Dynamically acting adjuvant ensemble

Abstract

The present invention relates to an adjuvant ensemble that combines two or more immune activators that induce diverse immune cell activation, and to uses thereof. This novel kinetically acting adjuvant ensemble maximizes the ultimate synergistic immune activation response by controlling the treatment order and time interval when combining immune activators that have different signaling systems in their immune activation mechanisms. The kinetic action is characterized by the second immune activator reacting to endogenous factors (enzymes, redox potential, GSH, and pH) and exogenous factors (redox, pH, temperature, photo / light, magnetism, ultrasound, and electrical response) within the immune cell, cleaving the chemical bond at the binding site, exposing the activation site and dynamically restoring function, or by sequentially releasing two or more immune activators from a carrier at a fixed time interval to induce immune activation.

Description

Technical Field

[0001] The present invention relates to a novel adjuvant ensemble containing two or more immunostimulatory substances that activate immune cells and whose action time is kinetically regulated. More specifically, after injection, the first immunostimulatory substance first binds to a receptor to primarily induce an immune response, and then the second immunostimulatory substance binds to the receptor and is designed such that an immune response is sequentially induced. The present invention relates to an adjuvant ensemble.

Background Art

[0002] An immune response is a series of reactions that activated immune cells have against exogenous and endogenous substances, i.e., antigens. When microorganisms such as bacteria and viruses, or foreign substances in the body flow into the body, immune cells recognize and activate them, secrete factors such as cytokines, and induce an inflammatory response. Recently, research on the mechanism at the stage of the innate immune response that acts non-specifically in the early stage of infection has been actively conducted. Among them, Toll-like receptors (TLRs) are receptors that can recognize pathogens in the early stage of inflammation and are known to recognize protoplasmic membrane components and nucleic acid components of pathogens and induce an immune response. Using this, various studies on Toll-like receptor ligands (TLR ligands) for activating immune cells have been actively conducted (US Patent Publication No. 2012-0294885).

[0003] Toll-like receptor agonists are agonists of Toll-like receptors within endosomes and are known to effectively induce not only humoral immunity but also cellular immunity. However, such multifunctional Toll-like receptor agonists are difficult to disperse in aqueous solutions due to their molecular structure. Moreover, they dissolve only in special organic solvents such as DMSO and methanol and do not dissolve in commonly used organic solvents, so there are limitations in manufacturing immunostimulatory drugs in various dosage forms. Therefore, they have been commercialized in a cream form (e.g., Aldara cream) by mixing various surfactants. In some studies, to overcome such problems, they were manufactured in the form of salts and made soluble in aqueous solutions. However, Toll-like receptor agonists manufactured in the form of salts are absorbed into blood vessels in the body and induce many side effects (e.g., cytokine storm, various non-specific hypersensitivity immune reactions, etc.) by inducing a systemic immune response in the blood vessels, so they are not easy to use at present. Also, due to such side effect problems, in order to be actually used for treatment, a concentration lower than the effective dose has to be administered, which is a factor in reducing efficacy. Some pharmaceutical companies have attempted to prevent direct absorption into blood vessels by introducing lipids showing lipophilic properties or by directly chemically bonding to high molecular chains having a huge size to overcome such problems. However, Toll-like receptor agonists manufactured by such methods still have the possibility of inducing toxicity by inducing non-specific immune reactions in the body because their active sites are still exposed to the outside.

[0004] Including such Toll-like receptor agonists, various innate immune inducers have been continuously reported to increase the effect of the innate immune response when used in combination. In particular, Toll-like receptor ligands having different signal transduction systems in the immune activation mechanism have been reported to increase the immune cell activation ability by 20 to 50 times or more when used in combination of two or more than when used alone. However, since innate immune inducers having different signal transduction systems have different mechanisms from each other, the effect of the immune activation reaction varies greatly depending on the order of treatment and / or the time interval between treatments. However, administering a plurality of innate immune inducers, that is, adjuvants, individually into the body at regular time intervals is not only impractical, but also the probability that the individually administered adjuvants stimulate the same immune cells to increase the immune activation effect is very low. Therefore, it is not easy to administer different types of innate immune inducers at different times (asynchronous treatment) in the medical field.

[0005] Therefore, if an adjuvant ensemble that can maximize the immune activation effect is developed by administering a plurality of adjuvants simultaneously (synchronous treatment) or by optimizing the time interval for each adjuvant injected into the body to bind to each receptor at regular time intervals for immune activation, the effect of the vaccine can be significantly improved by using this, and it is expected to have a very large ripple effect in the next-generation vaccine market.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The present invention has been made to solve the above-described problems in the prior art, and provides a kinetic acting adjuvant ensemble designed such that different adjuvants, for example, toll-like receptor agonists, saponins, antiviral peptides, inflammasome inducers, NOD ligands, cytosolic DNA sensor ligands, stimulator of interferon genes (STING) ligands, etc., can act in a defined order and at regular time intervals, and its uses, etc.

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

Means for Solving the Problems

[0008] The present invention provides an adjuvant ensemble composition that acts kinetically, wherein the composition contains two or more adjuvants. The first adjuvant is to first bind to an immune cell receptor and induce a primary immune response, and the second adjuvant is a conjugate to which a cleavable linker that can be cleaved at an activation site is bound, and sequentially binds to the immune cell receptor to induce a secondary immune response.

[0009] In one specific example of the present invention, a cleavable linker is attached to the activation site of the second adjuvant, maintaining an inactive state, and within 2 to 12 hours, more preferably within 3 to 9 hours, the cleavable linker that blocks the activation site is cleaved, and the activity of the immunostimulatory substance appears with a time delay. The adjuvant ensemble composition of the present invention can maximize the synergistic effect of the immunological reaction by providing a kinetic ensemble that acts at regular time intervals when two or more immunostimulatory substances are administered simultaneously. The kinetic ensemble can act at the molecular scale and / or the macro scale.

[0010] In another specific example of the present invention, the cleavable linker preferably contains any one or more bonds selected from the group consisting of disulfide, carbamate, hydrazine, ester, peptide, azide, amide, hydrazone, thioether, phosphodiester, thioketal, and combinations thereof. However, it is not limited thereto as long as the bond can be cleaved by endogenous factors (enzymes, redox potential, GSH, pH, etc.) and / or exogenous factors (redox, pH, temperature, photo / light, magnetism, ultrasonic, electrical responsive, etc.) in the living body.

[0011] In still another specific example of the present invention, 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, ultrasonic, magnetism, and light sources.

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

[0013] In still other specific examples of the present invention, one or more substances selected from the group consisting of cholesterol, lipid, protein, amino acid, peptide, and oligonucleotide are bound to the end of the cleavable linker, and the substances serve to block the activating moiety of the second adjuvant and can be various substances having a hydrophilic or lipophilic group.

[0014] In still other specific examples of the present invention, the second adjuvant is loaded in any one or more drug delivery carriers selected from the group consisting of nanoliposomes, nanoemulsions, nanomicelles, hydrogels, scaffolds, solid nanoparticles, and polymeric nanoparticles. The loading may be in a simple encapsulated form, regardless of binding, or may be in a form sandwiched between nanoparticle structures or in a bound form, but is not limited thereto as long as it contains the mRNA antigen and the immunostimulatory substance of the present invention.

[0015] In still other specific examples of the present invention, the drug delivery carrier further includes a first adjuvant.

[0016] In still other specific examples of the present invention, the drug delivery carrier further includes a ligand that reacts with a receptor present on the surface of immune cells or in endosomes or the cytosol.

[0017] In still other specific examples of the present invention, the drug delivery body further comprises any one or more immunostimulatory substances selected from the group consisting of toll-like receptor agonists, saponins, antiviral peptides, inflammasome inducers, NOD ligands, cytosolic DNA sensor ligands, stimulator of interferon genes (STING) ligands, outer wall components of pathogenic bacteria, alum, lipids, combinations thereof, and derivatives thereof.

[0018] In still other specific examples of the present invention, the second adjuvant may preferably be a toll-like receptor agonist, more preferably any one or more selected from the group consisting of toll-like receptor 1 agonist, 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, and toll-like receptor 9 agonist.

[0019] In still other specific examples of the present invention, the first adjuvant can be any one or more immunostimulatory substances selected from the group consisting of toll-like receptor agonists, saponins, antiviral peptides, inflammasome inducers, NOD ligands, cytosolic DNA sensor ligands, STING ligands, outer wall components of pathogenic bacteria, alum, lipids, combinations thereof, and derivatives thereof.

[0020] In still another specific example of the present invention, the immune cell is characterized by being any one or more selected from the group consisting of antigen-presenting cells (dendritic cells, macrophage), natural killer cells (NK cell), T cells, B cells, regulatory T cells, MDSC (myeoloid derived suppressor cells), and M2 macrophages.

[0021] In still another specific example of the present invention, the drug delivery body may be an ensemble designed such that two or more adjuvants are sequentially released, and is a structure including a core-shell having two or more layered structures such as liposomes, micelles, emulsions, self-assembled particles, polymer nanoparticles, etc., and may include an adjuvant released rapidly primarily (Firstly release of payload) and an adjuvant released secondly (Secondly release of payload).

[0022] In still another specific example of the present invention, the drug delivery body may contain a stimuli-responsive block, and the stimuli may include endogenous factors inside cells (enzymes, redox potential, GSH, pH, intracellular proteins, etc.), exogenous factors (redox, pH, temperature, photo / light, magnetism, ultrasonic waves, electrical responsive, etc.), and various physiological environments / immune factors in vivo. Further, the stimuli-responsive block may include two or more stimuli-responsive blocks and can sequentially react to different stimuli. Or it can also sequentially react according to the intensity of the stimuli.

[0023] Furthermore, the present invention provides a pharmaceutical composition for preventing or treating an infectious disease, cancer, metabolic syndrome, autoimmune disease or rare disease, which contains the adjuvant ensemble composition as an active ingredient.

[0024] In one specific example of the present invention, the pharmaceutical composition may further contain an antigen, a chemotherapeutic agent, or an immune checkpoint inhibitor, etc.

[0025] In another specific example of the present invention, the antigen is characterized by being 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, bacteria and a virus.

[0026] In still another specific example of the present invention, the pharmaceutical composition is characterized by suppressing cancer growth, metastasis, recurrence or resistance to a cancer treatment therapy.

[0027] Furthermore, the present invention provides a method for preventing or treating an infectious disease, cancer, metabolic syndrome, autoimmune disease or rare disease, which includes the step of administering to an individual a composition containing the adjuvant ensemble composition as an active ingredient.

[0028] Furthermore, the present invention provides the use of a composition containing the adjuvant ensemble composition as an active ingredient for preventing or treating an infectious disease, cancer, metabolic syndrome, autoimmune disease or rare disease.

[0029] Furthermore, the present invention provides the use of the adjuvant ensemble composition for producing a medicament used for the prevention or treatment of infectious diseases, cancer, metabolic syndrome, autoimmune diseases or rare diseases.

Advantages of the Invention

[0030] In the present invention, the novel adjuvant with the action time of the adjuvant kinetically regulated can not only increase the synergistic effect but also minimize the potential toxicity problem of the adjuvant as compared with the conventional simultaneous administration of two substances. In particular, an ensemble combining two or more immunostimulatory substances selected in a made-to-order manner is expected to be widely used as an effective preventive and therapeutic agent for various diseases such as anti-cancer vaccines and infectious diseases due to its improved immune-enhancing effect.

Brief Description of the Drawings

[0031]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18

Figure 19

Figure 20

Figure 21

Figure 22

Figure 23

Figure 24

Figure 25

Figure 26

Figure 27

Figure 28

Figure 29

Figure 30

Figure 31

[0032] [Best Mode for Carrying Out the Invention] When the inventors used two or more adjuvants in combination, they confirmed that the order and time difference act as important factors in the synergistic effect of the adjuvants. When inducing immune activation using a combined adjuvant, in order to maximize its effect, as a result of intensive research on an adjuvant ensemble system capable of kinetical control to adjust the activation time point of each adjuvant, the first adjuvant acts primarily, and the second adjuvant is maintained in a state where its activity is temporarily inhibited by linking a cleavable linker to the activation site. After in vivo administration, after reaching the target tissue or cell, that is, within 2 to 12 hours, the linker is cleaved and the activity of the adjuvant appears secondarily, and they invented an adjuvant ensemble composition capable of such kinetical control.

