Inhibitor of fibrosis progression

Abstract

This provides a new means for inhibiting the progression of fibrosis. [Solution] Provided is a pharmaceutical composition for suppressing the progression of fibrosis in systemic sclerosis, which comprises as an active ingredient an antibody against IL-31 receptor A. In another aspect, the present disclosure provides a pharmaceutical composition for suppressing Th2 bias to suppress the progression of fibrosis in systemic sclerosis, which comprises as an active ingredient an antibody against IL-31 receptor A. In a specific aspect, the antibody has neutralizing activity against IL-31 receptor A.

Description

[Technical Field]

[0001] The present disclosure relates to a pharmaceutical composition containing an antibody against IL-31 receptor A as an active ingredient. In one aspect, the present disclosure relates to an agent for suppressing the progression of fibrosis in systemic sclerosis, which comprises an antibody against IL-31 receptor A as an active ingredient. In another aspect, the present disclosure relates to a Th2-biased inhibitor for suppressing the progression of fibrosis in systemic sclerosis, which comprises an antibody against IL-31 receptor A as an active ingredient. [Background technology]

[0002] Systemic sclerosis (SSc) is a connective tissue disease characterized by excessive accumulation of extracellular matrix, such as collagen, in the skin and internal organs (Non-Patent Documents 1-2). The resulting fibrosis leads to tissue dysfunction and organ failure, which can be debilitating and life-threatening for patients. The etiology of SSc remains unknown, but three abnormalities—autoimmunity, vascular disorders, and fibrosis—are largely involved. Regarding immune abnormalities, extensive research has been conducted on T cell activation and polarization in both SSc patients and animal models (Non-Patent Document 3). For example, in the early stages of SSc, CD4 + It has been shown that T cells infiltrate into skin lesions (Non-Patent Document 4). These infiltrated T cells have been shown to highly express activation markers (Non-Patent Document 5). Similar T cell activation has also been found in peripheral blood (Non-Patent Document 6). Furthermore, activated CD4 +T cells are biased toward T helper (Th) type 2 (Non-Patent Documents 3 and 7). In fact, interleukin (IL)-4, IL-6, and IL-13, which are major Th2 cytokines (cytokines produced by Th2 cells), are overexpressed in the skin and serum of SSc patients (Non-Patent Documents 8-11). Th2 cytokines have also been reported to be associated with skin and lung fibrosis in bleomycin-induced SSc model (BLM-SSc) mice, a known SSc-like animal model (Non-Patent Documents 12-13). Mechanistically, these Th2 cytokines directly induce collagen production in fibroblasts (Non-Patent Documents 14-16). Furthermore, IL-4 and IL-6 act on naive CD4 + It induces the differentiation of T cells into Th2 cells, sustaining Th2 responses and profibrotic reactions (Non-Patent Documents 17-18). Collectively, these reports suggest that Th2 dominance is an important immunological feature of SSc that promotes fibrosis.

