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WO2014109617A1 - Composition pour la prévention ou le traitement de maladies auto-immunes, contenant htra2 comme principe actif - Google Patents

Composition pour la prévention ou le traitement de maladies auto-immunes, contenant htra2 comme principe actif Download PDF

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Publication number
WO2014109617A1
WO2014109617A1 PCT/KR2014/000361 KR2014000361W WO2014109617A1 WO 2014109617 A1 WO2014109617 A1 WO 2014109617A1 KR 2014000361 W KR2014000361 W KR 2014000361W WO 2014109617 A1 WO2014109617 A1 WO 2014109617A1
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htra2
autoimmune diseases
pharmaceutical composition
expression
preventing
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Korean (ko)
Inventor
임향숙
조미라
문영미
이선영
이재선
유준걸
손혜진
허양미
남민경
신현아
김재경
서현범
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Industry Academic Cooperation Foundation of Catholic University of Korea
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    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F21—LIGHTING
    • F21V—FUNCTIONAL FEATURES OR DETAILS OF LIGHTING DEVICES OR SYSTEMS THEREOF; STRUCTURAL COMBINATIONS OF LIGHTING DEVICES WITH OTHER ARTICLES, NOT OTHERWISE PROVIDED FOR
    • F21V21/00—Supporting, suspending, or attaching arrangements for lighting devices; Hand grips
    • F21V21/10—Pendants, arms, or standards; Fixing lighting devices to pendants, arms, or standards
    • F21V21/116—Fixing lighting devices to arms or standards
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/482—Serine endopeptidases (3.4.21)
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B05—SPRAYING OR ATOMISING IN GENERAL; APPLYING FLUENT MATERIALS TO SURFACES, IN GENERAL
    • B05B—SPRAYING APPARATUS; ATOMISING APPARATUS; NOZZLES
    • B05B17/00—Apparatus for spraying or atomising liquids or other fluent materials, not covered by the preceding groups
    • B05B17/08—Fountains
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21108—HtrA2 peptidase (3.4.21.108)
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F21—LIGHTING
    • F21S—NON-PORTABLE LIGHTING DEVICES; SYSTEMS THEREOF; VEHICLE LIGHTING DEVICES SPECIALLY ADAPTED FOR VEHICLE EXTERIORS
    • F21S8/00—Lighting devices intended for fixed installation
    • F21S8/08—Lighting devices intended for fixed installation with a standard
    • F21S8/085—Lighting devices intended for fixed installation with a standard of high-built type, e.g. street light
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F21—LIGHTING
    • F21V—FUNCTIONAL FEATURES OR DETAILS OF LIGHTING DEVICES OR SYSTEMS THEREOF; STRUCTURAL COMBINATIONS OF LIGHTING DEVICES WITH OTHER ARTICLES, NOT OTHERWISE PROVIDED FOR
    • F21V19/00—Fastening of light sources or lamp holders
    • F21V19/006—Fastening of light sources or lamp holders of point-like light sources, e.g. incandescent or halogen lamps, with screw-threaded or bayonet base
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F21—LIGHTING
    • F21V—FUNCTIONAL FEATURES OR DETAILS OF LIGHTING DEVICES OR SYSTEMS THEREOF; STRUCTURAL COMBINATIONS OF LIGHTING DEVICES WITH OTHER ARTICLES, NOT OTHERWISE PROVIDED FOR
    • F21V33/00—Structural combinations of lighting devices with other articles, not otherwise provided for
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F21—LIGHTING
    • F21W—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES F21K, F21L, F21S and F21V, RELATING TO USES OR APPLICATIONS OF LIGHTING DEVICES OR SYSTEMS
    • F21W2121/00—Use or application of lighting devices or systems for decorative purposes, not provided for in codes F21W2102/00 – F21W2107/00
    • F21W2121/02—Use or application of lighting devices or systems for decorative purposes, not provided for in codes F21W2102/00 – F21W2107/00 for fountains
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F21—LIGHTING
    • F21W—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES F21K, F21L, F21S and F21V, RELATING TO USES OR APPLICATIONS OF LIGHTING DEVICES OR SYSTEMS
    • F21W2131/00—Use or application of lighting devices or systems not provided for in codes F21W2102/00-F21W2121/00
    • F21W2131/10—Outdoor lighting
    • F21W2131/103—Outdoor lighting of streets or roads
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02B—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO BUILDINGS, e.g. HOUSING, HOUSE APPLIANCES OR RELATED END-USER APPLICATIONS
    • Y02B20/00—Energy efficient lighting technologies, e.g. halogen lamps or gas discharge lamps
    • Y02B20/72—Energy efficient lighting technologies, e.g. halogen lamps or gas discharge lamps in street lighting

Definitions

  • the present invention relates to a composition for preventing or treating autoimmune diseases comprising HtrA2 (High temperature requirement protein A2) protein as an active ingredient.
