 8 Agonists Confirmation of dynamic control of immune cell activation by the IL-1 receptor Cholesterol and resiquimod prepared in the same manner as in Example 11.1 were dissolved in water to form a disulfide. Nanoliposomes containing the conjugates linked by disulfide bonds were injected into immune cells. To confirm the effect of nanoliposomes on cellular immune function regulation, BMDCs were treated with nanoliposomes. After that, the amount of cytokines secreted and the costimulatory molecules (co-stimulatory molecules) secreted were measured in response to time. More specifically, the expression of Toll-like receptor 7 or 8 agonists was confirmed. Nanoliposomes or R848 were added at 1 × 10 to give a concentration of 1 μg / ml. 6 BMDC cells were treated with 100 μg / mL of ... The cell culture medium was centrifuged at 1,500 rpm for 3 minutes to separate the cells from the supernatant, Interleukin-10 (IL-10) and interleukin-1 contained in the supernatant The amount of IL-2 (IL-12) secreted was measured by ELISA, and the results are shown in Figure 7. In addition, the acquired cells were stained with fluorescent antibodies to confirm the maturity of the cells. After staining, dendritic cell co-immunoassays were performed using BD CantoII flow cytometry. The expression of stimulatory molecules was confirmed, and the results are shown in Figure 8.

[0106] As shown in FIG. 7, the cholesterol-R In the experimental group treated with nanoliposomes containing 848 conjugate (t-TLR7 / 8a), It was confirmed that the time point at which IL-10 was secreted was delayed by about 4 hours. The amount of nicotinamide continued to increase even after 24 hours.

[0107] As shown in FIG. 8, the co-stimulatory molecule, CD80, which indicates the maturity of dendritic cells, The results showed that the expression of CD86 in the R848-treated group was significantly higher than that in the control group. The expression levels of costimulatory molecules related to cholesterol-R848 did not increase over time, but The experimental group treated with nanoliposomes containing the conjugate (t-TLR7 / 8a) showed a significant increase in the expression of costimulatory molecules. It was confirmed that the expression level increased over time.

[0108] Through the above results, cholesterol-Toll-like receptor 7 or 8 agonist conjugates It is an immunomodulatory agent that can be dynamically controlled, and is a novel anti-toll receptor 7 or 8 agonist. Compared to its predecessor R848, it can induce an immune response after about four hours. In addition, it was confirmed that it can induce a sustained immune response. Sterol-toll-like receptor 7 or 8 agonist conjugates induce dendritic cell depletion (exh austion, i.e., a state in which no immune response is induced, but in a mature state. ing), that is, it is possible to maintain a state in which an immune response can be induced for a long period of time. Confirmed.

[0109] 13.2 Dynamically Acting Cholesterol Toll-Like Receptor 7 or 8 Agonists Confirmation of sustained immune response induction effect by nanoliposomes containing Cholesterol and resiquimod prepared in the same manner as in Example 11.1 were dissolved in water to form a disulfide. Nanoliposomes containing the conjugates bound by disulfide bonds To determine which concentrations of Toll-like receptor 7 or 8 agonists induce an immune response in Add 1 × 10 nanoliposomes or R848 to give a concentration of 1 μg / ml. 6 B MDC cells were treated with 1,000 mg of 1 ... After separating the cells from the supernatant by centrifugation at 500 rpm for 3 minutes, the obtained supernatant was used The amount of IL-12 secreted was measured. Next, the obtained cells were further divided into fresh medium and incubated for 4 The cell supernatant was collected at time intervals, and the amount of IL-12 secreted was confirmed by ELISA. The results are shown in Figure 9.

[0110] As shown in Figure 9, after changing to a new medium and culturing further, In the control group treated with R848, no further IL-12 was observed, whereas In the experimental group treated with nanoliposomes (t-TLR7 / 8a), IL-12 was increased for up to 16 hours. These results confirmed that the secretion of Toll-like receptor 7 or 8 agonists was sustained. Treatment with cholesterol alone rapidly induced depletion of dendritic cells, whereas treatment with cholesterol alone rapidly induced depletion of dendritic cells. -Toll-like receptor 7 or 8 agonist conjugates maintain the mature state of dendritic cells for a long period of time The researchers were able to confirm that the antibody produced a sustained immune response.