[0003] Factors suggested to be associated with Th2-dominant diseases include IL-4, IL-6, IL-13, and IL-31, an IL-6 family Th2 cytokine. Specifically, IL-31 has been shown to be highly expressed in Th2-dominant diseases such as allergic asthma, atopic dermatitis, and cutaneous T-cell lymphoma (Non-Patent Documents 19 to 24). In relation to SSc, increased expression of IL-31 has been reported in the fibrotic lungs of BLM-SSc mice (Non-Patent Document 25). A key pathological feature of SSc is the excessive production and accumulation of extracellular matrix proteins, such as collagen, by fibroblasts, which leads to progressive fibrosis of the skin and internal organs (Non-Patent Document 26). Few reports have investigated the role of IL-31 in the excessive production and accumulation of collagen in SSc. However, recent studies have shown that collagen production in skin fibroblasts derived from healthy individuals was promoted by IL-31 stimulation (Non-Patent Documents 27, 28). However, these reports did not show an increase in collagen production in skin fibroblasts derived from SSc patients by IL-31 stimulation. Thus, the role of IL-31 in fibrosis remains unclear. Furthermore, there have been no reports confirming that administration of anti-IL-31 receptor A antibodies can inhibit the progression of fibrosis. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Gabrielli A, Avvedimento EV, Krieg T. Scleroderma. N Engl J Med 2009; 360:1989-2003. [Non-patent document 2] Yoshizaki A. Pathogenic roles of B lymphocytes in systemic sclerosis. Immunol Lett 2018;195:76-82. [Non-patent document 3] O'Reilly S, Hugle T, van Laar JM. T cells in systemic sclerosis: a reappraisal. Rheumatology (Oxford) 2012;51:1540-9. [Non-patent document 4] Roumm AD, Whiteside TL, Medsger TA, et al. Lymphocytes in the skin of patients with progressive systemic sclerosis. Quantification, subtyping, and clinical correlations. Arthritis Rheum 1984;27:645-53. [Non-Patent Document 5] Kalogerou A, Gelou E, Mountantonakis S, et al. Early T cell activation in the skin from patients with systemic sclerosis. Ann Rheum Dis 2005;64:1233-5. [Non-Patent Document 6] Fiocco U, Rosada M, Cozzi L, et al. Early phenotypic activation of circulating helper memory T cells in scleroderma: correlation with disease activity. Ann Rheum Dis 1993;52:272-7. [Non-Patent Document 7] Barron L, Wynn TA. Fibrosis is regulated by Th2 and Th17 responses and by dynamic interactions between fibroblasts and macrophages. Am J Gastrointest Liver Physiol 2011;300:G723-8. [Non-Patent Document 8] Sato S, Hasegawa M, Takehara K. Serum levels of interleukin-6 and interleukin-10 correlate with total skin thickness score in patients with systemic sclerosis. J Dermatol Sci 2001;27:140–6.

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[0005] The invention of the present disclosure has been made in view of the above circumstances, and aims to provide, in one aspect, a new means for inhibiting the progression of fibrosis. In another aspect, the invention of the present disclosure aims to provide a new means for inhibiting Th2 bias, which is another important characteristic of the progression of fibrosis in systemic sclerosis. [Means for solving the problem]

[0006] As a result of extensive research, the present inventors have found that administration of an anti-IL-31 receptor A blocking antibody to an animal model of systemic sclerosis suppresses the progression of fibrosis and further found that administration of an anti-IL-31 receptor A antibody suppresses Th2 bias in T cells.

[0007] The present disclosure is based on such findings and specifically includes the embodiments exemplified below. [A1] An agent for suppressing the progression of fibrosis in systemic sclerosis, comprising an antibody against IL-31 receptor A as an active ingredient. [A2] The agent for suppressing the progression of fibrosis according to [A1], which suppresses Th2 bias. [A3] The agent for suppressing the progression of fibrosis according to any one of [A1] to [A2], wherein the antibody has neutralizing activity against IL-31 receptor A. [A4] The antibody, (1) An antibody comprising a heavy chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 3, and a light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 6; (2) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8; or (3) An antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 and a light chain having the amino acid sequence set forth in SEQ ID NO: 10. The agent for suppressing the progression of fibrosis according to any one of [A1] to [A3], [B1] A Th2 bias inhibitor containing an antibody against IL-31 receptor A as an active ingredient, which suppresses the progression of fibrosis in systemic sclerosis. [B2] The Th2 bias inhibitor according to [B1], wherein the antibody has neutralizing activity against IL-31 receptor A. [D1] A pharmaceutical composition for inhibiting the progression of fibrosis in systemic sclerosis, comprising an antibody against IL-31 receptor A as an active ingredient. [D2] A pharmaceutical composition for suppressing Th2 bias to inhibit the progression of fibrosis in systemic sclerosis, comprising an antibody against IL-31 receptor A as an active ingredient. [D3] The pharmaceutical composition according to any one of [D1] to [D2], wherein the antibody has neutralizing activity against IL-31 receptor A. [E1] A method for suppressing the progression of fibrosis in systemic sclerosis, comprising administering an antibody against IL-31 receptor A. [E2] A method for suppressing Th2 bias to inhibit the progression of fibrosis in systemic sclerosis, comprising administering an antibody against IL-31 receptor A. [E3] The method according to any one of [E1] to [E2], wherein the antibody has neutralizing activity against IL-31 receptor A. [F1] An antibody against IL-31 receptor A for use in inhibiting the progression of fibrosis in systemic sclerosis. [F2] An antibody against IL-31 receptor A for use in suppressing Th2 bias and inhibiting the progression of fibrosis in systemic sclerosis. [F3] The antibody according to any one of [F1] to [F2], which has neutralizing activity against IL-31 receptor A. [G1] Use of an antibody against IL-31 receptor A in the manufacture of an agent for inhibiting the progression of fibrosis in systemic sclerosis. [G2] Use of an antibody against IL-31 receptor A in the manufacture of a Th2 bias inhibitor for inhibiting the progression of fibrosis in systemic sclerosis. [G3] The use according to any one of [G1] to [G2], wherein the antibody has neutralizing activity against IL-31 receptor A. [Brief explanation of the drawings]