  • HtrA2 High temperature requirement protein A2
  • Immunity is one of the body's self-protective systems against all foreign polymers (antigens) that invade or are injected into living tissue.
  • the main component of the immune system is lymphocytes, which are white blood cells that are made in the bone marrow and circulate along the blood into lymph tissues and organs, mainly lymph nodes, spleen, and tonsils.
  • lymphocytes white blood cells that are made in the bone marrow and circulate along the blood into lymph tissues and organs, mainly lymph nodes, spleen, and tonsils.
  • B cells multiply rapidly when stimulated by an appropriate antigen, forming a clone that produces a special antibody (immunoglobulin) that neutralizes that antigen, and the antibodies that B cells produce circulate in body fluids.
  • Humoral immunity is performed.
  • T cells are produced in the thymus and migrate to lymphoid tissue, responsible for cell-mediated immunity that directly attacks antigens.
  • autoimmune diseases are diseases caused by abnormal reactions to magnetic cells
  • drugs mainly for suppressing autoimmune function are used.
  • many side effects are difficult to use continuously and do not sufficiently prevent relapses, so there is a limit to treatment for cure.
  • beta interferon is used, but the side effects are less expensive, the treatment costs are high, the life-long injection is inconvenient, and the effect of preventing recurrence is minimal.
  • the method of inhibiting the interaction between cells by the antibody administration to CD40 ligand, etc. has been used, but not very successful.
  • immunotherapies There are many other immunotherapies, but no one has yet been proven to show definite therapeutic efficacy.
  • autoimmune diseases can be effectively treated and harmless to the human body, there is a need for research on a therapeutic agent without side effects.
  • HtrA2 protein reduces the expression of STAT3, inhibits the expression of inflammatory cytokines, and regulates T cells (Treg).
  • the present invention was completed by confirming that it has a function of promoting the activity of and can effectively treat autoimmune diseases.
  • an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising HtrA2 protein as an active ingredient.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating autoimmune diseases comprising a polynucleotide encoding an HtrA2 protein as an active ingredient.
  • Another object of the present invention to provide an immunosuppressive agent comprising the composition as an active ingredient.
  • the present invention provides a pharmaceutical composition for the prevention or treatment of autoimmune diseases comprising HtrA2 protein as an active ingredient.
  • the HtrA2 protein may have an amino acid sequence represented by SEQ ID NO: 1.
  • the HtrA2 protein may be to reduce STAT3 expression and activity.
  • the HtrA2 protein may be to promote or increase the activity of regulatory T cells (Treg) and to reduce the concentration of total IgG and IgM in the blood.
  • Treg regulatory T cells
  • the HtrA2 protein may be to reduce or inhibit the expression of inflammatory cytokines IL-1b, IL-6 and IL-17.
  • the HtrA2 protein may inhibit the differentiation of osteoclasts.
  • the autoimmune disease may be selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, digestive diabetes, atopic dermatitis, autoimmune encephalomyelitis, asthma and Crohn's disease.
  • the present invention also provides a pharmaceutical composition for preventing or treating autoimmune diseases comprising a polynucleotide encoding an HtrA2 protein as an active ingredient.
  • the autoimmune disease may be selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, digestive diabetes, atopic dermatitis, autoimmune encephalomyelitis, asthma and Crohn's disease.
  • the polynucleotide may have a nucleotide sequence represented by SEQ ID NO: 2.
  • the polynucleotide may be contained in an expression vector.
  • the expression vector may be a plasmid or viral vector.
  • the present invention provides an immunosuppressive agent comprising the composition as an active ingredient.
  • the present invention has confirmed that the HtrA2 protein has a function of reducing the expression of STAT3, inhibiting the expression of inflammatory cytokines, and promoting the activity of regulatory T cells (Treg), thus effectively treating autoimmune diseases, and thus
  • the present invention is characterized by providing a composition for the prevention or treatment of autoimmune diseases comprising HtrA2 protein as an active ingredient.
