Autoantigen-specific nanofusion vaccine composition for treatment of systemic sclerosis for simultaneous immunomodulation and antifibrosis

Abstract

The present invention relates to an autoantigen-specific nanofusion vaccine composition for the treatment of systemic sclerosis, the composition being for simultaneous immunomodulation and antifibrosis. A nanofusion body carrying a vimentin peptide according to the present invention was found to inhibit tissue fibrosis in systemic sclerosis, reduce the amount of autoantibodies in serum, and regulate the expression of immune-regulatory cells and pathogenic cells associated with systemic sclerosis. The nanofusion body was also found to inhibit tissue fibrosis and to exhibit enhanced anti-fibrotic effects, when co-administered with a vimentin antibody, in an animal model reflecting the immune status of systemic sclerosis patients. In addition, the nano-vaccine was found to decrease the expression of fibrotic and pathogenic factors in tissues and was also found to improve disease activity not only in vimentin-specific systemic sclerosis but also in infection-associated systemic sclerosis.

Description

Autoantigen-specific nanofusion vaccine composition for the treatment of systemic sclerosis with simultaneous immunomodulation and antifibrosis

[0001] The present invention relates to an autoantigen-specific nano-fusion vaccine composition for the treatment of systemic sclerosis for simultaneous control of immunomodulation and antifibrosis.

[0002] An autoimmune disease is a condition in which the body's immune system attacks normal, healthy tissues, organs, or other body components. Many autoimmune diseases result from an abnormal immune response, leading to self-destruction. Popular examples include rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, scleroderma, polymyositis, dermatomyositis, anaphylactic purpura, and Sjögren's syndrome. Other conditions, such as primary biliary cirrhosis (PBC), chronic active hepatitis, and Hashimoto's thyroiditis, are all associated with autoimmune diseases.

[0003] Among these, systemic scleroderma or systemic sclerosis (SSc) is a progressive, debilitating autoimmune disorder, also known as dermal fibrosis, characterized by excessive protein deposition into the extracellular matrix by dermal fibroblasts. SSc is caused by activated fibroblasts overproducing connective tissue components such as collagen, causing changes in connective tissues such as the skin, blood vessels, gastrointestinal tract, lungs, kidneys, and muscle joints, resulting in functional defects. Depending on the affected area, SSc is broadly classified into a limited type, in which skin hardening occurs only in the hands below the elbows, feet below the knees, and face, and a diffuse type, in which skin hardening changes occur in more extensive areas, such as the skin above the elbows and knees, and in the trunk, accompanied by invasion of internal organs such as the kidneys and lungs. Patients with limited SSc have a 5-year survival rate of over 90%, whereas those with diffuse SSc have a poorer prognosis, with a 5-year survival rate of only 50-70%. Patients with diffuse skin disease often show upregulation of unique markers in the skin, such as type I interferon (IFN)-induced genes. The role of IFN in skin fibrosis is supported by recent reports of scleroderma occurring in patients receiving IFN therapy for chronic viral infections. SSc lesions can be characterized by symptoms such as tissue fibrosis, vasculopathy of small blood vessels, and a specific autoimmune disease mediated by autoantibodies.In particular, an autoimmune reaction is a phenomenon in which various immune diseases are caused by forming antibodies against substances existing within the body rather than external substances due to an abnormality in the immune system. Diseases caused by autoimmune diseases include SSc, systemic lupus erythematosus, rheumatoid arthritis, Sjogren syndrome, and myositis.

[0004] Meanwhile, vimentin, a structural protein encoded by the VIM gene in humans, is known to play a crucial role in supporting and anchoring the position of cellular organelles. The cytoskeleton of chondrocytes is primarily composed of actin microfilaments, tubulin microtubules, and vimentin intermediate filaments. Vimentin is known to be important for the structural formation of cells and tissues. However, in patients with autoimmune diseases or animal models, changes in vimentin protein expression levels and filament network organization have been observed, resulting in abnormal overexpression of vimentin, which has been reported to be associated with a damaged vimentin network. Furthermore, ankylosing spondylitis and tissue fibrosis are caused by chronic inflammation associated with repetitive tissue damage and autoimmune diseases. Chronic inflammation is induced by disruption of the existing tissue repair system, leading to the secretion of inflammatory cytokines, chemokines, and growth factors from immune cells. These factors induce the activation of fibroblasts and stellate cells, leading to their transformation into myofibroblasts and activated stellate cells, respectively. Afterwards, activated myofibroblasts and stellate cells induce the production of components of the extracellular matrix such as collagen, and due to excessive accumulation of extracellular matrix, the tissue hardens, inducing structural and functional damage. It has been reported that cell surface vimentin is specifically expressed in activated myofibroblasts and stellate cells. However, as of yet, there is no known composition that shows a clear preventive and therapeutic effect on SSc associated with mutation or overexpression of vimentin in patients with immune diseases such as systemic sclerosis. Therefore, SSc treatment is limited to somewhat blocking the progression of the disease by mixing various drugs that can suppress representative lesions such as inflammation and scleroderma.

[0005] Prophylactic vaccines are one of the most effective methods of disease prevention. Prophylactic vaccines are designed to prevent the spread of infection and their action relies on the induction of specific antibodies and memory B cells. In contrast, therapeutic vaccines are designed to eliminate the cause of a given disease. The activity of therapeutic vaccines primarily relies on antigen-specific CD8+ T cells primed to produce cytotoxic T lymphocytes (CTLs), which reject cancerous or infected cells.

[0006] Accordingly, the inventors of the present invention completed the present invention by producing a vimentin nano-fusion and confirming that the produced nano-fusion can be used as a vaccine for treating systemic sclerosis.

[0007] The purpose of the present invention is to provide a vaccine composition for preventing or treating systemic sclerosis, which comprises nanoparticles loaded with vimentin peptides as an active ingredient.

[0008] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating systemic sclerosis, which comprises nanoparticles loaded with vimentin peptides as an active ingredient.

[0009] Another object of the present invention is to provide a food composition for preventing or improving systemic sclerosis, which comprises nanoparticles loaded with vimentin peptides as an active ingredient.

[0010] Another object of the present invention is to provide a vaccine composition for preventing or treating systemic sclerosis, which comprises an immunomodulatory drug and an antigen-conjugated gold nanoparticle (Au nanoparticle) as active ingredients.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating systemic sclerosis, which comprises an immunomodulatory drug and an antigen-conjugated gold nanoparticle (Au nanoparticle) as active ingredients.

[0012] Another object of the present invention is to provide a food composition for preventing or improving systemic sclerosis, which comprises an immunomodulatory drug and antigen-bound gold nanoparticles (Au nanoparticles) as active ingredients.

[0013] Another object of the present invention is to provide a method for treating systemic sclerosis, comprising the step of administering a pharmaceutically effective amount of the above nanoparticles to a subject.

[0014] Another object of the present invention is to provide a method for treating systemic sclerosis, comprising the step of administering a pharmaceutically effective amount of the above gold nanoparticles to a subject.

[0015] In order to achieve the above purpose, the present invention provides a vaccine composition for preventing or treating systemic sclerosis, which comprises a nanoparticle loaded with a vimentin peptide as an active ingredient.

[0016] In addition, the present invention provides a pharmaceutical composition for preventing or treating systemic sclerosis, which comprises a nanoparticle loaded with a vimentin peptide as an active ingredient.

[0017] In addition, the present invention provides a food composition for preventing or improving systemic sclerosis, which comprises nanoparticles loaded with vimentin peptides as an active ingredient.

[0018] In addition, the present invention provides a vaccine composition for preventing or treating systemic sclerosis, which comprises an immunomodulatory drug and an antigen-conjugated gold nanoparticle (Au nanoparticle) as active ingredients.

[0019] In addition, the present invention provides a pharmaceutical composition for preventing or treating systemic sclerosis, which comprises an immunomodulatory drug and an antigen-conjugated gold nanoparticle (Au nanoparticle) as active ingredients.

[0020] In addition, the present invention provides a food composition for preventing or improving systemic sclerosis, which comprises an immunomodulatory drug and an antigen-bound gold nanoparticle (Au nanoparticle) as active ingredients.

[0021] In addition, the present invention provides a method for treating systemic sclerosis, comprising the step of administering a pharmaceutically effective amount of the above nanoparticles to a subject.

[0022] In addition, the present invention provides a method for treating systemic sclerosis, comprising a step of administering a pharmaceutically effective amount of the above gold nanoparticles to a subject.