[0008] [Figure 1] This figure shows that anti-IL-31RA antibody suppressed the progression of induced fibrosis in BLM-SSc mice. (A) BLM-SSc mice and PBS-treated control (PBS-ctrl) mice (n = 5 per group) were administered anti-mouse IL-31RA antibody (αIL-31RA) or isotype control IgG (Iso) on days 1, 8, and 15 and sacrificed for evaluation on day 22. (B) Dermal thickness and lung fibrosis scores were examined using histological sections obtained from each mouse. Representative histological sections of skin and lung stained with hematoxylin and eosin are shown (horizontal scale bar = 100 μm). The vertical bar with an arrow indicates dermal thickness. (C) mRNA expression of IL-4, IL-6, IL-10, IL-17A, TNF-α, TGF-β1, and IFN-γ in the skin and lungs of these mice was evaluated by real-time PCR. Bar graphs show the mean + standard deviation. *p<0.05, **p<0.01, and ***p<0.001. [Figure 2] This figure shows that anti-IL-31RA antibody suppressed Th2 bias in BLM-SSc mice. (A) The percentages of Th1, Th2, Th17, and Treg cells in the spleens of BLM-SSc mice administered anti-mouse IL-31RA antibody (αIL-31RA) or isotype control IgG (Iso) and control mice treated with PBS (PBS-ctrl) were analyzed by flow cytometry. After gating on the CD3+ population, splenocytes were identified as Th1 cells, IFN-γ+ CD4+ cells as Th1 cells, IL-4+ CD4+ cells as Th2 cells, and IL-17A+ CD4+ cells as Th17 cells. Among cells gated on the CD3+ CD4+ population, CD25+ Foxp3+ cells were identified as Treg cells. Bar graphs show the mean + standard deviation. (B) Serum concentrations of IL-4 and IL-6 were assessed by specific ELISA assays. Symbols represent individual mice. Horizontal lines represent the mean. *p<0.05, **p<0.01, and ***p<0.001. DETAILED DESCRIPTION OF THE INVENTION

[0009] In one aspect, the present disclosure provides a pharmaceutical composition for suppressing the progression of fibrosis in systemic sclerosis, and a pharmaceutical composition for suppressing Th2 bias for suppressing the progression of fibrosis in systemic sclerosis, which contain an antibody against IL-31 receptor A as an active ingredient (also referred to herein as an inhibitor of fibrosis progression in systemic sclerosis and an inhibitor of Th2 bias for suppressing the progression of fibrosis in systemic sclerosis, respectively). In one embodiment, the pharmaceutical composition of the present disclosure can suppress the progression of fibrosis in systemic sclerosis, for example, suppressing the progression of hardening of the skin and internal organs (e.g., lungs) and / or the progression of fibrosis. In one embodiment, the pharmaceutical composition of the present disclosure can suppress Th2 bias in systemic sclerosis, for example, suppressing the production of Th2 cytokines in systemic sclerosis and / or the progression of a Th2-dominant state in systemic sclerosis.

[0010] Lesions in which fibrosis occurs are called "fibrosis," and depending on the tissue in which fibrosis occurs, they are called skin fibrosis, pulmonary fibrosis, liver fibrosis, etc. In systemic sclerosis (SSc), this fibrosis occurs in the skin and internal organs.