[0111] 13.3. Dynamic interactions between cholesterol and Toll-like receptor 7 or 8 agonists Induction of CD4+ T cell differentiation to Th1 response by nanoliposomes containing cyclophosphamide conjugates confirmation) Nanoliposomes containing cholesterol-Toll-like receptor 7 or 8 agonist conjugates A sustained immune response caused by CD4 + To confirm whether it induces differentiation of T cells into Th1 responses, To do this, we first harvested spleens from C57BL / 6 mice, isolated them into single cells, and then transfected them with CD4 + Using a T cell isolation kit, CD4 + T cells were isolated. / ml) and a concentration of Toll-like receptor 7 or 8 agonist at 1 μg / ml 1 × 10 nanoliposomes or R848 6 BMDC cells were treated with 1 After 2 h of incubation, BMDCs and CD4 + The proportion of T cells The culture supernatant was collected after 5 days of culture and used for ELI. Interleukin 4 (IL-4) and interferon gamma (IFN-γ) using SA The secretion amount of was measured, and the results are shown in Figure 10.

[0112] As shown in FIG. 10, the amount of IFN-γ secreted was significantly higher in the rats treated with R848 and OVA than in the control rats treated with OVA. Control group (R848) and experimental group treated with nanoliposomes and OVA (t-TLR7 / 8a ), but the amount of IL-4 secreted was not significantly different from that in the control group, which used nanoliposomes and OVA. It was confirmed that the expression of t-TLR7 / 8a in the treated experimental group was decreased. Therefore, nanoliposomes containing cholesterol-Toll-like receptor 7 or 8 agonist conjugates were IL-12 secretion was enhanced in BMDCs treated with IL-12, which in turn promoted the secretion of CD4 + T thin We confirmed that the IFN-γ / IL-4 ratio increased by differentiating cells into Th1. I was able to acknowledge this.

[0113] [Example 14: Confirmation of synergistic effect by combined administration of adjuvant] 14.1. Toll-like receptor 3 agonists and kinetically acting cholesterol Synergy by combination of nanoliposomes containing Toll-like receptor 7 or 8 agonists (Confirmation of effectiveness) To confirm the synergistic effect of combined adjuvant administration, BMDCs were treated with poly(I:C) and R848, which are Toll-like receptor 3 agonists. More specifically, BMDCs were first treated with poly(I:C) and then incubated at 4°C for 1 h. Experimental groups treated with R848, a Toll-like receptor 7 or 8 agonist (R848 af ter poly(I:C)), and R848 was treated first, followed by poly(I:C) at time intervals. The experimental group treated with ly(I:C) (poly(I:C)) after R848), i.e. In other words, asynchronous treatment was implemented. As a control group, cells treated simultaneously with R848 and poly(I:C) were used. The amount of IL-12 secreted during simultaneous treatment was set as the reference point of 100, and the relative values ​​were calculated. In addition, the synchronous treatment group consists of As a control group, R848 and poly(I:C) were treated alone, and R848 and poly(I:C) were treated alone. A control group (R848 + poly(I:C)) was used that was simultaneously treated with poly(I:C). , treated with nanoliposomes containing cholesterol-resiquimod conjugates and poly(I:C). Next, we prepared the experimental group (t-TLR7 / 8a+poly(I:C)) in which the treatment was completed. After that, the cells were cultured for 36 hours to obtain the cell culture medium. After centrifugation at 400 nm for 3 min to separate the cells from the supernatant, the IL-1 The amount of 2 secreted was measured by ELISA, and the results are shown in Figure 11.

[0114] As shown in Figure 11, R848 was treated first, followed by poly(I:C). In the experimental group, the amount of IL-12 secreted was reduced compared to the control group. That is, between Toll-like receptor 3 agonists and Toll-like receptor 7 or 8 agonists. However, on the other hand, when poly(I:C) was first used, In the experimental group treated with R848, the animals were treated with R848 at intervals of 2 to 4 hours. The secretion of IL-12 was increased in the experimental group treated with IL-12 compared with the control group treated at the same time. , which was increased by about 130%. After the first adjuvant is activated, the second adjuvant is activated with a time lag of 2 to 4 hours. It was confirmed that when HUVANT is activated, the immune activation effect is maximized.

[0115] In addition, in the simultaneous treatment experiment, nanoliposomes and poly(I:C) were treated simultaneously. The highest expression of IL-2 was observed in the poly(I:C) group. ) is activated first, followed by cholesterol-toll-like receptor 7 or 8 agonist binding. Nanoliposomes containing the IgG are transported into cells, where cholesterol is sequestered by GILT. This converts resiquimod from an inactive state to an active state, thereby exerting its activity. It was confirmed that the results were the same as those of the experimental group treated at time intervals.

