WO2022235125A1 - Nanoparticules lipidiques-biopolymères à la surface desquelles sont liés un anticorps et un auto-antigène, et leur utilisation - Google Patents

Nanoparticules lipidiques-biopolymères à la surface desquelles sont liés un anticorps et un auto-antigène, et leur utilisation Download PDF

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WO2022235125A1
WO2022235125A1 PCT/KR2022/006518 KR2022006518W WO2022235125A1 WO 2022235125 A1 WO2022235125 A1 WO 2022235125A1 KR 2022006518 W KR2022006518 W KR 2022006518W WO 2022235125 A1 WO2022235125 A1 WO 2022235125A1
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nanoparticles
lipid
antibody
autoantigen
bound
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PCT/KR2022/006518
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English (en)
Korean (ko)
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오유경
박진원
비엣 르쿠옥
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서울대학교 산학협력단
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Priority claimed from KR1020220056180A external-priority patent/KR20220152512A/ko
Publication of WO2022235125A1 publication Critical patent/WO2022235125A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention relates to a lipid-biopolymer nanoparticle to which an antibody and an autoantigen are bound to a surface, a method for preparing the same, and a use thereof, and the like.
  • the present invention claims priority based on Korean Patent Application No. 10-2021-0058377 filed on May 6, 2021 and Korean Patent Application No. 10-2022-0056180, filed on May 6, 2022, The contents disclosed in the specification and drawings are incorporated herein by reference.
  • Autoimmune diseases are diseases in which an abnormal immune response occurs to normal body tissues or cells. Representatively, Rheumatoid arthritis, Inflammatory Bowel Disease, and Diabetes Mellitus type 1 ), and multiple sclerosis, which not only threatens the quality of life and life of the patient, but also causes great economic and emotional loss to the patient's family as well as society.
  • autoimmune diseases The etiology of most of these autoimmune diseases is unclear, and it is known that various genetic and environmental factors act in a complex way, and each patient has a different etiology. Therefore, in the treatment of autoimmune diseases, rather than achieving a cure by excluding the underlying factors of the patient, appropriate surgery and drug treatment are performed according to the patient's condition and symptoms, thereby alleviating the symptoms and delaying the progression of the disease, thereby improving the patient's life. The main objective is to improve the quality.
  • the etiology of autoimmune diseases is unclear, but as a result, the patient's immune system is characterized by an excessive immune response against abnormal tissues and cells. Attempts have been made to improve the patient's symptoms by inhibiting the response.
  • drug therapy has a number of problems, one of which is that the patient must be treated with the drug for a long period of time, or almost for the rest of his life.
  • Drug therapy for autoimmune diseases is symptomatic and not a fundamental treatment, so to relieve symptoms, you must take drugs continuously.
  • long-term drug use may not only put a psychological burden on the patient, but may also impair the quality of life due to drug interactions and side effects.
  • the currently used drug therapy lowers the systemic immune function, making the patient vulnerable to other infectious diseases and adversely affecting hematopoiesis.
  • antigen-specific immune tolerance is achieved using nanoparticles of a vaccine formulation by encapsulating autoantigens and immunosuppressive agents such as rapamycin in highly biocompatible polymers such as poly(lactic-co-glycolic acid) (PLGA).
  • PLGA poly(lactic-co-glycolic acid)
  • the immune system of our body can be largely divided into innate immune system and adaptive immune system, and the biggest difference between the two is specificity for antigen.
  • the former induces a non-specific but rapid immune response, and the latter shows a specific but delayed immune response.
  • the smooth mediation of these two immune responses maintains an effective and long-term immune response.
  • cells that play a role as mediators of the innate immune response and the adaptive immune response are antigen-presenting cells, and representatively, there are dendritic cells, macrophages, and B cells. . Therefore, it is effective to selectively modulate antigen-presenting cells to induce effective antigen-specific immune tolerance.
  • nanoparticles as a means to regulate the function of antigen-presenting cells in the body and suppress excessive immune responses to autoantigens.
  • nanoparticles capable of effectively performing the above functions without causing side effects in the body have not yet been discovered.
  • fullerene nanoparticles, platinum nanoparticles, manganese nanoparticles, etc. have been studied, but they exhibit toxicity in vivo and have poor biocompatibility.
  • the present invention has been devised to solve the above problems, and the surface is coated with a lipid membrane encapsulated with an immunomodulatory agent, and an autoantigen and antigen-presenting cell-specific antibody are bound to the surface of the biopolymer nanoparticles (antioxidants). This was completed by confirming that the nanoparticles) can prevent, improve, and treat autoimmune diseases by inhibiting excessive immune responses and inducing immune tolerance to autoantigens.
  • an object of the present invention is biopolymer nanoparticles; a lipid film coating the surface of the nanoparticles; an autoantigen bound to the surface of the lipid membrane; And an antigen-presenting cell-specific antibody or fragment thereof bound to the surface of the lipid membrane, to provide an antibody and an autoantigen bound to the surface of the lipid-biopolymer nanoparticles.
  • Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising the lipid-biopolymer nanoparticles bound to the surface of the antibody and the autoantigen as an active ingredient.
  • Another object of the present invention is to provide a food composition for the prevention or improvement of autoimmune diseases comprising the lipid-biopolymer nanoparticles bound to the surface of the antibody and autoantigen as an active ingredient.
  • Another object of the present invention is to provide a method for preparing lipid-biopolymer nanoparticles, wherein the antibody and the autoantigen are bound to the surface.
  • the present invention is antioxidant nanoparticles; a lipid film coating the surface of the nanoparticles; an autoantigen bound to the surface of the lipid membrane; And it provides an antigen-presenting cell-specific antibody or fragment thereof bound to the lipid membrane surface, wherein the antibody and the autoantigen are bound to the surface of the lipid-antioxidant nanoparticles.
  • the antioxidant nanoparticles may be one or more selected from the group consisting of polydopamine nanoparticles, tannin nanoparticles, and cerium oxide nanoparticles, but is not limited thereto.
  • the lipid membrane may be pegylated, but is not limited thereto.
  • the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG) , phosphocholine (PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), cholesterol (cholesterol), 1,2-dioleoyl-sn -glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and at least one selected from the group consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) may be included, but is not limited thereto.
  • DOTAP 1,2-dioleoyl-3-trimethylammonium-
  • the molar ratio of DPPC: DPPG may be 1 to 10: 1, but is not limited thereto.
  • the molar ratio of the DPPC: DPPG: DSPE-PEG2000-maleimide may be 100 to 1000: 10 to 500: 1, but is not limited thereto.
  • the antigen-presenting cell-specific antibody or fragment thereof may be an antibody or fragment thereof that specifically binds to dendritic cells or macrophages, but is not limited thereto.
  • the antigen-presenting cell-specific antibody or fragment thereof is CD80, CD86, CD123, CD303, CD304, CD68, CD11b, CD11c, BDCA-1, DC-SIGN, MHCII, F4/80, It may bind to one or more proteins selected from the group consisting of CD206, and CSF1-R, but is not limited thereto.
  • the antigen-presenting cell-specific antibody or fragment thereof is IgG, Fab', F(ab') 2 , Fab, Fv, recombinant IgG (rIgG), single chain Fv (scFv), and It may be at least one selected from the group consisting of diabody, but is not limited thereto.
  • the autoantigen may be a protein capable of inducing an autoimmune disease in an individual, a fragment thereof, or a variant thereof, but is not limited thereto.
  • the autoantigen is collagen, insulin, insulin B chain, proinsulin, myelin protein, myelin basic protein, myelin proteolipid protein, myelin oligodendrocytes It may be a protein derived from one or more selected from the group consisting of myelin oligodendrocyte glycoprotein, Hsp60, and Hsp65, a fragment thereof, or a variant thereof, but is not limited thereto.
  • the autoantigen; And the antigen-presenting cell-specific antibody or fragment thereof may have a thiol group or may be modified to have a thiol group, but is not limited thereto.
  • the lipid membrane comprises a lipid having a maleimide group, and the autoantigen; And the antigen-presenting cell-specific antibody or fragment thereof may be bound to a lipid membrane through binding of a thiol group and a maleimide group, but is not limited thereto.
  • the nanoparticles may further include an immunosuppressant, but is not limited thereto.
  • the immunosuppressant may be encapsulated in the lipid membrane, but is not limited thereto.
  • the immunosuppressive agent may be one or more selected from the group consisting of corticosteroids (Glucocorticoids), calcineurin inhibitors, antimetabolites, mTOR inhibitors, and vitamin D3, but is not limited thereto. .
  • corticosteroids Glucocorticoids
  • calcineurin inhibitors calcineurin inhibitors
  • antimetabolites mTOR inhibitors
  • vitamin D3 vitamin D3
  • the nanoparticles may have a diameter of 50 to 200 nm, but is not limited thereto.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of autoimmune diseases, comprising the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen as an active ingredient.
  • the present invention provides the use of the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles for the prevention or treatment of autoimmune diseases.
  • the present invention provides a use of the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles to induce immune tolerance to the autoantigen.
  • the present invention provides a method for preventing or treating an autoimmune disease, comprising administering the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles to an individual in need thereof.
  • the present invention provides a method for inducing immune tolerance to an autoantigen, comprising administering the antibody and the autoantigen bound to the surface of the lipid-antioxidant nanoparticles to a subject in need thereof.
  • the present invention provides the use of the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen for the preparation of a medicament for the prevention or treatment of autoimmune diseases.
  • the present invention provides the use of the antibody and the lipid-antioxidant nanoparticles bound to the surface of the autoantigen for the preparation of an agent for inducing immune tolerance to the autoantigen.
