IFN-γ or il-10 production inhibitor

Abstract

To provide a substance that effectively inhibits the production of IFN-γ or IL-10.SOLUTION: An IL-33 antagonist or IL-33 receptor antagonist is used as an active ingredient in an IFN-γ or IL-10 production inhibitor.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an agent for suppressing the production of interferon-γ (IFN-γ) and / or IL-10, which comprises as active ingredients a substance having an antagonistic effect against interleukin-33 (IL-33) (IL-33 antagonist) and / or a substance having an antagonistic effect against the IL-33 receptor (IL-33 receptor antagonist). [Background technology]

[0002] Kawasaki disease is a systemic vasculitis of unknown cause that mainly affects infants under the age of four, and the number of patients is increasing despite the recent declining birthrate. Symptoms in the acute phase reflect systemic vasculitis and are characterized by six clinical findings: fever, cervical lymphadenopathy, strawberry tongue, conjunctival congestion, amorphous rash, and changes in the extremities. The greatest problem with Kawasaki disease is that the most severe inflammation occurs in the coronary arteries, leading to coronary artery lesions (enlargement and aneurysm formation). Coronary artery lesions pose the risk of myocardial infarction and aneurysm rupture, and can cause sudden death at a young age.

[0003] For a long time, the standard treatment for Kawasaki disease was mainly intravenous immunoglobulin (IVIG) therapy and oral aspirin, but currently, multiple anti-inflammatory therapies such as steroids (prednisolone, methylprednisolone pulse), anti-tumor necrosis factor-α (TNF-α) monoclonal antibody preparations (infliximab: IFX), and cyclosporine have become treatment options. However, acute coronary artery lesions are still observed in approximately 8% of cases, and the rate of reduction in coronary artery lesions has remained stable in recent years.

[0004] The present inventors have participated in research into the elucidation of the pathology of Kawasaki disease and the development of new therapeutic agents, and contributed to the inclusion of IFX, the only molecular targeted therapy for Kawasaki disease, in the insurance coverage (Non-Patent Document 1). Although IFX is one of the effective treatments for IVIG-refractory Kawasaki disease, the success rate is only about 70%, which is not sufficient. In addition, from the viewpoint of side effects such as delayed myelination of central nerve cells and immunosuppression, active administration of IFX to infants at an age when Kawasaki disease is likely to occur is not recommended. For this reason, there is a need for treatment options that target major molecules other than TNF-α.

[0005] It has been reported that mTOR (mammalian target of rapamycin) inhibitors have a strong inhibitory effect on inflammation in Kawasaki disease (Patent Document 1). It has also been reported that adipose tissue-derived mesenchymal stem cells suppress the production of IL-17 and are effective against coronary artery lesions in Kawasaki disease model mice (Patent Document 2).

[0006] On the other hand, IL-33 is a cytokine belonging to the IL-1 family that is thought to play a role in inflammatory conditions. IL-33 is constitutively expressed in the nuclei of epithelial cells and vascular endothelial cells, and is released together with cell destruction due to tissue damage caused by infection or physical or chemical stress, and functions as an alarmin. It is also thought that the expression of IL-33 is increased by stimulation with lipopolysaccharide, etc., and that there is also a mechanism for secretion. IL-33 released outside the cell can activate intracellular signals by binding to the IL-33 receptor expressed on the cell. The IL-33 receptor is expressed in various immune cells and epithelial cells, and IL-33-induced intracellular signaling occurs in these cells. Recently, the present inventors have reported that IL-33 / IL-33 receptor may be a biomarker or a therapeutic target in the treatment of Kawasaki disease (Non-Patent Document 2). However, it was not known until now that the production of IFN-γ or IL-10 is suppressed when the signaling pathway of IL-33 / IL-33 receptor is inhibited. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2017-39719 A [Patent Document 2] JP 2015-71559 A [Non-patent literature]

[0008] [Non-Patent Document 1] Sci Rep. 2018 Jan 31;8(1):1994. [Non-Patent Document 2] Inflammation. 2023 Feb;46(1):480-490. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a substance that effectively suppresses the production of IFN-γ or IL-10. [Means for solving the problem]