[0116] 14.2. Toll-like receptor 4 agonists and kinetically acting cholesterol Synergy by combination of nanoliposomes containing Toll-like receptor 7 or 8 agonists (Confirmation of effectiveness) To confirm the synergistic effect of the combined adjuvant administration, the Toll-like receptor 4 (TLR4) receptor was activated. LPS, a toll-like receptor 7 or 8 agonist; and resiquimod, a toll-like receptor 7 or 8 agonist. Nanoliposomes containing cholesterol-Toll-like receptor 7 or 8 agonist conjugates were used The experiment was carried out in the same manner as in Example 14.1, and the results are shown in Figure 12.

[0117] As shown in Figure 12, LPS was treated, and R848 was administered at intervals of about 4 hours. The experimental group treated with LP showed the highest amount of IL-12 secretion, and the simultaneous treatment experiment also showed that LP Cholesterol-Toll-like receptor 7 or 8 agonist conjugates that interact dynamically with S The highest amount of IL-12 secretion was observed in the experimental group treated simultaneously with nanoliposomes containing I confirmed that.

[0118] The above results indicate that the order and time lag are important factors in the synergistic effect of adjuvants. In other words, it was confirmed that the combined adjuvant acts as a stimulant for immune activation. In order to increase the effect of inducing activation, activation of each adjuvant is required. Dynamic control that can adjust the timing is important. It was confirmed that.

[0119] [Example 15: Confirmation of immune activation efficacy by adjuvant ensemble] 15.1 Dynamically Acting Cholesterol Toll-Like Receptor 7 or 8 Agonists Nanoliposomes containing a toll-like receptor 3 agonist or toll-like receptor 4 agonist (Confirmation of immune activation efficacy by combination of agonists) Immune activation by combined administration of a primary adjuvant and a kinetically acting secondary adjuvant To confirm efficacy, poly(I:C) or LPS was used as the first adjuvant. As a secondary adjuvant, cholesterol-toll-like receptor 7 or 8 agonists were used. The experiment was carried out using nanoliposomes containing the sucrose conjugate. I:C), R848, containing cholesterol-toll-like receptor 7 or 8 agonist conjugate The control group was administered nanoliposomes containing LPS and R848 alone, while the other group was administered LPS and R848 simultaneously. Experimental group, experimental group administered poly(I:C) and R848 simultaneously, LPS and cholesterol Coadministration of nanoliposomes containing teratoma-toll-like receptor 7 or 8 agonist conjugates The experimental groups were poly(I:C) and cholesterol-Toll-like receptor 7 or 8. After preparing experimental groups in which the nanoliposomes containing the nicotinamide conjugate were administered simultaneously, The obtained cell culture medium was centrifuged at 1,500 rpm for 3 minutes. After isolating the heart and separating it into cells and supernatant, the supernatant was used to extract tumor necrosis The secretion of factors (TNF-α) and IL-6 was measured, and cells were labeled with fluorescent antibodies. Afterwards, dendritic cell co-stimulation was measured using BD CantoII flow cytometry. The expression of the molecule was confirmed to confirm the maturity of the cells. The results are shown in Figures 13 and 14. .

[0120] As shown in FIG. 13, compared with the control group in which each drug was administered alone, In the experimental group, the secretion of TNF-α and IL-6 increased, but the dynamics of the effect were not clear. Nanoliposomes containing cholesterol-Toll-like receptor 7 or 8 agonist conjugates and agonists The experimental group administered adjuvant showed significantly higher secretion of TNF-α and IL-6. I confirmed that.

[0121] Also, as shown in FIG. 14, the dynamic cholesterol-toll-like receptor Experimental group administered nanoliposomes containing 7 or 8 agonist conjugates in combination with adjuvant We confirmed that the expression level of costimulatory molecules was highest in

[0122] Based on the above results, it was found that the drug remained in an inactive state immediately after administration, and then after 2 to 4 hours, it was found to be inactivated. The combination of a kinetically controllable adjuvant and a general adjuvant to induce a specific immune response was used. By administering the adjuvants, the effect was significantly improved compared to the simple administration of each adjuvant in combination. It was confirmed that the immunoactivating effect was significantly increased.