  • the present invention provides a kit for preventing or treating autoimmune diseases, comprising the pharmaceutical composition.
  • the present invention provides a food composition for the prevention or improvement of autoimmune diseases, comprising the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen as an active ingredient.
  • the food composition includes a health functional food composition.
  • the present invention provides a health functional food for preventing or improving autoimmune diseases, comprising the lipid-antioxidant nanoparticles bound to the surface of the antibody and autoantigen as an active ingredient.
  • the lipid-antioxidant nanoparticles to which the antibody and the autoantigen are bound to the surface may satisfy one or more characteristics selected from the group consisting of, but are not limited thereto:
  • the lipid-antioxidant nanoparticles bound to the surface of the antibody and autoantigen may target lymph nodes or spleen, but are not limited thereto.
  • the autoimmune disease is rheumatoid arthritis, juvenile rheumatoid arthritis, systemic scleroderma, adult Still's disease, systemic lupus erythematosus, atopic dermatitis, Behcet's disease, multiple sclerosis, systemic sclerosis, Sjogren's disease Syndrome, primary biliary cirrhosis, celiac disease, inflammatory bowel disease, type 1 diabetes, autoimmune hemolytic anemia, Goodpasture syndrome, Graves disease, Hashimoto's thyroiditis, hyperthyroidism, myasthenia gravis, pemphigus, vasculitis, encephalomyelitis, pituitary, Vitiligo, asthma, primary biliary cirrhosis, optic nerve myelitis, pemphigus vulgaris, irritable bowel disease, Crohn's disease, colitis, ulcerative colitis, psoriasis, cardiomyopathy, mya
  • the present invention comprises the steps of (S1) producing antioxidant nanoparticles by inducing self-assembly of a biopolymer in a basic environment;
  • the antibody and autoantigen of claim 1 comprising the step of reacting the surface-coated antioxidant nanoparticles with an autoantigen and an antigen-presenting cell-specific antibody or fragment thereof with the surface of the lipid membrane-bound lipid-antioxidation
  • a method for preparing nanoparticles is provided.
  • the biopolymer may be one or more selected from the group consisting of polydopamine, tannin, and cerium oxide, but is not limited thereto.
  • the lipid membrane may be one in which an immunosuppressant is encapsulated, but is not limited thereto.
  • Lipid-antioxidant nanoparticles according to the present invention have autoantigens and antigen-presenting cell-specific antibodies modified on the surface to deliver the autoantigen specifically to antigen-presenting cells, and the surface is coated with a lipid membrane encapsulated with an immunomodulatory agent. Immune tolerance to autoantigens can be induced.
  • the nanoparticles according to the present invention are administered to an animal model of autoimmune disease, they effectively target lymph nodes and spleen, which are immune organs, and effectively inhibit excessive immune activation by antigen-presenting cells, thereby preventing, delaying, and It has been shown to be curable. Therefore, the nanoparticles according to the present invention are expected to be usefully utilized for the prevention or treatment of various autoimmune diseases, including encephalomyelitis.
  • LDPN Lipid-antioxidant nanoparticles
  • PN antioxidant nanoparticles
  • AbaLDPN-MOG pegylated lipid layers
  • PN antioxidant nanoparticles
  • LDPN lipid-antioxidant nanoparticles
  • LDPN-MOG autoantigen-modified lipid-antioxidant nanoparticles
  • AbaLDPN-MOG antibody and autoantigen-modified lipid-antioxidant nanoparticles
  • Figure 6 is flow cytometry the cell binding capacity of the nanoparticles after each treatment of CD80/86-expressing dendritic cells (dendritic cells) with nanoparticles modified with CD80/86-specific recombinant antibody or nanoparticles not modified with the antibody. It is the result of comparison by analysis.
  • Each of the nanoparticles was labeled with fluorescence (Cy5), and the degree of cell binding of the nanoparticles was confirmed through the fluorescence intensity of the cells.
  • FIG. 10 is a diagram showing an in vitro experiment for confirming the interaction inhibitory effect between dendritic cells and T cells of the nanoparticles according to the present invention.
  • the well plate was coated with a dendritic cell surface protein, and the nanoparticles of the present invention and T cells were treated together.
  • FIG. 11 is a result confirming the degree of inhibition of the interaction between dendritic cells and T cells through the IL-2 level in the culture medium when the nanoparticles according to the present invention are treated with T cells under the experimental conditions of FIG. 10 . According to the present invention, it was confirmed that the surface-modified lipid-antioxidant nanoparticles with the antibody block the interaction between dendritic cells and T cells to inhibit IL-2 secretion of T cells.
  • helper T cells CD3 and CD4 double positive; CD4 + T cells
  • cytotoxic T cells by splenocytes when the nanoparticles according to the present invention were treated together with T cells under the experimental conditions of FIG. (CD3 and CD8 double positive; CD8 + T cells) It is the result of confirming that expansion (expansion) is suppressed.
  • 15 is a diagram showing the mechanism of induction of immune-tolerance dendritic cells by nanoparticles according to the present invention.
  • 16 and 17 are results of confirming the distribution of nanoparticles in lymph nodes, which are target organs, by time after subcutaneous administration of the nanoparticles according to the present invention to mice. It was confirmed that the nanoparticles on the surface of which the recombinant antibody was modified had increased lymph node distribution and more continuously accumulated compared to the nanoparticles on which the antibody was not modified.
  • 20A shows an experimental schedule of an autoimmune disease animal model for confirming the humoral immunosuppressive effect of nanoparticles according to the present invention.
  • nanoparticles according to the present invention effectively inhibited the production of autoantibodies (anti-OVA IgG) in the serum of the animal model in an autoimmune disease model using ovalbumin. From this, it can be seen that the nanoparticles of the present invention have an effect of inhibiting ovalbumin-specific humoral immune response.
  • FIG. 21A to 21C are experimental schedules for confirming the prevention (FIG. 21A), progression inhibition (FIG. 21B), and therapeutic effect (FIG. 21C) of the nanoparticles according to the present invention for autoimmune disease (encephalomyelitis).
  • Figures 22a to 22c show changes in body weight over time and clinical trials after administering the nanoparticles according to the present invention to mice with encephalomyelitis for the purpose of preventing (FIG. 22A), inhibiting progression (FIG. 22B), or treating (FIG. 22C) of an autoimmune disease. It is the result of follow-up of adverse symptoms.
  • the nanoparticles according to the present invention were shown to improve the clinical symptoms of the subject and control the body weight to a normal level.
  • FIG. 23a to 23c are after administration of the nanoparticles according to the present invention to mice with encephalomyelitis for the purpose of preventing (FIG. 23A), inhibiting progression (FIG. 23B), or treating (FIG. 23C) of an autoimmune disease, dendritic cells (CD11c + MHCII) + ), macrophages (CD11b + F4/80 + ), helper T cells (CD4 + T cells or helper T cells; CD3 + CD4 + ), and cytotoxic T cells (CD8 + T cells or cytotoxic T cells; CD3 + CD8 + ) is the result of confirming the degree of invasion of the central nervous system. Regulatory T cells were analyzed using CD25 + FoxP3 + as a marker in helper T cells.
  • FIG. 24A to 24C show that when the nanoparticles according to the present invention are administered to mice with encephalomyelitis for the purpose of preventing (FIG. 24A), inhibiting progression (FIG. 24B), or treating (FIG. 24C) of an autoimmune disease, the ratio of regulatory T cells is It is the result of confirming the increase. In particular, it was also confirmed that the nanoparticles can inhibit the proportion of helper T cells expressing IFN- ⁇ or IL-17A that can damage tissues ( FIG. 24A ).
  • 25A to 25C are after administration of the nanoparticles according to the present invention to mice with encephalomyelitis for the purpose of preventing (FIG. 25A), inhibiting progression (FIG. 25B), or treating (FIG. 25C) of an autoimmune disease, cellularity by nanoparticles It is the result of confirming the ability to regulate the immune response. In mice administered with the nanoparticles according to the present invention, it was confirmed that the number of IFN- ⁇ splenocytes activated by the autoantigen (MOG peptide) decreased.
  • MOG peptide autoantigen
  • 26 is a result of confirming the invasion of immune cells into the central nervous system (myelin myelin) by immunofluorescent tissue method after administering the nanoparticles according to the present invention to an animal model of encephalomyelitis for the purpose of inhibiting disease progression.
  • FIG. 27 is a representative view showing the structure of the nanoparticles according to the present invention and the effect of inducing immune tolerance on autoantibodies thereof.
  • the antioxidant nanoparticles according to the present invention have a surface coated with a lipid membrane encapsulated with an immunomodulatory agent, and the surface is modified with autoantigens and antigen-presenting cell-specific antibodies through the lipid membrane.
  • the present invention provides antioxidant nanoparticles; a lipid film coating the surface of the nanoparticles; an autoantigen bound to the surface of the lipid membrane; and an antigen-presenting cell-specific antibody or fragment thereof bound to the surface of the lipid membrane, an antibody and an autoantigen bound to the surface of the lipid-antioxidant nanoparticles.
  • antioxidant nanoparticles refers to nanoparticles having an antioxidant function, and more preferably, nanoparticles capable of trapping active oxygen.
  • the antioxidant nanoparticles according to the present invention are biopolymer-based biopolymer nanoparticles, and may be nanoparticles containing a biopolymer or composed of a biopolymer.
  • a biopolymer is a natural polymer generated from the cells of a living organism or a polymer artificially synthesized by mimicking it, and is characterized by having excellent biocompatibility, biodegradability, and/or sustainability.