[0010] The present inventors have been conducting intensive research to solve the above problems. In the course of research, first, an experimental system was established in which human coronary artery endothelial cells (hereinafter sometimes referred to as "HCAEC") were co-cultured with human coronary artery smooth muscle cells (hereinafter sometimes referred to as "HCASMC") that had been necrotized after pre-stimulation with human IL-1β as an in vitro model of Kawasaki disease-related coronary arteritis. Next, an in vitro treatment model of Kawasaki disease-related coronary arteritis was prepared by treating the in vitro model of Kawasaki disease-related coronary arteritis with a test substance, and the results of the investigation showed that IFN-γ and IL-10 produced in the model could be effectively suppressed by treating the model with an IL-33 antagonist (anti-IL-33 antibody) or an IL-33 receptor antagonist (anti-ST2 antibody), thereby completing the present invention.

[0011] That is, the present invention is as follows. [1] An agent for suppressing the production of IFN-γ or IL-10, comprising an IL-33 antagonist or an IL-33 receptor antagonist. [2] The production inhibitor according to [1] above, wherein the IL-33 antagonist is an anti-IL-33 antibody, and the IL-33 receptor antagonist is an anti-ST2 antibody.

[0012] In another embodiment of the present invention, A method for preventing or ameliorating (treating) a disease or symptom associated with the production of IFN-γ or IL-10, comprising administering an IL-33 antagonist or an IL-33 receptor antagonist to a subject in need of such prevention or amelioration (treatment) of a disease or symptom associated with the production of IFN-γ or IL-10; an IL-33 antagonist or an IL-33 receptor antagonist for use in the prevention or amelioration (treatment) of a disease or condition associated with the production of IFN-γ or IL-10; Use of an IL-33 antagonist or an IL-33 receptor antagonist in the manufacture of a preventive or ameliorative (therapeutic) agent for a disease or symptom associated with the production of IFN-γ or IL-10; and Use of an IL-33 antagonist or an IL-33 receptor antagonist in the manufacture of an agent for suppressing the production of IFN-γ or IL-10; The following can be mentioned. Effect of the Invention

[0013] An IL-33 antagonist or an IL-33 receptor antagonist has an effect of effectively suppressing the production of IFN-γ or IL-10. Therefore, an agent containing an IL-33 antagonist or an IL-33 receptor antagonist as an active ingredient is useful for preventing or treating a disease or a symptom associated with the production of IFN-γ or IL-10. In addition, when, for example, an anti-IFN-γ antibody is used as an IFN-γ antagonist or an anti-IL-10 antibody is used as an IL-10 antagonist to suppress the production of IFN-γ or IL-10, the production of only one of IFN-γ or IL-10 can be suppressed, whereas when an anti-IL-33 antibody is used as an IL-33 antagonist or an anti-ST2 antibody is used as an IL-33 receptor antagonist, it is superior in that it can suppress the production of both IFN-γ and IL-10 while inhibiting the signal transduction pathway of IL-33 / IL-33 receptor. [Brief description of the drawings]