[0011] CD4 in the development of SSc + T cells play a key role, and CD4 + T cells are known to infiltrate skin lesions. +T cells are known to have subtypes such as Th1 cells, Th2 cells, Th17 cells, and regulatory T cells (Treg), and each subtype has its own characteristic cytokine secretion pattern. In particular, Th1 cells and Th2 cells maintain homeostasis by controlling each other's functions and maintaining a balanced relationship. It is said that when this balance (Th1 / Th2 balance) shifts towards one of the subtypes, diseases specific to each subtype occur. When the Th1 / Th2 balance shifts towards Th1 dominance, it is called "Th1 bias," and when it shifts towards Th2 dominance, it is called "Th2 bias." In addition to Th1 and Th2 cells, each CD4 + It is said that imbalances between T cell subtypes are also involved in the development of diseases. "Th2 bias" can also be expressed as "a bias toward Th2 type" or "a shift toward Th2 type immune responses," and is a condition that affects other CD4 + The same is true for T cell subtype bias.

[0012] As used herein, "fibrosis" refers to the phenomenon in which the skin or internal organs harden as a result of the accumulation of extracellular matrix such as collagen in the skin or internal organs. It is known that once fibrotic tissue has developed, it will never return to its original soft state.

[0013] As used herein, "suppressing the progression of fibrosis" and "suppressing the progression of fibrosis" refer to suppressing or inhibiting the progression of fibrosis compared to a control (e.g., a subject or subject population to which the fibrosis progression inhibitor of the present disclosure is not administered).

[0014] Various methods for assessing the degree of fibrosis are known.For example, skin fibrosis can be assessed by measuring the thickness of skin tissue samples under a microscope.For example, lung fibrosis can be assessed by measuring the severity of lung fibrosis using the pulmonary fibrosis score known as Ashcroft score.

[0015] As used herein, "suppressing Th2 bias" and "suppression of Th2 bias" refer to suppressing or inhibiting a shift in the Th1 / Th2 balance toward Th2 dominance compared to a control. The degree to which Th2 bias is suppressed is not limited, and may be, for example, suppressed, inhibited, or reduced by 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% compared to a control. In one embodiment, the control refers to the degree of Th2 bias in a subject or subject population (e.g., a systemic sclerosis patient or a systemic sclerosis patient population with a Th2-dominant disease) when the Th2 bias suppressant for suppressing fibrosis progression in systemic sclerosis of the present disclosure (the pharmaceutical composition of the present disclosure) is not administered. Alternatively, the shift in the Th1 / Th2 balance towards Th2 dominance may be suppressed, inhibited, or reduced to a level equivalent to or similar to that of a healthy person, for example, 2.0-fold, 1.9-fold, 1.8-fold, 1.7-fold, 1.6-fold, 1.5-fold, 1.4-fold, 1.3-fold, 1.2-fold, 1.1-fold, or 1.0-fold compared to a healthy person.

[0016] Methods for assessing the degree of Th1 or Th2 bias of Th cells (helper T cells) are known, and can be performed, for example, by measuring the expression levels of cytokines specific to Th1 cells and Th2 cells in a biological sample obtained from a subject and calculating the ratio between them. Similarly, Th17 / Treg bias can be assessed by calculating the ratio between the expression levels of cytokines specific to Th17 cells and Treg cells.

[0017] As used herein, "inhibiting the progression of fibrosis in systemic sclerosis" and "inhibiting the progression of fibrosis in systemic sclerosis" refer to suppressing or inhibiting the progression of systemic sclerosis compared to a control. In one embodiment, the control refers to the severity of systemic sclerosis in a subject or subject population (e.g., a systemic sclerosis patient or a systemic sclerosis patient population) when the agent for inhibiting the progression of fibrosis in systemic sclerosis (the pharmaceutical composition of the present disclosure) of the present disclosure is not administered. Methods for assessing the degree of progression of fibrosis in systemic sclerosis are known, and can be evaluated, for example, by the degree of fibrosis in the skin and / or internal organs (e.g., lungs), the degree of Th2 bias, or a combination thereof.

[0018] IL-31 is a new member of the IL-6 cytokine family and was initially reported as an inducer of mouse dermatitis. IL-31 is primarily produced by Th2 cells, but is also expressed in a variety of cells, including fibroblasts, keratinocytes, and macrophages. When IL-31 binds to the IL-31 receptor complex on the cell surface, it activates JAK / STAT, PI3K / AKT, and other intracellular signaling pathways, resulting in a broad immune response.