  • the nanoparticles are composed of biopolymers, even when administered in vivo, they have excellent biocompatibility and may not cause side effects such as toxicity.
  • Antioxidant nanoparticles according to the present invention preferably polydopamine nanoparticles, tannin nanoparticles, and characterized in that at least one selected from the group consisting of cerium oxide nanoparticles, most preferably polydopamine (polydopamine) nanoparticles can
  • autoimmune diseases refers to diseases caused by the immune system attacking normal cells or normal tissues inside, not by an external antigen. That is, the autoimmune disease refers to a disease caused by the activation of an excessive immune response to an autoantigen. Accordingly, in the present invention, autoimmune diseases include, without limitation, diseases that can be improved/treated by suppressing immune responses to autoantigens as well as diseases caused by an excessive immune response.
  • the autoimmune disease according to the present invention means a disease that can be induced by the production of autoantibodies against the autoantigen modified on the surface of the nanoparticles according to the present invention, and is not limited to specific diseases, but preferably Rheumatoid arthritis, juvenile rheumatoid arthritis, systemic scleroderma, adult Still's disease, systemic lupus erythematosus, atopic dermatitis, Behcet's disease, multiple sclerosis, systemic sclerosis, Sjogren's syndrome, primary biliary cirrhosis, celiac disease, inflammatory bowel disease, Type 1 diabetes, autoimmune hemolytic anemia, Goodpasture's syndrome, Graves' disease, Hashimoto's thyroiditis, hyperthyroidism, myasthenia gravis, pemphigus, vasculitis, encephalomyelitis, pituitary gland, vitiligo, asthma, primary biliary cirrhos
  • the antioxidant nanoparticles according to the present invention are preferably prepared by self-polymerization of a biopolymer under basic conditions, and are not limited to specific shapes or sizes.
  • the antioxidant nanoparticles may have a diameter of 50 to 200 nm, 50 to 150 nm, 50 to 100 nm, 100 to 200 nm, or 150 to 200 nm, but is not limited thereto.
  • the nanoparticles according to the present invention are characterized in that the surface is coated with a lipid membrane.
  • the present inventors have confirmed that when the nanoparticles are coated with a lipid membrane containing a lipid of a specific composition ratio, the stability of the nanoparticles is improved, and the nanoparticles can be stably bound to antibodies and autoantigens.
  • the lipid membrane may be pegylated.
  • the lipid membrane may include a lipid having a maleimide group.
  • the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG), phosphocholine (PC) , 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), cholesterol (cholesterol), 1,2-dioleoyl-sn-glycero-3- It may include at least one selected from the group consisting of phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
  • DOPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DOPE 1,
  • the 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol may be 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol), but limited thereto doesn't happen
  • the lipid membrane may include 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG).
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
  • the molar ratio of DPPC: DPPG is 1 to 10: 1, 1 to 8: 1, 1 to 5: 1, 1-3: 1, 1.5 to 10: 1, 1.5 to 8: 1, 1.5 to 5: 1, 1.5 to 3 : 1, 2 to 10 : 1, 2 to 8 : 1, 2 to 6 : 1, 2 to 4 : 1, or 2 to 3 : 1, but is not limited thereto.
  • the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), and 1,2-disteroyl-sn -glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide) may be included.
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
  • DSPE-PEG2000-maleimide 1,2-disteroyl-sn -glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000]
  • the molar ratio of the DPPC: DPPG: DSPE-PEG2000-maleimide is 100 to 1000: 10 to 500: 1, 100 to 1000: 50 to 500: 1, 200 to 1000: 100 to 500: 1, 300 to 1000: 100 to 500: 1,500 to 1000: 200 to 500: 1, 600 to 1000: 200 to 500: 1, 300 to 900: 100 to 500: 1, 300 to 900: 200 to 500: 1, 300 to 800: 200 to 500: 1, 400 to 800: 200 to 400: 1, 500 to 800: 200 to 400: 1, or 600 to 800: 250 to 350: 1, but is not limited thereto.
  • the 1,2-dipalmitoyl-sn-glycero-3-phosphocholine is represented by Formula 1 below, and the 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) is represented by Formula 2 , the phosphoglycerol (PG) is represented by Formula 3, the phosphocholine (PC) is represented by Formula 4, and the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol) )-2000] (DSPE-PEG2000-maleimide) may be represented by the following Chemical Formula 5, but is not limited thereto.
  • DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
  • PG phosphoglycerol
  • PC phosphocholine
  • the 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol) )-2000] DSPE
  • nanoparticles according to the present invention are characterized in that the surface is modified (bound, conjugated, supported, or attached) with an antigen-presenting cell-specific antibody or fragment thereof.
  • antibodies refers to specific protein molecules capable of specifically reacting with and binding to a specific antigen or epitope region thereof, and immunoglobulin molecules having antigen-binding ability (e.g., monoclonal antibodies, polyclonal antibodies, etc.), fragments of the immunoglobulin molecules (eg, IgG, Fab', F(ab') 2 , Fab, Fv, recombinant IgG (rIgG), single chain Fv (scFv), or diabodies, etc.) and the like.
  • immunoglobulin molecules having antigen-binding ability e.g., monoclonal antibodies, polyclonal antibodies, etc.
  • fragments of the immunoglobulin molecules eg, IgG, Fab', F(ab') 2 , Fab, Fv, recombinant IgG (rIgG), single chain Fv (scFv), or diabodies, etc.
  • the immunoglobulin molecule has a heavy chain and a light chain, each heavy and light chain comprising a constant region (region) and a variable region, wherein the light and heavy chain variable regions are capable of binding to an epitope of an antigen; a region “complementarity determining region (CDR)”; and four “framework regions” (FRs).
  • the CDRs of each chain are called sequentially CDR1, CDR2, CDR3, typically starting from the N-terminus, and are also identified by the chain in which the specific CDR is located.
  • a complete antibody has a structure with two full-length light chains and two full-length heavy chains, each light chain linked to the heavy chain by a disulfide bond.
  • the antibody may be an animal-derived antibody, a mouse-human chimeric antibody, a humanized antibody, or a human antibody.
  • antibody fragment refers to a functional fragment of the antibody capable of exhibiting the function of the antibody.
  • the antibody according to the present invention is an antibody that specifically binds to an antigen-presenting cell.
  • the antibody is a concept including a recombinant protein that performs a function similar to the antibody. That is, it is sufficient that the antigen-presenting cell-specific antibody according to the present invention is a recombinant protein capable of specifically binding to the antigen-presenting cell-specific protein, and does not necessarily have the structure of the antibody. More preferably, the antigen-presenting cell-specific antibody according to the present invention is an antibody capable of binding to a protein specifically expressed in the antigen-presenting cell.
  • the term “antigen presenting cells (APCs)” refers to a group of cells that process and present antigens so that specific lymphocytes such as T cells can recognize them. do. Natural types of APC include dendritic cells, macrophages, Langerhans cells, and B cells.
  • the antigen-presenting cell-specific antibody or fragment thereof is an antibody or fragment thereof that specifically binds to dendritic cells or macrophages. That is, the antigen-presenting cell-specific antibody or fragment thereof is an antibody or fragment thereof that binds to a protein specifically expressed in dendritic cells or macrophages.
  • the antigen-presenting cell-specific antibody or fragment thereof is sufficient as long as an antibody or fragment thereof capable of specifically binding to dendritic cells or macrophages is not limited to a specific type, and natural antibodies as well as artificially synthesized antibodies (recombinant antibody) can all be used.
  • the antibody or fragment thereof is selected from the group consisting of CD80, CD86, CD123, CD303, CD304, CD68, CD11b, CD11c, BDCA-1, DC-SIGN, MHCII, F4/80, CD206, and CSF1-R It is capable of binding to one or more selected proteins.
  • the antigen-presenting cell-specific antibody or fragment thereof according to the present invention is an antibody or fragment thereof that specifically binds to CD80 and/or CD86. More preferably, the antigen-presenting cell-specific antibody or fragment thereof according to the present invention is a recombinant protein (eg, antibody) or fragment thereof that specifically binds to CD80 and/or CD86, comprising the amino acid sequence of SEQ ID NO: 1 Or, more preferably, it may consist of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto, and variants of the amino acid sequence are included within the scope of the present invention.
  • the cell-penetrating peptide according to the present invention is a functional equivalent of a polypeptide constituting it, for example, some amino acid sequence of the polypeptide is modified by deletion, substitution or insertion, It is a concept including variants capable of functionally the same action as the polypeptide.
  • the recombinant protein (eg, antibody) or fragment thereof that specifically binds to CD80 and/or CD86 is 70% or more, more preferably 80% or more, even more preferably the amino acid sequence of SEQ ID NO: 1 may comprise an amino acid sequence having at least 90% sequence homology, most preferably at least 95% sequence homology.
  • % sequence homology for a polypeptide is determined by comparing two optimally aligned sequences to a comparison region, and a portion of the sequence of the polypeptide in the comparison region is a reference sequence (additional or additional may include additions or deletions (ie, gaps) compared to not including deletions).
  • autoantigens refers to an antigen of the subject itself that stimulates the immune system of the subject to produce autoantibodies. That is, autoantigens are inducers of autoimmune diseases, and autoimmune diseases are induced when an adaptive immune response to the autoantigens occurs. Autoantigens are not targeted by the immune system under normal conditions, but may act as antigens due to lack of immune resistance due to immune or environmental factors. Specific types of autoantigens are not limited, and proteins, peptides, enzyme complexes, ribonucleoproteins, DNA, phospholipids, and the like may all function as autoantigens. The specific type of autoantigen may vary depending on the type of autoimmune disease.