  • PIAS3 and suppressor of cytokine signaling 3 have been cited as inhibitory feedback of STAT3, and SOCS proteins have the activity of inhibiting ligand-induced responses by inhibiting JAKs,
  • the PIAS protein is known to have activity that inhibits the phosphorylation of STAT3 (Starr R, Hilton DJ. Negative regulation of the JAK / STAT pathway. Bioessays 1999; 21: 4752).
  • HtrA2 was also first identified as a mitochondrial serine protease, a protein that interacts with Mxi2.
  • HtrA2 is composed of mitochondrial targeting sequence (MTS), transmembrane domain (TM), protease domain, and PDZ domain, and exists in the intermembrane space of mitochondria.
  • the bacterium HtrA is likely to act as a chaperon at physiological temperature (37 ° C) and as a chaperon or protease.
  • apoptosis stimulation such as UV and staurosporine, HtrA2 is released into the cytoplasm from mitochondria together with Smac / DIABLO and cytochrome c to inhibit the action of inhibitor of apoptosis protein (IAP).
  • IAP inhibitor of apoptosis protein
  • HtrA2 is one of the regulatory factors that play a pivotal role in the apoptosis pathway.
  • linkage analysis and mutation screening confirmed that serine 276 mutated to cysteine in the HtrA2 gene of mnd2 (motor neuron degeneration 2) mice (see SEQ ID NO: 3 and SEQ ID NO: 4), and the loss of serine protease function of HtrA2. Due to this, mnd2 mice are reported to have a phenotype of smaller spleen and thymus, smaller body size, lower mobility, and an average lifespan of about 40 days than normal mice. This indicates that HtrA2 is effective in the prevention and treatment of degenerative brain diseases.
  • the present invention first identified that the HtrA2 protein can be used for the prevention or treatment of autoimmune diseases.
  • STAT3 was stained by immunofluorescence in spleen tissues of wild type and HtrA2 variant mice, thereby staining STAT3.
  • the expression levels of p-STAT3 tyr705 and ser727 were found to be more expressed in the spleen of HtrA2 mutant mice than the wild type, and the expression of STAT3 increased when HtrA2 mutants occurred. See FIG. 1).
  • the expression of the markers of T cells, B cells, dendritic cells and STAT3 in wild-type and HtrA2 variant mouse spleens was confirmed through immunofluorescence.
  • T cells, B cells, dendritic cells (Dendritic cells) were concentrated inside the follicles, and STAT3 was confirmed to be overactivated (see FIG. 2).
  • HtrA2 protein decreases the expression of STAT3, a transcription factor of h17 cells, promotes the activity of regulatory T cells (Treg), and inhibits the concentration of total IgG and IgM in the blood. It plays a role in the immune regulation (see Fig. 3a, 3b and 4).
  • the gene expression concentration of the inflammatory cytokines Il-1b, IL-6 and IL-17 isolated from the spleen was confirmed by realtime-PCR.
  • the HtrA2 protein of the present invention was inflammatory. It was found to inhibit the expression of the cytokines IL-1b, IL-6 and IL-17 (see FIGS. 5 and 7).
  • the present inventors in order to determine whether the HtrA2 protein of the present invention is effective in the treatment of arthritis, a type of autoimmune disease, osteoclasts in the bone marrow of HtrA2 variant mice and HtrA2 hetero mouse As a result of the differentiation experiment, it was found that HtrA2 influences osteoclast differentiation (see FIG. 6).
  • the present inventor s arthritis when inducing arthritis through injection of HtrA2 variant mouse or variant expression vector in order to determine what effect on the induction of arthritis when the function of HtrA2 is inhibited It was confirmed to be deeper (see FIG. 8).
  • GRIM19 gene associated with retinoid-interferon-induced mortality-19
  • HtrA2 is also involved in the expression of HtrA2
  • resulting in GRIM19 overexpression MRNA expression of HtrA2 was significantly increased in the cell line
  • overexpression of GRIM19 and HtrA2 expression in the spleen of GRIM19 overexpressing mice see FIGS. 9A and 9B.
  • the STAT3 protein was cut after reacting with HtrA2 and the amount thereof decreased, from which HtrA2 cleaved STAT3 It was found to have a reducing effect (see FIG. 11).