[0023] The nanoconjugate containing the vimentin peptide of the present invention was confirmed to suppress tissue fibrosis in systemic sclerosis, reduce the amount of autoantibodies in the serum, and regulate the expression of immune regulatory cells and systemic sclerosis pathogenic cells. Furthermore, in an animal model reflecting the immune status of systemic sclerosis patients, it was confirmed to suppress tissue fibrosis, and the tissue fibrosis inhibition effect was enhanced when co-administered with a vimentin antibody. Furthermore, the nanovaccine reduces the expression of tissue fibrogenic factors and systemic sclerosis pathogenic factors, and improves the disease activity in vimentin-specific systemic sclerosis and systemic sclerosis accompanied by infectious diseases, and thus can be usefully utilized in related industries.

[0024] Figure 1 is a diagram showing the structure and characteristics of a nano-fusion containing the vimentin peptide of the present invention.

[0025] A: Nano-fusion structure

[0026] B: Nano-fusion particle size distribution and surface charge

[0027] C: TEM image of nano-fusion

[0028] D: Drug loading efficiency; 1) Rapamycin loading efficiency, 2) Vimentin peptide UV-vis

[0029] Figure 2 is a diagram confirming the in vivo durability of the nano-fusion body of the present invention according to chitosan coding.

[0030] Figure 3 is a diagram showing the in vitro tDC increasing effect of rapamycin and vitamin D3 loaded on the nano-conjugate of the present invention confirmed by flow cytometry.

[0031] Figure 4 is a diagram showing the immune cell regulation effect of the nano-fusion (CNP-D / R / V) of the present invention in an animal model of systemic sclerosis confirmed by flow cytometry.

[0032] Figure 5 is a diagram illustrating a drug administration schedule for confirming the improvement of vimentin-specific systemic sclerosis by the nano-fusion (CNP-D / R / V) of the present invention.

[0033] Figure 6 is a diagram showing skin and lung tissue fibrosis confirmed by H&E staining in vimentin-specific systemic sclerosis following administration of the nano-fusion (CNP-D / R / V) of the present invention (A: staining result, B: quantification of staining result).

[0034] Figure 7 is a diagram showing the accumulation of collagen in skin and lung tissue in vimentin-specific systemic sclerosis following administration of the nano-fusion (CNP-D / R / V) of the present invention, confirmed by MT staining (A: staining result, B: quantification of staining result).

[0035] Figure 8 is a diagram showing the expression of fibrogenic factors in skin tissue in vimentin-specific systemic sclerosis following administration of the nano-fusion (CNP-D / R / V) of the present invention, confirmed by immunohistochemical staining (A: staining result, B: quantification of staining result).

[0036] Figure 9 is a diagram showing the expression of fibrogenic factors in lung tissue in vimentin-specific systemic sclerosis following administration of the nano-fusion (CNP-D / R / V) of the present invention, confirmed by immunohistochemical staining (A: staining result, B: quantification of staining result).

[0037] Figure 10 is a diagram showing the amount of autoantibodies in serum in vimentin-specific systemic sclerosis following administration of the nano-fusion (CNP-D / R / V) of the present invention, confirmed by ELISA.

[0038] Figure 11 is a diagram illustrating a drug administration schedule for confirming the improvement effect of the nano-fusion (CNP-D / R / V) of the present invention on patient immune-mimetic systemic sclerosis.

[0039] Figure 12 is a diagram showing the inhibition of skin and lung fibrosis in immune-mimetic systemic sclerosis following administration of the nano-fusion (CNP-D / R / V) of the present invention, confirmed by H&E staining (A: staining result, B: quantification of staining result).

[0040] Figure 13 is a diagram confirming the inhibition of skin and lung collagen accumulation in immune-mimetic systemic sclerosis following administration of the nano-fusion (CNP-D / R / V) of the present invention using MT staining (A: staining result, B: quantification of staining result).

[0041] Figure 14 is a diagram illustrating a drug administration schedule for confirming the combined effect of the nano-fusion (CNP-D / R / V) of the present invention with a vimentin antibody.

[0042] Figure 15 is a diagram showing the inhibition of skin and lung fibrosis in systemic sclerosis by combined administration of the nano-fusion (CNP-D / R / V) of the present invention and vimentin antibody, confirmed by H&E staining (A: staining result, B: quantification of staining result).

[0043] Figure 16 is a diagram confirming the inhibition of skin and lung collagen accumulation in systemic sclerosis by combined administration of the nano-fusion (CNP-D / R / V) of the present invention and vimentin antibody using MT staining (A: staining result, B: quantification of staining result).

[0044] Figure 17 is a diagram showing the characteristics and schematic diagram of LMP59 and SMP59, which are synthesized nano vaccines of the present invention (A: LMP59 characteristic confirmation, B: SMP59 characteristic confirmation, C: structural schematic diagram).

[0045] Figure 18 is a diagram illustrating a drug administration schedule for confirming the improvement of vimentin-specific systemic sclerosis by LMP59 of the present invention.

[0046] Figure 19 is a diagram confirming the tissue fibrosis inhibition effect of LMP59 of the present invention in vimentin-specific systemic sclerosis using H&E or MT staining (A: staining result, B: quantification of staining result).

[0047] Figure 20 is a diagram confirming the amount of autoantibodies in serum in vimentin-specific systemic sclerosis of LMP59 of the present invention.

[0048] Figure 21 is a diagram confirming the pathogenic T cell regulatory effect of LMP59 of the present invention in vimentin-specific systemic sclerosis.

[0049] Figure 22 is a diagram showing the expression of fibrosis factors and systemic sclerosis factors in skin tissue in vimentin-specific systemic sclerosis of LMP59 of the present invention confirmed by immunohistochemical staining (A: staining results, B: quantification of staining results).

[0050] Figure 23 is a diagram showing the expression of fibrotic factors and systemic sclerosis factors in lung tissue in vimentin-specific systemic sclerosis of LMP59 of the present invention confirmed by immunohistochemical staining (A: staining results, B: quantification of staining results).

[0051] Figure 24 is a diagram showing the inhibitory effect of systemic sclerosis tissue fibrosis according to the combined use of LMP59 and vimentin antibodies of the present invention, confirmed by H&E staining (A: staining result, B: quantification of staining result).

[0052] Figure 25 is a diagram confirming the amount of autoantibodies in serum according to the combined use of LMP59 and vimentin antibodies of the present invention.

[0053] Figure 26 is a diagram confirming the effect of regulating pathogenic T cells and Treg according to the combined use of LMP59 and vimentin antibodies of the present invention (A: quantification of pathogenic T cells, B: quantification of Treg).

[0054] Figure 27 is a diagram confirming the inhibition of tissue fibrosis in vimentin-specific systemic sclerosis of SMP59 of the present invention by H&E and MT staining (A: staining result, B: quantification of staining result).

[0055] Figure 28 is a diagram confirming the amount of autoantibodies in serum in vimentin-specific systemic sclerosis of SMP59 of the present invention.

[0056] Figure 29 shows the quantification of Treg and pathogenic T cell regulation in vimentin-specific systemic sclerosis of SMP59 of the present invention.

[0057] Figure 30 is a diagram showing the analysis of fibrotic factors in skin tissue in vimentin-specific systemic sclerosis of SMP59 of the present invention by confocal analysis (A: fluorescence expression results, B: fluorescence expression quantification).

[0058] Figure 31 is a diagram showing the analysis of fibrotic factors in lung tissue in vimentin-specific systemic sclerosis of SMP59 of the present invention by confocal analysis (A: fluorescence expression results, B: fluorescence expression quantification).

[0059] Figure 32 is a diagram confirming the effect of SMP59 of the present invention on suppressing spleen cell proliferation and simultaneous regulation of Th17 and Treg (A: quantification of spleen cell proliferation, B: confirmation of simultaneous regulation of Th17 and Treg).

[0060] Figure 33 is a diagram confirming the inhibition of tissue fibrosis by combined use of SMP59 of the present invention with vimentin antibody using H&E staining (A: staining result, B: quantification of staining result).

[0061] Figure 34 is a diagram confirming the amount of autoantibodies in serum according to the combination of SMP59 of the present invention with vimentin antibodies.

[0062] Figure 35 shows the quantitative results of the regulatory effects of pathogenic T cells and Treg according to the combined use of SMP59 of the present invention with vimentin antibody.

[0063] Figure 36 is a diagram illustrating a drug administration schedule for confirming the effectiveness of SMP59 of the present invention in improving systemic sclerosis accompanied by infectious diseases.

[0064] Figure 37 is a diagram confirming the tissue fibrosis inhibition effect of SMP59 of the present invention in infectious disease-associated systemic sclerosis using H&E and MT staining (A: staining result, B: quantification of staining result).

[0065] Figure 38 is a diagram confirming the amount of autoantibodies in serum in systemic sclerosis accompanied by an infectious disease of SMP59 of the present invention.