[0033] As shown in FIGS. 1 and 2, the adjuvant ensemble composition of the present invention can be adjusted at both the molecular scale and the macro scale, and a cleavable linker is bound to the activation site of the second adjuvant, maintaining an inactive state. Within 2 to 12 hours, more preferably within 3 to 9 hours, the cleavable linker that blocked the activation site is cleaved, and the activity of the immunostimulatory substance appears with a time delay, and the synergistic effect of the immunological reaction can be maximized.

[0034] Also, as shown in FIG. 3, the adjuvant ensemble composition of the present invention can maintain dendritic cells in a mature state, that is, a state capable of inducing an immune reaction, rather than in an exhausted state, that is, a state not inducing an immune reaction, for a long time, and can induce a sustained immune reaction. Also, when encountering naive T cells in the lymph nodes, it can effectively induce Th1 immune reaction-inducing cytokines such as IL-12 through strong immune activation induction together with the CD40-CD40L action during the process of presenting antigens.

[0035] As used herein, the term "adjuvant" refers to an immune modulator and generally encompasses substances that play a role in activating, inducing, or restoring the normal immune function of the immune system. The adjuvant refers to a substance used together with an antigen to enhance the immune response, and by being used together with the antigen, it can increase the production of antibodies and enhance humoral immunity and / or cellular immunity. The immune modulator preferably includes a toll-like receptor agonist, saponin, antiviral peptide, inflammasome inducer, NOD ligand, cytosolic DNA sensor ligand, STING (stimulator of interferon genes) ligand, emulsion, alum, incomplete Freund's adjuvant, Freund's adjuvant, or a combination thereof, and more preferably includes a toll-like receptor agonist.

[0036] As used herein, the term "toll-like receptor agonist" refers to a ligand that directly or indirectly acts on toll-like receptors, which are membrane proteins involved in innate immunity, and can mean a component that can trigger a signal transduction reaction via a signal transduction pathway through the generation of endogenous or exogenous ligands. In this specification, the toll-like receptor agonist may be a natural toll-like receptor agonist or a synthetic toll-like receptor agonist, and may be a toll-like receptor 1 agonist, 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, toll-like receptor 9 agonist, etc.

[0037] The toll-like receptor 1 agonist means a ligand capable of inducing a signal transduction reaction via TLR-1. As an example, it may be, but is not limited to, triacylated lipid peptide (LP); phenol-soluble modulin; lipid peptide of Mycobacterium tuberculosis; S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH; lipid peptide of Borrelia burgdorferi; trihydrochloride (Pam3Cys) lipid peptide that mimics the acetylated amino terminus of OspA lipid peptide, etc.

[0038] The toll-like receptor 2 agonist means a ligand capable of inducing a signal transduction reaction via TLR-2. As an example, it may be, but is not limited to, peptidoglycan, zymosan, HSP70, HMGB1, HA, bam3Cys-Lip, etc.

[0039] The toll-like receptor 3 agonist means a ligand capable of inducing a signal transduction reaction via TLR-3. As an example, it may be, but is not limited to, Poly(I:C), Poly(ICLC), Poly(IC12U), ampligen, etc. as poly IC systems.

[0040] The toll-like receptor 4 agonist means a ligand capable of inducing a signal transduction reaction via TLR-4. As an example, it may be, but is not limited to, outer membrane protein products of Shigella flexneri, AGP, CRX-527, MPLA, PHAD, 3D-PHAD, GLA, LPS, etc.

[0041] The toll-like receptor 5 agonist means a ligand capable of inducing a signal transduction reaction via TLR-5, and as an example, it may be, but is not limited to, flagellin and the like.

[0042] The toll-like receptor 6 agonist means a ligand capable of inducing a signal transduction reaction via TLR-6, and as an example, it may be, but is not limited to, diacyl lipopeptide, lipoteichoic acid and the like.

[0043] The aforementioned toll-like receptor 7 or 8 agonist means a ligand capable of inducing a signal transduction reaction via TLR-7 or 8. As an example, it may be an imidazoquinoline-based agonist, an 8-hydroxyadenine-based agonist, a pteridone-based agonist, a 2-aminopyrimidine-based agonist, a benzoazepine-based agonist, a 7-thia-8-oxoguanosine-based agonist, etc. The imidazoquinoline-based compound includes, but is not limited to, compounds or pharmaceutically acceptable salts of the types described in WO2018 196823, WO2011 049677, WO2011 027022, WO2017 102652, WO2019 040491, etc. Further, the 8-hydroxyadenine-based compound includes, but is not limited to, compounds or pharmaceutically acceptable salts of the types described in WO2012 080730, WO2013 068438, WO2019 036023, WO2019 035969, WO2019 035970, WO2019 035971, WO2019 035968, CN 108948016, US 2014 8846697, WO2016 023511, WO2017 133683, WO2017 133686, WO2017 133684, WO2017 133687, WO2017 076346, WO2018 210298, WO2018 095426, WO2018 068593, WO2018 078149, WO2018 041763, etc. The pteridone-based compound includes, but is not limited to, compounds or pharmaceutically acceptable salts of the types described in US 2010 0143301, WO2016 007765, WO2016 044182, WO2017 035230, WO2017 219931, WO2011 057148, CN 1087 94486, etc.The aminopyrimidine-based compounds include, but are not limited to, compounds of the types described in WO2010 133885, WO2012066335, WO2012 066336, WO2012 067268, WO2013 172479, WO2012 136834, WO2014 053516, WO2014 053595, US 2018 0215720, WO2012 156498, WO2014 076221, WO2016 141092, WO2018 045144, WO2015 014815, WO2018 233648, WO2014 207082, WO2014 056593, WO2018 002319, WO2013 117615, etc., or pharmaceutically acceptable salts thereof. The benzazepine-based compounds include, but are not limited to, compounds of the types described in WO2007 024612, WO2010 014913, WO2010 054215, WO2011 022508, WO2011 022509, WO2012 097177, WO2012 097173, WO2016 096778, WO2016 142250, WO2017 202704, WO2017 202703, WO2017 216054, WO2017 046112, WO2017 197624, etc., or pharmaceutically acceptable salts thereof. The thioxoguanosine-based compounds include, but are not limited to, compounds of the types described in WO2016 180691, WO2016 055553, WO2016 180743, WO2016 091698, etc., or pharmaceutically acceptable salts thereof. Additionally, it may also include toll-like receptor 7 or 8 compounds or pharmaceutically acceptable salts described in PCT / US2009 / 035563, PCT / US2015 / 028264, PCT / US2016 / 020499, WO2015 023598, PCT / US 2015 / 039776, etc.Alternatively, it may be, but is not limited to, imiquimod, resiquimod, dactolisib, gardiquimod, sumanirole, motolimod, vesatolimod, loxoribine, SM360320, CL264, 3M-003, IMDQ, Compound 54, etc., and includes all cases of toll-like receptor 7 or 8 agonists that can be easily inferred and used by those skilled in the art.

[0044] The toll-like receptor 9 agonist means a ligand that can induce a signal transduction reaction via TLR-9. As an example, it may be an immunostimulatory oligonucleotide, etc. The immunostimulatory oligonucleotide may contain one or more CpG motifs, but is not limited thereto.

[0045] As used herein, "saponin" is an amphipathic glycoside that acts as a surfactant. As an example, it may be, but is not limited to, QS21, Quil A, QS7, QS17, β-escin, digitonin, etc.

[0046] As used herein, "antiviral peptide" is a general term for peptides that exhibit an antiviral effect. As an example, it may be, but is not limited to, KLK (kallikrein), etc.

[0047] As used herein, "inflammasome inducer" is a general term for substances that induce inflammasome, which is a protein complex that recognizes and activates danger signals in the cytoplasm of eukaryotic cells. As an example, it may be, but is not limited to, TDB (trehalose-6,6-dibehenate), etc.

[0048] As used herein, the term "NOD ligand" generally refers to ligands that activate Nod-like receptors. By way of example, it may be, but is not limited to, M-TriLYS, N-glycosylated muramyldipeptide, etc.

[0049] As used herein, the term "cytosolic DNA sensor ligand" generally refers to ligands that activate cGAS, a DNA sensor. By way of example, it may be, but is not limited to, Poly(dA:dT), etc.

[0050] As used herein, the term "stimulator of interferon genes ligand" generally refers to ligands that activate STING, a sensor used by immune cells to detect cancer. By way of example, it may be, but is not limited to, cGAMP, di-AMP, di-GMP, etc.

[0051] As used herein, "cholesterol" refers to a type of lipid and generally denotes steroid-based organic substances having hydrophobic properties. The cholesterol may include all various analogs based on the cholesterol structure and compounds that can be obtained by chemically modifying a part of cholesterol. Preferably, it may include, but is not limited to, bile acids (cholic acid, deoxycholic acid, lithocholic acid, chenodeoxycholic acid), vitamin D, steroid hormones (testosterone, estradiol, cortisol, aldosterone, prednisolone, prednisone), etc. Further, the cholesterol is a substance that helps a toll-like receptor 7 or 8 agonist to be located on the surface and inside of nanoparticles in various forms, and can be replaced by lipid substances having a similar function, such as natural lipids (e.g., phospholipids), synthetic lipids, etc. It binds to the activation site of the toll-like receptor 7 or 8 agonist to make it in an inactivated state and prevent the toll-like receptor 7 or 8 agonist from being absorbed into blood vessels in the body. Therefore, there is no limitation as long as it is a known type of lipid.

[0052] As used herein, the term "cleavable linker" generally refers to a linker that contains a cleavable bond and can be cleaved under conditions such as low pH in the body, enzymes, glutathione, etc. in the physiological environments of the tumor microenvironment, intracellular endosomes and lysosomes; or by external stimuli, namely specific stimuli such as temperature, redox potential, ultrasonic waves, magnetic fields, near-infrared light, etc. Preferably, it means a linker containing bonds such as carbamate, disulfide, ester, peptide, azide, etc., or is not limited thereto as long as it is in a cleavable form. As an example of a cleavable linker, cleavable linker groups by enzymes include tobacco etch virus protease (TEV), trypsin, thrombin, cathepsin B, cathepsin D, cathepsin K, caspase 1, matrix metalloproteinase sequences, phosphodiester, phospholipid, ester, beta-galactose, etc.; cleavable linker groups by nucleophiles / bases include dialkyldialkoxysilane, cyanoethyl group, sulfone, ethylene glycolyl disuccinate, 2-N-acyl nitrobenzenesulfonamide, a-thiophenylester, unsaturated vinyl sulfide, sulfonamide after activation, malondialdehyde (MDA)-indole derivative, levulinoyl ester, hydrazone, acylhydrazone, alkyl thioester, etc.In addition, linker groups cleavable by a reducing agent include disulfide bridges, azo compounds, etc.; linker groups cleavable by an oxidizing agent include vicinal diols, selenium compounds, etc.; linker groups cleavable by an organometallic or metal catalyst include disulfide bridges, azo compounds, etc. Also, linker groups cleavable by an electrophilic / acidic agent include Paramethoxybenzyl derivative, tert-butylcarbamate analogue, dialkyl or diaryl dialkoxysilane, orthoester, acetal, aconityl, hydrazone, b-thiopropionate, phosphoramidate, imine, trityl, vinyl ether, polyketal, alkyl 2-(diphenylphosphino)benzoate derivatives, etc.; linker groups cleavable by light irradiation include 2-Nitrobenzyl derivatives, phenacyl ester, 8-quinolinyl benzenesulfonate, coumarin, phosphotriester, bis-arylhydrazone, bimane bi-thiopropionic acid derivative, etc.