  • HtrA2 As a result of investigating the mitochondrial activity regulation effect by HtrA2, the expression of genes involved in mitochondrial function such as Ndufb5, Cycs, ATP50, COx5a is significantly reduced in cells without HtrA2 From this, HtrA2 was found to be a factor that can control the expression of genes that induce or regulate mitochondrial ROS production and electron transport activity (see Figure 12).
  • the joints of the group injected with the HtrA2 overexpression vector are less destroyed than the control group. It was confirmed that the inflammatory cells were less invasive and the cartilage was also less damaged (see FIG. 14).
  • HtrA2 hetero mice had a greater weight change compared to the control (see Fig. 16), thereby reducing the function of HtrA2 Further exacerbate inflammatory bowel disease, suggesting that HtrA2 may be an important factor in treating inflammatory bowel disease.
  • the HtrA2 protein of the present invention can treat autoimmune diseases such as immune diseases.
  • the immune disease means a disease in which components of the mammalian immune system cause, mediate or otherwise contribute to the pathology of the mammal.
  • stimulation or interruption of an immune response may include any disease that has a compensatory effect on the progression of the disease, and in the present invention may include diseases caused by an overactive immune response. Examples of such immune diseases may include, but are not limited to, autoimmune diseases.
  • one of the most important traits of all normal individuals is that they do not deleteriously react with the antigenic substances that make up self, while non-self antigens can recognize and react to eliminate them.
  • Have the ability to The non-response of the body to autoantigens is called immunologic unresponsiveness or tolerance.
  • an immune response occurs to autoantigens, which causes the attack of one's own tissue.
  • the disease caused by this process is called an autoimmune disease. .
  • the autoimmune disease to which the composition can be applied in the present invention is not limited thereto, but may be selected from the group consisting of rheumatoid arthritis, systemic lupus erythematosus, digestive diabetes, atopic dermatitis, autoimmune encephalomyelitis, asthma and Crohn's disease. have.
  • the present invention can provide a pharmaceutical composition for preventing or treating autoimmune diseases containing HtrA2 protein as an active ingredient, and the HtrA2 protein included in the pharmaceutical composition of the present invention has a physiological activity substantially equivalent to that of the protein. It has a protein having. HtrA2 proteins having substantially equivalent physiological activity include the protein and its functional equivalents and functional derivatives.
  • the term “functional equivalent” refers to an amino acid sequence variant in which some or all of the native protein amino acids are substituted or a part of the amino acids are deleted or added, and have substantially the same physiological activity as the native HtrA2 protein.
  • “functional derivative” is meant a protein that has been modified to increase or decrease the physicochemical properties of the HtrA2 protein and has substantially the same physiological activity as the native HtrA2 protein.
  • the HtrA2 protein refers to a protein having an amino acid sequence represented by SEQ ID NO: 1.
  • the term 'treatment' unless stated otherwise, reverses, alleviates, inhibits, or prevents the disease or condition to which the term applies, or one or more symptoms of the disease or condition, As used herein, the term 'treatment' refers to the act of treating when 'treating' is defined as above.
  • a pharmaceutical composition for preventing or treating autoimmune diseases according to the present invention may include a pharmaceutically effective amount of HtrA2 protein alone or one or more pharmaceutically acceptable carriers, excipients or diluents.
  • the pharmaceutically effective amount herein refers to an amount sufficient to prevent, ameliorate and treat the symptoms of autoimmune diseases.
  • the pharmaceutically effective amount of HtrA2 protein according to the present invention is 0.5-100 mg / day / kg body weight, preferably 0.5-5 mg / day / kg body weight.
  • the pharmaceutically effective amount may be appropriately changed depending on the degree of symptoms of autoimmune disease, the age, weight, health condition, sex, route of administration and duration of treatment of the patient.
  • pharmaceutically acceptable refers to a composition which is physiologically acceptable and does not normally cause an allergic reaction such as gastrointestinal disorders, dizziness or the like when administered to a human.
  • carriers, excipients and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be further included.
  • compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
  • the formulations may be in the form of powders, granules, tablets, emulsions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, sterile powders.
  • composition for preventing or treating autoimmune diseases according to the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, the dosage of the active ingredient is the route of administration, the age, sex of the patient According to the present invention, the composition for preventing or treating autoimmune diseases according to the present invention may be appropriately selected according to various factors such as weight, severity of the patient, and the like. It can be administered in parallel.
  • the present invention can provide a pharmaceutical composition for preventing or treating autoimmune diseases comprising a polynucleotide encoding the HtrA2 protein as an active ingredient.