[0066] Figure 39 is a diagram quantifying the regulation of expression of pathogenic T cells in systemic sclerosis accompanied by infectious disease of SMP59 of the present invention.

[0067] Figure 40 quantifies the pathogenic T cell regulatory effect of SMP59 of the present invention in spleen cells.

[0068] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In the following description, detailed descriptions of well-known technologies to those skilled in the art may be omitted. Furthermore, in describing the present invention, detailed descriptions of related known functions or configurations may be omitted if it is determined that such detailed descriptions may unnecessarily obscure the gist of the present invention. Furthermore, the terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intentions of the user or operator, or the customs of the field to which the present invention pertains.

[0069] Therefore, definitions of these terms should be based on the overall content of this specification. Throughout this specification, whenever a part is said to "include" a component, this does not exclude other components, but rather implies the inclusion of additional components, unless otherwise specifically stated.

[0070] The present invention provides a vaccine composition for the prevention or treatment of systemic sclerosis, which comprises nanoparticles loaded with vimentin peptides as an active ingredient.

[0071] The term “prevention” used in the present invention means any act of suppressing symptoms of a specific disease or delaying its progression by administering the composition of the present invention.

[0072] The term “treatment” used in the present invention means any act of improving or beneficially altering the symptoms of a specific disease by administering the composition of the present invention.

[0073] The vaccine composition of the present invention can be administered alone, but preferably, it can be administered together with an immunostimulant. An immunostimulant is a substance that non-specifically promotes an immune response to an antigen during the initial activation process of immune cells. It refers to an agent or molecule that is not an immunogen for the host but enhances immunity by increasing the activity of cells of the immune system (Warren et al., 1986, Annu. Rev. Immunol,. 4:369). An immunostimulant that can be administered together with the vaccine composition of the present invention to enhance an immune response includes any of various immunostimulants. Typical immunostimulants include Freund's adjuvant, an aluminum compound, muramyl dipeptide, lipopolysaccharide (LPS), monophosphoryl lipid A, and quinol A, and are not limited thereto, but an example using Freund's adjuvant is disclosed as one of the preferred examples. The above-mentioned immunostimulant may be administered simultaneously with the vaccine composition or sequentially at an interval of time.

[0074] In addition, the vaccine composition of the present invention may additionally include a solvent, an excipient, etc. The solvent includes physiological saline solution, distilled water, etc., and the excipient includes aluminum phosphate, aluminum hydroxide, aluminum potassium sulfate, etc., but is not limited thereto, and may further include a material commonly used in vaccine production in the field to which the present invention pertains.

[0075] The vaccine composition of the present invention can be manufactured by methods commonly used in the technical field to which the present invention pertains. The vaccine composition of the present invention can be manufactured as an oral or parenteral preparation, and is preferably manufactured as an injectable solution as a parenteral preparation, and can be administered via the intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, nasal, or epidural route.

[0076] The vaccine composition of the present invention can be administered to a subject in an immunologically effective amount. The term “immunologically effective amount” refers to an amount sufficient to exhibit a preventive or therapeutic effect on systemic sclerosis or scleroderma and an amount that does not cause side effects or serious or excessive immune responses. The exact administration concentration varies depending on the specific immunogen to be administered, and can be easily determined by those skilled in the art based on factors well known in the medical field, such as the age, weight, health, sex of the subject to be vaccinated, the subject’s sensitivity to drugs, the administration route, and the administration method. The composition can be administered once or multiple times.

[0077] According to one embodiment of the present invention, the nanoparticle may include a core portion comprising at least one of a copolymer, a peptide, a fat-soluble vitamin, and a drug; a first shell portion comprising a surfactant; and a second shell portion comprising a polymer.

[0078] The copolymer may be, but is not limited to, poly(lactic-co-glycolic acid) (PLGA). The peptide may be a vimentin peptide, and the vimentin peptide may act as an antigen.

[0079] The "antigen" of the present invention is a substance capable of inducing an immune response and the production of antibodies, and is generally a collection of substances considered foreign in a living organism. Antigens are mainly pathogens or viruses, but proteins, polysaccharides, artificially synthesized substances, and autologous mutant cells (cancer cells, mutant proteins, etc.) can also act as antigens. The vimentin peptide of the present invention is a peptide of mutant vimentin that induces an autoimmune response in immune diseases, and is an antigen for producing antibodies that can specifically target the mutant peptide.

[0080] The fat-soluble vitamin may be, but is not limited to, vitamin D3.

[0081] The above drug may be Rapamycin.

[0082] The surfactant may be, but is not limited to, Pluronic F-68.

[0083] The structure of the above nanoparticle may be such that a first shell portion is coated on the surface of the core portion, and a second shell portion is coated on the outer surface of the first shell portion.

[0084] The above nanoparticles may have a size of 230 to 240 nm, preferably a size of 238.8 nm.

[0085] The zeta potential of the nanoparticles of the present invention may be 12 mV to 13 mV, preferably +12.5 mV.

[0086] According to one embodiment of the present invention, the vimentin peptide may include an amino acid sequence of sequence number 1, and the amino acid sequence of sequence number 1 may have a 5-' end connected to FITC (Fluorescein isothiocyanate) as a linker, and the linker sequence according to one embodiment may be an ACA base sequence in which three bases are connected, but any linker sequence that connects conventional proteins, base sequences, and compounds may be used without limitation.

[0087] According to one embodiment of the present invention, the nanoparticles may contain rapamycin or vitamin D3, and the rapamycin or vitamin D3 may increase the expression of tolerogenic dendritic cells (tDC).

[0088] According to one embodiment of the present invention, the composition may regulate the expression of immune cells, suppress the expression of Th2 or Th17, which are immune cells causing systemic sclerosis, and increase the expression of tDC.

[0089] According to one embodiment of the present invention, the composition may inhibit tissue fibrosis, and the inhibition of tissue fibrosis may be inhibiting a decrease in dermal thickness or collagen accumulation in the tissue, or may be inhibiting the expression of a fibrogenic factor or a factor related to systemic sclerosis in the tissue.

[0090] According to one embodiment of the present invention, the fibrotic factor or systemic sclerosis-related factor may be a factor selected from the group consisting of COL1 (collagen type I), a-SMA (Alpha smooth muscle actin), cell surface vimentin, IL-4, and IL-17.

[0091] According to one embodiment of the present invention, the tissue may be a tissue selected from the group consisting of skin, lung, liver, muscle, kidney, intestine and spleen, and is preferably skin or lung tissue, but is not limited thereto.

[0092] According to one embodiment of the present invention, the composition may reduce the amount of autoantibodies in serum, and the autoantibodies in serum may be total IgG, IgG2a, or IgG1.

[0093] According to one embodiment of the present invention, the composition may be administered in combination with a vimentin antibody.

[0094] In addition, the present invention provides a pharmaceutical composition for preventing or treating systemic sclerosis, which comprises a nanoparticle loaded with a vimentin peptide as an active ingredient.

[0095] The pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient. Any adjuvant known in the art may be used without limitation. However, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to enhance its effectiveness.

[0096] The pharmaceutical composition according to the present invention can be prepared in a form in which the active ingredient is mixed with a pharmaceutically acceptable carrier. Here, the pharmaceutically acceptable carrier includes carriers, excipients, and diluents commonly used in the pharmaceutical field. Pharmaceutically acceptable carriers that can be used in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0097] The pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories, or sterile injection solutions, each according to a conventional method.

[0098] When formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and such solid preparations can be prepared by mixing the active ingredient with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0099] The pharmaceutical composition according to the present invention can be administered to a subject via various routes. All modes of administration are contemplated, including oral, intravenous, intramuscular, subcutaneous, and intraperitoneal injection.

[0100] The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be selected in various ways depending on the subject, and it is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, pharmaceutical activity may not be observed, and if it exceeds 5,000 μg / ml, it may be toxic to the human body.

[0101] In addition, the present invention provides a food composition for preventing or improving systemic sclerosis, which comprises nanoparticles loaded with vimentin peptides as an active ingredient.

[0102] The term “improvement” as used herein means any action that at least reduces a parameter related to the condition being treated, for example, the severity of a symptom.

[0103] The food composition of the present invention may contain, in addition to containing the effective ingredient of the present invention, various flavoring agents or natural carbohydrates as additional ingredients, like conventional food compositions.

[0104] Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose, etc.; disaccharides such as maltose, sucrose, etc.; and polysaccharides such as dextrin, cyclodextrin, etc., and common sugars, and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As the above-mentioned flavoring agent, natural flavoring agent (thaumatin), stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agent (saccharin, aspartame, etc.) can be advantageously used. The food composition of the present invention can be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes, and health supplements.