[0053] As used herein, "combined administration" means administration together with a conjugate of a toll-like receptor 7 or 8 agonist and cholesterol and various substances such as an antigen, an immune checkpoint inhibitor, an immune antigen enhancer, an immune activator, and a chemotherapeutic agent, and there are no restrictions on the type and form thereof.

[0054] As used herein, the term "chemotherapeutic anti-cancer agent" refers to any compound known to those skilled in the art and used in the treatment of cancer without limitation. Examples thereof include, but are not limited to, Paclitaxel, Docetaxel, 5-Flurouracil, Alendronate, Doxorubicin, Simvastatin, Hydrazinocurcumin, Amphotericin B, Ciprofloxacin, Rifabutin, Rifampicin, Efavirenz, Cisplatin, Theophyline, Pseudomonas exotoxin A, Zoledronic acid, Trabectedin, Siltuximab, Dasatinib, Sunitinib, Apatinib, 5,6-Dimethylxanthenone-4-acetic acid, Silibinin, PF-04136309, Trabectedin, Carlumab, BLZ945, PLX3397, Emactuzumab, AMG-820, IMC-CS4, GW3580, PLX6134, N-acetyl-l-cystein, Vitamin C, bortezomib, aspirin, salicylates, Indolecarboxamide derivatives, quinazoline analogues, Thalidomide, prostaglandin metabolites, 2ME2, 17-AAG, Camptothecin, Topotecan, Pleurotin, 1-methylpropyl, 2-imidazolyl dissulphide, Tadalafil, Sildenafil, L-AME, Nitroaspirin, Celecoxib, NOHA, Bardoxolone methyl, D, L-1-methyl-tryptophan, Gemcitabine, Axitinib, Sorafenib, Cucurbitacin B, JSI-124, Anti IL-17 antibodies, Anti-glycan antibodies, Anti-VEGF antibodies, Bevacizumab, Antracycline, Tasquinimod, Imatinib, cyclophosphamide, and the like.

[0055] As used herein, the term "immune checkpoint inhibitor" generally refers to a cancer treatment method that activates the immune function of immune cells in the human body to fight cancer cells. Examples include, but are not limited to, anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-KIR, anti-LAG3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, anti-TIGIT, etc.

[0056] As used herein, the term "antigen" generally refers to all substances that cause an immune response in the body. Preferably, it is a pathogenic microorganism (such as bacteria, viruses, etc.), chemical substance, pollen, cancer cell, shrimp, etc., or a partial peptide or protein thereof. More preferably, it is a cancer antigen peptide. However, it is not limited thereto as long as it can cause an immune response in the body. The antigen may preferably be a protein, recombinant protein, glycoprotein, gene, peptide, polysaccharide, lipopolysaccharide, polynucleotide, cell, cell lysate, bacterium, virus, etc. More preferably, it may be a cancer antigen peptide. The glycoprotein may be an antibody, antibody fragment, structural protein, regulatory protein, transcription factor, toxin protein, hormone, hormone analog, enzyme, enzyme fragment, transport protein, receptor, receptor fragment, biodefense inducer, storage protein, movement protein, exploitive protein, reporter protein, etc. However, it is not limited thereto as long as it can act as an antigen in the living body and induce an immune response.

[0057] As used herein, "prevention" means all actions that suppress or delay the onset of diseases such as infectious diseases, cancer, metabolic syndrome, autoimmune diseases, and rare diseases by administering the composition according to the present invention.

[0058] As used herein, "treatment" means all actions that result in the improvement or beneficial change of symptoms of infectious diseases, cancer, metabolic syndrome, autoimmune diseases, rare diseases, etc. by administering the composition according to the present invention.

[0059] As used herein, "individual" or "subject" refers to a subject to whom the composition of the present invention can be administered, and there is no limitation on the subject.

[0060] As used herein, "infectious disease" generally refers to a disease induced by infection with foreign organisms such as viruses, bacteria, and fungi.

[0061] As used herein, the term "cancer" generically refers to various blood cancers, malignant solid tumors, etc. that can locally expand by invasion and systematically expand by metastasis. Although not particularly limited thereto, specific examples of cancer include colorectal cancer, adrenal cancer, bone cancer, brain cancer, breast cancer, bronchial cancer, colon cancer and / or rectal cancer, gallbladder cancer, gastrointestinal cancer, head and neck cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, nerve tissue cancer, pancreatic cancer, prostate cancer, parathyroid cancer, skin cancer, stomach cancer, thyroid cancer, etc. Other examples of cancer include adenocarcinoma, adenoma, basal cell carcinoma, cervical dysplasia and intraepithelial cancer, Ewing sarcoma, squamous cell carcinoma, salivary gland cell carcinoma, malignant brain tumor, blastoma, enteric gangliocytoma, hyperplastic corneal nerve cancer, islet cell cancer, Kaposi sarcoma, leiomyoma, leukemia, lymphoma, malignant carcinoid tumor, malignant melanoma, hypercalcemia malignancy, marfanoid habitus cancer, myeloid cancer, metastatic skin cancer, mucosal neuroma, myelodysplastic syndrome, multiple myeloma, fungating polyposis, neuroblastoma, osteosarcoma, osteogenic and other sarcomas, ovarian cancer, chromaffin cell tumor, polycythemia vera, primary brain tumor, small cell lung cancer, ulcerative and papillary squamous cell carcinoma, seminoma, soft tissue sarcoma, retinoblastoma, renal cell tumor or renal cell carcinoma (RCC), reticulum cell sarcoma, and Wilms tumor. Also included are astrocytoma, gastrointestinal stromal tumor (GIST), glioma or glioblastoma, hepatocellular carcinoma (HCC), pancreatic endocrine cancer, etc.

[0062] As used herein, the term "metabolic syndrome" means that an individual has three or more of five risk factors (hypertension, hyperglycemia, hypertriglyceridemia, low high-density lipoprotein cholesterol, and central obesity) that increase the risk of health problems including heart disease, diabetes, and stroke. By way of example, it includes metabolic diseases such as obesity, diabetes, hypertension, hyperlipidemia, heart disease, and gout, and generally refers to all diseases caused by metabolic syndrome.

[0063] As used herein, the term "autoimmune disease" generally refers to diseases caused by pathological reactions against autoantigens, including systemic autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and multiple sclerosis (MS), insulin-dependent diabetes mellitus (IDDM), Graves' disease, and allergy.

[0064] As used herein, the term "rare disease" generally refers to all diseases that affect only a small proportion of the population. Generally, they are genetic, have a very low incidence and prevalence, are difficult to diagnose, and there is no appropriate treatment method. Although the definition varies slightly from country to country, in Korea, based on Article 2 of the Rare Disease Management Act, "a rare disease means a disease with a diseased population of 20,000 or less, or a disease that is difficult to diagnose and the diseased population is unknown, and is defined as a disease determined according to the procedures and criteria specified by the Minister of Health and Welfare Ordinance." The World Health Organization (WHO) designates rare diseases when the prevalence is about 0.65 to 1 person or less per 1,000 people in the population, in the United States when the total number of patients is less than 200,000, and in the European Union (EU) when it is 5 people or less per 10,000 people.

[0065] As used herein, the “pharmaceutical composition” is characterized by being in the form of capsules, tablets, granules, injections, ointments, powders or beverages, and the pharmaceutical composition is characterized by being intended for humans. The pharmaceutical composition may be formulated and used, although not limited thereto, in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., topical agents, suppositories and sterile injection solutions, respectively, by conventional methods. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include, when administered orally, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, flavors, etc., and in the case of injections, buffers, preservatives, soothing agents, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used, and in the case of topical administration, bases, excipients, lubricants, preservatives, etc. may be used. The dosage form of the pharmaceutical composition of the present invention can be manufactured in various ways by mixing with pharmaceutically acceptable carriers as described above. For example, when administered orally, it can be manufactured in the form of tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be manufactured in unit dosage ampoules or multiple dosage forms. In addition, it can be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0066] On the other hand, examples of carriers, excipients and diluents suitable for formulation may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoic acid, propylhydroxybenzoic acid, talc, magnesium stearate or mineral oil, etc. Further, fillers, anticoagulants, lubricants, wetting agents, flavors, emulsifiers, preservatives, etc. may also be included.

[0067] The administration routes of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal. Oral or parenteral administration is preferred. The term "parenteral" as used in the present application includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can also be administered in the form of suppositories for rectal administration.

[0068] The pharmaceutical composition of the present invention can vary widely depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, diet, administration time, administration route, excretion rate, drug formulation and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, degree of illness, drug form, administration route and duration, but can be appropriately selected by those skilled in the art and can be administered at 0.0001 - 500 mg / kg or 0.001 - 500 mg / kg per day. Administration can be carried out once a day or divided into several times. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition or vaccine composition according to the present invention can be formulated into tablets, dragees, capsules, liquids, gels, syrups, slurries, suspensions.

[0069] Hereinafter, preferred examples are presented to assist in the understanding of the present invention. However, the following examples are provided only to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

Example

[0070] [Example 1: Synthetic method of a kinetically acting cholesterol - toll - like receptor agonist] Initially, it exists in an inactive form. However, after being transmitted into target cells such as the tumor microenvironment and immune cells in the body, it is converted to an active state by the physiological environment (low pH, enzymes, glutathione, etc.) and can exhibit immunostimulatory efficacy. To produce a kinetically controlled activity toll-like receptor agonist (TLR agonist), various toll-like receptor 7 or 8 agonists (imidazoquinoloine-based agonist, 8-hydroxyadenine-based agonist, pteridone-based agonist, 2-aminopyrimidine-based agonist, benzoazepine-based agonist, 7-thia-8-oxoguanosine-based agonist, etc.)'s active site, that is, the amine group (NH 2 ) site was conjugated with cholesterol by the chemical reactions of the following Reaction Formula 1 or 2. The binding of the toll-like receptor 7 or 8 agonist to cholesterol was through a cleavable bond such as a carbamate, disulfide, ester, peptide, or azide bond to bind with cholesterol (Sigma-Aldrich).

[0071]

Chemical Structure

[0072] Said R is a side chain containing an aliphatic group or an aromatic group, -NH-, -CO-, -CONH-, -CSNH-, -COO-, -CSO-, -SO 2 NH-, -SO 2-SO-, -O-, etc. may be included.

[0073] [Chemical formula]

[0074] Said R is a side chain containing an aliphatic group or an aromatic group, and may contain -NH-, -CO-, -CONH-, -CSNH-, -COO-, -CSO-, -SO 2 NH-, -SO 2 -SO-, -O-, etc. may be included.

[0075] [Example 2: Synthesis of 2-methyl-1-(3-nitroquinolin-4-ylamino)propan-2-ol] 2-Methyl-1-(3-nitroquinolin-4-ylamino)propan-2-ol (Compound 2) was synthesized using the method of the following Reaction Formula 3. More specifically, 1-amino-2-methylpropan-2-ol (14 g) and tetraethylamine (9.6 g) were added to dichloromethane (450 ml) to which Compound 1 (30 g) was added at 10-20°C, and the mixture was stirred for 2 hours to produce a mixture. Next, after evaporating the solvent under vacuum to concentrate the mixture, it was resuspended using methyl tert-butyl ether (150 ml). The resuspended mixture was separated using a filter and then concentrated under low pressure to obtain Compound 2 (32 g, 85.2%, yellow solid). The structure of the obtained Compound 2 was 1 verified using 1H NMR. 1 1H NMR (400 MHz, DMSO-d6): δ 9.91 (brs, 1H), 9.18 (s, 1H)), 8.46 (d, J = 8.0 Hz, 1H), 7.83 - 7.92 (m, 2H), 7.56 - 7.60 (m, 1H), 5.15 (s, 1H), 3.86 (d, J = 4.8 Hz, 2H), 1.15 (s, 6H).