[0014] [Figure 1] FIG. 1 shows a photograph (left) of an in vitro model of Kawasaki disease-related coronary arteritis prepared, and a schematic diagram of the in vitro model (right). [Diagram 2] HCAECs were co-cultured for 24 hours in the absence of HCASMCs ("-" in the figure) or in the presence of three types of HCASMCs (live HCASMCs ["live cells" in the figure], necrotic HCASMCs ["necrotic" in the figure], or necrotic HCASMCs prestimulated with human IL-1β and then subjected to necrosis ["necrotic + stimulated" in the figure]), and the culture supernatants were collected and the concentration of inflammatory cytokines (IL-6) was measured. The values ​​in the figure indicate the average concentrations of various inflammatory cytokines. [Diagram 3] FIG. 1 is a schematic diagram of an in vitro treatment model of coronary arteritis caused by Kawasaki disease using two types of test substances (anti-IL-33 antibody or anti-ST2 antibody). [Figure 4]HCAECs pretreated with four types of test substances (normal goat IgG control ["Control" in the figure], donated blood venoglobulin IH 5% [IVIG] ["IVIG" in the figure], anti-human IL-33 antibody ["anti-IL-33" in the figure], or anti-human ST2 antibody ["anti-ST2" in the figure]) were co-cultured for 24 hours with HCASMCs that had been pre-stimulated with human IL-1β and then necrotized. The culture supernatants were then collected and the concentrations of three types of inflammatory cytokines (IFN-γ [Figure 4A], IL-10 [Figure 4B], and IL-6 [Figure 4C]) were measured. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The inhibitor of the present invention is an agent (hereinafter sometimes referred to as "the inhibitor") containing an IL-33 antagonist and / or an IL-33 receptor antagonist (hereinafter sometimes referred to as "the IL-33 / IL-33 receptor antagonist of the present invention") specified for the use "to inhibit the production of IFN-γ or IL-10".

[0016] The present inhibitor may be used alone as livestock feed, food and beverage, cosmetics, quasi-drugs, or pharmaceuticals (preparations) containing the present IL-33 / IL-33 receptor antagonist as an active ingredient, or may be used in the form of a composition (livestock feed composition, food and beverage composition, cosmetic composition, quasi-topical composition, or pharmaceutical composition) by mixing with an additive. Examples of the food and beverage include health foods (functional foods, nutritional supplements, health supplements, nutritionally enhanced foods, nutritionally adjusted foods, supplements, etc.) and health functional foods (foods for specified health uses, foods with nutritional functions, foods with functional claims, etc.).

[0017] Examples of additives in the inhibitor of the present invention include physiologically acceptable ordinary carriers, binders, stabilizers, excipients, diluents, pH buffers, disintegrants, isotonicity agents, coating agents, solubilizers, lubricants, glidants, solubilizers, flavorings, sweeteners, solvents, gelling agents, nutrients, various oils, surfactants, preservatives, antioxidants, dispersants, chelating agents, thickeners, UV absorbers, emulsion stabilizers, pH adjusters, colorants, fragrances, etc. Specific examples of such additives include water, physiological saline, animal fats and oils, vegetable oils, lactose, starch, gelatin, crystalline cellulose, gum, talc, magnesium stearate, hydroxypropyl cellulose, polyalkylene glycol, polyvinyl alcohol, and glycerin.

[0018] [IL-33 antagonist] As used herein, the term "IL-33 antagonist" refers to a substance that has an antagonistic effect against IL-33 in mammalian cells (e.g., an effect of inhibiting the expression or function of IL-33). Examples of IL-33 antagonists include antibodies that specifically bind to IL-33 (anti-IL-33 antibodies), RNAs that have an RNA interference (RNAi) effect specifically against mRNA encoding IL-33 (hereinafter sometimes referred to as "RNAs targeting IL-33"), soluble ST2, and aptamers (nucleic acid molecules) that specifically bind to IL-33.

[0019] [Anti-IL-33 antibody] As used herein, the term "anti-IL-33 antibody" refers to an antibody that specifically binds to IL-33 in mammalian cells. Examples of anti-IL-33 antibodies include antibodies whose epitope is a part (e.g., 5 to 250 amino acid residues, 5 to 200 amino acid residues, 5 to 150 amino acid residues, 5 to 100 amino acid residues, 5 to 90 amino acid residues, 5 to 60 amino acid residues, 5 to 30 amino acid residues, or 5 to 10 amino acid residues) or the entirety of IL-33 in mammalian cells (e.g., human IL-33 consisting of the amino acid sequence of SEQ ID NO: 1).