[0123] 15.2 Dynamically Acting Cholesterol Toll-Like Receptor 7 or 8 Agonists Nanoliposomes containing a toll-like receptor 3 agonist or toll-like receptor 4 agonist (Confirmation of the sustained immune response induction effect by combination of agonists) Immune activation by combined administration of a primary adjuvant and a kinetically acting secondary adjuvant To confirm efficacy, poly(I:C) or LPS was used as the first adjuvant. As a secondary adjuvant, cholesterol-toll-like receptor 7 or 8 agonists were used. In this study, we performed experiments using nanoliposomes containing the IL-1 conjugate. S and R848, poly(I:C) and R848, LPS and cholesterol - Nanoliposomes containing Toll-like receptor 7 or 8 agonist conjugates or poly(I :C) and nanoribonucleotides containing cholesterol-Toll-like receptor 7 or 8 agonist conjugates The cell culture medium was harvested after 24 and 36 hours of treatment with ribosomal peptides. The nutrient solution was centrifuged at 1,500 rpm for 3 minutes to separate the cells from the supernatant, and the supernatant was The secretion levels of IL-12, IL-6, and TNF-α were determined using ELISA. The results are shown in Figure 15.

[0124] As shown in Figure 15, poly(I:C) or LPS and R848 were simultaneously treated. In the experimental group, the amount of secreted cytokines at 24 and 36 hours in all samples was Similar or decreased responses were observed in the presence of poly(I:C) or LPS and leucine-like All experimental groups were simultaneously treated with nanoliposomes containing receptor 7 or 8 agonists. The amount of cytokines secreted after 36 hours was significantly higher than that secreted after 24 hours in the same sample. The results confirmed that the amount of cyclooxygenase (CD) increased by 100 mg / kg / day. It has been shown that a sustained immune response can be induced by combined administration of an adjuvant and a first adjuvant. We were able to confirm that:

[0125] [Example 16: Preparation of adjuvant ensemble system] 16.1. Toll-like receptor 3 agonists and cholesterol-toll complexes acting dynamically Adjuvants composed of nanoliposomes containing leukocyte-like receptor 7 or 8 agonist conjugates (Production of the Complex) Cholesterol and resiquimod prepared in the same manner as in Example 11.1 were dissolved in water to form a disulfide. Nanoliposomes containing the conjugates linked by disulfide bonds and pol y (I:C) were mixed in a mass ratio of 4:1 and mixed in a vortex mixer. r, 3 seconds) to prepare a stable particle-form adjuvant complex. In the paper, the mass of the nanoliposome is determined by the mass of the Toll-like receptor contained in the nanoliposome. 7 or 8 Calculations were made in terms of the mass of the agonist.

[0126] (16.2. Confirmation of the degree of stabilization of the adjuvant complex) The adjuvant complex prepared in the same manner as in Example 16.1 was stabilized by electrical attraction. To confirm whether the complex was formed, poly(I:C) that did not form a complex was placed around the The remaining ions were analyzed by electrophoretic mobility shift analysis. This was confirmed using an electrochemical shift assay (EMSA). Agarose (1g) was added to E buffer, and dissolved by heating. The agarose gel was then prepared by pouring the mixture into a glass tube and solidifying it for about 30 minutes. 100bp), nanoliposomes containing cholesterol-resiquimod conjugates, poly(I C), and complex (K-nanoadjuvant) were sequentially treated, and then electrophoresed for 1 hour. The results are shown in Figure 16.

[0127] As shown in Figure 16, poly(I:C) and cholesterol-resiquimod conjugates The dynamically acting adjuvant complex was composed of nanoliposomes containing poly It was confirmed that all of the (I:C) was bound to form a stable complex. Ta.

[0128] 16.3. Confirmation of intracellular stability of adjuvant complexes To confirm whether the adjuvant complex remains a stable complex within cells, R848, FITC-conjugated cholesterol (chol-FITC), and loader Poly(I:C) with rhodamine (rhodamine-poly(I:C)) was used. The adjuvant complex was prepared in the same manner as in Example 16.1. BMDC (4 × 1 0 4 The cells were then treated with the adjuvant complex. After 4 h incubation at 4 °C and washing with PBS, endolysosomes were stained with LysoTrack The cells were stained with DeepRed and the nuclei with Hoechst 33342. Cell images were captured using taviosion PD. The results are shown in Figure 17. .

[0129] As shown in Figure 17, the fluorescence and Rhodograms of nanoliposomes containing chol-FITC The fluorescence of amine-poly(I:C) was confirmed at the same position, indicating that it was stable even inside the cells. It was confirmed that the determined complex was maintained.

