Composition for prevention or treatment of atopic dermatitis, comprising human monoclonal antibody specific to chi3l1

Abstract

The present invention relates to a composition for prevention or treatment of atopic dermatitis, comprising a human monoclonal antibody specific to CHI3L1, and specifically, the present invention relates to a pharmaceutical composition for prevention or treatment of atopic dermatitis, a cosmetic composition for prevention or alleviation of atopic dermatitis, and a health functional food for prevention or alleviation of atopic dermatitis, respectively comprising an anti-CHI3L1 antibody as an active ingredient. The monoclonal antibody specific to CHI3L1, according to the present invention, has the effects of reducing epidermal thickness increased by the development of atopic dermatitis, inhibiting mast cell infiltration, reducing the weight of lymph nodes, inhibiting the expression and production of inflammatory cytokines, and inhibiting the expression of p-STAT3 and CHI3L1, and thus may be usefully employed in the production of a medicine, cosmetics, and a health functional food capable of the effective prevention, alleviation, and treatment of atopic dermatitis.

Description

Composition for preventing or treating atopic dermatitis comprising a human monoclonal antibody specific for HI3L1

[0001] The present invention relates to a composition for preventing or treating atopic dermatitis comprising a human monoclonal antibody specific for CH13L1.

[0002] Atopic dermatitis is characterized by excessive infiltration of lymphocytes, macrophages, and granulated mast cells into the lesion, which increases the number of eosinophils in the patient's blood and the level of immunoglobulin E (IgE) in the serum. Atopic dermatitis has a complex pathogenesis that includes genetic, environmental, pharmacological, and immunological factors, but in particular, inflammatory factors such as nitric oxide (NO), interleukin-6 (IL-6), and interleukin-1β (IL-1β) are involved.

[0003] Immunosuppressants are used to treat atopic dermatitis, with steroids being the most commonly used. However, long-term use of steroids can cause various side effects, such as skin atrophy. Therefore, there is a growing need to develop new treatments for atopic dermatitis that are effective and free of side effects.

[0004] Meanwhile, chitinase-3-like protein 1 (CHI3L1) is a secreted heparin-binding glycoprotein expressed in various cell types, such as macrophages and neutrophils. CHI3L1 possesses cytokine and growth factor properties and is known to be associated with inflammatory diseases such as inflammatory bowel disease and several cancers. Furthermore, CHI3L1 has been linked to neuroinflammatory diseases such as atherosclerosis, liver damage, stroke, and Alzheimer's disease.

[0005] Meanwhile, there has been no research on the development of a treatment for atopic dermatitis using a specific antibody against CHI3L1 that can suppress the pathogenic action of CHI3L1.

[0006] Accordingly, the inventors of the present invention prepared a monoclonal antibody specific to CHI3L1, and confirmed that atopic dermatitis can be effectively prevented, improved, and treated when the monoclonal antibody specific to CHI3L1 prepared by the inventors is used, thereby completing the present invention.

[0007] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating atopic dermatitis, comprising an anti-CHI3L1 antibody as an active ingredient.

[0008] Another object of the present invention is to provide a cosmetic composition for preventing or improving atopic dermatitis, comprising an anti-CHI3L1 antibody as an active ingredient.

[0009] Another object of the present invention is to provide a health functional food for preventing or improving atopic dermatitis, comprising an anti-CHI3L1 antibody as an active ingredient.

[0010] In order to achieve the above-described purpose of the present invention, the present invention provides a pharmaceutical composition for preventing or treating atopic dermatitis, comprising an anti-CHI3L1 antibody as an active ingredient.

[0011] In one embodiment of the present invention, the anti-CHI3L1 antibody is a monoclonal antibody that specifically binds to CHI3L1, and may include (1) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 5; a CDR2 consisting of the amino acid sequence of SEQ ID NO: 6; and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 7; and a light chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 11; a CDR2 consisting of the amino acid sequence of SEQ ID NO: 12; and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 13, or (2) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 8; a CDR2 consisting of the amino acid sequence of SEQ ID NO: 9; and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 10; and a light chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 15; and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 16.

[0012] In one embodiment of the present invention, the anti-CHI3L1 antibody may include (a) a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 1; and (b) a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 3.

[0013] In one embodiment of the present invention, the anti-CHI3L1 antibody may include (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2; and (b) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4.

[0014] In one embodiment of the present invention, the anti-CHI3L1 antibody can prevent, improve, or treat atopic dermatitis by reducing the thickness of the skin epidermis increased by atopic dermatitis; inhibiting the infiltration of mast cells; reducing the weight of lymph nodes; inhibiting the expression and production of inflammatory cytokines; and inhibiting the expression of p-STAT3 and CHI3L1.

[0015] In one embodiment of the present invention, the inflammatory cytokine may be IL-1β, IL-4, CXCL8 or TSLP.

[0016] In addition, the present invention can provide a cosmetic composition for preventing or improving atopic dermatitis, comprising an anti-CHI3L1 antibody as an active ingredient.

[0017] Furthermore, the present invention can provide a health functional food for preventing or improving atopic dermatitis, which contains an anti-CHI3L1 antibody as an active ingredient.