[0019] The IL-31 receptor complex is a heterodimer consisting of IL-31 receptor A (IL-31RA) and oncostatin M receptor. Although oncostatin M receptor is also included in the receptor complex for oncostatin M, IL-31RA is unique to the IL-31 receptor. IL-31 primarily binds to IL-31RA, one of the two receptor subunits.

[0020] As used herein, the terms "anti-IL-31 receptor A antibody" and "antibody against IL-31 receptor A" are used interchangeably and refer to an antibody capable of specifically binding to IL-31 receptor A (IL-31RA). In the context of the presently disclosed invention, an antibody against IL-31 receptor A is preferably an antibody having neutralizing activity against IL-31 receptor A. As used herein, "neutralizing activity against IL-31 receptor A" refers to the activity of inhibiting the binding of IL-31 receptor A to its ligand, IL-31, and preferably refers to the activity of suppressing physiological activity based on IL-31 receptor A. Therefore, an "antibody having neutralizing activity against IL-31 receptor A" inhibits the binding of IL-31RA to IL-31, thereby suppressing, inhibiting, or blocking intracellular signaling mediated by IL-31RA.

[0021] As used herein, the term "antibody" refers to a molecule that specifically binds to a particular antigenic determinant (epitope), and includes various antibody structures, including, but not limited to, monoclonal antibodies (mAbs), polyclonal antibodies (pAbs), and antibody fragments.

[0022] In the context of the presently disclosed invention, the antibody against IL-31RA is preferably an antibody against mammalian IL-31RA, more preferably an antibody against human IL-31RA. An example of a neutralizing antibody against human IL-31RA is antibody A. Antibody A has been shown in clinical trials to improve the symptoms of atopic dermatitis. Antibody A comprises a heavy chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 3, and a light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 6. Antibody A also comprises a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8. Antibody A also comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 and a light chain having the amino acid sequence set forth in SEQ ID NO: 10. An example of a neutralizing antibody against mouse IL-31RA is an anti-mouse IL-31 receptor A function-blocking monoclonal antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 11 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 12. The nucleotide sequences of these heavy and light chain variable regions have been registered in the DDBJ / EMBL / GenBank international nucleotide sequence database under accession numbers LC554895 and LC554896, respectively.

[0023] All technical documents cited herein are incorporated herein by reference in their entirety. [Example]

[0024] The present disclosure will be specifically described below using examples, but the present disclosure is not limited to these examples.

[0025] Mice and experimental protocol Wild-type C57BL / 6 mice were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). Bleomycin (BLM, Nippon Kayaku Co., Ltd., Tokyo, Japan) was dissolved in phosphate-buffered saline (PBS) at a concentration of 1 mg / ml. To generate the BLM-SSc model (BLM-SSc) mice, the backs of female mice were shaved and subcutaneously injected daily with 200 μg of BLM, as previously described (Yoshizaki A, Iwata Y, Komura K, et al. CD19 regulates skin and lung fibrosis via Toll-like receptor signaling in a model of bleomycin-induced scleroderma. Am J Pathol 2008;172:1650-63). Mice treated with PBS instead of BLM (PBS-control mice) served as controls for BLM-SSc mice. To evaluate the effects of blocking IL-31 signaling, 200 μg of an anti-mouse IL-31 receptor A function-blocking monoclonal antibody (heavy chain variable region: SEQ ID NO: 11; light chain variable region: SEQ ID NO: 12; registered under DDBJ / EMBL / GenBank accession numbers LC554895 and LC554896, respectively) or a mouse IgG1κ isotype control (eBioscience, San Diego, CA, USA) was intraperitoneally injected every 7 days (days 1, 8, and 15). All mice used in this study were 6 weeks old. Five mice were tested in each group.

[0026] ELISA Serum samples were frozen at -80°C until use in the assay. Serum concentrations of IL-4 and IL-6 in mice were measured using ELISA kits (R&D Systems). All experiments were performed according to the manufacturer's instructions.