  • MAG Myelin Oligodendrocyte Glycoprotein
  • the types of autoantigens that can cause each autoimmune disease are known in the art (see, for example, Korean Patent Application Laid-Open No. 10-2020-0079507).
  • the autoantigen according to the present invention is sufficient as long as it stimulates the immune system to produce autoantibodies and can induce autoimmune diseases, and is not limited to specific types.
  • the autoantigen bound to the nanoparticles according to the present invention is one capable of inducing an autoimmune disease targeted by the nanoparticles (prevention, improvement, or treatment) by inducing the production of autoantibodies.
  • a person skilled in the art can select an appropriate autoantigen according to the type of autoimmune disease desired or according to the subject to which the nanoparticles will be administered, referring to known knowledge in the art.
  • the autoantigen according to the present invention may be a protein capable of inducing an autoimmune disease in an individual, a fragment thereof, or a variant thereof, but is not limited thereto.
  • the autoantigen is collagen, insulin, insulin B chain, proinsulin, myelin protein, myelin basic protein, myelin proteolipid protein, myelin oligodendrocyte glycoprotein (myelin oligodendrocyte glycoprotein), Hsp60, and may be a protein derived from one or more selected from the group consisting of Hsp65, a fragment thereof, or a variant thereof, but is not limited thereto.
  • the autoantigen; And the antigen-presenting cell-specific antibody or fragment thereof may have a thiol group or may be modified to have a thiol group.
  • the thiol group includes a free thiol group.
  • the thiol group is an autoantigen; And it may be bound to the amine group of the antigen-presenting cell-specific antibody or fragment thereof. That is, the thiol group is an autoantigen; And among the amino acids constituting the antigen-presenting cell-specific antibody or fragment thereof, it may be bound to an amino acid having a primary amine group, such as Lysine (Lys) or Arigine (Arg).
  • the thiol group may be present in the constant region of the light chain of the antibody or fragment thereof, but is not limited thereto.
  • the autoantigen; And antigen-presenting cell-specific antibody or fragment thereof may bind to the lipid membrane of nanoparticles through the thiol group, but is not limited thereto.
  • the bonding may be made through bonding of the thiol group and the maleimide group of the lipid membrane, but is not limited thereto.
  • the nanoparticles according to the present invention are characterized in that they further comprise an immunomodulatory agent, preferably an immunosuppressant agent.
  • the immunomodulatory agent may be encapsulated (bound, captured, or supported) on the lipid membrane coating the surface of the nanoparticles or inside the lipid membrane.
  • Immunosuppressants are drugs that suppress immune function to prevent the immune system from damaging healthy cells or tissues. It is mainly administered to prevent transplant rejection in patients undergoing organ transplantation or stem cell transplantation, and is also used to treat autoimmune diseases. That is, the immunosuppressive agent may induce immune tolerance of the immune system to the autoantigen.
  • the immunosuppressive agent is sufficient as long as it can suppress immune function, and is not limited to specific types, but specific examples include corticosteroids (Glucocorticoids), calcineurin (Calcineurine) inhibitors, antimetabolites, and mTOR inhibitors; and the like.
  • the immunosuppressive agent is dexamethasone, betamethasone, prednisone, prednisolone, methylprednisolone, triamcinolone A, deflazacort. Cyclosporine A), Tacrolimus, MPA (Mycophenolic acid), MMF (Mycophenolate mofetil), Azathioprine, Mizoribine, Everolimus, Rapamycin, Retinoic acid ( Retinoic acid), and may be selected from vitamin D3, but is not limited thereto.
  • the present invention provides a pharmaceutical composition for preventing, improving, or treating autoimmune diseases, comprising the lipid-antioxidant nanoparticles bound to the surface of the antibody and autoantigen according to the present invention as an active ingredient.
  • Such treatment includes inhibiting disease progression of an autoimmune disease.
  • the prevention, improvement, or therapeutic effect of the nanoparticles for autoimmune diseases is achieved by the immunosuppressive effect of the nanoparticles or the immune tolerance inducing effect on the autoantigens.
  • the lipid-antioxidant nanoparticles bound to the surface of the antibody and autoantigen may satisfy one or more characteristics selected from the group consisting of:
  • the nanoparticles when administered to a subject, they can target the lymph node or spleen, that is, migrate to the lymph node or spleen to exert the above effect.
  • Inhibiting the interaction between the antigen-presenting cell and the T cell means inhibiting the activation of the T cell by the antigen-presenting cell, preferably, the activation of the cytotoxic T cell. Furthermore, this includes induction of tolerogenic dendritic cells by the nanoparticles according to the present invention.
  • the (b) suppressing the tissue invasion of immune cells preferably means inhibiting the tissue invasion of dendritic cells, macrophages, and cytotoxic T cells.
  • the nanoparticles are for the purpose of preventing, improving, or treating encephalomyelitis, it means inhibiting the central nervous system invasion of the immune cells.
  • the (e) inflammatory cytokine may be one or more selected from the group consisting of TNF- ⁇ , IL-1 ⁇ , and IL-6, but may include without limitation as long as it is a cytokine that can induce inflammation. That is, the nanoparticles according to the present invention can inhibit the production and secretion of the inflammatory cytokines by activated immune cells.
  • the autoantibody of (f) may preferably be an autoantibody against an autoantigen supported on the nanoparticles. That is, the nanoparticles according to the present invention perform a function of inhibiting the generation of autoantibodies against the autoantigen by inducing immune tolerance against the antigen-presenting cell-specific delivery of the autoantigen.
  • the present invention provides a pharmaceutical composition for immunomodulation, comprising, as an active ingredient, the lipid-antioxidant nanoparticles bound to the surface of the antibody and the autoantigen according to the present invention.
  • the immunomodulation includes the use of immunosuppression against the autoantigen and the use of inducing immune tolerance against the autoantigen.
  • the content of the nanoparticles in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., for example, 0.0001 to 99.9% by weight, or 0.001 to 50% by weight based on the total weight of the composition may be, but is not limited thereto.
  • the content ratio is a value based on the dry amount from which the solvent is removed.
  • the pharmaceutical composition according to the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.
  • the excipient may be, for example, at least one selected from the group consisting of a diluent, a binder, a disintegrant, a lubricant, an adsorbent, a humectant, a film-coating material, and a controlled-release additive.
  • the pharmaceutical composition according to the present invention can be prepared according to a conventional method, respectively, in powders, granules, sustained-release granules, enteric granules, liquids, eye drops, elsilic, emulsions, suspensions, spirits, troches, fragrances, and limonaade.
  • tablets, sustained release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, perfusates, Warnings, lotions, pasta, sprays, inhalants, patches, sterile injection solutions, or external preparations such as aerosols can be formulated and used, and the external preparations are creams, gels, patches, sprays, ointments, warning agents , lotion, liniment, pasta, or cataplasma.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used.
  • water diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc.
  • water diluted hydrochloric acid, diluted sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (Twinester), polyoxyethylene monoalkyl ethers, lanolin ethers, Lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone,
  • sucrose solution other sugars or sweeteners may be used, and if necessary, a fragrance, colorant, preservative, stabilizer, suspending agent, emulsifier, thickening agent, etc. may be used.
  • Purified water may be used in the emulsion according to the present invention, and if necessary, an emulsifier, preservative, stabilizer, fragrance, etc. may be used.
  • the suspending agent according to the present invention includes distilled water, aqueous glucose solution, acacia, tragacantha, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906 , a suspending agent such as HPMC 2910 may be used, and if necessary, a surfactant, preservative, stabilizer, colorant, and fragrance may be used.
  • the injection according to the present invention includes distilled water for injection, glucose aqueous solution, 0.9% sodium chloride injection, Ringel injection, dextrose injection, dextrose + sodium chloride injection, PEG (PEG), lactated Ringel injection, ethanol, propylene glycol, non-volatile solvents such as oil-sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; Solubilizing aids such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tweens, nijeongtinamide, hexamine, and dimethylacetamide; Weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, buffers such as album
  • the suppository according to the present invention includes cacao fat, lanolin, witepsol, polyethylene glycol, glycerogelatin, methyl cellulose, carboxymethyl cellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cacao butter + Cholesterol, Lecithin, Lanet Wax, Glycerol Monostearate, Tween or Span, Imhausen, Monolene (Propylene Glycol Monostearate), Glycerin, Adeps Solidus, Butyrum Tego -G), Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydroxote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydro Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium, A, AS, B, C, D, E, I, T, Massa-MF, Masupol, Masupol-15, Neos
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract, for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc.
  • excipients for example, starch, calcium carbonate, sucrose ) or lactose, gelatin, etc.
  • lubricants such as magnesium stearate talc are also used.
  • Liquid formulations for oral administration include suspensions, internal solutions, emulsions, syrups, etc.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories.
  • Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • the nanoparticles according to the present invention are dispersed in distilled water or isotonic solution and administered.
  • the isotonic solution is not limited to a specific type, and may include without limitation as long as it is isotonic with the body fluid of the subject to be administered, but is preferably selected from aqueous glucose solution and physiological saline (NaCl solution).
  • aqueous glucose solution and physiological saline (NaCl solution).
  • NaCl solution physiological saline
  • the concentration (w/w%) of the aqueous glucose solution may be 4 to 6%, and most preferably, the concentration may be 5%, but is not limited thereto.
  • the physiological saline concentration (w/w%) may be 0.5 to 1%, and most preferably, the concentration may be 0.9%, but is not limited thereto.