[0018] The monoclonal antibody specific to CHI3L1 according to the present invention can effectively prevent, improve and treat symptoms of atopic dermatitis by reducing the increased thickness of the skin epidermis caused by atopic dermatitis, inhibiting mast cell infiltration, reducing the weight of lymph nodes, inhibiting the expression and production of inflammatory cytokines and inhibiting the expression of p-STAT3 and CHI3L1. Therefore, the anti-CHI3L1 antibody of the present invention, which is a monoclonal antibody specific to CHI3L1, can be usefully used in the manufacture of pharmaceuticals, cosmetics and health functional foods that can effectively prevent, improve and treat atopic dermatitis.

[0019] Figure 1 shows the results of confirming the atopic dermatitis inhibition effect according to the anti-CHI3L1 antibody treatment of the present invention, (a) shows a group in which atopic dermatitis was induced by applying a PA solution to the back skin of mice, a group treated with an anti-CHI3L1 antibody (0.1 or 0.2 mg / kg) and a group treated with an anti-IL-4R antibody (0.2 mg / kg) 2 hours before application of the PA solution, and a photograph of the back skin tissue of each group of mice observed under a microscope after H&E staining, mast cell staining and neutrophil (Ly6G+ cell) cell staining, (b) shows an analysis of the clinical score of atopic dermatitis symptoms (n=10), (c) shows a quantitative analysis of the skin epidermal thickness (n=10), (d) shows the number of mast cells (n=10), (e) shows the number of neutrophils (Ly6G+ cells) (n=10), and (f) shows a quantitative analysis of the serum IgE concentration (n=10). The results are presented. All data are expressed as mean ± standard deviation, and all results indicate * P < 0.05; ** P < 0.01; ###,*** P < 0.001.

[0020] Figure 2 shows the results of measuring the size and weight of lymph nodes isolated from each mouse group used in the experiment of Figure 1 (a), and (b) to (d) show the results of analyzing the cell number ratios of CD4+ T cells, CD8+ T cells, and CD11c+ dendritic cells in each mouse group.

[0021] Figure 3 shows the results of analyzing the mRNA levels of CHI3L1, IL1B, IL4, Cxcl1, and Tslp expressed in skin tissues isolated from each mouse group used in the experiment of Figure 1 using RT-qPCR (a) and the results of analyzing the protein levels of CHI3L1, IL1B, IL4, Cxcl1, and Tslp using ELISA (b).

[0022] FIG. 4 shows the results of confirming the skin inflammation inhibition effect in an atopic dermatitis-like RHS model according to the anti-CHI3L1 antibody treatment of the present invention. After culturing RHS model cells with an atopic dermatitis-inducing cocktail for 6 days together with anti-CHI3L1 antibody (500 ng / ml) or anti-IL-4R antibody (500 ng / ml), (a) shows a photograph of the epidermis and dermis observed under a microscope after H&E staining (n=5, scale bar: 200 μm), and the bar graph on the right shows the results of measuring the epidermal thickness, (b) shows the results of measuring the mRNA levels of CHI3L1, IL1B, IL4, Cxcl8, and Tslp in the RHS skin model of each group through RT-qPCR, and (c) shows the results of measuring the protein levels of CHI3L1, IL1B, IL4, Cxcl8, and Tslp by ELISA.

[0023] Figure 5 confirms the inhibitory effect of atopic dermatitis-related cytokines according to the treatment with the anti-CHI3L1 antibody of the present invention in HaCaT cells. HaCaT cells were pretreated with the indicated concentrations of anti-CHI3L1 antibody or anti-IL4R antibody, and 24 hours later, the cells were treated with an atopic dermatitis-inducing cocktail (IL-4 30 ng / ml, IL-13 30 ng / ml, TNF-α 3.5 ng / ml) for 4 hours, respectively. (a) shows the analysis of the mRNA expression levels of CHI3L1, IL1B, IL4, Cxcl8, and Tslp by RT-qPCR, and (b) shows the analysis of the protein levels of CHI3L1, IL1B, IL4, Cxcl8, and Tslp by ELISA.

[0024] FIG. 6 shows the results of confirming the inhibitory effect of the anti-CHI3L1 antibody according to the present invention on the STAT3 signaling pathway. (a) shows the results of analyzing the expression levels of CHI3L1 and STAT3 and the expression levels of p-STAT3 by Western blot after treating mice with PA solution three times a week for four weeks, and applying anti-CHI3L1 antibody or anti-IL-4R antibody to the back skin two hours before applying the PA solution after the first week, (b) shows the images of immunohistochemical analysis using anti-CHI3L1 antibody in the skin tissue of each mouse (n=5, scale bar: 100 μm), (c) shows the results of analyzing the expression levels of CHI3L1 and STAT3 and the expression levels of p-STAT3 by Western blot after culturing RHS model cells with an atopic dermatitis-inducing cocktail for six days with anti-CHI3L1 antibody or anti-IL-4R antibody (500 ng / ml) at each concentration. (d) Pretreatment of HaCaT cell lines with the indicated concentrations of anti-CHI3L1 antibody or anti-IL4R antibody, and 24 hours later, the cells were treated with an atopic dermatitis-inducing cocktail (IL-4 30 ng / ml, IL-13 30 ng / ml, TNF-α 3.5 ng / ml) for 4 hours, and the results of Western blot analysis of the expression levels of CHI3L1 and STAT3 and the expression levels of p-STAT3 are shown.

[0025] Figure 7 shows the results of analyzing the mRNA expression levels of TGF-β1, NCF2, CTSS, CXCL8, and CD14 in CHI3L1-overexpressing HaCaT cell lines.