[0020] [IL-33 targeting RNA] As used herein, "IL-33-targeting RNA" refers to a single-stranded or double-stranded RNA that can anneal to a part or all of an mRNA encoding IL-33 in a mammalian cell and can suppress the expression of an mRNA encoding IL-33 in a mammalian cell and / or suppress the translation of an mRNA encoding IL-33 in a mammalian cell. An IL-33-targeting RNA is generally an RNA that contains a nucleotide sequence that is at least 80% (preferably at least 85%, more preferably at least 88%, even more preferably at least 90%, even more preferably at least 92%, even more preferably at least 94%, even more preferably at least 96%, even more preferably at least 98%, and most preferably 100%) identical to a part (e.g., 5 to 100 nucleotide residues, 5 to 80 nucleotide residues, 5 to 60 nucleotide residues, 5 to 50 nucleotide residues, 5 to 40 nucleotide residues, 5 to 30 nucleotide residues) or the whole of an mRNA encoding IL-33 (e.g., human IL-33 consisting of the amino acid sequence of SEQ ID NO: 1) in a mammalian cell.

[0021] [Soluble ST2] As used herein, "soluble ST2" refers to a secreted form of ST2 (also referred to as IL1RL1 [interleukin 1 receptor like 1]) that lacks the transmembrane domain and intracellular domain. Soluble ST2 may be any polypeptide that contains a part or all of the extracellular domain of ST2 (for example, a polypeptide consisting of amino acid residues 19 to 328 in human ST2 [soluble ST2] consisting of the amino acid sequence of SEQ ID NO: 2) and binds to IL-33, and may be optionally modified, for example, with polyethylene glycol or a constant domain of an antibody. In particular, soluble ST2 (sST2-Fc) bound to a constant domain of immunoglobulin is preferred.

[0022] [IL-33 receptor antagonist] As used herein, the term "IL-33 receptor antagonist" refers to a substance that has an antagonistic effect against IL-33 receptor in mammalian cells (e.g., an effect of inhibiting the expression or function of IL-33 receptor). IL-33 receptor is composed of a heterodimer of ST2 and IL-1RAcP (IL-1 receptor accessory protein), and intracellular signal transduction is activated by binding of IL-33 to the heterodimer. Therefore, more specifically, the term "IL-33 receptor antagonist" refers to a substance that has an antagonistic effect against either one or both of ST2 and IL-1RAcP. Examples of IL-33 receptor antagonists include antibodies that specifically bind to IL-33 receptor (anti-IL-33 receptor antibodies), RNAs that have an RNA interference (RNAi) effect specifically against mRNAs that code for IL-33 receptor (hereinafter sometimes referred to as "RNAs that target IL-33 receptor"), and aptamers (nucleic acid molecules) that specifically bind to IL-33 receptor.

[0023] [Anti-IL-33 receptor antibody] As used herein, the term "anti-IL-33 receptor antibody" refers to an antibody that specifically binds to an IL-33 receptor (i.e., ST2 and / or IL-1RAcP) in a mammalian cell. Examples of the anti-IL-33 receptor antibody include antibodies whose epitope is a part (e.g., 5 to 350 amino acid residues, 5 to 300 amino acid residues, 5 to 250 amino acid residues, 5 to 200 amino acid residues, 5 to 150 amino acid residues, 5 to 100 amino acid residues, 5 to 90 amino acid residues, 5 to 60 amino acid residues, 5 to 30 amino acid residues, 5 to 10 amino acid residues) or the entirety of an IL-33 receptor in a mammalian cell (e.g., human ST2 [soluble ST2] consisting of the amino acid sequence of SEQ ID NO: 2, or human ST2 [membrane-type ST2] consisting of the amino acid sequence of SEQ ID NO: 3).