[0105] In addition, the food composition may contain, in addition to the extract as an active ingredient, various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain fruit pulp for producing natural fruit juice, fruit juice drinks, and vegetable drinks.

[0106] The functional food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of preventing or treating systemic sclerosis. The term "health functional food composition" in the present invention refers to a food manufactured and processed using raw materials or ingredients having functionality useful to the human body according to Act No. 6727 on Health Functional Foods, and means to be consumed for the purpose of obtaining a useful effect for health purposes such as regulating nutrients for the structure and function of the human body or physiological effects. The health functional food of the present invention may include conventional food additives, and whether it is suitable as a food additive is determined by the specifications and standards for the relevant item according to the general provisions and general test methods of the Food Additives Codex approved by the Ministry of Food and Drug Safety, unless otherwise specified. Items listed in the "Food Additives Codex" include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Examples thereof include natural additives such as persimmon pigment, licorice extract, crystalline cellulose, high-molecular weight pigment, and guar gum; mixed preparations such as sodium L-glutamate preparations, noodle additive alkaline agents, preservative preparations, and tar color preparations. For example, a health functional food in tablet form can be prepared by mixing the active ingredient of the present invention with excipients, binders, disintegrants, and other additives, granulating the mixture using a conventional method, and then adding a lubricant, etc. to compress and molding, or directly compressing and molding the mixture. In addition, the health functional food in tablet form can contain a maturing agent, etc., if necessary. Among health functional foods in capsule form, hard capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a conventional hard capsule, and soft capsules can be prepared by filling a mixture of the active ingredient of the present invention with additives such as excipients into a capsule base such as gelatin. The above soft capsule may contain a plasticizer such as glycerin or sorbitol, a coloring agent, a preservative, etc., as needed.The ring-shaped health functional food can be prepared by molding a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can be coated with white sugar or other coating agents, or the surface can be coated with a substance such as starch or talc. The granular health functional food can be manufactured into a granular form using a mixture of the active ingredient of the present invention with excipients, binders, disintegrants, etc. using a conventionally known method, and, if necessary, can contain a flavoring agent, a flavoring agent, etc.

[0107] In addition, the present invention provides a vaccine composition for preventing or treating systemic sclerosis, which comprises an immunomodulatory drug and an antigen-conjugated gold nanoparticle (Au nanoparticle) as active ingredients.

[0108] According to one embodiment of the present invention, the gold nanoparticles of the present invention have a size of 1 to 1,000 nm and a shape of, but not limited to, a sphere, a rod, a cube, etc. As the type of ligand, hydrophilic and amphiphilic single molecules and polymers having thiol, phosphate, and amine functional groups can be used, but are not limited thereto.

[0109] The above may be a case where an amino acid or ligand is bound to the surface of the gold nanoparticle to form a brush or mushroom structure, preferably glutathione (GSH) or an arginine ligand is bound, and more preferably, an immunomodulatory drug and an antigen may be additionally bound.

[0110] The immunomodulatory drug and antigen of the present invention can be included in the gold nanoparticles by 1) direct binding to the gold nanoparticles, or 2) electrostatic binding (charge interaction) with an amino acid (glutathione) or a ligand (arginine ligand).

[0111] According to one embodiment of the present invention, the immunomodulatory drug may be a compound represented by the following chemical formula 1.

[0112] [Chemical Formula 1]

[0113]

[0114] The compound of the above chemical formula 1 of the present invention is a compound named metformin, and is a compound with CAS number 657-24-9.

[0115] According to one embodiment of the present invention, the antigen may be a vimentin peptide, and the vimentin peptide may include an amino acid sequence of SEQ ID NO: 2.

[0116] According to one embodiment of the present invention, the composition may suppress the expression of T cells causing systemic sclerosis, and the T cells causing systemic sclerosis may be IL-17 positive, IL-4 positive, or IL-13 positive T cells.

[0117] According to one embodiment of the present invention, the composition may reduce the amount of autoantibodies in serum, and the autoantibodies in serum may be total IgG, IgG1 or IgG2a.

[0118] According to one embodiment of the present invention, the composition may inhibit tissue fibrosis, and the inhibition of tissue fibrosis may be inhibition of the thickness of the skin dermis layer or lung fibrosis.

[0119] According to one embodiment of the present invention, suppressing the tissue fibrosis may be suppressing the expression of a fibrotic factor or a systemic sclerosis factor in the tissue, and the fibrotic factor or systemic sclerosis factor may be a factor selected from the group consisting of IL-17, IL-4, cell surface vimentin, alpha smooth muscle actin (a-SMA), and collagen type I A1 (COL1A1) and CD19.

[0120] According to one embodiment of the present invention, the tissue may be a tissue selected from the group consisting of skin, lung, liver, muscle, kidney, intestine and spleen, and is preferably skin or lung tissue, but is not limited thereto.

[0121] According to one embodiment of the present invention, the composition may increase the expression of Treg.

[0122] According to one embodiment of the present invention, the systemic sclerosis may further include vimentin-specific systemic sclerosis or infection-associated systemic sclerosis.

[0123] According to one embodiment of the present invention, the composition may be administered in combination with a vimentin antibody.

[0124] In addition, the present invention provides a pharmaceutical composition for preventing or treating systemic sclerosis, which comprises an immunomodulatory drug and an antigen-conjugated gold nanoparticle (Au nanoparticle) as active ingredients.

[0125] In addition, the present invention provides a food composition for preventing or improving systemic sclerosis, which comprises an immunomodulatory drug and an antigen-bound gold nanoparticle (Au nanoparticle) as active ingredients.

[0126] In addition, the present invention provides a method for treating systemic sclerosis, comprising the step of administering a pharmaceutically effective amount of the above nanoparticles to a subject.

[0127] The treatment method of the present invention comprises administering the nanoparticles to a subject in a therapeutically effective amount. It is preferred that the specific therapeutically effective amount for a specific subject be applied differently depending on various factors including the type and degree of response to be achieved, the specific composition including whether other agents are used in some cases, the age, body weight, general health, sex and diet of the subject, the time of administration, the route of administration and the secretion rate of the composition, the treatment period, drugs used together or simultaneously with the specific composition, and similar factors well known in the medical field. The daily dosage is 0.0001 to 100 mg / kg, preferably 0.01 to 100 mg / kg, based on the amount of the pharmaceutical composition of the present invention, and can be administered 1 to 6 times a day. However, it is obvious to those skilled in the art that the dosage or administration of each active ingredient should be such that the content of each active ingredient is not excessively high and does not cause side effects. Therefore, it is preferred that the effective amount of a composition suitable for the purpose of the present invention be determined in consideration of the aforementioned matters.

[0128] The above object is applicable to any mammal, which includes not only humans and primates, but also livestock such as cows, pigs, sheep, horses, dogs and cats.

[0129] The nanoparticles of the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes. All modes of administration are conceivable, including oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection.

[0130] In addition, the present invention provides a method for treating systemic sclerosis, comprising a step of administering a pharmaceutically effective amount of the above gold nanoparticles to a subject.

[0131] Hereinafter, the present invention will be described in more detail with reference to examples. These examples are intended merely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

[0132] <Example 1> Production and characterization of oral nanoconjugates

[0133] <1-1> Fabrication of nano-conjugates

[0134] A nanoconjugate loaded with the vimentin peptide of the present invention and coated with chitosan was fabricated. Specifically, CNP-D / R / V, a nanoconjugate of the present invention, was fabricated by loading poly(lactic-co-glycolic acid) nanoparticles with FITC (Fluorescein isothiocyanate)-linked vimentin peptide (SEQ ID NO: 1: RLRSSVPGV, peptides with FITC for imaging purposes and peptides without FITC for other purposes), rapamycin, and vitamin D3, and coating the nanoparticles micellized with the surfactant Pluronic F-68 with chitosan. The nanoconjugate was fabricated using a traditional oil-in-water emulsion method. The hydrophobic PLGA core is surrounded by F-68 surfactant. In this form, the surface potential is negatively charged due to the influence of PLGA. Therefore, when chitosan is mixed in after particle production, chitosan is coated on the surface again due to electrical attraction. The specific structure of the produced nano-fusion is schematically illustrated in Figure 1A.

[0135] The fabricated CNP-D / R / V was confirmed to have a size of 238.8 nm and a zeta potential of +12.5 mV (Fig. 1B). In addition, compared to PLGA nanoparticles (PLGA-NP), the size of the nanoconjugate was confirmed to have increased slightly (Figs. 1B and 1C). The initial loading rate of rapamycin analyzed by HPLC was 69.9%, and the initial loading rate of vimentin peptide analyzed based on the fluorescence amount measurement of FITC was confirmed to be 90.7% (Fig. 1D).