[0026] Figure 8 shows the results confirming the involvement of CXCL8 in anti-CHI3L1 antibody-mediated skin inflammatory response, (a) showing the results of protein association network analysis of CHI3L1 and predicted target proteins, (b) showing the results of confirming the mRNA levels of CHI3L1 and Cxcl8 by RT-qPCR after treating HaCaT cell lines with recombinant CHI3L1 protein (100 ng / ml) for 24 hours, (c) showing the results of transfecting HaCaT cell lines with human CHI3L1 siRNA (20 nM) and analyzing the mRNA levels of CHI3L1 and Cxcl8 by RT-qPCR, (d) showing the results of treating HaCaT cell lines with recombinant CXCL8 protein (50 ng / ml) and, 24 hours later, with an atopic dermatitis-inducing cocktail (IL-4 30 ng / ml, IL-13 30 ng / ml, TNF-α 3.5 ng / ml) for 4 hours, The results of RT-qPCR analysis of mRNA levels of CHI3L1, IL1B, IL4, Cxcl8, and Tslp are shown. (e) HaCaT cell lines were transfected with human CXCL8 siRNA (20 nM), and 24 hours later, treated with an atopic dermatitis-inducing cocktail (IL-4 30 ng / ml, IL-13 30 ng / ml, TNF-α 3.5 ng / ml) for 4 hours, and the results of RT-qPCR analysis of mRNA levels of CHI3L1, IL1B, IL4, Cxcl8, and Tslp are shown.

[0027] Figure 9 shows (a) the CHI3L1 protein level in serum and the results of ROC analysis for CHI3L1, (b) the CXCL8 protein level in serum and the results of ROC analysis for CXCL8, and (c) the results of analysis of the correlation between CXCL8 and CHI3L1, for sera obtained from normal individuals and patients with atopic dermatitis.

[0028] The present invention is characterized by providing a composition for preventing or treating atopic dermatitis, which contains a monoclonal antibody specific for CHI3L1 (Chitinase-3-like 1) as an active ingredient.

[0029] Chitinase-3-like 1 (CHI3L1) is a secreted heparin-binding glycoprotein expressed in various cell types, such as macrophages and neutrophils. It has cytokine and growth factor properties and is known to be closely associated with inflammatory diseases such as rheumatoid arthritis, inflammatory bowel disease, psoriasis, and several cancers. In addition, CHI3L1 is known to be crucially involved in neuroinflammatory diseases such as atherosclerosis, liver damage, stroke, and Alzheimer's disease, and high genetic variation in the CHI3L1 promoter region has been reported to be associated with atopy.

[0030] Therefore, the development of a disease treatment agent through inhibition of CHI3L1 is under study, and in the present invention, the inventors have for the first time demonstrated that a monoclonal antibody specific to CHI3L1, newly synthesized previously, can effectively prevent, improve, and treat atopic dermatitis.

[0031] The monoclonal antibody specific to CHI3L1 according to the present invention is a new monoclonal antibody specific to CHI3L1 developed and synthesized by the present inventors, and the antibody refers to a monoclonal antibody that specifically binds to YKL-40 disclosed in the registered patent no. 10-2142499 of the present inventors.

[0032] Chitinase 3-like 1 (CHI3L1) is a 40 kDa secreted glycoprotein belonging to the chitinase-like proteins (CLPs) and also known as YKL-40. Chi3L1 is a highly conserved chitin-binding protein, and its three-dimensional crystal structure shows a typical folding structure of chitinase family proteins, but chitinase activity is characterized by the deletion of essential amino acid residues in the enzymatic domain. Chi3L1 is known to be overexpressed in cancer or tumor-associated macrophages, and its expression level is known to be high in the serum of patients with metastatic cancer or chronic inflammatory diseases. However, its relationship with atopic dermatitis has not been studied to date.

[0033] Meanwhile, the inventors of the present invention have for the first time discovered that a monoclonal antibody specific to CHI3L1 has preventive, ameliorating, and therapeutic effects on atopic dermatitis.

[0034] In one embodiment of the present invention, in the dorsal skin of mice in which atopic dermatitis was induced by PA (phthalic anhydride) treatment, the group treated with the monoclonal antibody specific to CHI3L1 of the present invention showed a decrease in the clinical score of atopic dermatitis compared to the group that was not treated with the antibody, and the thickness of the thickened epidermis was also reduced, and the number of mast cell infiltration and the number of neutrophil cells were also significantly reduced. In addition, it was confirmed that the symptoms of atopic dermatitis were improved and treated by the monoclonal antibody specific to CHI3L1 of the present invention, such as suppression of excessive IgE production, which is one of the characteristics of atopic dermatitis. In addition, it was confirmed that the prevention, improvement, and treatment effects of atopic dermatitis by the monoclonal antibody specific to CHI3L1 of the present invention were more excellent than those of the group treated with the IL-4R antibody, which is known to have a conventional atopic dermatitis treatment effect.

[0035] In another embodiment of the present invention, the monoclonal antibody specific to CHI3L1 of the present invention (anti-CHI3L1 antibody) was analyzed to see whether it suppresses the expression and production of inflammatory cytokines in skin tissues of mice induced with atopic dermatitis. As a result, it was found that the expression levels of mRNA and protein of CHI3L1, IL-1β, IL-4, IL-6, CXCL1, and TSLP were all significantly suppressed by treatment with the antibody of the present invention.