[0076] [Chemical formula]

[0077] [Example 3: Synthesis of 1-(3-Aminoquinolin-4-ylamino)-2-methylpropan-2-ol] 1-(3-Aminoquinolin-4-ylamino)-2-methylpropan-2-ol (Compound 3) was synthesized using the method of Reaction Scheme 4 below. More specifically, Compound 2 (32 g), methanol (500 ml), and Pd / C catalyst (3.2 g) were mixed in a reactor at 10 - 20 °C. Then the mixture was degassed and flushed three times with hydrogen. After the hydrogen was vaporized to maintain 1 atm, the mixture was stirred at room temperature for 5 hours. Then the mixture was resuspended with methyl tert-butyl ether (100 ml). The resuspended mixture was separated using a filter and then concentrated under low pressure to obtain Compound 3 (27 g, 95.4%, yellow solid). The structure of the obtained Compound 3 was 1 verified using 1H NMR. 1 1H NMR (400 MHz, DMSO-d6): δ 8.37 (s, 1H)), 7.99 - 8.01 (m, 1H), 7.72 - 7.74 (m, 1H), 7.32 - 7.39 (m, 2H), 5.04 (s, 2H), 4.77 (brs, 1H), 4.67 - 4.70 (m, 1H), 4.12 (brs, 2H), 1.15 (s, 6H).

[0078] [Chemical formula]

[0079] [Example 4: Synthesis of 1-(2-Ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol] 1-(2-Ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl)-2-methylpropan-2-ol (Compound 4) was synthesized using the method of the following Reaction Formula 5. More specifically, after adding Compound 3 (27 g) and 2-ethoxyacetic acid (30 ml) to a reactor at 10 - 20°C, the mixture was stirred at 120 - 130°C for 5 hours. Then, the mixture was cooled to 20 - 25°C, and saturated sodium carbonate (150 ml) was added. Next, the reaction product was extracted using a mixed solution of dichloromethane and methanol (10 / 1, v / v). The extracted organic solution layer was washed with brine, and then water was removed using sodium sulfate (10 g). Next, the organic solution layer with water removed was filtered using a filter and then concentrated under low pressure to obtain Compound 4 (30 g, 85.8%, yellow gel). The structure of the obtained Compound 4 was 1 verified using 1H NMR. 1 1H NMR (DMSO-d6 400 MHz): δ 9.18 (s, 1H)), 8.63 (d, J = 8.0 Hz, 1H), 8.13 (dd, J = 1.6, 8.0 Hz, 1H), 7.63 - 7.71 (m, 2H), 4.91 (s, 2H), 4.78 (brs, 2H), 3.54 (q, J = 6.8 Hz, 2H), 1.10 - 1.18 (m, 9H).

[0080] [Chemical Formula]

[0081] [Example 5: Synthesis of 2-(Ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinoline 5-oxide] 2-(Ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-5-oxide (Compound 5) was synthesized using the method of the following Reaction Formula 6. More specifically, after adding Compound 4 (30 g), dichloromethane (350 ml), and metachloroperbenzoic acid (26 g) to a reactor at 10 - 20 °C, the mixture was stirred at room temperature for 4 hours. Next, after adding a saturated sodium carbonate solution (150 ml) and a sodium sulfate solution (150 ml) to the stirred mixture, the reactants were extracted using a mixed solution of dichloromethane and methanol (10 / 1, v / v). The moisture in the extracted organic solution layer was removed using sodium sulfate (30 g), then filtered using a filter, and then concentrated under low pressure. Thereafter, the concentrated reactant was resuspended using ethyl acetate (50 ml), separated using a filter, and then dried under low pressure to obtain Compound 5 (30 g, 94.9%, yellow solid). The structure of the obtained Compound 5 was 1 verified using 1H NMR. 1 1H NMR (DMSO-d6 400 MHz): δ 9.04 (s, 1H), 8.79 (d, J = 8.4 Hz, 1H), 8.71 (d, J = 8.4 Hz, 1H), 7.77 - 7.80 (m, 2H), 4.93 (s, 2H), 4.73 (brs, 2H), 3.54 (q, J = 6.8 Hz, 2H), 1.12 - 1.18 (m, 9H).

[0082] [Chemical formula]

[0083] [Example 6: Synthesis of 1-(4-amino-2-ethoxymethyl)-1H-imidazo[4.5-c]quinolin-1-yl)-2-methylpropan-2-ol] 1-(4-Amino-2-ethoxymethyl)-1H-imidazo[4.5-c]quinolin-1-yl)-2-methylpropan-2-ol (Compound 6) was synthesized using the method of the following Reaction Scheme 7. More specifically, to a reactor at 10 - 20 °C were added Compound 5 (30 g), DCM (600 ml), 4-methylbenzene-1-sulfonyl chloride (18.2 g), and aqueous ammonia (NH 3 ·H 2 O, 180 ml), and the mixture was stirred at room temperature for 16 hours. Next, distilled water was added to the stirred mixture, and then the mixture was separated using a mixed solution of dichloromethane and methanol (10 / 1, v / v). The separated organic solution layer was washed with brine and then dried over anhydrous sodium sulfate (50 g) to remove moisture. The organic solution layer from which moisture had been removed was filtered using a filter, concentrated under low pressure, and the concentrated reaction product was resuspended for 30 minutes using a mixed solution of methyl tert-butyl ether and methanol (15 / 1, v / v). Then, after separation using a filter, it was dried under low pressure to obtain Compound 6 (18 g, 60%, yellow solid). The structure of the obtained Compound 6 was verified using 1 1H NMR. 1 1H NMR (DMSO-d6 400 MHz): δ 8.27 (d, J = 8.0 Hz, 1H), 7.59 (d, J = 7.6 Hz, 1H), 7.40 (t, J = 7.2 Hz, 1H), 7.21 (t, J = 7.2 Hz, 1H), 6.57 (brs, 2H), 4.89 (s, 2H), 4.68 (brs, 2H), 3.52 (q, J = 6.8 Hz, 2H), 1.11 - 1.17 (m, 9H).

[0084]

Chemical formula

[0085] [Example 7: Synthesis of 10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo[a]phenanthren-3-yl 2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl carbamate] Using the method of Reaction Scheme 8 below, 10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo[a]phenanthren-3-yl 2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-yl carbamate (Compound 8) was synthesized. First, Compound 7 (TCI, 50 g, Cholesterol chloroformate) was purified using column chromatography filled with 250 g of silica gel (0% - 20% ethyl acetate in n-hexane) to obtain pure Compound 7 (30 g). Next, Compound 6 (15 g) and dichloromethane (198.9 g) were added to a reactor at 10 - 20 °C, and then pure Compound 7 (30 g) and tetraethylamine (9.6 g) were added in sequence, followed by stirring at 20 - 25 °C for 16 hours. After adding water to the stirred mixture, dichloromethane was added to extract the reactants. The extracted organic solution layer was washed with brine and then dried over anhydrous sodium sulfate (195 g) to remove moisture. The organic solution layer with removed moisture was filtered using a filter and then concentrated under low pressure. Next, the concentrated reactant was resuspended using a mixed solution of methyl tert-butyl ether and methanol (10 / 1, v / v). Then, after separation using a filter, it was dried under low pressure to obtain Compound 8 (10.2 g, 55.1%, white solid). The structure of the obtained Compound 8 was 1 verified using 1H NMR. 1 Through the 1H NMR results, it was confirmed that a conjugate in which resiquimod (R848) and cholesterol were linked by a carbamate bond was produced. 1 1H NMR (CDCl3 400 MHz): δ 8.13 - 8.19 (m, 2H), 7.59 - 7.63 (m, 1H), 7.46 - 7.50 (m, 1H), 5.42 - 5.43 (m, 1H), 4.92 (brs, 2H), 4.72 - 4.80 (m, 3H), 3.68 (q, J = 6.8 Hz, 2H), 3.24 (s, 1H), 2.51 - 2.59 (m, 1H), 2.36 - 2.47 (m, 1H), 1.96 - 2.11 (m, 3H), 1.81 - 1.95 (m, 2H), 1.45 - 1.75 (m, 9H), 1.02 - 1.35 (m, 27H), 0.94 (d, J = 6.4 Hz, 3H), 0.89 (d, J = 6.4 Hz, 6H), 0.71 (s, 3H).

[0086]

Chem.

[0087] [Example 8: Synthesis of Bis(2,5-dioxopyrrolidin-1-yl) 2,2'-disulfanediylbis(ethane-2,1-diyl) dicarbonate] Bis(2,5-dioxopyrrolidin-1-yl) 2,2'-disulfanediylbis(ethane-2,1-diyl) dicarbonate (Compound 9) was synthesized using the method of the following Reaction Scheme 9. First, Compound 7 (TCI, 70 g) was subjected to column chromatography filled with 350 g of silica gel (0% - 20% ethyl acetate in n-hexane) to obtain pure Compound 7 (40 g). Next, pure Compound 7 (40 g) and dichloromethane (100 ml) were added to a reactor at 10 - 15 °C, and then a dichloromethane (250 ml) solution containing bis(2-hydroxyethyl) disulfide and pyridine (21 g) were added in sequence. After the mixture was stirred at room temperature for 2 hours, distilled water (200 ml) was added. Then, dichloromethane (150 ml) was added three times to extract the reactants. The extracted organic solution layer was washed with brine and then dried over anhydrous sodium sulfate (20 g) to remove moisture. The moisture-removed reactant was filtered using a filter and then concentrated under reduced pressure. Then, column chromatography (silica gel, 300 g, 10% - 30% ethyl acetate in n-hexane) was used to obtain Compound 9 (20 g, 39.6%, yellow gel). The structure of the obtained Compound 9 was 1 verified using 1H NMR. 1 1H NMR (CDCl 3 400 MHz): δ 5.41 - 5.42 (m, 1H), 4.46 - 4.56 (m, 1H), 4.41 (t, J = 6.8 Hz, 2H), 3.91 (t, J = 6.0 Hz, 2H), 2.98 (t, J = 6.8 Hz, 2H), 2.91 (t, J = 6.0 Hz, 2H), 2.35 - 2.47 (m, 2H), 1.79 - 2.08 (m, 6H), 1.43 - 1.73 (m, 7H), 1.25 - 1.42 (m, 5H), 1.06 - 1.22 (m, 7H), 0.97 - 1.03 (m, 5H), 0.93 (d, J = 6.4 Hz, 3H), 0.88 (dd, J = 1.6, 6.8 Hz, 6H), 0.69 (s, 3H).

[0088]

Chemical Structure

[0089] [Example 9: Synthesis of Compound 10] Compound 10 was synthesized using the method of Reaction Scheme 10 below. More specifically, after adding Compound 9 (20 g) and dichloromethane (200 ml) to a reactor at 10 - 15°C, bis(2,5-dioxopyrrolidin-1-yl) carbonate (18 g) and tetraethylamine (10.7 g) were added in sequence. The mixture was stirred at room temperature for 3 hours, then distilled water (300 ml) was added, and then dichloromethane (150 ml) was added three times to extract the reactants. The extracted organic solution layer was washed with brine and then dehydrated using anhydrous sodium sulfate (20 g). The dehydrated reactant was filtered using a filter, and then the filtrate was concentrated under low pressure. Then, column chromatography (silica gel, 200 g, 5% - 20% ethyl acetate in n-hexane) was used to obtain Compound 10 (18 g, 72%, yellow gel). The structure of the obtained Compound 10 was 1 verified using 1H NMR. 1 1H NMR (CDCl 3 400 MHz): δ 5.39 - 5.40 (m, 1H), 4.57 (t, J = 6.8 Hz, 2H), 4.43 - 4.52 (m, 1H), 4.37 (t, J = 6.4 Hz, 2H), 2.96 - 3.03 (m, 4H), 2.84 (s, 4H), 2.34 - 2.44 (m, 2H), 1.78 - 2.04 (m, 5H), 1.42 - 1.73 (m, 7H), 1.22 - 1.40 (m, 5H), 1.06 - 1.20 (m, 7H), 0.95 - 1.04 (m, 5H), 0.91 (d, J = 6.0 Hz, 3H), 0.86 (d, J = 6.4 Hz, 6H), 0.67 (s, 3H).