[0130] [Example 17: Confirmation of efficacy of adjuvant complex] 17.1. Efficacy of adjuvant complexes in inducing sustained immune responses in tumor-draining lymph nodes (Check the performance) The adjuvant complex prepared in the same manner as in Example 16.1 was used to investigate the effect of the adjuvant complex on tumor-draining lymph nodes. To confirm the efficacy of inducing immune responses, R848 (25 μg), p poly(I:C) (6.25 μg), or the same amount of R848 and poly(I:C) The adjuvant complex containing the SIINFEKL antigen was mixed with the SIINFEKL antigen and administered subcutaneously as a single inoculation. Lymph nodes were excised 6, 12, 24, 48, and 72 hours after inoculation, and the excised lymph nodes were The sections were suspended in cell lysis buffer (CellLytic MT cell lysis). The cell-disrupted solution was centrifuged at 10,000xg for 10 minutes at 4°C. The supernatant was then separated to remove impurities. The amount of IL-12p70 present in the cells was confirmed by ELISA, and the results are shown in Figure 18.

[0131] As shown in FIG. 18, the control group treated simultaneously with R848 and poly(I:C) showed The secretion of IL-12p70 in lymph nodes decreased after 12 hours of treatment with the adjuvant complex. The experimental group was found to secrete IL-12p70 continuously for up to 48 hours. The results show that the kinetically acting adjuvant complex is composed of a first adjuvant and a second adjuvant. It has been confirmed that adjuvant induces a sustained immune response in the body because it acts at different times I was able to do it.

[0132] 17.2. Confirmation of Antitumor Efficacy of Adjuvant Complexes To confirm the antitumor efficacy of the adjuvant complex prepared in the same manner as in Example 16.1 5 × 10 B16OVA melanoma cells were cultured in a 10-well plate. 5 The cells were subcutaneously inoculated into the right flank of mice. Next, R848 (25μg), p poly(I:C) (6.25 μg), or the same amount of R848 and poly(I:C) The adjuvant complex containing the SIINFEKL antigen was mixed with the SIINFEKL antigen and administered subcutaneously once every three days. The tumor size and survival status were then continuously monitored. Diameter x minor axis diameter 2 The results are shown in Figure 19.

[0133] As shown in FIG. 19, two of five mice administered the adjuvant complex had 3 The mice survived for up to 6 days, and tumor volume was effectively suppressed. The use of mechanically acting adjuvant complexes significantly increases the anticancer effect. We were able to confirm that this is possible.

[0134] 17.3. Adjuvant complexes in tumor-draining lymph nodes and immune responses in the tumor microenvironment Confirmation of induction efficacy) Tumor-draining lymph nodes (TDLs) of the adjuvant complex prepared in the same manner as in Example 16.1 N) and the efficacy of inducing an immune response in the tumor microenvironment (TME). 16OVA melanoma cells 5 x 10 5 The cells were subcutaneously injected into the right flank of mice to induce tumorigenesis. Next, we created an animal model of cancer by injecting R848 (25 μg), poly(I:C ) (6.25 μg), or an adjuvant containing the same amount of R848 and poly(I:C) The complex was mixed with SIINFEKL antigen and administered as a single subcutaneous inoculation three times at three-day intervals. Three days after the final inoculation, tumor tissues and tumor-draining lymph nodes were excised. To analyze immune cells collected in lymph nodes, tumor tissue and lymph nodes were mechanically disrupted. After that, collagenase D (1 mg / ml) was added. The cells were resuspended in medium and incubated in a shaking incubator at 37°C for 40 minutes. Then, the cells were filtered through a 70 μm cell filter. The cells were filtered using a cell strainer, washed twice with PBS, and then isolated into a single The cells were acquired. The acquired single cells were stained with various fluorescently conjugated antibodies. The results were then analyzed using BD Canto II flow cytometry. The results are shown in Figures 20 to 23.

[0135] As shown in Figures 20 to 23, the kinetically acting adjuvant complexes enhanced tumor clearance. It was confirmed that mature dendritic cells were generated in the draining lymph nodes. In tumor tissues and tumor-draining lymph nodes of mice treated with the complex, R848 and poly(I: C) compared with the experimental group administered + Very frequent polyactivity in T cells IFN-γ + Granzyme-B + and CD69. CD8 in the experimental group treated simultaneously with 48 and poly(I:C) + In T cells, T cel Increased expression levels of PD-1, TIM-3, and LAG-3, indicating L-cell exhaustion However, the experimental group treated with the adjuvant complex showed similar results to the control group (treated with PBS). In addition, it was confirmed that CD4 + T cells, CD8 + T cells, natural killer cells (Natural Killer cell; NK cell) and Natural Killer cell In natural killer T cells, expression of CD69 is induced. Myeloid derived suppressor cells, which represent immune inhibitory cells, were It was confirmed that the production of MDSCs was suppressed. Through this, the adjuvant complex of the present invention suppresses T cell exhaustion in vivo. This not only enhances the immune activation effect but also prolongs the duration of the immune response. This shows a significantly higher immunomodulatory effect than simply administering an adjuvant in combination. We were able to confirm that this was the case.