[0027] Histological analysis Skin and lung tissues were fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin for histological evaluation. Dermal thickness, defined as the distance between the epidermal-dermal junction and the dermal-fatty junction, was measured. The severity of pulmonary fibrosis was assessed semiquantitatively as described by Ashcroft et al. (Ashcroft T, Simpson JM, Timbrell V. Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol 1988;41:467-70.). Briefly, the grading criteria were as follows: Grade 0 = normal lung; Grade 1 = minimal fibrous thickening of the alveolar or bronchiolar walls; Grade 3 = moderate wall thickening without obvious damage to the lung structure; Grade 5 = increased fibrosis with clear damage to the lung structure and formation of fibrous bands or small fibrous masses; Grade 7 = severe structural deformation and large fibrous areas; and Grade 8 = complete fibrous obliteration of the field. Grades 2, 4, and 6 were used as intermediate grades between the above criteria.

[0028] RNA isolation and real-time polymerase chain reaction (PCR) Total RNA was isolated from tissues using RNeasy spin columns (Qiagen, Crawley, UK) according to the manufacturer's instructions. Total RNA from each sample was reverse transcribed into cDNA. Gene expression was quantified by SYBR green real-time PCR using an ABI Prism 7000 sequence detector (Applied Biosystems, Foster City, CA, USA). GAPDH was used as an endogenous control to normalize the amount of cDNA loaded. Relative values ​​(fold differences) were calculated using the comparative Ct method as previously reported (Yoshizaki A, Iwata Y, Komura K, et al. CD19 regulates skin and lung fibrosis via Toll-like receptor signaling in a model of bleomycin-induced scleroderma. Am J Pathol 2008;172:1650-63.). Primer sequences were as follows: Il4-forward 5'-CAACGAAGAACACCACAGAG-3' (SEQ ID NO: 13), Il4-reverse 5'- GGACTTGGACTCATTCATGG-3' (SEQ ID NO: 14); Il6-forward 5'- GATGGATGCTACCAAACTGGAT-3' (SEQ ID NO: 15), Il6-reverse 5'- CCAGGTAGCTATGGTACTCCAGA-3' (SEQ ID NO: 16); Ifng-forward 5'-TCAAGTGGCATAGATGTGGAAGAA-3' (SEQ ID NO: 17); Ifng-reverse 5'-TGGCTCTGCAGGATTTTCATG-3' (SEQ ID NO: 18); Il17a-forward 5'-CAGCAGCGATCATCCCTCAAAG-3' (SEQ ID NO: 19), Il17a-reverse 5'-CAGGACCAGGATCTCTTGCTG-3' (SEQ ID NO: 20), Il10-forward 5'-TTTGAATTCCCTGGGTGAGAA-3' (SEQ ID NO: 21); Il10-reverse 5'-ACAGGGGAGAAATCGATGACA-3' (SEQ ID NO: 22); Tgfb1-forward 5'-GCAACATGTGGAACTCTACCAGAA-3' (SEQ ID NO: 23), Tgfb1-reverse 5'-GACGTCAAAAGACAGCCACTCA-3' (SEQ ID NO: 24); Tnfa-forward 5'-ACCCTCACACTCAGATCATCTTC-3' (SEQ ID NO: 25), Tnfa-reverse 5'-TGGTGGTTTGCTACGACGT-3' (SEQ ID NO: 26); and Gapdh-forward 5'-CGTGTTCCTACCCCCAATGT-3' (SEQ ID NO: 27), Gapdh-reverse 5'-TGTCATCATACTTGGCA GGTTTCT-3' (SEQ ID NO: 28).

[0029] Flow cytometry As previously reported (Wen X, He L, Chi Y, et al. Dynamics of Th17 cells and their role in Schistosoma japonicum infection in C57BL / 6 mice. PLoS Negl Trop Dis 2011;5:e1399.), CD4 + T cell differentiation was analyzed. Briefly, to detect Th1, Th2, or Th17 cells, 2 × 10 splenocytes were cultured. 6Cells were suspended in RPMI 1640 medium at 1000 cells / ml and stimulated with 25 ng / ml PMA (Adipogen, San Diego, CA, USA) and 1 mg / ml ionomycin (Sigma, St. Louis, MO, USA) for 6 hours at 37°C in 5% CO2 in the presence of 2 μM monensin (Invitrogen, Carlsbad, CA, USA). The surface of the samples was then stained with anti-CD3-PE mAb and anti-CD4-FITC mAb (eBioscience). After fixation and permeabilization with Cytofix / Cytoperm buffer (BD PharMingen, San Diego, CA, USA), the intracellular contents of the samples were stained with APC-conjugated mAbs (eBioscience) against IFN-γ, IL-4, or IL-17A to detect Th1, Th2, or Th17 cells, respectively. Isotype-matched APC-conjugated mAbs (eBioscience) were used as controls. To detect Treg cells, we used the Mouse Regulatory T Cell Staining Kit (eBioscience) according to the manufacturer's protocol. Briefly, 2 × 10 6 Splenocytes suspended at 1000 cells / ml were surface-stained with anti-CD3-PE / Cy7 mAb, anti-CD4-FITC mAb, and anti-CD25-APC mAb (eBioscience). After fixation and permeabilization with Cytofix / Cytoperm, the cells were intracellularly stained with anti-Foxp3-PE mAb or rat IgG2a-PE as a control. Samples were analyzed using a FACS Verse flow cytometer (BD Biosciences, San Diego, CA, USA).