[0090] [Chemical formula]

[0091] [Example 10: Synthesis of 2-((2-((10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo[a]phenanthren-3-yloxy)carbonyloxy)ethyl)disulfanyl)ethyl 2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-ylcarbamate] Using the method of Reaction Scheme 11 below, 2-((2-((10,13-dimethyl-17-(6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclo[a]phenanthren-3-yloxy)carbonyloxy)ethyl)disulfanyl)ethyl 2-(ethoxymethyl)-1-(2-hydroxy-2-methylpropyl)-1H-imidazo[4,5-c]quinolin-4-ylcarbamate (Compound 11) was synthesized. More specifically, after adding Compound 6 (15 g) and dichloromethane (198.9 g) to a reactor at 10 - 20 °C, Compound 10 (40.5 g) and tetraethylamine (9.6 g) were added in sequence. The mixture was stirred at 20 - 25 °C for 16 hours, and then distilled water (225 ml) was added. Next, dichloromethane (99.45 g) was added 5 times to extract the reactant. The extracted organic solution layer was washed with brine and then dehydrated using anhydrous sodium sulfate (195 g). Next, the dehydrated reactant was filtered using a filter, and the filtrate was concentrated under low pressure. Then, Compound 11 (10.8 g, 37.4%, white solid) was obtained using column chromatography (silica gel, 100 g, 10% - 50% ethyl acetate in n-hexane). The structure of the obtained Compound 11 was 1 verified using 1H NMR. 1 Through the 1H NMR results, it was confirmed that a conjugate in which R848 and cholesterol were cross-linked by a disulfide bond was produced. 1 1H NMR (CDCl 3400 MHz): δ 8.15 - 8.17 (m, 2H), 7.60 - 7.64 (m, 1H), 7.47 - 7.51 (m, 1H), 5.39 - 5.40 (m, 1H), 4.93 (s, 2H), 4.81 (s, 2H), 4.56 (t, J = 6.4 Hz, 2H), 4.45 - 4.54 (m, 1H), 4.41 (t, J = 6.4 Hz, 2H), 3.68 (q, J = 6.8 Hz, 2H), 3.13 (s, 1H), 3.09 (t, J = 6.4 Hz, 2H), 3.01 (t, J = 6.4 Hz, 2H), 2.34 - 2.47 (m, 2H), 1.92 - 2.06 (m, 3H), 1.79 - 1.90 (m, 2H), 1.23 - 1.72 (m, 21H), 1.06 - 1.21 (m, 7H), 0.96 - 1.05 (m, 5H), 0.93 (d, J = 6.4 Hz, 3H), 0.88 (dd, J = 1.6, 6.4 Hz, 6H), 0.69 (s, 3H).

[0092]

Chem.

[0093] [Example 11: Production of Nanoparticles Containing a Conjugate of a Cholesterol-Toll-Like Receptor 7 or 8 Agonist] Since the conjugate of the cholesterol-Toll-like receptor 7 or 8 agonist contains cholesterol, it can be easily produced in various nanoparticle forms, thereby maximizing the interaction with immune cells.

[0094] (11.1. Production of Nanoliposomes Containing a Toll-Like Receptor 7 or 8 Agonist Conjugated with Cholesterol) To produce a nanoliposome containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate, a nanoliposome was produced using a cholesterol-resiquimod conjugate, which is one of the cholesterol-Toll-like receptor 7 or 8 agonist conjugates produced in the same manner as in Example 1. More specifically, 5 mg of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine, Avanti), 1.5 mg of DDAB (dimethyldioctadecylammonium bromide, Avanti), and 1 mg of the cholesterol-resiquimod conjugate were dissolved in 0.5 ml of chloroform to produce a mixture. The mixture was evaporated to remove chloroform using a rotary evaporator (30 minutes) to produce a thin film in the form of multiple layers, to which 2 ml of phosphate buffered saline (PBS) was added and stirred at room temperature for 2 hours. Next, after obtaining a monolayer liposome through a homogenization step using a tip sonicator (amplitude: 20%, 2 minutes), a stable nanoliposome containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate was produced through a mini extruder for homogenization of the nanoliposome. The produced nanoliposome was quantified for the amount of the cholesterol-Toll-like receptor 7 or 8 agonist contained therein using ultraviolet and visible light spectroscopy (UV-Vis spectrophotometer).

[0095] (11.2. Production of Nanoemulsion Containing Toll-like Receptor 7 or 8 Agonist Bound with Cholesterol) To produce a nanoemulsion containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate, a cholesterol-resiquimod conjugate, which is one of the cholesterol-Toll-like receptor 7 or 8 agonist conjugates produced by the same method as in Example 1, was used to produce a nanoemulsion. More specifically, after adding 1 mg of DOPC, 240 μg of cholesterol (Sigma-Aldrich), and 240 μg of cholesterol-resiquimod conjugate to 1 ml of chloroform and dissolving them to produce a mixture. Next, the mixture was transferred to a round-bottom flask, and then the chloroform was completely evaporated using a rotary evaporator to produce a thin-film form. Next, after adding and dissolving Squalene (5% v / v), Tween 80 (0.5% v / v), and Span 85 (0.5% v / v) in 2 ml of phosphate buffer solution, the solution was added onto the lipid film, dispersed for 1 minute using a Tip sonicator, and stirred for about 2 hours using a tube revolve to produce a nanoemulsion containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate, and then stored in a 4°C refrigerator until use.

[0096] (11.3. Production of Nanomicelles Composed of Toll-like Receptor 7 or 8 Agonist Bound with Cholesterol and Saponin) To produce a nanomicelle composed of a cholesterol-Toll-like receptor 7 or 8 agonist conjugate and saponin, a cholesterol-resiquimod conjugate, which is one of the cholesterol-Toll-like receptor 7 or 8 agonist conjugates produced in the same manner as in Example 1, was used to produce the nanomicelle. More specifically, phosphatidylcholine:saponin:cholesterol-resiquimod conjugate was mixed at a weight ratio of 5:3:2, and then added to ether to a concentration of 14 mg / ml and dissolved to produce an ether solution containing lipids. Next, saponin was dissolved in 4 ml of distilled water at a concentration of 1.5 mg / ml, placed in a 20-ml glass bottle, the glass bottle was stoppered with a rubber stopper, and then stored in a 55°C water jacket. Next, 1 ml of the ether solution containing lipids was added to the glass bottle containing saponin at a rate of 0.2 ml / min using a syringe pump and stirred for 2 hours. At this time, the tip of the syringe needle was positioned below the surface of the aqueous solution containing saponin, and a second needle was inserted into the rubber stopper for ventilation. Next, the glass bottle was transferred to room temperature and stirred for 3 days for stabilization, and a nanomicelle composed of resiquimod bound with cholesterol and saponin was produced.

[0097] (11.4. Production of Polymer Nanoparticles Composed of Toll-like Receptor 7 or 8 Agonist Bound with Cholesterol) To produce polymer nanoparticles composed of a cholesterol-Toll-like receptor 7 or 8 agonist conjugate, polymer nanoparticles were produced using a cholesterol-resiquimod conjugate, which is one of the cholesterol-Toll-like receptor 7 or 8 agonist conjugates produced in the same manner as in Example 1. More specifically, 60 mg of a PLGA polymer (Eudragit) with a lactide to glycolide composition ratio of 50:50 was dissolved in 1 ml of chloroform solvent. Next, 5 mg of the cholesterol-resiquimod conjugate was added to the solvent, and the cholesterol-resiquimod conjugate and the polymer were dissolved using an ultrasonic bath (Emerson Model CPX5800H-E). Next, while adding 200 μl of the dissolved solution to 10 ml of a 2.5% aqueous PVA solution one by one, it was dispersed for 1 minute using a tip sonicator (Sonics&Materials Model VCX 750). At this time, the output of the disperser was 750 watts, the vibration intensity was 20 kHz, and the amplitude was set to 20%. Next, the aqueous solution produced to completely evaporate the organic solvent in which PLGA was dissolved was stirred at 600 rpm at room temperature for 8 hours or more. To remove unreacted polymer and cholesterol-resiquimod conjugate, centrifugation was performed at 12,000 rpm for 12 minutes using a centrifuge (Centrifuge, Hanil, Combi-514R), and after removing the supernatant, 10 ml of ultrapure water was added and dispersed in a ultrasonic disperser for 30 seconds. After repeating the above process three times, it was dried using a freeze-drying method and stored at -20°C.

[0098] [Example 12: Confirmation of the effect of chemically cleaving the bond of a cholesterol-Toll-like receptor 7 or 8 agonist that acts kinetically by γ-interferon-inducible lysosomal thiol reductase present in endolysosome] (12.1. Intracellular uptake of nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist that acts kinetically) To confirm whether the nanoliposomes containing the conjugate of cholesterol and resiquimod produced by the same method as in Example 11.1 and bound by a disulfide bond move into cells via endocytosis, experiments were conducted using bone marrow-derived dendritic cells (BMDC). More specifically, Dynasore (40 μg), which suppresses endocytosis, was treated on BMDC and cultured for 1 hour. Next, nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate were treated, and cell culture supernatants were obtained at 4, 8, 12, and 24 hours, respectively. Next, the obtained cell culture supernatants were centrifuged at 1,500 rpm for 3 minutes to separate the cells and the supernatant, and the amount of interleukin-12 (IL-12) contained in the supernatant was measured by enzyme-linked immunosorbent assay (ELISA). Thereafter, all experiments were repeated at least 3 times, and the results were shown as the mean value ± standard deviation. Statistical significance was confirmed by Student's t-test, and it was determined that there was statistical significance if P < 0.05. The results are shown in Figure 4.

[0099] As shown in Figure 4, it was confirmed that a very low amount of IL-12 was secreted in the group treated with the endocytosis inhibitor. Through the above results, it was possible to confirm that the nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate normally move into cells and show an immune activation reaction.

[0100] (12.2. Confirmation of the presence or absence of the expression of gamma interferon-inducible lysosomal thiol reductase in BMDC and macrophages) To confirm whether gamma-interferon-inducible lysosomal thiol reductase (GILT) is expressed in bone marrow-derived dendritic cells (BMDCs) and macrophages, experiments were conducted using BMDCs and RAW 264.7 cells, which are macrophages. More specifically, total ribonucleic acid (RNA) from BMDCs and RAW 264.7 cells was isolated using Trizol reagent. Next, complementary deoxyribonucleic acid (cDNA) was synthesized using the Maxime RT PreMix tool with the isolated RNA as a template. Next, real-time polymerase chain reaction (RT-PCR) was performed using the synthesized cDNA, and the PCR products were subjected to agarose gel electrophoresis to confirm the expression level of GILT. The results are shown in Figure 5.

[0101] As shown in Figure 5, it was confirmed that GILT is expressed in dendritic cells and macrophages as antigen-presenting cells.

[0102] (12.3. Confirmation of the presence or absence of chemical bond cleavage of cholesterol-Toll-like receptor 7 or 8 agonists that act kinetically by GILT) To confirm whether the disulfide bond, which is a chemical bond between cholesterol and the toll-like receptor 7 or 8 agonist, is cleaved by GILT present in the endosome, 50 μl of the nanoliposome containing the conjugate in which cholesterol and resiquimod were bound by a disulfide bond, produced in the same manner as in Example 11.1, was placed in a 5 ml tube. Next, cysteine dissolved in PBS with or without GILT was mixed with the nanoliposome, and samples were obtained at different times while culturing in a 37°C shaking incubator. Next, the obtained samples were rapidly frozen using liquid nitrogen and stored in a -20°C freezer. All the samples obtained after 12 hours were freeze-dried in a vacuum freeze-dryer at 10 pascals and at -80°C for 24 hours. Next, the freeze-dried samples were analyzed by liquid chromatography-mass spectrometry (LC-MS) to quantify the amount of resiquimod (R848) separated from the cholesterol-toll-like receptor 7 or 8 agonist conjugate. The results are shown in Figure 6.