[0136] In addition, to confirm the cytokines secreted in tumor and lymph node tissues, The tumor tissue was then lysed in CellLytic MT cell lysis buffer. The cells were suspended in 100 ml of ethanol and then mechanically disrupted. Then, the cells were centrifuged at 10,000xg for 10 minutes at 4°C. The lymph node tissue was then mechanically disrupted and collagenase was added to obtain the supernatant. The cells were then resuspended in medium supplemented with ZeD (1 mg / ml). After culturing in a shaking incubator at 37°C for 40 minutes, the cells were filtered through a 70 μm cell strainer. The mixture was then washed twice with PBS and then filtered using a 1,500 ml rainer. After centrifugation at 400 rpm for 3 min to obtain single cells, the cells were distributed onto plates and incubated at 37°C for 24 h. The cell culture was then centrifuged to obtain the supernatant. The secretion levels of IL-12p70 and IFN-γ were confirmed by ELISA. The results are shown in Figures 24 and 25.

[0137] As shown in Figures 24 and 25, the adjuvant complex was found to be effective in tumor tissue and tumor secretion. The results showed that IL-12p70 and IFN-γ secretion was effectively induced in both the draining lymph nodes and the lymph nodes. Confirmed.

[0138] The results suggest that the kinetically acting adjuvant complex promotes the proliferation of various immune cells. Improved performance, increased cytokine production, and CD8 + Inducing T cell exhaustion By increasing lytic granule production without + T cell effector factor (e It was confirmed that the function of the effector was improved.

[0139] [Example 18: Confirmation of efficacy of adjuvant complex using IL-12 neutralizing antibody] The adjuvant complex enhances immune cell proliferation and immune activation by IL-12 To confirm whether this is mediated by sustained production of p70, we first Chromoma cells 5 x 10 5 The cells were inoculated subcutaneously into the right flank of mice to create an animal model of cancer. Next, we used anti-mouse IL-12p75 antibody before inoculating the cancer animal model with the adjuvant complex. (300 μg) was administered intraperitoneally five times at three-day intervals. The immunization complex was mixed with SIINFEKL antigen and given as a single subcutaneous inoculation three times at three-day intervals. The tumor size and survival status were then continuously monitored, and the results are shown in FIG.

[0140] As shown in FIG. 26, in the experimental group treated with anti-IL-12, the adjuvant complex The results confirmed that IL-12 inhibits the Toll-like receptor (TLR) 3 agonist and cholesterol-toll-like receptor 7 or 8 agonist combinations Innate immune stimulation of nanoliposome-based, kinetically acting adjuvant complexes and has been confirmed to play an important role during adaptive immune responses.

[0141] [Example 19: Confirmation of the effect of inducing antitumor immune response via local inoculation of adjuvant complex ] 19.1. Confirmation of antitumor efficacy against metastatic cancer via local injection of adjuvant complex approval) To confirm whether the adjuvant complex exerts antitumor effects against metastatic cancer through local injection To incubate 5 x 10 TC-1 cells 5 The cells were subcutaneously inoculated into the right flank of mice to create a cancer model. After 4 days, the cells were then secondary cultured with 2.5 × 10 TC-1 cells. 5 Cells from mice The adjuvant complex was then administered subcutaneously to the left flank of the animal model. The mice were mixed with the antigen and given a single subcutaneous injection at 3-day intervals for four times. Then, three days after the last injection, the tumor was Tumor tissue and tumor-draining lymph nodes were removed. Tumor size and viability were monitored continuously. The results are shown in Figure 27.

[0142] As shown in FIG. 27, in the experimental group inoculated with the adjuvant complex, only the primary tumor The results showed that the growth of secondary tumors was also effectively suppressed.