  • composition according to the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined.
  • the nanoparticles according to the present invention based on the weight of the antioxidant nanoparticles, 1 to 100 mg / kg, 1 to 90 mg / kg, 1 to 80 mg / kg, 1 to 70 mg relative to the individual weight /kg, 1-60 mg/kg, 1-50 mg/kg, 1-40 mg/kg, 1-30 mg/kg, 1-20 mg/kg, 1-10 mg/kg, 1-5 mg/kg kg, 10-100 mg/kg, 20-100 mg/kg, 30-100 mg/kg, 40-100 mg/kg, 10-80 mg/kg, 10-60 mg/kg, 20-60 mg/kg , 30 to 60 mg / kg, or may be administered in a dose of 40 to 60 mg / kg, but is not limited thereto.
  • the nanoparticles may be administered multiple times until the desired effect is achieved as well as a single administration.
  • the administration method of the nanoparticles according to the present invention may vary depending on the purpose.
  • the nanoparticles according to the present invention when the nanoparticles according to the present invention are administered for the purpose of preventing autoimmune diseases, the nanoparticles may be administered in advance in the absence of symptoms of the disease, and the nanoparticles according to the present invention inhibit the progression of autoimmune diseases
  • the nanoparticles may be administered immediately after the disease is identified.
  • the nanoparticles according to the present invention are administered for the purpose of treating an autoimmune disease, the nanoparticles may be administered immediately after symptoms of the autoimmune disease appear.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount capable of obtaining the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention pertains.
  • the pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration can be envisaged, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal (intrathecal) injection, sublingual administration, buccal administration, rectal insertion, vaginal It can be administered according to internal insertion, ocular administration, ear administration, nasal administration, inhalation, spraying through the mouth or nose, skin administration, transdermal administration, and the like.
  • the pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with several related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.
  • “individual” means a subject in need of treatment for a disease, and more specifically, human or non-human primates, mice, rats, dogs, cats, horses, cattle, etc. means the mammals of
  • administration means providing a predetermined composition of the present invention to a subject by any suitable method.
  • prevention means any action that suppresses or delays the onset of a target disease
  • treatment means that the target disease and its metabolic abnormalities are improved or It means any action that is advantageously changed
  • improvement means any action that reduces a parameter related to a desired disease, for example, the degree of a symptom by administration of the composition according to the present invention.
  • the "improvement” includes “inhibiting the progression of a disease”.
  • the present invention provides a food composition for preventing or improving autoimmune diseases, comprising the nanoparticles according to the present invention as an active ingredient.
  • the food composition includes a health functional food composition. That is, the main object of the present invention is to provide a functional dietary and pharmaceutical composition comprising the nanoparticles as an active ingredient.
  • the biopolymer nanoparticles of the present invention When the nanoparticles of the present invention are used as food additives, the biopolymer nanoparticles can be added as they are or used together with other foods or food ingredients, and can be appropriately used according to a conventional method.
  • the mixed amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).
  • the nanoparticles of the present invention may be added in an amount of 15% by weight or less, or 10% by weight or less based on the raw material.
  • the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount greater than the above range.
  • the health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, as in a conventional beverage.
  • the above-mentioned natural carbohydrates are monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol.
  • natural sweeteners such as taumatine and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like can be used.
  • the proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g per 100 mL of the composition of the present invention.
  • the composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may contain a carbonation agent used for carbonated beverages, and the like.
  • the composition of the present invention may contain fruit for the production of natural fruit juice, fruit juice beverage, and vegetable beverage. These components may be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0.01-0.20 parts by weight per 100 parts by weight of the composition of the present invention.
  • health functional food is the same term as food for special health use (FoSHU), and refers to foods with high medical and medical effects processed to efficiently exhibit bioregulatory functions in addition to nutritional supply. Meaning, the food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, etc. to obtain a useful effect in the prevention or improvement of autoimmune diseases.
  • the health functional food of the present invention can be prepared by a method commonly used in the art, and during the manufacture, it can be prepared by adding raw materials and components commonly added in the art.
  • unlike general drugs there are no side effects that may occur when taking the drug for a long period of time by using food as a raw material, and it can be excellent in portability.
  • the present invention provides a kit for preventing, improving, or treating autoimmune diseases, comprising the nanoparticles according to the present invention.
  • the “kit” is not limited to a specific form or type, and a kit of a type commonly used in the art may be used.
  • the kit according to the present invention may further include a storage solution of the nanoparticles, an administration tool, a suspension for administration, etc. in addition to the nanoparticles, and may further include instructions on the characteristics of the nanoparticles, manufacturing methods, etc. can
  • the present invention comprises the steps of (S1) producing antioxidant nanoparticles by inducing self-assembly of a biopolymer in a basic environment;
  • the antibody and autoantigen of claim 1 comprising the step of reacting the surface-coated antioxidant nanoparticles with an autoantigen and an antigen-presenting cell-specific antibody or fragment thereof with the surface of the lipid membrane-bound lipid-antioxidation
  • a method for preparing nanoparticles is provided.
  • the biopolymer in step (S1) is as described above.
  • the biopolymer may be one or more selected from the group consisting of polydopamine, tannin, and cerium oxide, but is not limited thereto.
  • the step (S1) may specifically include the following steps:
  • the biopolymer solution may be preferably selected from the group consisting of polydopamine solution, tannin solution, and cerium oxide solution, preferably polydopamine aqueous solution, more preferably dopamine hydrochloride. It may be a solution.
  • the solvent of the biopolymer solution may be distilled water, but is not limited thereto.
  • the basic solution is used for the purpose of inducing self-assembly of the biopolymer in a basic environment.
  • the specific type of the basic solution is not limited, but may be preferably selected from sodium hydroxide solution, potassium hydroxide solution, calcium hydroxide solution, and the like.
  • the biopolymer solution is titrated to pH 5 to 10, pH 6 to 10, pH 7 to 10, pH 8 to 10, pH 9 to 10, or pH 9.5 to 10 with a basic solution.
  • a basic solution may be, but is not limited thereto.
  • the self-assembly of the step (S1-2) may be accomplished by magnetically stirring the biopolymer solution.
  • the magnetic stirring may be carried out at 10 to 35 °C, 10 to 30 °C, 10 to 27 °C, 15 to 30 °C, 20 to 30 °C, 20 to 27 °C, or 23 to 27 °C, 1 to 40 hours, 1 to 35 hours, 1 to 30 hours, 1 to 25 hours, 5 to 35 hours, 10 to 35 hours, 15 to 35 hours, 15 to 30 hours, 20 to 30 hours, or 20 to 25 hours may be performed, but is not limited thereto.
  • step (S1) of the manufacturing method may optionally further include the following steps after the step (S1-2):
  • the washing of step (S1-3) can be made by suspending the biopolymer nanoparticles in distilled water and then centrifuging, but is not limited thereto, and the nanoparticle washing method known in the art can be used without limitation.
  • the nanoparticle washing method known in the art can be used without limitation.
  • the filtration of step (S1-4) may be made using a polycarbonate filter, but is not limited thereto.
  • the biopolymer nanoparticles may be stored in a suspended state in distilled water, but is not limited thereto.
  • the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), phosphorylglycerol (PG), phosphocholine ( PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide), cholesterol (cholesterol), 1,2-dioleoyl-sn-glycero- It may include at least one selected from the group consisting of 3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
  • DOPC 3-phosphocholine
  • DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanol
  • the lipid membrane is 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), and 1,2-disteroyl-sn- It may be prepared by mixing glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide) in the molar ratio described above, dissolving it in an organic solvent and concentrating under reduced pressure.
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
  • 1,2-disteroyl-sn- It may be prepared by mixing glycero-3-phosphoethanolamine-N-[maleimide(polyethyleneglycol)-2000] (DSPE-PEG2000-maleimide) in the
  • the organic solvent may be any one or more selected from the group consisting of chloroform, hexane, ethyl acetate, methanol, dichloromethane, carbon tetrachloride, benzene, DMSO and DMF, preferably chloroform and methanol solution,
  • the chloroform and methanol solution is chloroform: methanol (v/v) 1 to 10: 1, 1 to 8: 1, 1 to 6: 1, 1 to 5: 1, 2 to 10: 1, 3 to 10: 1, 2 to 8: 1, 3 to 7: 1, or may be mixed in a ratio of 3 to 6: 1, but is not limited thereto.
  • the lipid membrane and the antioxidant nanoparticles have a weight ratio (w/w) of the lipid membrane: antioxidant nanoparticles of 1 to 20: 27, 1 to 18: 27, 1 to 16: 27, 1 to 14: 27, 1 to 12: 27, 1 to 10: 27, 1 to 8: 27, 1 to 6: 27, or 1 to 4: 27, but is not limited thereto, and the nanoparticles may be coated with the lipid. It is enough to have
  • the lipid membrane may be an immunomodulatory agent, more preferably an immunosuppressive agent encapsulated therein.
  • the immunosuppressant may be encapsulated in the lipid membrane by dissolving each lipid component in an organic solvent during the preparation of the lipid membrane and adding together when mixing.
  • the immunosuppressive agent has a weight ratio (w/w) of the immunosuppressant: antioxidant nanoparticles (preferably polydopamine nanoparticles) of 0.0001 to 0.01: 1, 0.0001 to 0.01: 1, 0.001 to 0.01: 1, or 0.001 to 0.005:1 may be added, but is not limited thereto.