[0030] statistical analysis Data are expressed as mean ± standard deviation. Statistical analysis was performed using the Mann-Whitney U test for comparisons between two groups. P values ​​less than 0.05 were considered significant. All analyses were performed using GraphPad Prism 7.03 (GraphPad Software, La Jolla, CA, USA).

[0031] Anti-IL-31RA function-blocking mAb suppressed fibrosis and Th2 bias in BLM-SSc mice. To clarify the role of IL-31 in the development of SSc, we evaluated fibrosis and Th2 bias in BLM-SSc mice treated with anti-IL-31RA function-blocking mAb. BLM was injected subcutaneously daily, and anti-IL-31RA mAb was administered weekly for 3 weeks (Fig. 1A). Compared with isotype control IgG, anti-IL-31RA mAb significantly reduced dermal thickness in BLM-SSc mice (p<0.01; Fig. 1B). Furthermore, anti-IL-31RA mAb significantly reduced lung fibrosis scores in BLM-SSc mice (p<0.001; Fig. 1B). Regarding cytokine mRNA expression in skin and lung tissues, anti-IL-31RA mAb suppressed the expression of IL-4, IL-6, IL-10, and TGF-β1, which were upregulated in BLM-SSc mice (Fig. 1C). Furthermore, anti-IL-31RA mAb significantly reduced the proportion of Th2 cells (p<0.01) and the Th2 / Th1 ratio (p<0.05) but not the Th17 / Treg ratio in splenic T cells of BLM-SSc mice (Fig. 2A). Anti-IL-31RA mAb also ameliorated the excessive production of serum IL-4 and IL-6 in BLM-SSc mice (p<0.01 and p<0.001, respectively; Fig. 2B). Overall, anti-IL-31RA mAb significantly reduced BLM-induced fibrosis and Th2 bias. [Industrial Applicability]

[0032] The invention of the present disclosure has revealed the effect of administering an anti-IL-31RA antibody in an animal model of systemic sclerosis fibrosis. In particular, a pharmaceutical composition of the present disclosure containing an anti-IL-31RA antibody suppresses the progression of fibrosis, for example, in the skin or lungs, and is therefore useful as an agent for suppressing the progression of fibrosis in systemic sclerosis. Furthermore, a pharmaceutical composition of the present disclosure containing an anti-IL-31RA antibody suppresses Th2 bias, and is therefore useful as an agent for suppressing Th2 bias for suppressing the progression of fibrosis in systemic sclerosis.

Claims

1. An agent for suppressing the progression of fibrosis in systemic sclerosis, comprising an antibody against IL-31 receptor A as an active ingredient, The antibody (1) An antibody comprising a heavy chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 1, CDR2 having the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 3, and a light chain variable region comprising CDR1 having the amino acid sequence set forth in SEQ ID NO: 4, CDR2 having the amino acid sequence set forth in SEQ ID NO: 5, and CDR3 having the amino acid sequence set forth in SEQ ID NO: 6; (2) an antibody comprising a heavy chain variable region having the amino acid sequence set forth in SEQ ID NO: 7 and a light chain variable region having the amino acid sequence set forth in SEQ ID NO: 8; or (3) An antibody comprising a heavy chain having the amino acid sequence set forth in SEQ ID NO: 9 and a light chain having the amino acid sequence set forth in SEQ ID NO:

10. It is an inhibitor of the progression of fibrosis.

2. The agent for suppressing the progression of fibrosis according to claim 1, which suppresses Th2 bias.

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