[0136] <1-2> Confirmation of in vivo persistence of nanoconjugates

[0137] To confirm the in vivo persistence of the vimentin peptide fusion of the present invention, the intestinal retention times of PLGA-NP and chitosan-coated nanoparticles (Chitosan-coated-NP, CNP) were determined. Specifically, dye-loaded PLGA-NP or CNP was orally administered to mice, and the fluorescence expression of the dye was confirmed 3 and 6 hours later using a bioimaging system.

[0138] As a result, as shown in Fig. 2, compared to PLGA-NP, CNP was confirmed to remain in the intestine even after 6 hours, confirming that chitosan-coated nanoparticles have excellent in vivo persistence.

[0139] <Example 2> Confirmation of the immune cell regulation effects of rapamycin and vitamin D3

[0140] <2-1> Confirmation of in vitro immune tolerance dendritic cell regulation

[0141] It was confirmed whether rapamycin and vitamin D3 loaded on the nano-fusion of the present invention increase tolerogenic dendritic cells (tDC). Specifically, 1x10 mouse-derived spleen cells were cultured in a culture medium containing 5% fetal bovine serum. 6Diluted to the cell number, they were spread on well plates, and cultured under mature dendritic cell (mDC) differentiation conditions, treated with 1 nM rapamycin or 100 nM vitamin D3. To confirm differentiation into tDC, CD11c-positive, CD274-positive, and IL-10-positive cells were analyzed by flow cytometry. As controls, the nil group, which is an untreated control group, and the mDC group cultured only under mature dendritic cell differentiation conditions (mDC condition) were used.

[0142] As a result, as shown in Fig. 3, it was confirmed that the number of mature dendritic cells significantly increased in the group treated with rapamycin or vitamin D3 compared to the nil group and the mDC group.

[0143] <2-2> Confirmation of immune cell regulation in vivo

[0144] It was confirmed whether the nano-conjugate loaded with the vimentin peptide of the present invention also regulates immune cells in vivo. Specifically, after removing hair from B6 mice using clippers, 50 μl of vimentin protein at a concentration of 4 mg / ml and 50 μl of complete Freund's adjuvant (CFA) were mixed and injected subcutaneously into the base of the mouse tail. After 2 weeks, 50 μl of vimentin protein at a concentration of 4 mg / ml and 50 μl of incomplete Freud's adjuvant (IFA) were mixed and injected subcutaneously into the base of the mouse tail. After 24 hours, the hair on the back of the neck was removed using clippers, and systemic sclerosis was induced by subcutaneously injecting 50 μg / 100 μl bleomycin in PBS daily for 2 weeks. Starting one week prior to the induction of systemic sclerosis, 300 μl of CNP-D / R / V was administered orally three times a week for six weeks. At the end of the experiment, mice were humanely sacrificed, and splenocytes were obtained from the mice. The expression of Th2 and Th17 cells, which are pathogenic cells of systemic sclerosis, and the expression of tDCs were confirmed by flow cytometry.

[0145] As a result, as shown in Fig. 4, compared to the vehicle group, in the group administered CNP-D / R / V, Th2 and Th17, which are immune cells that cause systemic sclerosis, were significantly reduced, and immune tolerant dendritic cells were significantly increased, confirming that the nano-fusion containing the vimentin peptide of the present invention regulates immune cells in systemic sclerosis.

[0146] <Example 3> Confirmation of Vimentin-Specific Improvement of Systemic Sclerosis with Nano-Conjugate

[0147] <3-1> Confirmation of inhibition of tissue fibrosis

[0148] The nanoconjugate containing the vimentin peptide of the present invention was confirmed to have an effect of improving vimentin-specific systemic sclerosis immunosensitized to vimentin. Specifically, after removing hair from B6 mice using clippers, 50 μl of vimentin protein at a concentration of 4 mg / ml and 50 μl of CFA were mixed and injected subcutaneously into the base of the mouse tail. Two weeks later, 50 μl of vimentin protein at a concentration of 4 mg / ml and 50 μl of IFA were injected, and 24 hours later, the hair on the back of the neck was removed using clippers, and systemic sclerosis was induced by subcutaneous injection of 50 μg / 100 μl bleomycin in PBS daily for two weeks. After inducing systemic sclerosis, 300 μl of CNP-D / R / V was orally administered three times a week for a total of six weeks, starting from one week before (Fig. 5). At the end of the experiment, the mice were humanely sacrificed, and skin and lung tissues were obtained. The skin dermal layer thickness and lung histological indices were determined by H&E staining and Masson's Trichrome (MT) staining, and the degree of collagen deposition in the tissues was quantified. Wild-type mice (WT) and the vehicle group, which was administered an equivalent amount of solvent instead of CNP-D / R / V, were used as controls.

[0149] As a result, as shown in Fig. 6, compared to the vehicle group, the group administered CNP-D / R / V showed a decrease in skin dermal layer thickness and a decrease in lung tissue index, confirming that damage caused by tissue fibrosis was protected. In addition, as a result of examining collagen accumulation in tissues, it was confirmed that collagen accumulation was significantly reduced in skin and lung tissues (Fig. 7).

[0150] <3-2> Confirmation of suppression of expression of factors related to tissue fibrosis and systemic sclerosis

[0151] It was confirmed whether the nano-conjugate containing the vimentin peptide of the present invention regulates the expression of factors related to fibrosis and systemic sclerosis in tissues. Specifically, the skin and lung tissues obtained in Example 3-1 were sectioned, and the expression of COL1 (collagen type I), a-SMA (alpha smooth muscle actin), cell surface vimentin (CSV), IL-4, and IL-17, which are factors related to fibrosis and systemic sclerosis, was confirmed by immunohistochemical staining.

[0152] As a result, as shown in FIGS. 8 and 9, in skin and lung tissues, compared to the vehicle group, the group administered CNP-D / R / V showed a significant decrease in the expression of fibrosis and systemic sclerosis-related factors, COL1 (collagen type I), a-SMA (Alpha smooth muscle actin), cell surface vimentin (CSV), IL-4, and IL-17, confirming that the nano-fusion of the present invention inhibits tissue fibrosis.

[0153] <3-3> Confirmation of autoantibody control in serum

[0154] Blood was obtained from the mice sacrificed in Example 3-1 above, and the amounts of total IgG, IgG2a, and IgG1, which are vimentin-specific autoantibodies, were analyzed by ELISA analysis to confirm the autoimmune suppression effect by autoantibodies.

[0155] As a result, as shown in Fig. 10, when the autoantibodies in serum, vimentin-specific antibodies, were confirmed, the autoantibodies, total IgG, IgG2a, and IgG1, were significantly increased in the Vehicle group compared to the WT group, but when CNP-D / R / V was administered, the amount of increased autoantibodies in serum was confirmed to be significantly reduced.

[0156] <Example 4> Confirmation of improvement in patient's immune-mimetic systemic sclerosis

[0157] It was confirmed whether the nanoconjugate containing the vimentin peptide of the present invention improves systemic sclerosis reflected in the immune response of the patient. Specifically, peripheral blood mononuclear cells (PBMCs) derived from a patient with systemic sclerosis were injected intraperitoneally one week before inducing immunosensitization with vimentin in NSG mice. After that, hair was removed using clippers, and a mixture of 50 μl of vimentin protein at a concentration of 4 mg / ml and 50 μl of CFA was subcutaneously injected into the base of the tail of the mice. After two weeks, a mixture of 50 μl of vimentin protein at a concentration of 4 mg / ml and 50 μl of IFA was injected, and 24 hours later, the hair on the back of the neck was removed using clippers, and 50 μg / 100 μl of bleomycin was subcutaneously injected daily for two weeks to induce systemic sclerosis. Starting one week before the induction of systemic sclerosis, 300 μl of CNP-D / R / V was administered orally three times a week for a total of six weeks (Fig. 11). At the end of the experiment, the mice were humanely sacrificed, and skin and lung tissues were obtained. Skin dermal layer thickness and lung histological indices were determined by H&E staining and Masson's Trichrome (MT) staining, and the degree of collagen deposition in the tissues was quantified. The vehicle group, which was administered an equivalent amount of solvent instead of CNP-D / R / V, was used as a control group.

[0158] As a result, as shown in Fig. 12, compared to the vehicle group, the group administered CNP-D / R / V showed a decrease in the thickness of the skin dermis layer and a decrease in the lung tissue index, confirming that damage caused by tissue fibrosis was protected. In addition, as a result of confirming collagen accumulation in the tissue, it was confirmed that collagen accumulation was significantly reduced in the skin and lung tissues (Fig. 13), confirming that the nano-fusion of the present invention improves systemic sclerosis even in a model reflecting the immune status of the patient.