[0024] [IL-33 receptor-targeting RNA] As used herein, "RNA targeting IL-33 receptor" means RNA of a single- or double-stranded structure that can anneal to a part or all of the mRNA encoding the IL-33 receptor (i.e., ST2 and / or IL-1RAcP) in a mammalian cell, and that can suppress the expression of the mRNA encoding ST2 and / or IL-1RAcP in a mammalian cell, and / or suppress the translation of the mRNA encoding ST2 and / or IL-1RAcP in a mammalian cell. RNA targeting the IL-33 receptor is usually RNA that contains a nucleotide sequence that is at least 80% (preferably at least 85%, more preferably at least 88%, even more preferably at least 90%, even more preferably at least 92%, even more preferably preferably at least 94%, even more preferably preferably at least 96%, even more preferably preferably at least 98%, and most preferably 100%) identical to a portion (e.g., 5 to 100 nucleotide residues, 5 to 80 nucleotide residues, 5 to 60 nucleotide residues, 5 to 50 nucleotide residues, 5 to 40 nucleotide residues, 5 to 30 nucleotide residues) or the entirety of an mRNA encoding ST2 and / or IL-1RAcP in a mammalian cell (e.g., human ST2 [soluble ST2] consisting of the amino acid sequence of SEQ ID NO: 2, or human ST2 [membrane-type ST2] consisting of the amino acid sequence of SEQ ID NO: 3).

[0025] [antibody] The origin, type, class, form, and the like of the "antibody" in this specification is not particularly limited, and examples of the antibody include: an antibody derived from a human; an antibody derived from a non-human animal such as a mouse or a rat; a polyclonal antibody, an oligoclonal antibody (a mixture of several to several tens of antibodies), a monoclonal antibody; a chimeric antibody or a humanized antibody in which a partial region of the antibody (for example, a constant region) is replaced with a region derived from a different biological species; an F(ab')2 antibody fragment obtained by digesting a monoclonal antibody with pepsin, a Fab' antibody fragment obtained by reducing an F(ab')2 antibody fragment, an Fab fragment obtained by digesting a monoclonal antibody with papain, and other antibody fragments; and a single chain antibody (single chain Fv; scFv) in which an antibody heavy (H) chain variable region and an antibody light (H) chain variable region are linked by an amino acid bridge; and the like. The antibody can be obtained by any method known in the art. For example, when obtaining a monoclonal antibody, the hybridoma method, the phage display method, a genetic engineering method, or the like. Alternatively, a commercially available product (e.g., an anti-human IL-33 antibody [Table 1, manufactured by R&D Systems], an anti-human ST2 antibody [Table 2, manufactured by R&D Systems]) can also be used.

[0026] The IL-33 / IL-33 receptor antagonist, which is the active ingredient of the inhibitor, has the effect of effectively suppressing the production of IFN-γ and IL-10. Therefore, the inhibitor can be advantageously used as a preventive or ameliorating agent for diseases or symptoms associated with the production of IFN-γ or IL-10 (i.e., an agent containing the IL-33 / IL-33 receptor antagonist, specified for the use "to prevent or ameliorate diseases or symptoms associated with the production of IFN-γ or IL-10").

[0027] [Diseases or symptoms associated with the production of IFN-γ or IL-10] As used herein, "diseases or symptoms associated with the production of IFN-γ or IL-10" means one or more selected from diseases or symptoms caused by the production (increase) of IFN-γ or IL-10 (i.e., diseases or symptoms resulting from the production [increase] of IFN-γ or IL-10); diseases or symptoms in which the production (increase) of IFN-γ or IL-10 occurs; and diseases or symptoms in which the production (increase) of IFN-γ or IL-10 occurs as a result of the onset of a disease or symptom.

[0028] In this specification, specific diseases or symptoms in the "diseases or symptoms associated with the production of IFN-γ or IL-10" include, for example, Kawasaki disease coronary arteritis (see the following Example), myocarditis (see Heart Fail Rev. 2014 Mar;19(2):227-36., J Am Coll Cardiol. 2004 Sep 15;44(6):1292-7., Journal of the Japanese Society of Internal Medicine, Vol. 96, No. 10, October 10, 2007), and macrophage activation syndrome (see Ann Rheum Dis. 2023 Jun;82(6):857-865.).

[0029] [Prevent or improve disease or symptoms] As used herein, "preventing or ameliorating a disease or symptom" means one or more selected from preventing the onset of a disease or symptom; delaying the onset of a disease or symptom; preventing or delaying the progression of the severity of a disease or symptom; improving the level of severity of a disease or symptom; and ameliorating a disease or symptom.