[0036] These results imply that the monoclonal antibody specific for CHI3L1 of the present invention can regulate the expression of inflammatory mediators involved in the development of atopic dermatitis.

[0037] Therefore, through these results, the inventors of the present invention were able to confirm that the monoclonal antibody specific to CHI3L1 of the present invention can reduce the thickness of the thickened skin epidermis when atopic dermatitis develops, suppress the infiltration of mast cells, reduce the weight of lymph nodes, and suppress the expression and production of inflammatory cytokines, and thus can be used as a new therapeutic agent for atopic dermatitis.

[0038] Meanwhile, STAT3 (Signal transducer and activator of transcription 3) is known to be an important transcription factor in inflammation, and recent studies have shown that hyperphosphorylation of STAT3 is observed in atopic dermatitis patients compared to normal people, and that administration of a Janus kinase (JAK) inhibitor improves disease symptoms. In addition, it is known that the TSLP (thymic stromal lymphopoietin) receptor, an interleukin (IL)-7-like cytokine receptor, plays an important role in inducing the development of atopic disease by activating the STAT3 pathway.

[0039] Therefore, inhibitors that can suppress STAT3 activity may be used as treatments for atopic dermatitis.

[0040] Accordingly, the inventors of the present invention analyzed whether the monoclonal antibody specific to CHI3L1 of the present invention affects the STAT3 signaling pathway known to be related to the onset of atopic dermatitis, and as a result, confirmed that it can inhibit STAT3 signaling pathway activation by inhibiting STAT3 phosphorylation.

[0041] Furthermore, in another embodiment of the present invention, in order to analyze factors related to CHI3L1-mediated atopic dermatitis, HumanBase protein-protein interaction network analysis was performed, and as a result, it was found that CHI3L1 was associated with TGF-β1, NCF2, CTSS, CXCL8, and CD14, and in particular, it was confirmed that CXCL8 increased to the highest level of expression by CHI3L1 treatment, and thus CXCL8 could be determined to be the factor most closely related to CHI3L1-mediated atopic dermatitis.

[0042] Accordingly, the present inventors conducted an experiment to verify whether CXCL8 is involved in the CHI3L1-mediated skin inflammatory response, and as a result, it was shown that CXCL8 treatment increased the mRNA levels of IL-1β, IL-6, CXCL8, TSLP, and CHI3L1, which are cytokines associated with atopic dermatitis, whereas when the expression of CXCL8 was suppressed by si-CXCL8 treatment, the increased expression levels of the above cytokines were decreased.

[0043] Additionally, analysis results performed on serum samples from patients with atopic dermatitis showed that the expression levels of CHI3L1 and CXCL8 were increased in the serum of patients with atopic dermatitis compared to normal individuals.

[0044] Through this, the inventors of the present invention were able to find that CHI3L1 and CXCL8 have significantly increased expression compared to the normal control group when atopic dermatitis occurs, and that this increased expression has high sensitivity and specificity, so they can be usefully used as biomarkers for the diagnosis of atopic dermatitis.

[0045] Therefore, the present invention can provide a pharmaceutical composition for preventing or treating atopic dermatitis, comprising an anti-CHI3L1 antibody as an active ingredient.

[0046] In the present invention, "antibody" refers to an immunoglobulin molecule that immunologically reactives with a specific antigen, and is a concept encompassing both polyclonal and monoclonal antibodies. Furthermore, the term encompasses forms produced through genetic engineering, such as chimeric antibodies (e.g., humanized murine antibodies) and heterologous antibodies (e.g., bispecific antibodies).

[0047] As used herein, "monoclonal antibody" is a term known in the art and refers to a highly specific antibody directed against a single antigenic site. Unlike polyclonal antibodies, which typically contain different antibodies directed against different epitopes (epitopes), monoclonal antibodies are directed against a single determinant on an antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical assays utilizing antigen-antibody binding, and furthermore, because they are synthesized through hybridoma culture, they have the advantage of being free from contamination by other immunoglobulins.

[0048] Typically, immunoglobulins have heavy and light chains, each of which comprises a constant region and a variable region (also known as a "domain"). The light chain variable region and the heavy chain variable region comprise three variable regions called "complementarity determining regions" (hereinafter referred to as "CDRs") and four "framework regions." The CDRs are primarily responsible for binding to epitopes on an antigen.

[0049] The CDRs of each chain are typically called CDR1, CDR2, and CDR3 sequentially starting from the N-terminus, and can also be identified by the chain on which a particular CDR is located.

[0050] The term "heavy chain" above may refer to both a full-length heavy chain and fragments thereof, which comprises a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and three constant region domains CH1, CH2, and CH3. The term "light chain" above may refer to both a full-length light chain and fragments thereof, which comprises a variable region domain VL comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a constant region domain CL.

[0051] The term "variable" as used herein refers to a specific region of sequence that differs between antibodies and is used to determine the binding specificity of each antibody for a specific antigen. The variability of an antibody may not be uniformly distributed throughout the antibody's variable domain, but rather may be concentrated in the CDRs. These CDRs may have unique sequences for each monoclonal antibody, and some or all of the CDRs may interact to allow a single monoclonal antibody to recognize a specific epitope.