[0143] (19.2. Confirmation of the effect of suppressing pulmonary metastasis via local inoculation of adjuvant complex) To confirm whether local administration of the adjuvant complex inhibits cancer metastasis, 4T1 cells 5 x 10 5 The cells were subcutaneously inoculated into the right flank of mice to create an animal model of cancer. Then, 5 days later, tumor lysate (10 μg) and adjuvant complex were administered. The mixture was mixed and administered as a single subcutaneous inoculation four times at 3-day intervals. Mice were then euthanized 30 days later. After that, a mixture of India ink (47 ml) and PBS (3 ml) was injected into the organ. The stained lungs were excised, washed with PBS, and then fixed with a fixative (70 % ethane (40 ml), 4% formaldehyde (1 ml) and acetic acid (0.5 ml) The lung metastatic nodules were counted by visual observation. The results are shown in Figure 28. .

[0144] As shown in Figure 28, many metastatic tumor nodules were observed in the lungs in the control group, whereas the adjuvant group showed no significant changes in the lungs. It was confirmed that no lung metastasis was observed in the experimental group administered with Bunt complex.

[0145] (19.3. Orthotopic tumor control of metastatic cancer via local inoculation of adjuvant complex Confirmation of antitumor efficacy against orthotopic tumors via local inoculation of the adjuvant complex To confirm whether the compound exhibited antitumor efficacy, C57BL / 6 mice were temporarily anesthetized with respiratory anesthesia. Next, the right side of the chest skin was incised, and 5 × 10 TC-1 cells were injected into the right side of the lung lobe. 5 cell Then, after 3 days, the adjuvant complex was mixed with the Long-E7 peptide. The mice were then subcutaneously inoculated four times at 3-day intervals. Then, three days after the last inoculation, the mice were euthanized. Thereafter, the lungs were removed, sectioned, and stained with hematoxylin and eosin. The results are shown in Figure 29.

[0146] As shown in Figure 29, tumor cells inoculated directly into the lungs did not cause any significant changes compared to the untreated control group. In the experimental group, which received the adjuvant complex, tumor growth was completely suppressed. was confirmed to be inhibited.

[0147] Based on the above results, it is possible to achieve systemic antitumor activity even with the local administration of the dynamically acting adjuvant complex. It was confirmed that the antibody generated an immune response and effectively exhibited an antitumor effect.

[0148] [Example 20: Adjuvant complex and immune checkpoint inhibitor therapy or chemotherapy Confirmation of antitumor efficacy in combination with Immunostimulatory effects of the adjuvant complex enhance the therapeutic effects of various anti-cancer treatments. An experiment was carried out to see if this could be improved.

[0149] 20.1. Combination of adjuvant complex and immune checkpoint inhibitor therapy Confirmation of antitumor efficacy Adjuvant complexes and immune checkpoint inhibitor therapy (ICBT therapy) To confirm the antitumor efficacy of the combination of 5 Thin The cells were subcutaneously inoculated into the right flank of mice to create a cancer animal model. The E7 complex and the Long-E7 peptide were mixed and administered subcutaneously six times at three-day intervals. Anti-PD-L1 was then administered intraperitoneally eight times at 2-day intervals. Three days after the last administration, tumor The expression level of PD-L1 in the tissue is checked, and the tumor size and survival status are continuously monitored. The results are shown in Figure 30.

[0150] As shown in Figure 30, the adjuvant complex was combined with immune checkpoint inhibitor therapy. When combined, PD-L1 expression was significantly increased and cancer growth was effectively suppressed. We also confirmed that the combination of adjuvant complexes and immune checkpoint inhibitor therapy When combined, the animals survived for more than 120 days. The IL-1 complex induces increased activity of cytotoxic lymphocytes and increased IFN-γ secretion in the tumor microenvironment By increasing PD-L1 expression, it regulates immune responses within the tumor microenvironment. This will further increase the anti-cancer effect of immune checkpoint inhibitor therapy. We were able to confirm that:

[0151] 20.2. Confirmation of Antitumor Efficacy Through Combination of Adjuvant Complexes and Chemotherapy Antibody therapy via combination of adjuvant complexes and chemotherapy To confirm tumor efficacy, 5 × 10 TC-1 cells were initially 5 The cells were then inoculated into the right flank of the mice. The animals were then inoculated with the adjuvant complex and Lon after 4 days to create a cancer animal model. The g-E7 peptide was mixed and administered subcutaneously in a single infusion five times at 3-day intervals. The liposomal formulation (80 μg) was intravenously injected twice at an interval of 6 days. The survival of the mice was continuously monitored, and the results are shown in FIG.

[0152] As shown in Figure 31, the combination of adjuvant complexes and chemotherapy significantly improved the survival of cancer cells. It was also confirmed that the growth of the adjuvant complex was effectively suppressed. When combined with POI therapy, patients survived for more than 160 days. These results suggest that the combination of adjuvant complexes and chemotherapy can be used to effectively treat cancer. I was able to confirm that it was treatable.