[0030] The diseases or symptoms to which the term is applied are those associated with the production of IFN-γ or IL-10, and do not include diseases or symptoms that are not associated with the production of IFN-γ or IL-10, even if the specific name of the disease or symptom is the same.

[0031] The subject to which the present inhibitor is administered may be any subject (mammal) that requires inhibition of the production of IFN-γ or IL-10.

[0032] The above-mentioned mammals include humans and non-human mammals (e.g., cows, pigs, sheep, goats, mice, rats, hamsters, guinea pigs, etc.). Other embodiments of non-human mammals include livestock (e.g., cows, pigs, sheep, goats, etc.).

[0033] The administration form of the inhibitor of the present invention may be oral administration in the form of powder, granules, tablets, capsules, syrup, etc., or parenteral administration in the form of topical skin preparation, transdermal preparation, transmucosal preparation, nasal preparation, enteral preparation, injection, suppository, inhalant, patch, etc.

[0034] The dosage of the present inhibitor and the dosage of the present IL-33 / IL-33 receptor antagonist contained in the present inhibitor are appropriately determined depending on age, body weight, sex, symptoms, drug sensitivity, etc., and are, for example, in the range of 1 μg to 200 mg / kg (body weight) / day. The present inhibitor is administered once or multiple times (for example, 2 to 4 times) per day, and the dosage may be adjusted depending on the degree of effect.

[0035] The inhibitor may contain, in addition to the IL-33 / IL-33 receptor antagonist, a substance that has the effect of suppressing the production of IFN-γ or IL-10. However, since the IL-33 / IL-33 receptor antagonist alone exhibits an excellent effect of suppressing the production of IFN-γ or IL-10, it is preferable that the inhibitor does not contain, in addition to the IL-33 / IL-33 receptor antagonist, a substance that has the effect of suppressing the production of IFN-γ or IL-10 (e.g., a compound, a protein [e.g., an antibody such as an anti-IFN-γ antibody or an anti-IL-10 antibody], DNA, RNA).

[0036] The present invention will be described in more detail below with reference to examples, but the technical scope of the present invention is not limited to these examples. EXAMPLES

[0037] 1. Method [Necrotic smooth muscle model] Human coronary artery smooth muscle cells (HCASMC) (Lonza) were routinely cultured in a humidified environment at 37°C with 5% CO2. The medium used was smooth muscle cell growth medium (SmGM-2 BulletKit, Lonza). HCASMC were stimulated with recombinant human IL-1β (R&D Systems) (10 ng / mL) for 4 hours. As a comparative control, an experiment was also performed in which HCASMC were cultured for 4 hours in the absence of recombinant human IL-1β (unstimulated). HCASMC were washed twice with hydroxyethylpiperazineethanesulfonic acid (HEPES), and the cell mass was frozen at -80°C for 30 minutes, then thawed in a 37°C water bath to cause necrosis. The necrotic cell mass was cultured at 4 × 10 4 Smooth muscle cell growth medium was added to obtain a density of cells / mL.

[0038] [In vitro model of Kawasaki disease-induced coronary arteritis] Human coronary artery endothelial cells (HCAEC) (Lonza) were routinely cultured in a humidified, 5% CO2, 37°C environment. Endothelial cell growth medium (EGM-2 BulletKit, Lonza) was used as the medium. 4 × 10 cells were cultured per well in a 24-well plate. 4 HCAECs were seeded and cultured for 24 hours to allow attachment. Three types of HCASMCs (live HCASMCs, necrotic HCASMCs, or necrotic HCASMCs prestimulated with human IL-1β) were added at 2 × 10 4 The cells were seeded in the upper chamber of the transwell insert at a density of 1000 μL, placed on the well seeded with HCAECs, and co-cultured for 24 hours to create an in vitro model of Kawasaki disease coronary arteritis (Figure 1). After culture, the culture supernatant was collected. As a control, the upper chamber of the transwell insert without HCASMCs seeded was placed on the well seeded with HCAECs, and a similar experiment was performed.