[0052] The term "phage display technology" used in the present invention refers to a technology for selecting antibodies that exhibit antigen binding ability by selecting only phages that exhibit significant binding ability to a target antigen from a phage library. In the above technology, "panning" refers to a process for selecting only phages that express peptides on their surface that have the property of binding to a target molecule (antibody, enzyme, cell surface receptor, etc.) from a phage library that expresses peptides on the outer wall (coat) of the phage. By repeating this process 3 to 10 times, antibodies that exhibit significant binding ability to a target antigen can be selected, and the selected antibodies can be manufactured into humanized monoclonal antibodies.

[0053] The monoclonal antibody of the present invention may include variants of the amino acid sequence set forth in the attached sequence listing within the range capable of specifically binding to Chi3L1. For example, the amino acid sequence of the monoclonal antibody may be changed to improve the binding affinity and / or other biological properties of the monoclonal antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the monoclonal antibody. Such amino acid mutations may be made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; Phenylalanine, tryptophan, and tyrosine are biologically functional equivalents. When introducing mutations, the hydrophobicity index of an amino acid can be considered.

[0054] Additionally, equivalent or similar biological activity can be achieved by substitution between amino acids having similar hydrophilicity values.

[0055] Specifically, the anti-CHI3L1 antibody according to the present invention is a monoclonal antibody that specifically binds to CHI3L1, and comprises (1) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 5; a CDR2 consisting of the amino acid sequence of SEQ ID NO: 6; and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 7; and a light chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 11; a CDR2 consisting of the amino acid sequence of SEQ ID NO: 12; and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 13, or (2) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 8; a CDR2 consisting of the amino acid sequence of SEQ ID NO: 9; and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 10; and a light chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 14, a CDR2 consisting of the amino acid sequence of SEQ ID NO: 15; and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 16.

[0056] In addition, the anti-CHI3L1 antibody according to the present invention may include (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1; and (b) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3.

[0057] In addition, the anti-CHI3L1 antibody according to the present invention may include (a) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2; and (b) a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 4.

[0058] In the following examples of the present invention, experiments were performed using the 3A10-F1 (H1) antibody, a humanized monoclonal antibody that specifically binds to CHI3L1, as an anti-CHI3L1 antibody according to the present invention.

[0059] The pharmaceutical composition of the present invention may additionally include a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable" means non-toxic to cells or humans exposed to the composition. Any carrier known in the art, such as buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, bases, excipients, lubricants, etc., may be used without limitation.

[0060] In addition, the pharmaceutical composition of the present invention can be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, according to conventional methods. Furthermore, it can be used in the form of external preparations for skin in the form of ointments, lotions, sprays, patches, creams, powders, suspensions, gels, or gels. Carriers, excipients, and diluents that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulating, the composition may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0061] Solid preparations for oral administration include, for example, tablets, pills, powders, granules, and capsules, and these solid preparations can be prepared by mixing one or more excipients, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition to excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include, for example, suspensions, oral solutions, emulsions, and syrups, and in addition to diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives can be included.

[0062] Formulations for parenteral administration include, for example, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0063] The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. The term "administration" as used herein refers to introducing a given substance into a subject using an appropriate method, and the composition can be administered via any common route as long as it can reach the target tissue. Examples include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration.

[0064] In addition, the present invention can provide a biomarker composition for diagnosing atopic dermatitis, which comprises CXCL8 protein or a gene encoding the same as an active ingredient, and can also provide a composition for diagnosing atopic dermatitis, which comprises an agent capable of specifically detecting CXCL8 protein or a gene encoding the same.

[0065] Furthermore, the present invention can provide a cosmetic composition for preventing or improving atopic dermatitis, which comprises an anti-CHI3L1 antibody as an active ingredient.

[0066] The anti-CHI3L1 antibody, which is an effective ingredient contained in the cosmetic composition of the present invention, is as described above.

[0067] The cosmetic composition of the present invention can be manufactured into any formulation commonly manufactured in the art, and for example, can be formulated into a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, pack, massage cream, and spray, but is not limited thereto. More specifically, it can be manufactured into a formulation of a flexible toner, a nourishing toner, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

[0068] When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.

[0069] When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

[0070] When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth, etc. can be used as a carrier component.

[0071] When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and particularly in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether may be additionally included.

[0072] When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester may be used as a carrier component.

[0073] When the cosmetic composition of the present invention is soap, a surfactant-containing cleansing formulation, or a surfactant-free cleansing formulation, it can be applied to the skin and then wiped off, removed, or washed off with water. As specific examples, the soap is liquid soap, powder soap, solid soap, and oil soap, the surfactant-containing cleansing formulation is cleansing foam, cleansing water, cleansing towel, and cleansing pack, and the surfactant-free cleansing formulation is cleansing cream, cleansing lotion, cleansing water, and cleansing gel, but is not limited thereto.

[0074] In addition, the present invention can provide a food composition for preventing or improving atopic dermatitis, which comprises an anti-CHI3L1 antibody as an active ingredient, and can also provide a health functional food for preventing or improving atopic dermatitis, which comprises the composition.

[0075] The anti-CHI3L1 antibody included as an effective ingredient in the health functional food according to the present invention is as described above.

[0076] The health functional food of the present invention can be used by adding the anti-CHI3L1 antibody of the present invention as is or together with other foods or food ingredients, and can be used appropriately according to a conventional method.