  • the polynucleotide encoding the HtrA2 protein may include DNA or RNA, and preferably has a DNA sequence represented by SEQ ID NO: 2.
  • the polynucleotide is introduced into an expression vector, such as a plasmid or viral vector, by targeting known methods and then the expression vector is targeted to the phenotype by infection or transduction by various methods known in the art. It can be introduced into the cell.
  • an expression vector such as a plasmid or viral vector
  • Plasmid expression vectors are FDA's approved gene delivery methods for human use that deliver plasmid DNA directly to human cells (Nabel, E. G., et al., Science, 249: 1285-1288, 1990). Plasmid DNA has the advantage that it can be homogeneously purified unlike viral vectors.
  • mammalian expression plasmids known in the art can be used. For example, but not limited to, pRK5 (European Patent No. 307,247), pSV16B (International Patent Publication No. 91/08291), pVL1392 (PharMingen), and the like are representative.
  • Plasmid expression vectors comprising polynucleotides according to the present invention are methods known in the art, such as, but not limited to, transient transfection, microinjection, transduction Cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection ), Electroporation, gene gun, and other known methods for introducing DNA into cells (Wu et al., J. Bio. Chem., 267). : 963-967, 1992; Wu and Wu, J. Bio. Chem., 263: 14621-14624, 1988).
  • the vector capable of expressing HtrA2 can be administered by a known method.
  • it may be administered topically, parenterally, orally, nasal, intravenously, intramuscularly, subcutaneously or by other suitable means.
  • the present invention can provide a medicament for the prevention or treatment of autoimmune diseases, including a composition containing an expression vector comprising a polynucleotide encoding an HtrA2 protein as an active ingredient, and the present invention further provides an HtrA2 protein or the protein.
  • An immunosuppressive agent comprising a polynucleotide encoding an as an active ingredient can be provided.
  • the HtrA2 protein according to the present invention has a mechanism in which the HtrA2 protein reduces the expression of STAT3, a transcription factor of Th17 cells, inhibits the expression of inflammatory cytokines, and regulates an excessive immune response by promoting the activity of regulatory T cells (Treg). Since it is made through, it is effective to effectively treat autoimmune diseases caused by inflammatory reactions and the regulation of various immune responses.
  • STAT3 a transcription factor of Th17 cells
  • Figure 2 shows the results of the expression of T cells, B cells, dendritic cells and markers of STAT3 in wild-type and HtrA2 variant mouse spleens by immunofluorescence.
  • 3A and 3B show the cell populations of Treg (CD4 + CD25 + Foxp3 +) in wild-type and HtrA2 variant mouse spleens measured by flow cytometry.
  • FIG. 4 shows the results of realtime-PCR identification of AID and Blimp-1, which are transcription factors of plasma cells capable of producing antibodies, and IgG and IgM, which are markers of antibodies, in cells isolated from wild-type and HtrA2 variant mouse spleens. .
  • 5 is a result of measuring the gene expression concentration by realtime-PCR in cells isolated from inflammatory cytokines Il-1b and IL-6 in the spleen.
  • Figure 6 shows the results of analysis of osteoclast differentiation in bone marrow of HtrA2 variant mice and HtrA2 heteromouse by TRAP staining.
  • 9a and 9b are immunofluorescence staining photographs and graphs showing the expression inducing effect of HtrA2 by GRIM19 overactivity.
  • 10 is a graph showing cytoplasmic migration of HtrA2 by GRIM19 overexpression.
  • FIG. 11 is an electrophoretic photograph showing the effect of inhibiting and degrading Stat3 activity by HtrA2.
  • FIG. 12 is a graph showing the effect of HtrA2 on the expression of genes involved in mitochondrial ROS production.
  • FIG. 13 is a graph showing the effect of inhibiting rheumatoid arthritis using HtrA2.
  • FIG. 14 is an immunostaining photograph showing the effect of HtrA2 inhibiting joint and cartilage damage in a model of rheumatoid arthritis inducing HtrA2 overexpression.
  • FIG. 15 is an immunostaining photograph showing the effect of HtrA2 inhibiting the expression of inflammatory cytokines in the joint in a rheumatoid arthritis model inducing HtrA2 overexpression.
  • 16 is a graph showing the effect of HtrA2 on inflammatory bowel disease.