  • step (S3) reacting the antigen-presenting cell-specific antibody or fragment thereof with the nanoparticles is an autoantigen in the suspension of nanoparticles obtained from step (S2);
  • a solution of antigen-presenting cell-specific antibody or fragment thereof may be added and stirred.
  • the stirring may be performed at 10 to 35 °C, 10 to 30 °C, 10 to 27 °C, 15 to 30 °C, 20 to 30 °C, 20 to 27 °C, or 23 to 27 °C, 1 to 30 hours, 1 to 25 hours, 1 to 20 hours, 1 to 15 hours, 5 to 30 hours, 10 to 25 hours, 10 to 20 hours, or 10 to 15 hours, but is not limited thereto.
  • the antigen-presenting cell-specific antibody or fragment thereof has a weight ratio (w/w) of the antibody or fragment: antioxidant nanoparticles (preferably polydopamine nanoparticles) of 0.01 to 1:1, 0.01 to 0.9:1, 0.01 to 0.8:1, 0.01 to 0.7:1, 0.01 to 0.6:1, 0.05 to 1:1, 0.07 to 1:1, 0.09 to 1:1, 0.1 to 1:1, 0.2 to 1:1, 0.3 to 1:1, 0.2 to 0.8:1, 0.2 to 0.6:1, or 0.4 to 0.6:1 may be added, but is not limited thereto.
  • antioxidant nanoparticles preferably polydopamine nanoparticles
  • the autoantigen has a weight ratio (w/w) of the autoantigen: antioxidant nanoparticles (preferably polydopamine nanoparticles) of 0.01 to 1:1, 0.02 to 1:1, 0.05 to 1 : 1, 0.07 to 1:1, 0.09 to 1:1, 0.01 to 0.8:1, 0.01 to 0.6:1, 0.01 to 0.5:1, 0.01 to 0.3:1, 0.05 to 0.3:1, or 0.09 to 0.2: 1 may be added, but is not limited thereto.
  • antioxidant nanoparticles preferably polydopamine nanoparticles
  • the preparation method includes the autoantigen;
  • the method may further include modifying the antigen-presenting cell-specific antibody or fragment thereof to have a thiol group.
  • the method of adding the thiol group is known in the art, but it may be preferably carried out by adding a traut reagent to a solution of the autoantigen or antibody and reacting.
  • Antioxidant nanoparticles (hereinafter, “AbaLDPN-MOG”) were prepared whose surface was coated with a lipid layer containing an immune tolerance-inducing drug, and autoantigens and recombinant antibodies were bound to the surface of the lipid layer.
  • the overall manufacturing process is shown in FIG. 1 .
  • Polydopamine nanoparticles were synthesized through self-polymerization of dopamine under alkaline conditions.
  • dopamine hydrochloride was dissolved in 25 mL of triple distilled water (TDW) to prepare a final concentration of 2 mg/mL, and then the dopamine hydrochloride aqueous solution was titrated to pH 9.68 using 1N sodium hydroxide solution, Magnetic stirring was carried out at room temperature (25 °C) for 24 hours.
  • TDW triple distilled water
  • a black polydopamine nanoparticle precipitate was collected by centrifugation at 13,500 g for 20 minutes, and washed with triple distilled water until the supernatant became transparent.
  • the polydopamine nanoparticles were filtered through a 450 nm polycarbonate filter and stored at 4° C. as a suspension in distilled water.
  • PN or antioxidant nanoparticles polydopamine nanoparticles.
  • 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DPPC
  • 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol DPPG
  • 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneglycol)-2000] DSPE-PEG2000-maleimide
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DPPG 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol
  • DSPE-PEG2000-maleimide 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneglycol)-2000]
  • DSPE-PEG2000-maleimide 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(poly
  • 1,2-dipalmitoyl-sn-glycero-3-phosphocholine DPPC
  • 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol DPPG
  • 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneglycol)-2000] DSPE-PEG2000-maleimide
  • DSPE-PEG2000-maleimide 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneglycol)-2000]
  • DSPE-PEG2000-maleimide 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneglycol)-2000]
  • DSPE-PEG2000-maleimide 1,2-disteroyl-sn-glycero-3-phosphoethanolamine -N-[maleimide(polyethyleneg
  • the prepared lipid thin film was hydrated by adding the polydopamine aqueous solution prepared in Example 1-1 or an isotonic 5% glucose aqueous solution.
  • the drug-encapsulated lipid-antioxidant nanoparticles (LDPN) were stored at 4°C.
  • Autoantigen myelin oligodendrocyte glycoprotein, MOG
  • traut reagent was added to a final concentration of 2 mg/mL to 10 mg/mL of an autoantigen not containing a thiol group, reacted at 25° C. for about 1 hour, followed by thiolation and purification. Autoantigens that already have a thiol group can be used immediately.
  • the purified autoantigen was treated with a lipid-antioxidant nanoparticle suspension and reacted with nanoparticles at 25 °C for 12 hours, and centrifuged at 13,500 g for 20 minutes to bind autoantigen-bound lipid-antioxidant nanoparticles (LDPN).
  • LDPN autoantigen-bound lipid-antioxidant nanoparticles
  • Recombinant antibody and autoantigen were added to the nanoparticles prepared in Example 1-3 and vigorously stirred.
  • Recombinant antibody (10 mg/mL; CD80/86-specific antibody) and autoantigen (10 mg/mL) were reacted under traut reagent 2 mg/mL conditions, followed by thiolation and purification.
  • a recombinant protein Abatacept (Orencia, Bristol-Myers Squibb; BMS) was used as the CD80/86-specific antibody.
  • Abatacept is a protein in which human cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) is fused with the Fc portion of human IgG1, and can specifically bind to CD80 and CD86.
  • CTL-4 cytotoxic T-lymphocyte-associated antigen 4
  • Abatacept has the form of a homodimer, and the monomer is known to have a 357 amino acid sequence (SEQ ID NO: 1).
  • Belatacept (Nulojix, BMS) may be used.
  • Autoantigens or antibodies that already have a thiol group can be used immediately. Purified autoantigens and antibodies were treated with lipid-antioxidant nanoparticle suspension to react with nanoparticles at 25° C.
  • AbaLDPN-MOG lipid-bound autoantigen and recombinant antibody- Antioxidant nanoparticles
  • polydopamine nanoparticles (control) prepared by the method of Example 1-1 and nanoparticles prepared by the method of Examples 1-3, 1-4, or 1-5 were prepared, and 10% fetal calf
  • serum fetal bovine serum
  • serum containing 100 units/ml penicillin the size of AbaLDPN-MOG was measured using a dynamic light scattering method (ELS8000 instrument, Photal, Osaka, Japan).
  • ELS8000 instrument Photal, Osaka, Japan
  • the zeta potential in aqueous solution was measured using laser Doppler electrophoresis, and it was confirmed that there was no significant difference between the respective compositions.
  • the immunomodulatory drug (dexamethasone) encapsulated in AbaLDPN-MOG prepared in Example 1 and the autoantigen bound to the nanoparticles were quantified using high performance liquid chromatography (HPLC, Agilent).
  • nanoparticles are dispersed in methanol, and all drugs encapsulated in the lipid layer are precipitated by ultrasonic dispersion for 30 minutes, followed by centrifugation at 27,000 x g for 20 minutes to dissolve the drug. Only the supernatant was analyzed.
  • the mobile phase was prepared in a composition of tertiary distilled water: acetonitrile in a volume ratio of 70:30, and the detection wavelength was 254 nm.
  • a C18 reverse-phase chromatography column (C18 reverse-phase HPLC column, Phenomenex) was used for the column, and was carried out at 25°C.
  • Example 1 the nanoparticles prepared in Example 1 were freeze-dried, and Fourier transformation-infrared spectroscopy (FT-IR) (FT/IR-400, JASCO) was performed to confirm the lipid thin film coating of the nanoparticles. did. Infrared spectroscopy was measured using attenuated total reflection (ATR). As a result, it was confirmed that a strong peak appeared at 2900 to 3000 cm -1 . This is due to the CH stretching vibration that many fatty acids of phospholipids have. In the case of Raman spectroscopy (LabRAM HR. Evolution, HORIBA), freeze-dried nanoparticles were used similarly to the above and analyzed using a 532 nm laser. As a result, it was confirmed that the D band and G band, which are the characteristics of the polymer compound having aromatic rings, appeared in common in all compositions (FIG. 4).
  • FT-IR Fourier transformation-infrared spectroscopy
  • Example 1 The particle size and shape of AbaLDPN prepared in Example 1 were evaluated, and in the case of elemental analysis, energy dispersive X-ray spectroscopy (EDS) mounted on a transmission electron microscope was used.
  • EDS energy dispersive X-ray spectroscopy
  • the analysis results are shown in FIG. 5 . Specifically, as a result of transmission electron microscopy, it was confirmed that the shape of the particle was similar to the result of size analysis of the nanoparticles using the dynamic light scattering method. In addition, as a result of elemental analysis using EDS, it was confirmed that the detection of carbon, nitrogen, and oxygen, which are major constituent elements of antioxidant nanoparticles, was the most dominant.
  • the detection of phosphorus refers to the phospholipid layer, and the detection of sulfur is by a protein bound to the surface.
  • the binding ability of the LDPN nanoparticles and AbaLDPN-MOG nanoparticles prepared in Example 1 to antigen-presenting cells expressing CD80/86 was confirmed by flow cytometry, confocal microscopy, and transmission electron microscopy, and under ex vivo conditions. Selectivity for target cells was evaluated.
  • the binding ability of LDPN and AbaLDPN-MOG prepared in Example 1 to CD80/86-expressing dendritic cells was confirmed by flow cytometry and confocal microscopy.