[0077] There are no specific restrictions on the types of foods mentioned above. Examples of foods to which the composition can be added include beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and include all health foods in the conventional sense.

[0078] In addition to the above, the composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination.

[0079] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to more specifically illustrate the present invention, and the scope of the present invention is not limited to these examples.

[0080]

[0081] Experimental Methods and Materials

[0082] Preparation of experimental animals

[0083] Animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC) of the Chungbuk National University Laboratory Animal Research Center (CBNUA-1693-21-01). Every effort was made to minimize animal suffering and reduce the number of animals used. All mice were housed in cages of three per cage, in an automatically controlled environment with temperature (21-25°C), relative humidity (45-65%), and a 12-h light-dark cycle, with free access to food and water.

[0084]

[0085] Preparation of an animal model of atopic dermatitis symptoms

[0086] Atopic dermatitis-like skin inflammation was induced in HR-1 (8-week-old, n=10 / each group) mice by applying AOO (vehicle; acetone:olive = 4:1) or 5% phthalic anhydride (PA) solution (100 μL of AOO solution, 3 times a week for 4 weeks) to the dorsal skin. After application of the PA solution, the anti-CHI3L1 antibody of the present invention (0.1 or 0.2 mg / kg; dissolved in saline) or anti-IL-4R antibody (0.2 mg / kg; dissolved in saline) was injected subcutaneously under the dorsal skin. The mice were then sacrificed, and blood samples were collected. In addition, the anti-IL-4R antibody used in this experiment was purchased from Origene (#AP20570PU-N, RRID:AB_10756826, Rockville, MA).

[0087]

[0088] Serum samples from patients with atopic dermatitis

[0089] Human serum samples from patients diagnosed with atopic dermatitis and normal controls were obtained from the Chungbuk National University Hospital Biobank and the Korea Institute of Radiology Radiological Sciences Radiation Biobank. All studies using human samples were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of Chungbuk National University Hospital (IRB No. CBNU-201902-BR-786-01).

[0090]

[0091] Body weight and lymph node weight measurement

[0092] Body weight changes were measured once a week for 4 weeks using an electronic scale (Mettler Toledo, Greifensee, Switzerland). The weights of lymph nodes collected from sacrificed mice were also measured using the same method.

[0093]

[0094] Preparation of a reconstituted human skin model (RHS) similar to atopic dermatitis

[0095] Epidermis and dermis, including reconstituted human skin (RHS) models (Neoderm-ED), were purchased from TEGO Science Inc. (Seoul, Korea). The RHS model exhibits an atopic dermatitis (AD)-like inflammatory response because it was composed of an inflammatory cocktail described according to the company's specifications with some modifications. The inflammatory cocktail is an AD-inducing cocktail consisting of 30 ng / mL IL-4, 30 ng / mL IL-13, and 3.5 ng / mL TNF-α (PeproTech Inc., Rocky Hill, NJ). Antibodies against the cocktail were added or not to the culture medium for 6 days. The culture medium was changed every 48 hours. RHS models were transiently transfected with CHI3L1 siRNA or CXCL8 siRNA using RNAiMAX reagent in OPTI-MEM according to the manufacturer's specifications (Invitrogen). CHI3L1 siRNA and CXCL8 siRNA were purchased from Origene (Rockville, MA).

[0096]

[0097] Immunohistochemistry (IHC)

[0098] Dorsal skin removed from mice and RHS tissues was fixed in 4% formalin, embedded in paraffin wax, and sectioned at 5 μm thickness. The skin sections were then stained with hematoxylin and eosin (H&E). Epidermal thickness was measured under a light microscope (Olympus, Tokyo, Japan), and mast cells were stained with toluidine blue solution according to the manufacturer's instructions.

[0099]

[0100] ELISA analysis

[0101] IgE and cytokines in skin tissue and serum from the RHS model were measured using ELISA kits purchased from KOMA Biotech (Seoul, Korea) and R&D Systems (Minneapolis, MN) according to the manufacturers' instructions.

[0102] Whole blood from mice was placed in BD Microtainer blood collection tubes (#365967, BD Biosciences, East Rutherford, NJ) and centrifuged at 1,000 g for 15 minutes to isolate serum. Additionally, 100 mg of mouse skin tissue was homogenized using PRO-PREP™ protein extraction solution (#17081, iNtRON, Seongnam, Korea) and lysed by incubation on ice for 60 minutes. The skin tissue was then centrifuged at 13,000 g for 20 minutes at 4°C, and a total of 100 μg of the separated protein was applied to the ELISA kit. Serum IgE and cytokines from skin tissue or RHS models were measured using ELISA kits purchased from KOMA Biotech (Seoul, Korea) and R&D Systems (Minneapolis, MN) according to the manufacturers' instructions, and cytokine concentrations were normalized to the total protein amount.

[0103]

[0104] Western blotting

[0105] 100 mg of skin tissue or RHS samples were collected and homogenized in lysis buffer (Pro-prep protein extraction buffer; iNtRON, Seongnam, Korea). Nuclear extracts were also obtained from the samples using a nuclear extraction kit (Abcam, Cambridge, MA). The membrane was incubated with specific antibodies [rabbit polyclonal antibodies against CHI3L1 (dilution 1:1000; #A3166, RRID:AB_2764957, Abclonal, Wuhan, China), STAT3 (dilution 1:1000; #9139, RRID:AB_331757, Cell Signaling Technology, Beverly, MA), and phospho-STAT3 (dilution 1:1000; #9145, RRID:AB_2491009, Cell Signaling Technology) and mouse monoclonal antibody against β-actin (dilution 1:5000; #sc-517582, RRID:AB_2833259, Santa Cruz Biotechnology, Dallas, TX)] for 4 h at room temperature. The intensity of protein detection bands was measured using a Fusion SOLO S image acquisition system (Vilber Lourmat, Eberhardzell, Germany), and band intensities were measured using ImageJ software (NIH, Bethesda, MA).