  • 17 is a graph showing the effect of HtrA2 on inflammatory cytokine secretion in colorectal cancer cell lines.
  • STAT3 a transcription factor of Th17 cells, was stained by immunofluorescence in spleen tissues of wild-type and HtrA2 variant mice, and the expression of STAT3 in HtrA2 variant mice compared to wild-type mice was examined.
  • the HtrA2 variant mouse (Stock number 004608) was purchased from Jaxson lab and has a recessive mutation on the chromosome of a gene called mnd2, located in mouse chromosome 6.
  • T cells, B cells, dendritic cells, markers and expression of STAT3 in wild-type and HtrA2 variant mouse spleens were confirmed by immunofluorescence in tissues for observation.
  • Treg CD4 + CD25 + Foxp3 +
  • activated immune cells such as effector T cells
  • the Lymphocyte gate was not different, but it was confirmed that the CD4 + cell population was 13% higher than the wild type in the HtrA2 variant mouse, but the percentage of Treg was 5.74%, which was about 4% lower than the wild type (Fig. 3a and 3a). 3b).
  • Realtime-PCR identified AID and Blimp-1, the transcription factors of plasma cells capable of producing antibodies, and IgG and IgM, markers of antibodies, from cells isolated from the spleens of 38-day-old wild-type and HtrA2 variant mice. It was.
  • IL-1b and TNF- ⁇ which are known to be representative inflammatory cytokines, was increased by realtime-PCR in cells isolated from the spleen. IL-1b and TNF- ⁇ were increased in HtrA2 mutant mice. As a result, it can be seen that IL-1b, TNF- ⁇ , which is important for inflammation, may cause an abnormal form of HtrA2 (FIG. 5).
  • osteoclast differentiation was performed in bone marrow of HtrA2 variant mice and HtrA2 heteromouse.
  • the present inventors transfected the HtrA2 overexpression vector and the mock vector into LBRM cells, which are T cell lymphoma cell lines, and then gave TCR stimulation for 3 days and Expression of 17 was measured by real time PCR.
  • GRIM19 is a complex1 protein in mitochondria that is involved in ROS production and cell survival. GRIM19 is known to interact with mature HtrA2. To determine whether GRIM19 is involved in the expression of HtrA2, the expression of HtrA2 in GRIM19 overexpressing cell lines produced by injecting GRIM19 and expression vectors into T cell line LBRM cell line was examined.
  • mRNA expression of HtrA2 was significantly increased in GRIM19 overexpressing cell line, and in the spleen of GRIM19 overexpressing mouse (GRIM19 transgenic mice, GRIM19 Tg) to confirm the expression of GRIM19 and HtrA2 in spleen.
  • GRIM19 transgenic mice GRIM19 Tg
  • GRIM19 overexpression and HtrA2 expression were correlated in the spleen of GRIM19 overexpressing mice (FIGS. 9A and 9B).
  • HtrA2 was more present in the cytoplasm of mouse spleen cells over-expressed GRIM19, and as a result, it was found that HtrA2 matures and migrates to the cytoplasm and functions in the GRIM19 overexpression environment (Fig. 10).
  • STAT3 overexpression of STAT3 protein from E. coli and HtrA2 overexpression of E. coli was reacted for 16 hours at 37 ° C, followed by electrophoresis of the protein, and Western blot of STAT3 protein.
  • HtrA2 has an effect of reducing the amount by cleaving STAT3.
  • RNAs from HtrA2 + / + MEF cell lines and HtrA2-/-cell lines were confirmed.
  • HtrA2 was found to be a factor that can regulate the expression of genes that induce or regulate mitochondrial ROS production and electron transport activity.
  • HtrA2 In order to determine what effect HtrA2 has on arthritis, the present inventors measured the arthritis index by injecting HtrA2 overexpression vector into a CIA mouse model derived from type 2 collagen. To this end, arthritis was induced by intradermal injection of collagen type 2 into DBA1 / J mice, and HtrA2 overexpression vectors were injected into the mice by hydrodynamic injection after arthritis induction. Hydrodynamic injection was performed by injecting a vector diluted in physiological saline into the tail vein by using a physical force to enter the cytoplasm through the cell membrane.
  • the histological examination of the articular tissues of the arthritis mouse animal model was performed.
  • the HtrA2 overexpression vector and the control group were fixed with 10% formalin after induction of arthritis, decalcified bone, and a block was prepared with paraffin. From this, a joint section (7 ⁇ m) was prepared and stained with hematoxylin and eosin. In addition, toluidin blue and safranin O were stained to confirm the degree of cartilage destruction.