[0103] As shown in Figure 6, it was confirmed that the disulfide bond between cholesterol and R848 was cleaved by GILT, and R848 was detected in a separated form.

[0104] Through the above results, it was confirmed that the nanoliposome containing the conjugate in which cholesterol and the toll-like receptor 7 or 8 agonist were linked by a disulfide bond moved into the cell through endocytosis, and the bond was cleaved by GILT present in the endosome within the cell, resulting in the separation of cholesterol and the toll-like receptor 7 or 8 agonist, showing an immunostimulatory effect.

[0105] [Example 13: Confirmation of the effect of inducing an immune response by a kinetically acting cholesterol-toll-like receptor 7 or 8 agonist] (13.1. Confirmation of the activation of immune cells kinetically controlled by nanoliposomes containing a cholesterol-toll-like receptor 7 or 8 agonist) To confirm the effect of the nanoliposomes containing a conjugate in which cholesterol and resiquimod are linked by a disulfide bond, produced in the same manner as in Example 11.1, on the regulation of the immune function of immune cells, after treating BMDCs with the nanoliposomes, the cytokine secretion levels and the expression of co-stimulatory molecules over time were confirmed. More specifically, nanoliposomes or R848 were each added to 1 × 10 6 BMDC cells at a concentration of the toll-like receptor 7 or 8 agonist of 1 μg / ml, and cell culture supernatants were obtained at 4-hour intervals starting from 4 hours later. The obtained cell culture supernatants were centrifuged at 1,500 rpm for 3 minutes to separate the cells and the supernatant, and the secretion levels of interleukin-10 (IL-10) and interleukin-12 (IL-12) contained in the supernatant were measured using ELISA. The results are shown in Fig. 7. In addition, to confirm the maturity of the obtained cells, the cells were stained with fluorescent antibodies, and then the expression of co-stimulatory molecules on dendritic cells was confirmed using BD CantoII flow cytometry. The results are shown in Fig. 8.

[0106] As shown in Fig. 7, it was confirmed that the time point at which IL-10 was secreted was delayed by about 4 hours in the experimental group treated with nanoliposomes containing the cholesterol-R848 conjugate (t-TLR7 / 8a) compared to the experimental group treated with R848 alone. It was also confirmed that the amount of cytokine continued to increase even after 24 hours.

[0107] As shown in Fig. 8, as a result of confirming the expression of co-stimulatory molecules, namely CD80 and CD86, which indicate the maturity of dendritic cells, in the experimental group treated with R848 alone, the expression level of the co-stimulatory molecules did not increase over time, but in the experimental group treated with nanoliposomes containing the cholesterol-R848 conjugate (t-TLR7 / 8a), it was confirmed that the expression level of the co-stimulatory molecules increased over time.

[0108] Through the above results, it was confirmed that the cholesterol-Toll-like receptor 7 or 8 agonist conjugate is an immunomodulator with controllable dynamics, and can not only induce an immune response after about 4 hours, but also induce a sustained immune response compared with R848, a conventional Toll-like receptor 7 or 8 agonist. Also, it was confirmed that the cholesterol-Toll-like receptor 7 or 8 agonist conjugate can maintain dendritic cells in a mature state, i.e., a state capable of inducing an immune response, rather than in an exhausted state, i.e., a state not capable of inducing an immune response, for a long period of time.

[0109] (13.2. Confirmation of the effect of inducing a sustained immune response by nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist that acts kinetically) To confirm whether nanoliposomes containing a conjugate in which cholesterol and resiquimod are bound by a disulfide bond and manufactured in the same manner as in Example 11.1 can continuously induce an immune response, nanoliposomes or R848 were each adjusted to a concentration of 1 μg / ml of the Toll-like receptor 7 or 8 agonist and added to 1×10 6 BMDC cells, and after 12 hours, the cell culture supernatant was obtained. The obtained cell culture supernatant was centrifuged at 1,500 rpm for 3 minutes to separate the cells and the supernatant, and then the amount of IL-12 secreted was measured using the obtained supernatant. Next, the obtained cells were further dispensed into fresh medium, the cell supernatant was obtained at 4-hour intervals, and the amount of IL-12 secreted was confirmed using ELISA. The results are shown in Figure 9.

[0110] As shown in Fig. 9, looking at the results after further culturing in alternation with a new medium (After), in the control group treated with R848, IL-12 was no longer observed, but in the experimental group (t-TLR7 / 8a) treated with nanoliposomes, it was confirmed that the secretion of IL-12 continued until 16 hours. Through the above results, when treating with toll-like receptor 7 or 8 agonist alone, the depletion state of dendritic cells is induced quickly, but it can be confirmed that the cholesterol-toll-like receptor 7 or 8 agonist conjugate maintains the mature state of dendritic cells for a long time and induces a sustained immune response.

[0111] (13.3. Confirmation of the effect of inducing the differentiation of CD4+ T cells into Th1 response by nanoliposomes containing a cholesterol and toll-like receptor 7 or 8 agonist conjugate that acts kinetically) To confirm whether the sustained immune response by nanoliposomes containing a cholesterol-toll-like receptor 7 or 8 agonist conjugate induces the differentiation of CD4 + T cells into Th1 response, first, spleens were collected from C57BL / 6 mice, made into single cells, and then CD4 + T cells were separated using a CD4 + T cell isolation kit. Next, together with OVA (10 μg / ml), nanoliposomes or R848 were each added to 1×10 6 BMDC cells at a concentration of 1 μg / ml of the toll-like receptor 7 or 8 agonist and cultured for 12 hours. Then, they were dispensed into a 96-well plate at a ratio of BMDC to CD4 + T cells of 1:10 and co-cultured. After 5 days of culture, the culture supernatant was obtained, and the secretion levels of interleukin 4 (IL-4) and interferon gamma (IFN-γ) were measured using ELISA. The results are shown in Fig. 10.

[0112] As shown in Fig. 10, the secretion level of IFN-γ showed no significant difference compared with the control group (R848) treated with R848 and OVA and the experimental group (t-TLR7 / 8a) treated with nanoliposomes and OVA. However, it was confirmed that the secretion level of IL-4 decreased in the experimental group (t-TLR7 / 8a) treated with nanoliposomes and OVA. Through the above results, it was confirmed that the secretion of IL-12 was promoted in BMDCs treated with nanoliposomes containing cholesterol-Toll-like receptor 7 or 8 agonist conjugates, thereby differentiating CD4 + T cells into Th1 cells, and increasing the ratio of IFN-γ / IL-4 could be confirmed.

[0113] [Example 14: Confirmation of Synergistic Effect by Combined Administration of Adjuvants] (14.1. Confirmation of Synergistic Effect by the Combination of a Toll-like Receptor 3 Agonist and Nanoliposomes Containing a Kinetically Acting Cholesterol-Toll-like Receptor 7 or 8 Agonist) To confirm the synergistic effect by combined adjuvant administration, BMDCs were treated with poly(I:C) and R848, which are Toll-like receptor 3 agonists known as adjuvants. More specifically, an experimental group (R848 after poly(I:C)) in which BMDCs were first treated with poly(I:C) and then treated with R848, which is a Toll-like receptor 7 or 8 agonist, at an interval, and an experimental group (poly(I:C) after R848) in which R848 was first treated and then poly(I:C) was treated at an interval, that is, asynchronous treatment was performed. As a control group for asynchronous treatment, cells treated with R848 and poly(I:C) simultaneously were used, and relative values were converted with the amount of IL-12 secreted at the time of simultaneous treatment as the reference point 100. As a synchronous treatment group, a control group in which R848 and poly(I:C) were each treated alone as a control group and a control group in which R848 and poly(I:C) were treated simultaneously (R848 + poly(I:C)) were used, and an experimental group (t-TLR7 / 8a + poly(I:C)) treated with nanoliposomes containing a cholesterol-Resiquimod conjugate and poly(I:C) was prepared. Next, after the treatment was completed, the cells were cultured for 36 hours to obtain cell culture supernatants. The obtained cell culture supernatants were centrifuged at 1,500 rpm for 3 minutes to separate the cells and the supernatant, and then the secretion amount of IL-12 contained in the supernatant was measured using ELISA. The results are shown in Fig. 11.

[0114] As shown in Fig. 11, it was confirmed that in the experimental group that first treated R848 and then treated with poly(I:C), the secretion amount of IL-12 decreased compared with the control group treated simultaneously. That is, it was confirmed that there is no synergistic effect between the toll-like receptor 3 agonist and the toll-like receptor 7 or 8 agonist. However, conversely, in the experimental group that first treated with poly(I:C) and then treated with R848, the secretion of IL-12 increased in the experimental group treated with R848 at 2- to 4-hour intervals, and this was confirmed to be a value increased by about 130% compared with the control group treated simultaneously. Through the above results, it was possible to confirm that when the first adjuvant is treated and the second adjuvant is activated with a time difference of 2 to 4 hours after the first adjuvant is activated, the immunostimulatory effect is maximized.

[0115] Also, in the simultaneous treatment experiment, it was confirmed that the experimental group that simultaneously treated nanoliposomes and poly(I:C) showed the highest expression level of IL-2. This is because poly(I:C) is first activated, and then the nanoliposomes containing the cholesterol-toll-like receptor 7 or 8 agonist conjugate move into the cells, and cholesterol is separated by GILT, so that resiquimod is converted from an inactivated state to an activated state, and it was confirmed that the same results as the experimental group treated at time intervals were shown by showing activity.

[0116] (14.2. Confirmation of Synergistic Effect by Combination of Toll-Like Receptor 4 Agonist and Kinetically Acting Nanoliposomes Containing Cholesterol-Toll-Like Receptor 7 or 8 Agonist) To confirm the synergistic effect by administration of the combined adjuvant, an experiment was conducted in the same manner as in Example 14.1 using LPS, a toll-like receptor 4 agonist, resiquimod, a toll-like receptor 7 or 8 agonist, and nanoliposomes containing a cholesterol-toll-like receptor 7 or 8 agonist conjugate. The results are shown in Fig. 12.

[0117] As shown in Fig. 12, in the experimental group treated with LPS and then treated with R848 at time intervals of about 4 hours, the highest IL-12 secretion was shown. Also, in the co-treatment experiment, it was confirmed that the highest IL-12 secretion was shown in the experimental group co-treated with nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate that acts kinetically with LPS.

[0118] Through the above results, it was confirmed that the order and time difference act as important factors in the synergistic effect of adjuvants. That is, when inducing immune activation using a combination adjuvant, in order to increase its effect, it was confirmed that kinetically controlling the activation timing of each adjuvant is important.

[0119] [Example 15: Confirmation of Immunostimulatory Efficacy by Adjuvant Ensemble] (15.1. Confirmation of Immunostimulatory Efficacy by the Combination of Nanoliposomes Containing a Cholesterol-Toll-like Receptor 7 or 8 Agonist Conjugate That Acts Kinetically and a Toll-like Receptor 3 Agonist or a Toll-like Receptor 4 Agonist) To confirm the immunostimulatory efficacy by the combined administration of a first adjuvant and a second adjuvant that acts kinetically, poly(I:C) or LPS was used as the first adjuvant, and experiments were conducted using nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate as the second adjuvant. A control group in which LPS, poly(I:C), R848, and nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate were each administered alone to BMDCs, and experimental groups in which LPS and R848 were co-administered, poly(I:C) and R848 were co-administered, LPS and nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate were co-administered, and poly(I:C) and nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate were co-administered were each prepared, and then cultured for 36 hours to obtain cell culture supernatants. The obtained cell culture supernatants were centrifuged at 1,500 rpm for 3 minutes to separate into cells and supernatants, and then the supernatants were used to measure the secretion levels of tumor necrosis factor (TNF-α) and IL-6 contained in the supernatants. The cells were labeled with fluorescent antibodies and then the expression of co-stimulatory molecules on dendritic cells was confirmed using BD CantoII flow cytometry to confirm the maturity of the cells. The results are shown in FIGS. 13 and 14.