[0106]

[0107] Quantitative real-time PCR analysis

[0108] Total RNA from skin and RHS tissues was extracted using the RiboEX RNA Extraction Kit (#301-001, GeneAll Biotechnology, SEOUL, Korea), and cDNA was synthesized using the High Performance RNA-to-cDNA kit (Applied Biosystems, Foster City, CA). Quantitative real-time PCR was performed using RT-LAMP 2X Master Mix (SYBR green) (#EBT-1722, ELPIS Biotech, Daejeon, Korea) and specific primers in a StepOnePlus™ PCR system (Applied Biosystems). The values ​​obtained for target gene expression were normalized to 18S rRNA and quantified relative to the expression of the control sample. The primers used for quantitative real-time PCR are shown in Table 1 below.

[0109]

[0110]

[0111] Cell culture and transfection

[0112] The HaCaT human keratinocyte cell line was purchased from the American Type Culture Collection (RRID: CVCL_0038, ATCC, Manassas, VA). Cells were cultured at 37°C in DMEM medium supplemented with 10% FBS, penicillin (100 units / mL), and streptomycin sulfate (100 μg / mL) in a humidified atmosphere of 5% carbon dioxide. Cells were incubated with various concentrations of anti-CHI3L1 antibody (100–500 ng / mL in saline) and then stimulated with an atopic dermatitis cocktail. STAT3 siRNA and CXCL8 siRNA were purchased from Origene (Rockville, MA), and HaCaT cells were transiently transfected with STAT3 siRNA or CXCL8 siRNA using RNAiMAX reagent in OPTI-MEM according to the manufacturer's instructions.

[0113]

[0114] Flow cytometry

[0115] Lymph nodes were stained with anti-CD11c+ (#553802; RRID:AB-395061, #550261; RRID:AB_398460, BD bioscience), anti-CD4 (#553051; RRID:AB_398528, #553730; RRID:AB_395014, BD Bioscience), anti-CD8a (#553035; RRID:AB_398527, #553033; RRID:AB_394571, BD Bioscience), and anti-CD8b (#569946, #568907, BD Bioscience). Additionally, APC rat IgG1 (#554686, RRID:AB_398577, BD biosciences), PE rat IgG2a (#553930, RRID:AB_479719, BD bioscience), PE hamster IgG (#553954, RRID:AB_395158, BD bioscience), and APC hamster IgG1 (#553974, RRID:AB_398541, BD bioscience) were used as isotype controls, and intracellular staining was performed using the Fixation / Permeabilization Solution kit (#554722, BD Bioscience) according to the manufacturer's specifications. Samples were analyzed using a BD FACS Calibur™ FlowCytometer (BD Biosciences), and data were extracted using CellQuest Pro v6.0 software (BD Biosciences).

[0116]

[0117] Protein-protein interaction networks and analysis of atopic dermatitis patients

[0118] The protein-protein interaction network of CHI3L1 was analyzed using the HumanBase database (https: / hb.flaironinstitute.org / ; RRID:SCR_016145) and GeneMANIA (https: / genemania.org / ; RRID:SCR_005709), which are publicly available sources of protein-protein interaction information.

[0119]

[0120] Statistical analysis

[0121] Experiments were performed three times, and all experiments were repeated at least three times. All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA). Group differences were analyzed using one-way ANOVA and Tukey's multiple comparison test. All values ​​are expressed as mean ± SD, and significance was set at p < 0.05 for all tests.

[0122]

[0123] ingredient

[0124] Acetone, formalin, hematoxylin, eosin, methanol, and BSA were purchased from Sigma Ltd (St. Louis, MO, USA).

[0125]

[0126] <Example 1>

[0127] Preparation of anti-CHI3L1 antibodies that specifically bind to CHI3L1: Phage display library panning.

[0128] Antibodies to Chi3L1 that showed significant binding affinity to Chi3L1 were selected using phage display technology. Binding affinity to hChi3L1 was confirmed using ELISA with hChi3L1 phage. Briefly, 30 ng of hChi3L1 antigen was immobilized per well, and the wells with immobilized antigen were blocked with MPBS (5% skim milk in PBS), followed by addition of hChi3L1 phage and mChi3L1 phage for reaction. To detect bound phage, the wells were washed four times with PBST (0.05% Tween 20 in PBS), and then anti-M13-HRP (1:5,000) was added for reaction. Finally, after washing four times with PBST, TMB (3,3',5,5'-Tetramethylbenzidine) substrate was added to induce a color reaction, and the reaction was terminated by adding 2N H2SO4. The absorbance of each well was measured at a wavelength of 450 nm.

[0129] As a result, seven clones with antibodies against hChi3L1 were selected from the Fab-I library and Fab-II library, and among them, 3A10-F1 (H1), which showed the lowest EC50 value, was selected for further experiments.