[0039] [Measurement of cytokine concentrations in cultured cell supernatants] The inflammatory cytokine (IL-6) concentration in the collected culture supernatant was measured using a flow cytometer (BD Cytometric Bead Array Human Th1 / Th2 Cytokine Kit II) (BD Biosciences) (Figure 2). The detection limit for IL-6 was 3.0 pg / mL.

[0040] [Statistical analysis] Differences in the results were analyzed using non-repeated measures ANOVA and Mann-Whitney U-test. In all statistical analyses, statistical significance was set at p<0.05. Statistical analysis and calculations were performed using Excel Statistics for Windows, v.3.00 (BellCurve). The same was performed in Example 2 below.

[0041] 2.Results When HCAECs were co-cultured with necrotic HCASMCs, the concentration of inflammatory cytokines (IL-6) in the culture supernatant was significantly increased compared to when they were co-cultured with live HCASMCs (Fig. 2). In addition, when HCAECs were co-cultured with necrotic HCASMCs after pre-stimulation with human IL-1β, the concentration of inflammatory cytokines (IL-6) in the culture supernatant was further significantly increased compared to when they were co-cultured with necrotic HCASMCs (Fig. 2). These results indicate that the experimental system in which HCAECs are co-cultured with necrotic HCASMCs after pre-stimulation with human IL-1β is useful as an in vitro model of Kawasaki disease-induced coronary arteritis. EXAMPLES

[0042] Next, an in vitro therapeutic model of Kawasaki disease-related coronary arteritis was prepared by treating HCAECs with test substances in an experimental system in which HCAECs were co-cultured with necrotic HCASMCs that had been pre-stimulated with human IL-1β.

[0043] 1. Method [In vitro treatment model of coronary arteritis caused by Kawasaki disease using the test substance] HCAECs (Lonza) were routinely cultured in a humidified, 5% CO2, 37°C environment. Endothelial cell growth medium (EGM-2 BulletKit, Lonza) was used as the medium. 4 × 10 cells were cultured per well in a 24-well plate. 4 HCAECs were seeded and cultured for 24 hours to allow them to adhere. After pretreatment for 30 minutes with four types of test substances (5 μg / mL normal goat IgG control [manufactured by R&D Systems], 2 mg / mL donated blood venoglobulin IH 5% [manufactured by Japan Blood Products Organization], 5 μg / mL anti-human IL-33 antibody [Table 1, #AF3625, manufactured by R&D Systems], or 5 μg / mL anti-human ST2 antibody [Table 2, #AF523, manufactured by R&D Systems]), 2 × 10 HCAECs were seeded and cultured for 24 hours to allow them to adhere. After pretreatment with four types of test substances (5 μg / mL normal goat IgG control [manufactured by R&D Systems], 2 mg / mL donated blood venoglobulin IH 5% [manufactured by Japan Blood Products Organization], 5 μg / mL anti-human IL-33 antibody [Table 1, #AF3625, manufactured by R&D Systems], or 5 μg / mL anti-human ST2 antibody [Table 2, #AF523, manufactured by R&D Systems]), necrotized HCASMCs prepared in Example 1 after prestimulation with human IL-1β were added to the cells at 2 × 10 4 The cells (500 μL) were seeded in the upper chamber of the transwell insert at a density of 100 μg / ml, placed on top of the well seeded with HCAECs, and co-cultured for 24 hours to create an in vitro treatment model of Kawasaki disease coronary arteritis (Figure 3). After incubation, the culture supernatant was collected.

[0044] [Table 1] The underlined portion in the table indicates the immunogen sequence (Ser112-Thr270) of the anti-human IL-33 antibody used in this example. The anti-human IL-33 antibody used in this example has an effect of inhibiting the signal transduction pathway of IL-33 / IL-33 receptor (i.e., an IL-33 antagonist) as is clear from the literature "Cell Rep. 2022 Aug 30; 40(9): 111281.", the literature "J Immunol. 2016 Sep 15; 197(6): 2409-20.", the literature "Clin Exp Allergy. 2012 Feb; 42(2): 218-28.", and the like.