[0120] As shown in FIG. 13, compared with the control group administered alone, the secretion levels of TNF-α and IL-6 increased in the experimental groups administered concurrently, but it was confirmed that the experimental groups administered a combination of nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate that acts kinetically and an adjuvant showed significantly higher secretion levels of TNF-α and IL-6.

[0121] Also, as shown in FIG. 14, it was confirmed that the experimental group administered a combination of nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate that acts kinetically and an adjuvant showed the highest expression level of co-stimulatory molecules.

[0122] Through the above results, it was confirmed that by co-administering an adjuvant capable of kinetically controlling to maintain an inactivated state immediately after administration and inducing a delayed immune response about 2 to 4 hours later and a general adjuvant, a significantly increased immune activation effect can be shown as compared with the case of simply co-administering each adjuvant alone.

[0123] (15.2. Confirmation of the effect of inducing a sustained immune response by a combination of a nanoliposome containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate that acts kinetically and a Toll-like receptor 3 agonist or a Toll-like receptor 4 agonist) To confirm the immune activation efficacy by co-administration of a first adjuvant and a second adjuvant that acts kinetically, poly(I:C) or LPS was used as the first adjuvant, and a nanoliposome containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate was used as the second adjuvant for the experiment. More specifically, BMDCs were treated with LPS and R848, poly(I:C) and R848, LPS and a nanoliposome containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate, or poly(I:C) and a nanoliposome containing a cholesterol-Toll-like receptor 7 or 8 agonist conjugate, and cell culture supernatants were obtained after 24 hours and 36 hours. The obtained cell culture supernatants were centrifuged at 1,500 rpm for 3 minutes to separate into cells and supernatants, and then the secretion amounts of IL-12, IL-6, and TNF-α contained in the supernatants were confirmed using ELISA. The results are shown in Fig. 15.

[0124] As shown in Fig. 15, in the experimental groups treated simultaneously with poly(I:C) or LPS and R848, the amounts of cytokines secreted at 24 hours and 36 hours were similar or decreased in all samples. However, in the experimental groups treated simultaneously with poly(I:C) or LPS and nanoliposomes containing a cholesterol-Toll-like receptor 7 or 8 agonist, it was confirmed that the amount of cytokines secreted 36 hours later was increased compared to the amount of cytokines secreted 24 hours later in all samples. Through the above results, it was confirmed that a sustained immune response can be induced through the combined administration of a kinetically acting second adjuvant and a first adjuvant.

[0125] [Example 16: Preparation of an Adjuvant Ensemble System] (16.1. Preparation of an Adjuvant Complex Composed of Nanoliposomes Containing a Toll-like Receptor 3 Agonist and a Kinetically Acting Cholesterol-Toll-like Receptor 7 or 8 Agonist Conjugate) Nanoliposomes containing a conjugate in which cholesterol and resiquimod are linked by a disulfide bond, prepared in the same manner as in Example 11.1, and poly(I:C) were mixed at a mass ratio of 4:1, and an adjuvant complex in a stable particle form was prepared using a vortex mixer (3 seconds). In this specification, the mass of the nanoliposomes was calculated by converting it to the mass of the Toll-like receptor 7 or 8 agonist contained in the nanoliposomes.

[0126] (16.2. Confirmation of the Degree of Stabilization of the Adjuvant Complex) To confirm whether the adjuvant complex produced in the same manner as in Example 16.1 formed a complex stabilized by electrical attraction, it was confirmed using electrophoresis mobility shift assay (EMSA) whether poly(I:C) that did not form a mixture remained on the periphery. More specifically, agarose (1 g) was added to TAE buffer, melted by heating, poured into a mold, and solidified for about 30 minutes to produce an agarose gel. Next, after sequentially treating a size marker (100 bp), nanoliposomes containing a cholesterol-resiquimod conjugate, poly(I:C), and a complex (K-nanoadjuvant), electrophoresis was performed for 1 hour. The results are shown in Figure 16.

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

[0128] (16.3. Confirmation of intracellular stability of adjuvant complex) To confirm whether the adjuvant complex maintained a stable complex state even intracellularly, an adjuvant complex was produced in the same manner as in Example 16.1 using R848, cholesterol conjugated with FITC (chol-FITC), and poly(I:C) conjugated with rhodamine (Rhodamine-poly(I:C)). Next, BMDC (4×10 4 cells / well) was dispensed into an ibidi μ-slide-8-well microscopy chamber and treated with the adjuvant complex. Then, after culturing at 37 °C for 4 hours and washing with PBS, endosomes were stained with LysoTracker DeepRed and nuclei were stained with Hoechst 33342. Next, cell images were acquired using Deltaviosion PD. The results are shown in Figure 17.

[0129] As shown in Fig. 17, since the fluorescence of the nanoliposomes containing chol-FITC and the fluorescence of Rhodamine-poly(I:C) were confirmed at the same position, it was confirmed that a stable complex was maintained even inside the cells.

[0130] [Example 17: Confirmation of the Efficacy of the Adjuvant Complex] (17.1. Confirmation of the Efficacy of Inducing a Sustained Immune Response in Tumor-Draining Lymph Nodes of the Adjuvant Complex) To confirm the efficacy of the adjuvant complex produced by the same method as in Example 16.1 in inducing an immune response in tumor-draining lymph nodes, C57BL / 6 mice were subcutaneously inoculated with a single dose of R848 (25 μg), poly(I:C) (6.25 μg), or an adjuvant complex containing the same amounts of R848 and poly(I:C) mixed with the SIINFEKL antigen. Next, the lymph nodes were removed 6, 12, 24, 48, and 72 hours after inoculation. The removed lymph nodes were suspended in CellLytic MT cell lysis buffer and then mechanically disrupted. The cell-disrupted solution was centrifuged at 10,000 x g for 10 minutes at 4°C to obtain a supernatant with impurities removed. Next, the amount of IL-12p70 contained in the obtained supernatant was confirmed using ELISA. The results are shown in Fig. 18.

[0131] As shown in Fig. 18, it was confirmed that in the control group treated with R848 and poly(I:C) simultaneously, the secretion of IL-12p70 in the lymph nodes decreased 12 hours later, while in the experimental group treated with the adjuvant complex, IL-12p70 was continuously secreted until 48 hours. Through the above results, it was confirmed that the kinetically acting adjuvant complex, since the action time points of the first adjuvant and the second adjuvant are different, can continuously induce an immune response in the body.

[0132] (17.2. Confirmation of the Anti-Tumor Efficacy of the Adjuvant Complex) To confirm the anti-tumor efficacy of the adjuvant complex produced by the same method as in Example 16.1, primarily 5×10 5Cells were subcutaneously inoculated into the right flank of mice to establish a cancer animal model. Next, an adjuvant complex containing R848 (25 μg), poly(I:C) (6.25 μg), or the same amount of R848 and poly(I:C) was mixed with the SIINFEKL antigen and subcutaneously administered once every three days. Next, the tumor size and the presence or absence of survival were continuously confirmed. The tumor volume was calculated as "long axis diameter × short axis diameter 2 / 2". The results are shown in Figure 19.

[0133] As shown in Figure 19, it was confirmed that 2 out of 5 mice administered with the adjuvant complex survived until 36 days, and the tumor volume was also effectively suppressed. Through the above results, it was confirmed that the anti-cancer effect can be significantly increased by using the adjuvant complex that acts kinetically.

[0134] (17.3. Confirmation of the Efficacy of the Adjuvant Complex in Inducing Immune Responses in Tumor-Draining Lymph Nodes and Tumor Microenvironment) To confirm the efficacy of the adjuvant complex prepared by the same method as in Example 16.1 in inducing immune responses in tumor-draining lymph nodes (TDLN) and tumor microenvironment (TME), first, 5 × 10 5Cells were subcutaneously inoculated into the right flank of mice to establish a cancer animal model. Next, an adjuvant complex containing R848 (25 μg), poly(I:C) (6.25 μg), or the same amount of R848 and poly(I:C) was mixed with the SIINFEKL antigen and subcutaneously inoculated once at 3-day intervals three times. Next, 3 days after the last inoculation, tumor tissues and tumor-draining lymph nodes were excised. Next, to analyze the immune cells collected in the tumor tissues and lymph nodes, the tumor tissues and lymph nodes were mechanically disrupted and then resuspended in a medium supplemented with collagenase D (1 mg / ml) and cultured in a shaker incubator at 37°C for 40 minutes. Then, filtration was performed using a 70-μm cell strainer, and washing was performed twice with PBS to obtain single cells. The obtained single cells were stained using various antibodies conjugated with fluorescence and then analyzed using BD Canto II flow cytometry. The results are shown in FIGS. 20 to 23.

[0135] As shown in FIGS. 20 to 23, it was confirmed that the kinetically acting adjuvant complex generates mature dendritic cells in tumor-draining lymph nodes. More specifically, in the tumor tissues and tumor-draining lymph nodes of mice administered with the adjuvant complex, compared with the experimental group administered with R848 and poly(I:C) simultaneously, CD8 + T cells induced multifunctional IFN-γ + granzyme-B + and CD69 at a very high frequency. Also, in the CD8 + T cells of the experimental group treated with R848 and poly(I:C) simultaneously, the expression levels of PD-1, TIM-3, and LAG-3, which indicate the exhausted state of T cells, increased, but in the experimental group treated with the adjuvant complex, it was confirmed that they were similar to the control group (PBS treatment). Also, CD4 + T cells, CD8 +In T cells, natural killer (NK) cells, and natural killer T (NKT) cells, it was confirmed that the expression of CD69 was induced and the generation of myeloid-derived suppressor cells (MDSCs), which are representative of immunosuppressive cells, was suppressed. Through the above results, it was confirmed that the adjuvant complex of the present invention not only suppresses the depletion state of T cells and enhances the immune activation effect in vivo, but also significantly exhibits a higher immunomodulatory effect compared to simply administering the adjuvant in combination by extending the duration of the immune response.

[0136] In addition, to confirm the cytokines secreted in tumor tissues and lymph node tissues, the obtained tumor tissues were suspended using CellLytic MT cell lysis and then mechanically disrupted. Next, centrifugation was performed at 10,000 x g for 10 minutes at 4°C to obtain the supernatant. Next, the lymph node tissues were mechanically disrupted and resuspended in a medium supplemented with collagenase D (1 mg / ml). Next, the resuspended solution was cultured in a shaker incubator at 37°C for 40 minutes and then filtered using a 70-μm cell strainer. Next, after washing twice with PBS, centrifugation was performed at 1,500 rpm for 3 minutes to obtain single cells, which were then dispensed into plates and cultured at 37°C for 24 hours. The cell culture solution was centrifuged to obtain the supernatant. Next, the secretion levels of IL-12p70 and IFN-γ contained in the obtained supernatant were confirmed using ELISA. The results are shown in FIGS. 24 and 25.

[0137] As shown in FIGS. 24 and 25, it was confirmed that the adjuvant complex effectively induces the secretion of IL-12p70 and IFN-γ in both tumor tissues and tumor-draining lymph nodes.

[0138] Through the above results, the kinetically acting adjuvant complex improves the proliferation ability of various immune cells, increases the cytokine production, and CD8+ By increasing the production of lytic granules without inducing the exhaustion state of T cells, it was confirmed that the effector function of CD8 + T cells can be improved.