[0159] <Example 5> Confirmation of the combined effect of nanoconjugate and vimentin antibody

[0160] It was confirmed whether the nano-conjugate containing the vimentin peptide of the present invention improves systemic sclerosis when used in combination with a vimentin antibody. Specifically, while producing a vimentin-specific systemic sclerosis animal model in the same manner as in Example 3-1, CNP-D / R / V and vimentin antibody (vim ab) (the vimentin-neutralizing antibody of the present invention was HzVSF_V13 of Korean Patent Publication No. 10-1912375, administered subcutaneously twice a week for a total of 6 weeks at a concentration of 9 mg / kg) were co-administered at the time of drug administration (Fig. 14). Thereafter, at the end of the experiment, the mice were humanely sacrificed, and skin and lung tissues were obtained. The skin dermal layer thickness and lung histological indices were confirmed by H&E staining and Masson's Trichrome (MT) staining, and the degree of collagen deposition in the tissues was quantified. As control groups, a vehicle group administered with the same amount of solvent instead of CNP-D / R / V and vimentin antibody, a group administered CNP-D / R / V alone, and a group administered vimentin antibody alone were used.

[0161] As a result, as shown in Fig. 15, compared to the vehicle group, in the group administered CNP-D / R / V or vimentin antibody alone, the dermal layer thickness and lung histological index were significantly reduced, and in the group administered CNP-D / R / V and vimentin antibody in combination, the dermal layer thickness and lung histological index were significantly reduced compared to the groups administered each alone, confirming that the combined use of CNP-D / R / V and vimentin antibody showed a synergistic effect in improving systemic sclerosis.

[0162] In addition, as shown in Fig. 16, in terms of collagen accumulation within the tissue, the group administered CNP-D / R / V and vimentin antibody in combination significantly reduced collagen accumulation within the tissue compared to the group administered CNP-D / R / V or vimentin antibody alone, confirming a synergistic effect in the effect of inhibiting tissue fibrosis.

[0163] <Example 6> Synthesis of nanovaccine for subcutaneous injection

[0164] <6-1> LMP59 synthesis

[0165] 2.2 mM sodium citrate (SC) was dissolved in 150 ml of distilled water, and then 0.1 ml of tannic acid (TA; 2.5 mM) and 1 ml of potassium carbonate (PC; 150 mM) were mixed. The mixture was heated to 70°C, and 100 μl of tetrachloroauric acid (25 mM) dissolved in 1 ml of distilled water was added and reacted for 15 minutes. After the reaction was completed, the mixture was cooled to room temperature, and the formed gold nanoparticles were centrifuged at 2500 rpm for 5 minutes using a centrifugal filter (30k MWCO) to remove unreacted SC, TA, and PC. After that, glutathione (GSH) in an amount 200 times that of the gold nanoparticles was added and reacted for 15 minutes. After that, unreacted GSH was removed using a centrifugal filter (30k MWCO). Then, arginine-ligand reduced with sodium borohydride was added and reacted for 15 minutes. After the reaction was completed, unreacted arginine-ligand was removed, and in order to load metformin, 7.7 mM metformin was slowly added and reacted at room temperature for 15 minutes. Afterwards, 200 μg of vimentin peptide 59-78 (SEQ ID NO: 2: GVYATRSSAVRLRSSVPGVR) was added and reacted for 15 minutes. After the reaction was completed, unreacted metformin and vimentin peptide were removed to produce LMP59.

[0166] The characteristics of the fabricated LMP59 were confirmed to have an SPR peak of 523 nm and a hydrated size of 25 nm. The zeta potential at this time was -45 mV (Fig. 17A). The loading amount of metformin loaded on the gold nanoparticles was 10%, and the loading amount of the vimentin peptide acting as an antigen was 50%. A schematic diagram of the specific LMP59 is shown in Fig. 17C. As illustrated in Fig. 17C, it was confirmed that in the LMP59 of the present invention, metformin and vimentin peptides were directly bound to the gold nanoparticles or incorporated into the gold nanoparticles through electrostatic bonding (charge interaction) with glutathione or arginine ligands.

[0167] <6-2> SMP59 synthesis

[0168] After dissolving SC (2.2 mM) in 150 ml of distilled water, 0.1 ml of TA (2.5 mM) and 1 ml of PC (150 mM) were mixed. The mixture was heated to 70°C, and 100 μl of tetrachloroauric acid (25 mM) dissolved in 1 ml of distilled water was added and reacted for 15 minutes. After the reaction was completed, the mixture was cooled to room temperature, and the formed gold nanoparticles were centrifuged at 2500 rpm for 5 minutes using a centrifugal filter (30k MWCO) to remove unreacted SC, TA, and PC. After that, 200 times the molar number of gold nanoparticles of poly(ethylene glycol) methyl ether thiol was added and reacted for 15 minutes. After that, unreacted Poly(ethylene glycol) methyl ether thiol was removed, and in order to support metformin, 7.7 mM metformin was added and reacted for 15 minutes. After that, 250 μg of vimentin peptide 59-78 (SEQ ID NO: 2: GVYATRSSAVRLRSSVPGVR) was added and reacted for 15 minutes, and after the reaction was completed, unreacted metformin and vimentin peptide were removed to produce SMP59.

[0169] The characteristics of the fabricated SPM59 were confirmed to have an SPR peak of 540 nm and a hydrated size of 34 nm. The zeta potential at this time was -26.8 mV (Fig. 17B). The loading amount of metformin loaded on the gold nanoparticles was 15%, and the loading amount of vimentin peptide acting as an antigen was 50%. A schematic diagram of the specific SMP59 is shown in Fig. 17C.

[0170] <Example 7> Confirmation of Vimentin-Specific Improvement of Systemic Sclerosis by LMP59

[0171] The nanovaccine containing the vimentin peptide of the present invention was confirmed to have a vimentin-specific improvement effect on systemic sclerosis. Specifically, after hair was removed from B6 mice using clippers, 50 μl of vimentin protein at a concentration of 4 mg / ml and 50 μl of CFA were mixed and injected subcutaneously into the lower back of the mice. Two weeks later, 50 μl of vimentin protein at a concentration of 4 mg / ml and 50 μl of IFA were injected. Systemic sclerosis was induced using bleomycin by daily subcutaneous injection of 50 μl / 100 μl PBS for 3 weeks. To avoid overlapping administration schedules of the systemic sclerosis-inducing and -treating drugs, 200 μl of LMP59 was injected subcutaneously twice a week for 3 weeks to confirm the effect of vimentin peptide on improving systemic sclerosis starting from the 3rd week of systemic sclerosis induction (Fig. 18). Afterwards, at the end of the experiment, the mice were humanely sacrificed, and skin and lung tissues were obtained. The skin dermal layer thickness and lung fibrosis index were determined by H&E staining and Masson's Trichrome (MT) staining, and the area of ​​collagen areas in the tissues was quantified. In addition, blood was obtained and the amount of autoantibodies in the serum and the expression of systemic sclerosis-causing cells in spleen cells were confirmed. The wild-type mouse (WT) group and the vehicle group, which were induced with systemic sclerosis and injected with the same amount of solvent alone, were used as controls.

[0172] As a result, as shown in Figure 19, compared to the vehicle group, the LMP59-administered group showed a significant decrease in the thickness of the dermal layer and a significant decrease in the pulmonary fibrosis index. Furthermore, a significant decrease in the area of ​​collagen regions within the tissue was confirmed.

[0173] In addition, as a result of confirming the level of vimentin-specific antibodies, which are autoantibodies in the serum, it was confirmed that the total IgG and IgG1 autoantibodies were significantly increased in the Vehicle group compared to the WT group, but when LMP59 was administered, the amount of increased autoantibodies in the serum was significantly reduced (Fig. 20).

[0174] As a result of confirming the expression of T cells in the spleen of systemic sclerosis pathogens, as shown in Figure 21, compared to the vehicle group, the group administered LMP59 showed a significant decrease in the expression of IL-4, IL-17, and IL-13 positive T cells, confirming that LMP59 has an improving effect even in vimentin-specific systemic sclerosis.

[0175] <Example 8> Confirmation of inhibition of fibrosis-related factors in tissues

[0176] It was confirmed whether the nano-vaccine containing the vimentin peptide of the present invention suppresses the expression of fibrogenic factors in tissues in vimentin-specific systemic sclerosis. Specifically, the skin tissue and lung tissue obtained in Example 7 were sectioned, and the expression of systemic sclerosis and fibrogenic factors IL-17, IL-4, cell surface vimentin (CSV), and fibrogenic factors alpha-smooth muscle actin (α-SMA) and collagen type I A1 (COL1A1) in each tissue was confirmed by immunohistochemical staining.