[0045] [Table 2] The underlined portion in the table indicates the immunogen sequence (Lys19-Phe328) of the anti-human ST2 antibody used in this Example. As is clear from the literature "J Allergy Clin Immunol. 2008 Jun;121(6):1484-90." and the like, the anti-human ST2 antibody used in this Example has the effect of inhibiting the signal transduction pathway of IL-33 / IL-33 receptor (i.e., an IL-33 receptor antagonist).

[0046] [Measurement of cytokine concentrations in cultured cell supernatants] The concentrations of three inflammatory cytokines (IFN-γ, IL-10, and IL-6) in the collected culture supernatants were measured using a flow cytometer (BD Cytometric Bead Array Human Th1 / Th2 Cytokine Kit II) (BD Biosciences) (Figure 4). The detection limits for IFN-γ, IL-10, and IL-6 were 7.1 pg / mL, 2.8 pg / mL, and 3.0 pg / mL, respectively.

[0047] 2.Results [IFN-γ concentration] In an experimental system (Figure 3) in which HCAECs were co-cultured with necrotized HCASMCs after pre-stimulation with human IL-1β, pretreatment with 5% IVIG, an immunoglobulin preparation used as an anti-inflammatory agent, showed little change in the IFN-γ concentration in the culture supernatant compared to pretreatment with a normal goat IgG control (compare "IVIG" and "control" in Figure 4A). On the other hand, in the above experimental system, pretreatment with an anti-human IL-33 antibody, an IL-33 antagonist, or an anti-human ST2 antibody, an IL-33 receptor antagonist, showed a significant decrease in the IFN-γ concentration in the culture supernatant compared to pretreatment with a normal goat IgG control (compare "anti-IL-33" or "anti-ST2" and "control" in Figure 4A).

[0048] [IL-10 concentration] In the above experimental system, pretreatment with donated venoglobulin IH5%, anti-human IL-33 antibody, or anti-human ST2 antibody significantly reduced the IL-10 concentration in the culture supernatant compared to pretreatment with normal goat IgG control (Figure 4B: Comparison of "IVIG", "anti-IL-33", or "anti-ST2" with "control"). In addition, in the above experimental system, pretreatment with anti-human IL-33 antibody reduced the IL-10 concentration in the culture supernatant compared to pretreatment with donated venoglobulin IH5% (Figure 4B: Comparison of "anti-IL-33" with "IVIG"). In addition, in the above experimental system, pretreatment with anti-human ST2 antibody reduced the IL-10 concentration in the culture supernatant compared to pretreatment with donated venoglobulin IH5% (Figure 4B: Comparison of "anti-ST2" with "IVIG").

[0049] [IL-6 concentration] In the above experimental system, pretreatment with 5% donated blood venoglobulin IH, anti-human IL-33 antibody, or anti-human ST2 antibody reduced the IL-6 concentration in the culture supernatant compared to pretreatment with normal goat IgG control, and in particular, pretreatment with 5% donated blood venoglobulin IH showed a significant decrease (Figure 4C: comparison of ``IVIG,'' ``anti-IL-33,'' or ``anti-ST2'' with ``control'').

[0050] [summary] It has been shown that treatment with an IL-33 antagonist (anti-IL-33 antibody) or an IL-33 receptor antagonist (anti-ST2 antibody) in an in vitro model of Kawasaki disease-related coronary arteritis can effectively suppress the production of IFN-γ and IL-10 in the model, indicating that the IL-33 antagonist (anti-IL-33 antibody) or the IL-33 receptor antagonist (anti-ST2 antibody) is effective against diseases associated with the production of IFN-γ or IL-10, such as Kawasaki disease-related coronary arteritis. [Industrial Applicability]

[0051] The present invention contributes to the prevention or treatment of diseases or symptoms associated with the production of IFN-γ or IL-10.

Claims

1. An agent for suppressing the production of IFN-γ or IL-10, comprising an IL-33 antagonist or an IL-33 receptor antagonist.

2. The production inhibitor according to claim 1, wherein the IL-33 antagonist is an anti-IL-33 antibody, and the IL-33 receptor antagonist is an anti-ST2 antibody.

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