  • the nanoparticles were surface-modified with a recombinant antibody capable of specifically binding to CD80/86 according to Example 1.
  • spleen-derived dendritic cells from C57BL/6 (8 weeks old) were obtained using flow cytometry (FACS Aria Sorting).
  • flow cytometry FACS Aria Sorting
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IL-4 interleukin-4
  • IMDM Iscove's Modified Dulbecco's Medium
  • dendritic cells were treated with Cy5-labeled nanoparticles as polydopamine to a concentration of 400 ⁇ g/mL. After 1 hour, the nanoparticles are removed, and the Cy5 fluorescence intensity of the dendritic cells is checked by flow cytometry.
  • the experimental group treated with AbaLDPN-MOG nanoparticles on the surface of which the antibody was modified exhibited higher fluorescence intensity than the experimental group treated with LDPN-MOG.
  • the above results show that the nanoparticle modification of the recombinant antibody increases the binding capacity of the nanoparticles to dendritic cells.
  • dendritic cells and nanoparticles were cultured in the same manner as above, and cells from which nanoparticles were removed were fixed in 10% formalin/PBS (v/v) for 1 hour. Afterwards, nuclei were stained with Hoechst at 2 ⁇ g/mL for 15 minutes and observed under a microscope.
  • the binding ability of LDPN and AbaLDPN-MOG prepared in Example 1 to CD80/86-expressing dendritic cells was confirmed by transmission electron microscopy.
  • the nanoparticles were surface-modified with a recombinant antibody capable of specifically binding to CD80/86 according to Example 1.
  • nanoparticles were treated to be 400 ⁇ g/mL as polydopamine. After 1 h, each cell was collected and fixed with Karnovsky's solution for 2 h, then washed 3 times with cold 0.05 M sodium carcodylate buffer, and the pellet was washed with 1% osmium tetroxide at 4 °C for 2 h. (osmium tetroxide) was post-fixed. The fixed pellet was washed three times with cold triple distilled water, then stained with 0.5% uranyl acetate at 4°C overnight, and ethanol (30%, 50%, 70%, 80%, 90% and 100%). 3) was dehydrated.
  • the in vitro conditions of FIG. 10 mimic the interaction between dendritic cells and T cells, and are implemented by coating a 96-well plate with anti-CD3 antibody (aCD3Ab) and CD80 Fc fusion protein (CD80 Fc).
  • ACD3Ab and CD80 Fc were each prepared in PBS at a concentration of 10 ⁇ g/mL, covered with a well plate, and stored at 4°C for 12 hours. After 12 hours, when the coating of the well plate is completed, after washing once with PBS, spleen cells prepared from C57BL/6 (8 weeks old) were seeded at a density of 3 ⁇ 10 6 cells per well, and the nanoparticles were 400 as polydopamine. It was processed so that it might become microgram/mL. After 48 hours, the medium in which the cells were cultured was obtained, and IL-2 was detected by performing ELISA (R&D systems, DY402-05) according to the manufacturer's protocol.
  • the group treated with nanoparticles (LDPN-MOG or AbaLDPN-MOG) containing the immunomodulatory drug dexamethasone had IL-2 secretion by T cells compared to the group treated with dexamethasone and antigen directly (Dexa+MOG). was remarkably suppressed (FIG. 11).
  • AbaLDPN-MOG nanoparticles whose surface was modified with a dendritic cell-specific antibody inhibited IL-2 secretion of T cells more effectively than LDPN-MOG on which the antibody was not modified.
  • the antibody-modified lipid-antioxidant nanoparticles according to the present invention effectively block the interaction between T cells and dendritic cell surface proteins coated on a well plate, thereby inhibiting T cell activation. .
  • the nanoparticles according to the present invention inhibited IL-2 secretion of T cells more effectively compared to CTLA-4 Fc used as a positive control.
  • nanoparticles according to the present invention The effect of inhibiting the interaction between dendritic cells and T cells was evaluated through the proliferation and division levels of T cells in the in vitro environment shown in FIG. 10 .
  • the experiment was carried out under the same conditions as in FIG. 10, and similarly, aCD3Ab and CD80 Fc were coated on a 96 well plate and implemented. Cells were washed once with PBS before seeding.
  • Splenocytes were prepared by staining with carboxyfluorescein succinimidyl ester (CFSE).
  • CFSE carboxyfluorescein succinimidyl ester
  • the nanoparticles according to the present invention significantly inhibited the division and proliferation of T cells compared to the control groups (untreated control group, MOG-treated group, dexamethasone and MOG-treated group).
  • control groups untreated control group, MOG-treated group, dexamethasone and MOG-treated group.
  • AbaLDPN-MOG nanoparticles whose surface was modified with a dendritic cell-specific antibody inhibited the division and proliferation of T cells more effectively than LDPN-MOG on which the antibody was not modified.
  • Analysis of T cell expansion index through fluorescence intensity was performed with flowJo v10 program.
  • CD3 and CD4 double positive cells meaning helper T cells; and CD3 and CD8 double-positive cells, which mean cytotoxic T cells, respectively, were analyzed, and it was confirmed that AbaLDPN-MOG nanoparticles whose surface was modified with a dendritic cell-specific antibody effectively inhibited both types of T cells.
  • splenocytes were treated with nanoparticles corresponding to 400 ⁇ g/mL as polydopamine for 1 hour, and the cells were cultured for 72 hours with the nanoparticles removed. After activating the cells by treating the cultured splenocytes with 1 ⁇ g/mL LPS, the cells were checked by flow cytometry 6 hours later. In order to analyze the protein present in the cytoplasm, it was treated with LPS at a concentration of 5 ⁇ g/mL with brefeldin A to prevent the leakage of the protein into the cytoplasm. Fluorescent antibody staining of cells was performed according to the protocol of the corresponding product (BioLegend 424401).
  • CD11c and MHCII Only double-positive cells of CD11c and MHCII were selected from spleen cells, and the mean fluorescence intensity of fluorescence for their target proteins was analyzed.
  • the cells treated with the antioxidant-nanoparticles according to the present invention decreased the levels of CD80, CD86, and MHCII, which are cell membrane proteins related to the activation of dendritic cells, and the inflammatory cytokine TNF- It was confirmed that the secretion of ⁇ , IL-1 ⁇ , and IL-6 was also suppressed. In particular, the effect was more pronounced in cells treated with lipid-antioxidant nanoparticles (AbaLDPN-MOG), the surface of which was modified with a recombinant antibody.
  • the above results show that the nanoparticles according to the present invention can induce immune-tolerance dendritic cells by effectively blocking T cell activation by CD80, CD86, and MHCII in dendritic cells (FIG. 15).
  • Lymph nodes and spleen which are secondary immune organs, are the places where antigen-presenting cells, including dendritic cells, are abundantly distributed, and are major target organs for the nanoparticles for immunomodulation according to the present invention.
  • the present inventors confirmed through preclinical tomographic imaging, flow cytometry and immunofluorescence histology that the nanoparticles were targeted to the secondary immune organs of the animal model when the nanoparticles were subcutaneously injected into an animal model.
  • Cy5-labeled nanoparticles were injected subcutaneously in the lumbar spine of C57BL/6 (8 weeks old). Nanoparticles were administered as polydopamine at 50 mg/kg, and when 6, 24, 48, and 72 hours had elapsed after administration, Cy5 fluorescence intensity in lymph nodes was measured.
  • the spleen-targeting ability of the recombinant antibody (CD80/86-specific recombinant antibody) and autoantigen-modified lipid-antioxidant nanoparticles prepared according to Example 1 on the surface was confirmed by analyzing dendritic cells in the lymph nodes.
  • the nanoparticles according to the present invention were administered to mice in the same manner as in Experimental Example 6-2, and then lymph nodes of the mice were isolated and confirmed by flow cytometry.
  • lymph node targeting ability of the recombinant antibody (CD80/86-specific recombinant antibody) and the lipid-antioxidant nanoparticles modified on the surface of the autoantigen was confirmed by immunofluorescence histology.
  • the lymph nodes of the mouse were isolated and histological analysis was performed using a confocal microscope and an Automated Multimodal Tissue Analysis System (PerkinElmer, Vectra). did.
  • the isolated lymph nodes were fixed in 10% formalin/PBS (v/v) for 24 hours, and then the cryoprotection process was performed in 30% sucrose solution for 24 hours.
  • the tissue embedding medium was OCT compound tissue-TEK, and was stored at -80 °C after processing using liquid nitrogen.
  • the cryosection slide was prepared using a cryosection (Leica), and the thickness of the tissue section was 5 ⁇ m.
  • the efficacy of the nanoparticles according to the present invention was confirmed in an animal model of autoimmune disease using ovalbumin (OVA).
  • OVA ovalbumin
  • the effects of nanoparticles on the humoral immune response were evaluated by analyzing the concentration of autoantibodies in serum, and the modulating effect of the cellular immune response was evaluated by ELISPOT.
  • An autoimmune disease animal model (12 weeks old) using C57BL/6 was constructed so that an immune response to an autoantigen (ovalbumin, OVA), not a pathogenic antigen, occurs.
  • OVA autoantigen
  • CFA complete freund's adjuvant
  • OVA invivogen
  • the concentration of the autoantibody in the body of the animal model was measured.