[0177] As a result, as shown in Figure 22, compared to the vehicle group, the group administered LMP59 showed a significant decrease in the expression of IL-17, IL-4, CSV, aSMA, and COL1A1, which are systemic sclerosis and fibrosis factors in skin tissue, thereby confirming an improvement in skin fibrosis.

[0178] In addition, in lung tissue, it was confirmed that the expression of the above factors was significantly reduced in the group administered LMP59 compared to the vehicle group (Fig. 23), confirming that the LMP59 of the present invention suppresses the expression of fibrosis and systemic sclerosis factors in skin and lung tissue.

[0179] <Example 9> Confirmation of the combined effect of nano vaccine and vimentin antibody

[0180] It was confirmed whether the nano-vaccine containing the vimentin peptide of the present invention improves systemic sclerosis when used in combination with a vimentin antibody. Specifically, a vimentin-specific systemic sclerosis animal model was created in the same manner as in Example 7, and LPM59 and a vimentin antibody (Patent No. 10-1912375; HzVSF_V13) were co-administered at the time of drug administration. Specifically, starting from the third week after the end of the systemic sclerosis induction period, the antibody drug was administered twice a week at a concentration of 9 mg / kg for a total of three weeks, and LPM59 and the antibody were co-administered. Thereafter, skin and lung tissues were stained with H&E to confirm the thickness of the skin dermis layer and the pulmonary fibrosis index, and the amounts of total IgG, IgG1, and IgG2a, which are vimentin-specific autoantibodies in the serum, and the T cell subtypes in spleen cells were analyzed.

[0181] As a result, as shown in Figure 24, it was confirmed that when LMP59 was administered in combination with vimentin antibody, the skin dermal layer thickness and lung tissue fibrosis index were significantly reduced compared to when LMP59 was administered alone.

[0182] In addition, as a result of comparing the amount of autoantibodies in serum, it was confirmed that the amount of total IgG, IgG1, and IgG2a, which are vimentin-specific antibodies, was significantly reduced in the group administered LMP59 in combination with vimentin antibodies compared to the group administered LMP59 alone (Figure 25).

[0183] In addition, in the results of T cell subtype analysis, IL-4 and IL-17 positive cells, which are T cells that cause systemic sclerosis, were decreased by combined administration of LMP59 and vimentin antibody compared to the LMP59 only administration group, and Tregs, which are immune regulatory cells, were confirmed to increase by combined administration of LMP59 and vimentin antibody (Fig. 26), confirming that when LMP59 of the present invention is combined with vimentin antibody, the vimentin-specific systemic sclerosis improvement effect increases.

[0184] <Example 10> Confirmation of Vimentin-Specific Improvement of Systemic Sclerosis by SMP59

[0185] The nanovaccine containing the vimentin peptide of the present invention was confirmed to have a vimentin-specific improvement effect on systemic sclerosis. Specifically, an animal model was prepared using the same method as in Example 7, and SMP59 was administered instead of LMP59. Afterwards, at the end of the experiment, the mice were humanely sacrificed, and skin and lung tissues were obtained. The skin dermal layer thickness and lung fibrosis index were confirmed by H&E staining and Masson's Trichrome (MT) staining, and the area of ​​collagen areas in the tissues was quantified. In addition, blood was obtained, and the amount of autoantibodies in the serum and the expression of systemic sclerosis pathogenic cells in spleen cells were confirmed. The control group was a wild-type mouse (WT) group and a vehicle group in which systemic sclerosis was induced and only an equal amount of solvent was injected.

[0186] As a result, as shown in Figure 27, compared to the vehicle group, the SMP59-administered group showed a significant decrease in the thickness of the dermal layer and a significant decrease in the pulmonary fibrosis index. In addition, a significant decrease in the area of ​​collagen regions within the tissue was confirmed.

[0187] In addition, as a result of confirming the level of vimentin-specific antibodies, which are autoantibodies in the serum, it was confirmed that the total IgG and IgG1 autoantibodies were significantly increased in the Vehicle group compared to the WT group, but when SMP59 was administered, the amount of increased autoantibodies in the serum was significantly reduced (Fig. 28).

[0188] As a result of confirming the expression of T cells, which are pathogenic for systemic sclerosis, in the spleen, as shown in Figure 29, compared to the vehicle group, the group administered SMP59 showed a significant increase in the expression of Treg, which is an immune regulatory cell, and a significant decrease in the expression of IL-4, IL-17, and IL-13 positive T cells.

[0189] <Example 11> Confirmation of Vimentin-Specific Suppression of Systemic Sclerosis Tissue Fibrosis by SMP59

[0190] The present invention was confirmed to confirm whether the nano-vaccine containing the vimentin peptide suppresses tissue fibrosis in vimentin-specific systemic sclerosis. Specifically, skin and lung tissues obtained from the mice sacrificed in Example 10 were sectioned and analyzed by confocal analysis, and the tissue expression of vimentin antigen reactive area, CD19, and CSV was quantified by fluorescence.

[0191] As a result, as shown in Figures 30 and 31, compared to the vehicle group, in the group administered SMP59, the fluorescence expression of the vimentin antigen reaction area was significantly reduced, and the fluorescence expression of CD19 and CSV was significantly reduced, confirming that SMP59 inhibits fibrosis of skin and lung tissue.

[0192] <Example 12> Confirmation of the simultaneous Th17 and Treg regulation effect of nano vaccines

[0193] We confirmed whether the nanovaccine containing the vimentin peptide of the present invention simultaneously regulates Th17 and Tregs in spleen cells. Specifically, we created a vimentin-specific systemic sclerosis model in SKG mice that spontaneously developed autoimmune disease using the same method as Example 7, and examined whether the SMP59 nanovaccine could directly reduce the proliferative response of inflammatory cells in isolated spleen cells.

[0194] As a result, as shown in Fig. 32A, it was confirmed that the SMP59 nano vaccine of the present invention inhibited the proliferation of spleen cells compared to the vehicle group, and in the vimentin-specific systemic sclerosis model produced in the same manner as Example 7, spleen cells from the group administered the SMP59 nano vaccine were isolated and treated with the same vimentin protein as the peptide included in the nano vaccine under Th0 and Th17 cell differentiation conditions. As a result, it was confirmed that the vimentin protein re-sensitization inhibited the differentiation of Th17 and promoted the differentiation of Treg in the vaccine-administered group (Fig. 32B).

[0195] <Example 13> Confirmation of the combined effect of nano vaccine and vimentin antibody

[0196] It was confirmed whether the nano-vaccine containing the vimentin peptide of the present invention, when used in combination with a vimentin antibody, improves systemic sclerosis. Specifically, a vimentin-specific systemic sclerosis animal model was created using the same method as in Example 7, and SPM59 and vimentin antibody at 9 mg / kg were administered twice a week for a total of 3 weeks, starting from the 3rd week after the end of the systemic sclerosis induction period. Thereafter, skin and lung tissues were stained with H&E to confirm the thickness of the skin dermis layer and the pulmonary fibrosis index, and the amounts of total IgG and IgG2a, which are vimentin-specific autoantibodies in the serum, and T cell subtypes in spleen cells were analyzed.

[0197] As a result, as shown in Figure 33, it was confirmed that when SMP59 was administered in combination with vimentin antibody, the skin dermal layer thickness and lung tissue fibrosis index were significantly reduced compared to when SMP59 was administered alone.

[0198] In addition, as a result of comparing the amount of autoantibodies in serum, it was confirmed that the amount of total IgG and IgG2a, which are vimentin-specific antibodies, was significantly reduced in the group administered with vimentin antibodies compared to the group administered SMP59 alone (Figure 34).

[0199] In addition, in the results of T cell subtype analysis, IL-4 and IL-17 positive cells, which are systemic sclerosis pathogenic T cells, were decreased by combined administration of SMP59 and vimentin antibody compared to the SMP59 only administration group, and Tregs, which are immune regulatory cells, were confirmed to increase by combined administration of SMP59 and vimentin antibody (Fig. 35), confirming that when SMP59 of the present invention is combined with vimentin antibody, the vimentin-specific systemic sclerosis improvement effect increases.