[0139] [Example 18: Confirmation of the efficacy of the adjuvant complex using an anti-IL-12 neutralizing antibody] To confirm whether the enhanced proliferation and immunostimulatory ability of immune cells by the adjuvant complex are mediated by the continuous production of IL-12p70, primarily 5×10 5 cells of B16OVA melanoma cells were subcutaneously inoculated into the right flank of mice to establish a cancer animal model. Next, before inoculating the adjuvant complex into the cancer animal model, anti-mouse IL-12p75 (300 μg) was intraperitoneally administered 5 times at 3-day intervals. Next, it was administered 5 times, and 3 days later, the adjuvant complex was mixed with the SIINFEKL antigen and subcutaneously inoculated once a single time 3 times at 3-day intervals. Next, the tumor size and the presence or absence of survival were continuously confirmed. The results are shown in FIG. 26.

[0140] As shown in FIG. 26, it was confirmed that the antitumor effect of the adjuvant complex decreased in the experimental group treated with anti-IL-12. Through the above results, it was confirmed that IL-12 plays an important role between the innate immune stimulation and the adaptive immune response of the kinetically acting adjuvant complex composed of a toll-like receptor 3 agonist and a cholesterol-toll-like receptor 7 or 8 agonist conjugate-containing nanoliposome.

[0141] [Example 19: Confirmation of the effect of inducing an antitumor immune response through local inoculation of the adjuvant complex] (19.1. Confirmation of the antitumor efficacy against metastatic cancer through local inoculation of the adjuvant complex) To confirm whether the adjuvant complex exhibits antitumor efficacy against metastatic cancer through local inoculation, 5×10 5 cells of TC-1 cells were subcutaneously inoculated into the right flank of mice to establish a cancer animal model. Next, 4 days later, secondarily 2.5×10 5Cells were subcutaneously inoculated into the left flank of mice. Next, the adjuvant complex was mixed with the SIINFEKL antigen in a cancer animal model and single inoculated subcutaneously 4 times at 3-day intervals. Next, 3 days after the last inoculation, tumor tissues and tumor-draining lymph nodes were excised. Tumor size and the presence or absence of survival were continuously confirmed. The results are shown in Fig. 27.

[0142] As shown in Fig. 27, it was confirmed that in the experimental group inoculated with the adjuvant complex, the growth of not only primary tumors but also secondary tumors was effectively suppressed.

[0143] (19.2. Confirmation of the effect of suppressing lung metastasis through local inoculation of the adjuvant complex) To confirm whether local inoculation of the adjuvant complex suppresses cancer metastasis, primarily 5×10 5 cells were subcutaneously inoculated into the right flank of mice to produce a cancer animal model. Next, 5 days later, tumor lysate (10 μg) and the adjuvant complex were mixed and single inoculated subcutaneously 4 times at 3-day intervals. Next, 30 days later, after euthanizing the mice, a solution of India ink (47 ml) and PBS (3 ml) was intravenously injected into the organs to stain the lungs. The stained lungs were excised, washed with PBS, and then immersed in a fixing solution (70% ethanol (40 ml), 4% formaldehyde (1 ml), and acetic acid (0.5 ml)) for fixation, and lung metastatic nodules were counted by visual observation. The results are shown in Fig. 28.

[0144] As shown in Fig. 28, a large number of lung metastatic tumor nodules were confirmed in the control group, but it was confirmed that no lung metastasis was observed in the experimental group administered with the adjuvant complex.

[0145] (19.3. Confirmation of the antitumor efficacy against orthotopic tumors for metastatic cancer through local inoculation of the adjuvant complex) To confirm whether local inoculation of the adjuvant complex exhibits antitumor efficacy against orthotopic tumors, primarily C57BL / 6 mice were anesthetized with respiratory anesthesia. Next, after incising the right side of the chest skin, 5×105 Cells were inoculated. Next, 3 days later, the adjuvant complex was mixed with the Long-E7 peptide and subcutaneously singly inoculated 4 times at 3-day intervals. Next, 3 days after the last inoculation, the mice were euthanized, and then the lungs were excised. The excised lungs were sectioned and stained with hematoxylin and eosin. The results are shown in Figure 29.

[0146] As shown in Figure 29, it was confirmed that the tumor cells directly inoculated into the lungs grew aggressively in the control group without any treatment, while the tumor growth was completely inhibited in the experimental group administered with the adjuvant complex.

[0147] Through the above results, it was confirmed that the local administration method of the kinetically acting adjuvant complex can also generate a systemic anti-tumor immune response and effectively show an anti-tumor effect.

[0148] [Example 20: Confirmation of anti-tumor efficacy through the combination of an adjuvant complex and immune checkpoint inhibitor therapy or chemotherapy] An experiment was conducted to confirm whether the treatment effect against various anti-cancer treatments can be improved through the immune activation effect of the adjuvant complex.

[0149] (20.1. Confirmation of anti-tumor efficacy through the combination of an adjuvant complex and immune checkpoint inhibitor therapy) To confirm the anti-tumor efficacy through the combination of an adjuvant complex and immune checkpoint inhibitor therapy (ICBT therapy), primarily 5×10 5 cells were subcutaneously inoculated into the right flank of mice to establish a cancer animal model. Next, 4 days later, the adjuvant complex and the Long-E7 peptide were mixed and subcutaneously singly inoculated 6 times at 3-day intervals. Next, anti-PD-L1 was intraperitoneally administered 8 times at 2-day intervals. 3 days after the last administration, the expression level of PD-L1 in the tumor tissue was confirmed, and the tumor size and the presence or absence of survival were continuously confirmed. The results are shown in Figure 30.

[0150] As shown in Fig. 30, it was confirmed that when the adjuvant complex was combined with the immune checkpoint inhibitor therapy, the expression of PD-L1 was significantly increased and the growth of cancer was also effectively suppressed. In addition, it was confirmed that when the adjuvant complex was combined with the immune checkpoint inhibitor therapy, the mice survived for 120 days or more. Through the above results, it was confirmed that the adjuvant complex increased the expression of PD-L1 by inducing an increase in the activity of cytotoxic lymphocytes and an increase in IFN-γ secretion in the tumor microenvironment, and further increased the anti-cancer effect of the immune checkpoint inhibitor therapy by regulating the immune response in the tumor microenvironment.

[0151] (20.2. Confirmation of anti-tumor efficacy through the combination of adjuvant complex and chemotherapy) To confirm the anti-tumor efficacy through the combination of the adjuvant complex and chemotherapy, first, 5×10 5 TC-1 cells were subcutaneously inoculated into the right flank of mice to establish a cancer animal model. Next, 4 days later, the adjuvant complex and the Long-E7 peptide were mixed and subcutaneously inoculated once a single time at 3-day intervals for 5 times. Next, doxorubicin liposome formulation (80 μg) was intravenously injected twice at 6-day intervals. Next, the tumor size and the presence or absence of survival were continuously confirmed. The results are shown in Fig. 31.

[0152] As shown in Fig. 31, it was confirmed that when the adjuvant complex was combined with chemotherapy, the growth of cancer was effectively suppressed. In addition, it was confirmed that when the adjuvant complex was combined with the immune checkpoint inhibitor therapy, the mice survived for 160 days or more. Through the above results, it was confirmed that cancer can be effectively treated through the combined therapy of the adjuvant complex and chemotherapy.

[0153] The above description of the present invention is for illustrative purposes, and those with ordinary knowledge in the technical field to which the present invention pertains should be able to understand that the present invention can be easily modified into other specific forms without changing the essential features with the technical idea of the present invention. Therefore, the embodiments described above should be understood to be illustrative in all respects and not restrictive.

Industrial Applicability

[0154] The kinetically acting adjuvant ensemble composition of the present invention can not only maximize the synergistic effect of the immunological reaction by providing a kinetic ensemble that acts at regular time intervals when two or more immunologically active substances are administered simultaneously, but also can be easily incorporated with various functional drugs into the drug carrier and regulated to be secreted sequentially, so that it can be applied not only to various diseases that can be treated with adjuvants, but also can significantly increase the therapeutic effect.

Claims

**Claim 1** A kinetically acting adjuvant ensemble composition, wherein the composition comprises two or more adjuvants, a first adjuvant that first binds to an immune cell receptor and induces a primary immune response, wherein the first adjuvant is any one or more immunostimulatory substances selected from the group consisting of triacylated lipid peptides (LP), phenol-soluble modulins, lipid peptides of Mycobacterium tuberculosis, S-(2,3-bis(palmitoyloxy)-(2-RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser-(S)-Lys(4)-OH, lipid peptides of Borrelia burgdorferi, trihydrochloride (Pam3Cys) lipid peptides, peptidoglycan, zymosan, HSP70, HMGB1, HA, bam3Cys-Lip, alum, derivatives of alum, polyIC systems, ampligen, outer membrane protein preparations of Shigella flexneri, AGP, CRX-527, MPLA, PHAD, 3D-PHAD, GLA, LPS, flagellin, diacyl lipopeptide, lipoteichoic acid, QS21, Quil A, QS7, QS17, squalene, β-escin, digitonin, KLK, incomplete Freund's adjuvant, Freund's adjuvant, TDB (trehalose-6,6-dibehenate), M-TriLYS, N-glycosylated muramyl dipeptide, Poly(dA:dT), cGAMP, di-AMP, and di-GMP, a second adjuvant that is a toll-like receptor 7 or 8 agonist to which a cleavable linker is attached at the activation site, and that sequentially binds to an immune cell receptor to induce a secondary immune response, The toll-like receptor 7 or 8 agonist is any one selected from the group consisting of imidazoquinoline agonists, hydroxyadenine agonists, pteridone agonists, aminopyrimidine agonists, benzazepine agonists, and thioxoguanosine agonists, A cleavable linker is bound to the activation site of the second adjuvant, maintaining an inactivated state, and the cleavable linker that blocked the activation site within 2 to 12 hours is cleaved, and the activity of the immunostimulant appears with a time delay. characterized by an adjuvant ensemble composition.

2. The adjuvant ensemble composition according to claim 1, wherein the cleavable linker contains any one or more bonds selected from the group consisting of disulfide, carbamate, hydrazine, ester, peptide, azide, amide, hydrazone, thioether, phosphodiester, thioketal, and combinations thereof.

3. The adjuvant ensemble composition according to claim 1, wherein the cleavable linker further contains ethylene oxide or ethylene glycol at both ends or one end.

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

5. The adjuvant ensemble composition according to claim 1, wherein any one or more substances selected from the group consisting of cholesterol, lipid, protein, amino acid, peptide, and oligonucleotide are bound to the end of the cleavable linker.

6. The adjuvant ensemble composition according to claim 1, wherein the second adjuvant is loaded on any one or more drug delivery bodies selected from the group consisting of nanoliposomes, nanoemulsions, nanomicelles, hydrogels, scaffolds, solid nanoparticles, and polymer nanoparticles.

7. The adjuvant ensemble composition according to claim 6, wherein the drug delivery body further comprises a first adjuvant.

8. The adjuvant ensemble composition according to claim 6, wherein the drug delivery body further comprises a ligand that reacts with a receptor present on the surface of immune cells or in endosomes or the cytosol.

9. The drug delivery body is The adjuvant ensemble composition according to claim 6, further comprising any one or more immunostimulatory substances selected from the group consisting of toll-like receptor agonists, saponins, antiviral peptides, inflammasome inducers, NOD ligands, CDS ligands (cytosolic DNA sensor ligands), STING (stimulator of interferon genes) ligands, outer wall components of pathogenic bacteria, alum, lipids, combinations thereof, and derivatives thereof.

10. The adjuvant ensemble composition according to claim 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, MDSC (myeloid-derived suppressor cells), and M2 macrophages.

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

12. The adjuvant ensemble composition according to claim 11, further comprising an antigen, a chemotherapeutic agent, or an immune checkpoint inhibitor.

13. The adjuvant ensemble composition according to claim 12, 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.

14. The adjuvant ensemble composition according to claim 11, which is characterized by suppressing cancer growth, metastasis, recurrence, or resistance to a cancer treatment therapy.

15. Use of the adjuvant ensemble composition according to claim 1 for producing an agent for improving the immune activation of the adjuvant in the composition according to claim 1.

Images