  • the nanoparticles of the present invention (AbaLDPN-OVA) loaded with an autoantigen (AbaLDPN-OVA) were subcutaneously injected three times with an interval of 7 days, and the tail of the individual every week from the time point 14 days elapsed from the start date of induction of the autoimmune disease model. 10 ⁇ L of blood was obtained from the vein. Blood was well diluted in 5 mM EDTA/PBS aqueous solution containing 10% FBS, and then centrifuged at 2,000 g for 10 minutes to separate red blood cells. The separated serum samples were diluted according to the sensitivity of the ELISA, and ELISA was performed using an OVA-coated well plate.
  • the level of autoantibody (anti-OVA IgG) in the mouse administered with the nanoparticles of the present invention was significantly reduced compared to other controls, in particular, the recombinant antibody was modified on the surface of the lipid - It was confirmed that the level of autoantibodies in the mice receiving the antioxidant nanoparticles decreased more significantly than the mice receiving the nanoparticles in which the antibody was not modified.
  • the above results show that the nanoparticles according to the present invention can effectively suppress the humoral immune response for a long period of time despite continuous antigen exposure, and that the humoral immunosuppressive effect of the nanoparticles surface-modified with antibodies and autoantigens is particularly excellent. back it up
  • the efficacy of the nanoparticles according to the present invention was evaluated using an experimental autoimmune encephalomyelitis (EAE) model. Efficacy evaluation was made by confirming the degree of central nervous system invasion and demyelination of immune cells, including changes in the subject's clinical symptoms (clinical score) and body weight.
  • EAE experimental autoimmune encephalomyelitis
  • the experimental autoimmune encephalomyelitis (EAE) model is an experimental animal model widely used in the study of multiple sclerosis in humans. Fragment peptides 35 to 55 of myelin oligodendrocyte glycoprotein (MOG) present in the nervous system of healthy individuals ( It is a model constructed by inducing an autoimmune response to MOG 35-55 ).
  • MOG myelin oligodendrocyte glycoprotein
  • MOG 35-55 MOG peptide
  • CFA Heat inactivated M. tuberculosis, 4 mg/mL
  • PTX pertussis toxin
  • the administration schedule differed depending on the experimental purpose (prevention, progression inhibition, and treatment of autoimmune diseases).
  • the nanoparticles were administered twice at intervals of 1 week from 2 weeks before inducing the disease model (FIG. 21a).
  • the nanoparticles were administered twice at intervals of 1 week (FIG. 21b), and when symptoms began to appear when the purpose of the treatment of autoimmune disease was to start, the subjects with a clinical score of 0.5 were randomly selected, and the control group and the experimental group , and the nanoparticles were administered by subcutaneous injection 4 times every 3 days to 50 mg/kg as polydopamine ( FIG. 21c ).
  • mice treated with the lipid-antioxidant nanoparticles effectively improved clinical symptoms and normally controlled body weight, and in particular, AbaLDPN-MOG on which the recombinant antibody was modified on the surface of LDPN-MOG was not treated. It was confirmed that it showed more encouraging results compared to that ( FIGS. 22a to 22c ).
  • Antioxidant according to the present invention The ability to improve the central nervous system invasion of immune cells by nanoparticles was evaluated by flow cytometry.
  • nanoparticles were administered to the autoimmune disease model in the same manner as in Experimental Example 8-1, and after 28 days from the start date of the experiment, the spinal cord was extracted from each individual.
  • the spinal cord was isolated into single cells using 1 mg/mL collagenase (Sigma-Aldrich) and a cell strainer (40 ⁇ m) (SPL), and was used after hemolysis of red blood cells in hypotonic buffer.
  • Each immune cell was basically selected to express CD45, CD11c + MHCII + for dendritic cells, CD11b + F4/80 + for macrophages, CD3 + CD4 + for helper T cells, and cytotoxic T
  • CD3 + CD8 + and in the case of regulatory T cells, CD25 + FoxP3 + in helper T cells were analyzed as markers.
  • helper T cells they were analyzed to commonly label CD3 + CD4 + and express either IFN- ⁇ or IL-17A. The labeling method was performed in the same manner as the flow cytometry method.
  • FIGS. 23A-23C The results are shown in FIGS. 23A-23C.
  • the antioxidant nanoparticles according to the present invention were administered for the purpose of prevention, progression inhibition, or treatment of autoimmune diseases, it was confirmed that the invasion of immune cells into the central nervous system was reduced compared to the untreated control group.
  • the effect was more pronounced in the AbaLDPN-MOG administration group than in the LDPN-MOG administration group. More specifically, AbaLDPN-MOG inhibited invasion into the central nervous system most effectively for dendritic cells, macrophages, and cytotoxic T cells, and LDPN-MOG and AbaLDPN-MOG showed similar effects on helper T cells.
  • FIGS. 24a to 24c shows that the antioxidant nanoparticles according to the present invention have an effect of increasing the ratio of regulatory T cells capable of regulating an excessive immune response.
  • the ratio of helper T cells expressing IFN- ⁇ or IL-17A, which can damage tissues was most significantly decreased in the experimental group administered with AbaLDPN-MOG.
  • Antioxidant according to the present invention The ability of nanoparticles to modulate antigen-specific cellular immune response of immune cells was evaluated using ELISPOT.
  • nanoparticles were administered to the autoimmune disease model in the same manner as in Experimental Example 8-1, and splenocytes were extracted from the subjects after 28 days from the start date of the experiment, under the treatment of 5 ⁇ g/mL MOG peptide.
  • ELISPOT (BD, Mouse IFN- ⁇ ELISPOT Set, 551083) was performed. Specific experiments were carried out according to the protocol of the product.
  • the nanoparticles according to the present invention When the antioxidant nanoparticles according to the present invention are administered for the purpose of preventing, inhibiting progression, or treating autoimmune diseases, the nanoparticles activate the splenocytes of each animal model by autoantigen (MOG peptide) to produce IFN- ⁇ . It was confirmed that secretion was reduced. That is, the nanoparticles can effectively inhibit the cellular immune response of immune cells activated by the MOG peptide, and in particular, in the case of lipid-antioxidant nanoparticles modified with recombinant antibody, lipid-antioxidant nanoparticles that are not modified with recombinant antibody It has been shown to more effectively modulate the immune response.
  • MOG peptide autoantigen
  • Antioxidant according to the present invention The ability of the nanoparticles to improve the central nervous system invasion of immune cells and the inhibitory effect of demyelination was analyzed using immunofluorescence histology.
  • nanoparticles were administered to the autoimmune disease model in the same manner as in Experimental Example 8-1, and after 28 days from the start date of the experiment, the spinal cord was extracted from each individual. The extracted spinal cord was treated in the same manner as in Experimental Example 6-3, and immunofluorescence histology was performed.
  • CD45 is a marker of immune cells that invaded the central nervous system
  • MBP myelin basic protein
  • mice receiving the antioxidant nanoparticles according to the present invention showed a remarkably reduced central nervous system invasion of immune cells compared to the untreated control group. appear.
  • the above results show that the antioxidant nanoparticles according to the present invention can protect the myelin sheath in an animal model of encephalomyelitis and regulate the immune response by blocking the invasion of the central nervous system by immune cells.
  • the lipid-antioxidant nanoparticles to which the antibody and the autoantigen according to the present invention are bound to the surface effectively target the spleen or lymph node when administered to a subject, bind specifically to antigen-presenting cells, and are encapsulated in the lipid membrane. It was confirmed that not only effectively delivered the immunosuppressive agent, but also induces immune tolerance to autoantigens ( FIG. 27 ). That is, the nanoparticles according to the present invention can inhibit the generation of autoantibodies against autoantigens, suppress hyperactivity of immune cells, and suppress invasion of normal tissues by immune cells, thus preventing and treating various autoimmune diseases. It is expected to be used for this purpose.
  • Lipid-antioxidant nanoparticles according to the present invention have autoantigens and antigen-presenting cell-specific antibodies modified on the surface to deliver the autoantigen specifically to antigen-presenting cells, and the surface is coated with a lipid membrane encapsulated with an immunomodulatory agent. Immune tolerance to autoantigens can be induced.
  • the nanoparticles according to the present invention are administered to an animal model of autoimmune disease, they effectively target lymph nodes and spleen, which are immune organs, and effectively inhibit excessive immune activation by antigen-presenting cells, thereby preventing, delaying, and It has been shown to be curable. Therefore, the nanoparticles according to the present invention are expected to be usefully utilized for the prevention or treatment of various autoimmune diseases, including encephalomyelitis.

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Abstract

Les nanoparticules anti-oxydantes lipidiques selon la présente invention comportent un auto-antigène et un anticorps spécifique d'une cellule présentatrice d'antigènes modifiés à leur surface, et peuvent ainsi délivrer des auto-antigènes spécifiquement en direction de cellules présentatrices d'antigènes, et sont revêtues à leur surface d'une membrane lipidique encapsulant un immunomodulateur, et peuvent ainsi induire une immunotolérance aux auto-antigènes. En particulier, il a été confirmé que les nanoparticules selon la présente invention, lorsqu'elles sont administrées à des modèles animaux de maladies auto-immunes, peuvent cibler efficacement les ganglions lymphatiques et la rate, qui sont des organes du système immunitaire, pour inhiber efficacement une activation immunitaire excessive par des cellules présentatrices d'antigènes et, ainsi, prévenir, retarder et traiter les maladies auto-immunes. Par conséquent, on s'attend à ce que les nanoparticules selon la présente invention soient efficacement utilisées pour prévenir ou traiter diverses maladies auto-immunes, dont l'encéphalomyélite.
PCT/KR2022/006518 2021-05-06 2022-05-06 Nanoparticules lipidiques-biopolymères à la surface desquelles sont liés un anticorps et un auto-antigène, et leur utilisation WO2022235125A1 (fr)

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