[0200] <Example 14> Confirmation of improvement in systemic sclerosis accompanied by infection with SMP59

[0201] The nanovaccine containing the vimentin peptide of the present invention was confirmed to be effective in improving systemic sclerosis accompanied by infection. Specifically, after hair was removed from B6 mice using clippers, 50 μl of vimentin protein at a concentration of 4 mg / ml and 50 μl of CFA were mixed and injected subcutaneously into the base of the mouse tail. Two weeks later, 50 μl of vimentin protein at a concentration of 4 mg / ml and 50 μl of IFA were injected, and 24 hours later, the hair on the back of the neck was removed using clippers. Systemic sclerosis was induced by subcutaneous injection of bleomycin (50 μl / 100 μl PBS) daily for 3 weeks. In addition, to induce infection after bleomycin administration, an ACE2 vector expressing angiotensin-converting enzyme 2 (ACE2) and a spike vector expressing the spike protein of SARS-CoV-2 were injected at a concentration of 100 μg / 50 μl twice a week. One week after inducing systemic sclerosis, 200 μl of SMP59 was injected subcutaneously twice a week for a total of 5 weeks (Fig. 36). At the end of the experiment, the mice were humanely sacrificed, and skin and lung tissues were obtained. The skin dermal layer thickness and lung fibrosis index were confirmed by H&E staining and Masson's Trichrome (MT) staining, and the area of ​​collagen areas in the tissues was quantified. In addition, blood was obtained and the amount of autoantibodies in the serum and the expression of systemic sclerosis pathogenic cells in lung tissue cells were confirmed.

[0202] In addition, spleen tissues were stimulated with LPS and vimentin protein, treated with 3H-thymidine 3 days later, and differentiation patterns were determined using gamma counting on the 4th day. The wild-type mouse (WT) group and the vehicle group, which were induced with systemic sclerosis and injected with the same volume of solvent, were used as controls.

[0203] As a result, as shown in Figure 37, compared to the vehicle group, the thickness of the skin dermis and the lung fibrosis index were significantly reduced in the group administered SMP59, and the fibrosis areas of skin tissue and lung tissue were significantly reduced.

[0204] In addition, as a result of comparing the amount of autoantibodies in serum, it was confirmed that the amount of total IgG, a vimentin-specific antibody, was significantly reduced in the group administered SMP59 (Figure 38).

[0205] As a result of the analysis of the T cell subtypes that are pathogenic for systemic sclerosis in lung tissue, compared to the vehicle group, the number of IL-4 and IL-17 positive cells, which are pathogenic for systemic sclerosis T cells in lung tissue, was reduced in the group administered SMP59 (Fig. 39), and it was confirmed that the expression of IL-4 and IL-17 positive cells, which are pathogenic for systemic sclerosis T cells, was significantly reduced in cells restimulated with LPS and vimentin in spleen tissue (Fig. 40).

[0206] Therefore, it was confirmed that the nanoconjugate containing the vimentin peptide of the present invention suppresses tissue fibrosis in systemic sclerosis, reduces the amount of autoantibodies in the serum, and regulates the expression of immune regulatory cells and systemic sclerosis pathogenic cells. Furthermore, it was confirmed that it suppresses tissue fibrosis in an animal model reflecting the immune status of systemic sclerosis patients, and that the tissue fibrosis inhibition effect is enhanced when co-administered with a vimentin antibody. In addition, it was confirmed that the nanovaccine reduces the expression of tissue fibrogenic factors and systemic sclerosis pathogenic factors, and improves the disease activity in vimentin-specific systemic sclerosis and systemic sclerosis accompanied by infectious diseases.

Claims

1. A vaccine composition for the prevention or treatment of systemic sclerosis, comprising nanoparticles loaded with vimentin peptides as an active ingredient.

2. In paragraph 1, A composition wherein the above nanoparticles are poly(lactic-co-glycolic acid, PLGA) nanoparticles.

3. In paragraph 2, A composition wherein the PLGA nanoparticles are coated with chitosan.

4. In paragraph 1, A composition wherein the above vimentin peptide comprises an amino acid sequence of sequence number 1.

5. In paragraph 4, A composition in which the amino acid sequence of the above sequence number 1 is connected at the 5-' end to FITC (Fluorescein isothiocyanate) by a linker.

6. In paragraph 1, The above nanoparticles are a composition containing rapamycin or vitamin D3.

7. In paragraph 6, A composition wherein the above rapamycin or vitamin D3 increases the expression of tolerogenic dendritic cells (tDC).

8. In paragraph 1, The above composition is a composition that regulates the expression of immune cells.

9. In paragraph 8, A composition that regulates the expression of the above immune cells, suppresses the expression of Th2 or Th17, which are immune cells that cause systemic sclerosis.

10. In paragraph 8, A composition that regulates the expression of the above immune cells by increasing the expression of Treg.

11. In paragraph 1, The composition above is a composition that inhibits tissue fibrosis.

12. In paragraph 11, A composition that suppresses the above tissue fibrosis by suppressing a decrease in dermal thickness or collagen accumulation within the tissue.

13. In paragraph 11, A composition that suppresses the above tissue fibrosis by suppressing the expression of a fibrotic factor or a systemic sclerosis-related factor in the tissue.

14. In paragraph 13, A composition wherein the above fibrotic factor or systemic sclerosis-related factor is a factor selected from the group consisting of COL1 (collagen type I), a-SMA (Alpha smooth muscle actin), cell surface vimentin, IL-4, and IL-17.

15. In paragraph 11, A composition wherein the above tissue is a tissue selected from the group consisting of skin, lung, liver, muscle, kidney, intestine, and spleen.

16. In paragraph 1, The composition above is a composition that reduces the amount of autoantibodies in serum.

17. In paragraph 16, A composition wherein the autoantibody in the serum is total IgG, IgG2a or IgG1.

18. In paragraph 1, A composition wherein the above composition is administered in combination with a vimentin antibody.

19. A pharmaceutical composition for the prevention or treatment of systemic sclerosis, comprising nanoparticles loaded with vimentin peptide as an active ingredient.

20. A food composition for preventing or improving systemic sclerosis, comprising nanoparticles loaded with vimentin peptide as an active ingredient.

21. A vaccine composition for the prevention or treatment of systemic sclerosis, comprising an immunomodulatory drug and an antigen-conjugated gold nanoparticle (Au nanoparticle) as active ingredients.

22. In paragraph 21, The above immunomodulatory drug is a composition represented by the following chemical formula 1: [Chemical Formula 1] 23. In paragraph 21, A composition wherein the above antigen is a vimentin peptide.

24. In paragraph 23, A composition wherein the above vimentin peptide comprises an amino acid sequence of sequence number 2.

25. In paragraph 21, The composition above is a composition that suppresses the expression of T cells that are a cause of systemic sclerosis.

26. In paragraph 25, A composition wherein the above-mentioned systemic sclerosis pathogenic T cell is an IL-17 positive, IL-4 positive or IL-13 positive T cell.

27. In paragraph 21, The composition above is a composition that reduces the amount of autoantibodies in serum.

28. In paragraph 27, A composition wherein the autoantibody in the serum is total IgG, IgG1 or IgG2a.

29. In paragraph 21, The composition above is a composition that inhibits tissue fibrosis.

30. In paragraph 29, A composition that inhibits tissue fibrosis, which inhibits the thickness of the skin dermis layer or lung fibrosis.

31. In paragraph 29, A composition that suppresses the above tissue fibrosis by suppressing the expression of a fibrotic factor or a systemic sclerosis factor in the tissue.

32. In paragraph 31, A composition wherein the above fibrotic factor or systemic sclerosis factor is a factor selected from the group consisting of IL-17, IL-4, cell surface vimentin, a-SMA (Alpha smooth muscle actin), COL1A1 (collagen type I A1), and CD19.

33. In paragraph 29, A composition wherein the above tissue is a tissue selected from the group consisting of skin, lung, liver, muscle, kidney, intestine, and spleen.

34. In paragraph 21, The composition above is a composition that increases the expression of Treg.

35. In paragraph 21, A composition wherein the above systemic sclerosis further includes vimentin-specific systemic sclerosis or systemic sclerosis with infection.

36. In paragraph 21, A composition wherein the above composition is administered in combination with a vimentin antibody.

37. A pharmaceutical composition for the prevention or treatment of systemic sclerosis, comprising an immunomodulatory drug and an antigen-conjugated gold nanoparticle (Au nanoparticle) as active ingredients.

38. A food composition for preventing or improving systemic sclerosis, comprising an immunomodulatory drug and antigen-conjugated gold nanoparticles (Au nanoparticles) as active ingredients.

39. A method for treating systemic sclerosis, comprising administering to a subject a pharmaceutically effective amount of the nanoparticle of paragraph 1.

40. A method for treating systemic sclerosis, comprising administering to a subject a pharmaceutically effective amount of the gold nanoparticles of Article 21.

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