[0153] The above description of the invention is given by way of example only and is intended to be illustrative and not restrictive of the invention as claimed. A person skilled in the art may, without changing the technical idea or essential features of the present invention, make any modification in another specific form. You can see that it can be easily transformed. Therefore, the embodiments described above are illustrative in all respects and are not limiting. It should be understood as follows. [Industrial Applicability]

[0154] The dynamically acting adjuvant ensemble compositions of the present invention comprise two or more immunologically active agents. When two chemicals are administered simultaneously, the dynamic ensemble that acts at a certain time interval is called By providing these, we can not only maximize the synergistic effect of the immunological response, but also It is also possible to easily incorporate various functional drugs into the drug delivery system and regulate them to be secreted sequentially. This not only makes it applicable to a variety of diseases that can be treated with adjuvants, but also , the therapeutic effect can be significantly increased.

Claims

1. 1. A kinetically acting adjuvant ensemble composition comprising: The composition comprises two or more adjuvants; The first adjuvant first binds to immune cell receptors to induce a primary immune response; The second adjuvant is a conjugate having a cleavable linker bound to an activation site, which sequentially binds to an immune cell receptor to induce a secondary immune response. Adjuvant ensemble compositions.

2. The kinetic action is The second adjuvant is characterized in that a cleavable linker is bound to the activation site of the second adjuvant, the second adjuvant is maintained in an inactive state, and the cleavable linker blocking the activation site is cleaved within 2 to 12 hours, and the activity of the immunoactivator appears in a time-delayed manner. The adjuvant ensemble composition of claim 1.

3. The adjuvant ensemble composition 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.

4. The adjuvant ensemble composition of claim 1, wherein the cleavable linker further comprises ethylene oxide or ethylene glycol at both or one of its termini.

5. The adjuvant ensemble composition 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.

6. The adjuvant ensemble composition according to 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.

7. The adjuvant ensemble composition according to claim 1, characterized in that the second adjuvant is loaded into any one or more drug delivery vehicles selected from the group consisting of nanoliposomes, nanoemulsions, nanomicelles, hydrogels, scaffolds, solid nanoparticles and polymeric nanoparticles.

8. The adjuvant ensemble composition according to claim 7, wherein the drug delivery vehicle further comprises a first adjuvant.

9. The adjuvant ensemble composition according to claim 7, wherein the drug delivery vehicle further comprises a ligand that reacts with a receptor present on the surface of an immune cell or in an endosome or cytosol.

10. The drug delivery system is The adjuvant ensemble composition according to claim 7, further comprising at least one immune activator 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, a STING (stimulator of interferon genes) ligand, an outer wall component of a pathogen, an alum, lipids, a combination thereof, and a derivative thereof.

11. The adjuvant ensemble composition according to claim 1, wherein the second adjuvant is a toll-like receptor agonist.

12. 2. The adjuvant ensemble composition according to claim 1, wherein the first adjuvant is at least one immune activator 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, a STING (stimulator of interferon genes) ligand, an outer wall component of a pathogen, an alum, lipids, a combination thereof, and a derivative thereof.

13. The adjuvant ensemble composition according to claim 1, characterized in that the immune cells are at least one selected from the group consisting of antigen-presenting cells (dendritic cells, macrophages), natural killer cells (NK cells), T cells, B cells, regulatory T cells, myeloid derived suppressor cells (MDSCs), and M2 macrophages.

14. The adjuvant ensemble composition according to claim 1, which is for the prevention or treatment of one or more diseases selected from the group consisting of infectious diseases, cancer, metabolic syndrome, autoimmune diseases, and rare diseases.

15. The adjuvant ensemble composition of claim 14, further comprising an antigen, a chemical anticancer agent, or an immune checkpoint inhibitor.

16. The adjuvant ensemble composition according to claim 15, characterized in that the antigen is one or more selected from the group consisting of proteins, recombinant proteins, glycoproteins, genes, peptides, polysaccharides, lipopolysaccharides, polynucleotides, cells, cell lysates, bacteria and viruses.

17. The adjuvant ensemble composition according to claim 14, characterized in that it suppresses cancer proliferation, metastasis, recurrence or resistance to anti-cancer treatment.

18. Use of the adjuvant ensemble composition of claim 1 for producing a medicament for enhancing the immune activation of an adjuvant.

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