  • HtrA2 when inducing inflammatory bowel disease-inducing animals (IBD) by drinking 2.5% dextran sulfate sodium (DSS) water in C57BL / 6 wild-type mice and HtrA2 hetero mutant for 5 days The weight change of the test animal was observed.
  • IBD inflammatory bowel disease-inducing animals
  • DSS dextran sulfate sodium
  • HtrA2 may be an important factor in the treatment of inflammatory bowel disease because the deterioration of HtrA2 worsens inflammatory bowel disease.
  • HtrA2 can regulate inflammatory cytokine secretion in HT-29 cells, a colorectal cancer cell line, it is inflammatory in colorectal cancer cell line into which HtrA2 is inserted and in a culture medium obtained after 3 days after stimulating ConA in mock The amount of cytokine TNF- ⁇ was confirmed by ELISA.
  • HtrA2 can reduce the inflammatory environment by inhibiting the production of inflammatory cytokine TNF- ⁇ in colorectal cancer cells.

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Abstract

La présente invention concerne une composition pour la prévention ou le traitement de maladies auto-immunes, contenant la protéine A2 à exigence de température élevée (HtrA2) comme principe actif. L'HtrA2, selon la présente invention, réduit l'expression de STAT3, qui est un facteur de transcription des lymphocytes Th17, inhibe l'expression de cytokines inflammatoires et favorise l'activité d'un lymphocyte T régulateur (Treg), régulant ainsi une réponse immunitaire excessive par l'intermédiaire d'un mécanisme et, par conséquent, il est possible de traiter efficacement des maladies auto-immunes provoquées par la régulation anormale d'une réponse inflammatoire et de diverses réponses immunitaires.
PCT/KR2014/000361 2013-01-14 2014-01-13 Composition pour la prévention ou le traitement de maladies auto-immunes, contenant htra2 comme principe actif Ceased WO2014109617A1 (fr)

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KR1020130003938A KR101463325B1 (ko) 2013-01-14 2013-01-14 HtrA2 단백질을 유효성분으로 포함하는 자가면역질환의 예방 또는 치료용 조성물
KR10-2013-0003938 2013-01-14

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
CN106687135A (zh) * 2014-09-15 2017-05-17 豪夫迈·罗氏有限公司 使用pd‑1轴结合拮抗剂和il‑17结合拮抗剂治疗癌症的方法
CN113265387A (zh) * 2021-01-22 2021-08-17 中国人民解放军军事科学院军事医学研究院 一种成熟HtrA2突变体N196C及其制备方法和应用

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WO2016159695A1 (fr) * 2015-03-31 2016-10-06 가톨릭대학교 산학협력단 Composition pour la prévention ou le traitement de maladies auto-immunes, contenant ssu72 en tant que principe actif

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WO2000070033A1 (fr) * 1999-05-19 2000-11-23 Smithkline Beecham Corporation Htra2 murin
US20070031872A1 (en) * 2004-01-27 2007-02-08 Rejko Krueger A141S and G399S mutation in the Omi/HtrA2 protein in Parkinson's disease
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US20070031872A1 (en) * 2004-01-27 2007-02-08 Rejko Krueger A141S and G399S mutation in the Omi/HtrA2 protein in Parkinson's disease
US7947436B2 (en) * 2004-12-13 2011-05-24 Alethia Biotherapeutics Inc. Polynucleotides and polypeptide sequences involved in the process of bone remodeling

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ZURAWA-JANICKA, D. ET AL.: "HtrA Proteins as Targets in Therapy of Cancer and Other Diseases", EXPERT OPINION ON THERAPEUTIC TARGETS, vol. 14, no. 7, 2010, pages 665 - 679 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106687135A (zh) * 2014-09-15 2017-05-17 豪夫迈·罗氏有限公司 使用pd‑1轴结合拮抗剂和il‑17结合拮抗剂治疗癌症的方法
CN113265387A (zh) * 2021-01-22 2021-08-17 中国人民解放军军事科学院军事医学研究院 一种成熟HtrA2突变体N196C及其制备方法和应用
CN113265387B (zh) * 2021-01-22 2022-05-20 中国人民解放军军事科学院军事医学研究院 一种成熟HtrA2突变体N196C及其制备方法和应用

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