[0130] In addition, antibodies showing significant binding ability to mChi3L1 were selected, and the binding ability to mChi3L1 was confirmed through ELISA for mChi3L1 phage. Briefly, 30 ng of mChi3L1 antigen was immobilized per well, and the wells with immobilized antigens were blocked with MPBS (5% skim milk in PBS), and then hChi3L1 phage and mChi3L1 phage were added and reacted. To detect the bound phage, after washing four times with PBST (0.05% Tween 20 in PBS), Anti-M13-HRP (1:5,000) was added and reacted. Finally, after washing four times with PBST, TMB (3,3',5,5'-Tetramethylbenzidine) substrate was added to induce a color reaction, and the reaction was terminated by adding 2N H2SO4, and the absorbance of each well was measured at a wavelength of 450 nm. As a result, six antibodies were selected from the Fab-II library, while no clones were formed from the Fab-I or scFv libraries. Among them, the antibody with the lowest EC50 value was 4E3-F2 (M3).

[0131] The amino acid sequence information of the 3A10-F1 (H1) antibody and the 4E3-F2 (M3) antibody, which are anti-CHI3L1 antibodies of the present invention that specifically bind to the above-mentioned final selected CHI3L1, is as shown in Table 2 below. In addition, in the examples below, the 3A10-F1 (H1) antibody was used in the experiments.

[0132] <h2 style=";text-align:left;direction:ltr">항체서열정보서열번호3A10-F1 (H1)항체의 중쇄 항체의 중쇄 항체의 항체의 경쇄 DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSFPLTFGQGTKVEIK43A10-F1 (H1) CDRH1NYAMS53A10-F1 (H1) CDRH2GISGSGGTTYYADSVKG63A10-F1 (H1) CDRH3VGTFDV74E3-F2 (M3) CDRH1SYDIH84E3-F2 (M3) CDRH2IISPYLGITIYAQKFQG94E3-F2 (M3) CDRH3RYFYYQSEAFDY103A10-F1 (H1) CDRL1RASQTISSWLN113A10-F1 (H1) CDRL2AASRLQS123A10-F1 (H1) CDRL3QQSYSTPLT134E3-F2 (M3) CDRL1RASQSISNYLN144E3-F2 (M3) CDRL2AASTLQS154E3-F2 (M3) CDRL3QQSYSFPLT16<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0133] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0134] <h2 style=";text-align:left;direction:ltr"> <실시예 2><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0135] Analysis of the Effect of Anti-CHI3L1 Antibodies on the Progression of Atopic Dermatitis

[0136] The present inventors conducted an analysis to determine whether anti-CHI3L1 antibody treatment could prevent, improve, and treat atopic dermatitis (AD). To this end, we analyzed epidermal thickness and histological changes associated with anti-CHI3L1 antibody treatment in mice induced with AD.

[0137] As a result, as shown in Fig. 1a, changes in epidermal thickness and histological changes were observed by anti-CHI3L1 antibody treatment, and in the PA (phthalic anhydride) treatment group, clinical atopic dermatitis symptoms (clinical scores) such as erythema, edema, and erosion increased compared to the control group, whereas the anti-CHI3L1 antibody treatment group of the present invention was shown to alleviate clinical atopic dermatitis symptoms induced by PA (Fig. 1b). In addition, the anti-CHI3L1 antibody treatment group of the present invention was shown to have a decrease in epidermal thickness increased by PA treatment (Fig. 1c), and the number of mast cell infiltration was also shown to have decreased (Fig. 1d).

[0138] In addition, the effect of the anti-CHI3L1 antibody of the present invention on neutrophils was confirmed. When PA-treated mice were compared with control mice, the number of Ly6G+ cells (neutrophils) increased, but the group treated with the anti-CHI3L1 antibody effectively reduced the increase in such neutrophils (Fig. 1e).

[0139] Additionally, it is well known that one of the characteristics of atopic dermatitis is excessive IgE production. Therefore, serum IgE concentrations were measured in mice induced with PA and mice treated with anti-CHI3L1 antibodies, and it was confirmed that the serum IgE level increased by PA could be significantly reduced by dose-dependent administration of the anti-CHI3L1 antibody of the present invention (Fig. 1f).

[0140] Furthermore, lymph nodes are known to participate in the process of regulating immune activity in atopic dermatitis. Accordingly, the inventors of the present invention measured the lymph node weight of mice in which atopic dermatitis was induced by PA treatment and found that the lymph node weight increased. On the other hand, the group treated with the anti-CHI3L1 antibody of the present invention showed a decrease in the lymph node weight increased by PA (Fig. 2a). According to lymph node analysis, PA treatment increased CD4+ T cells, CD8+ T cells, and CD11c+ dendritic cells, but it was confirmed that the increase in these cells was suppressed by treatment with the anti-CHI3L1 antibody of the present invention (Figs. 2b-2d).

[0141] In addition, in a PA-induced atopic dermatitis mouse model, the preventive, ameliorating, and therapeutic effects of atopic dermatitis by treatment with the anti-CHI3L1 antibody of the present invention were shown to be superior to those by treatment with the IL-4R antibody (see Figs. 1 and 2).

[0142]

[0143] <Example 3>

[0144] Analysis of the effects of anti-CHI3L1 antibodies on inflammatory cytokine release

[0145] To determine the effect of anti-CHI3L1 antibody on the release of inflammatory cytokines in the skin tissue of mice with atopic dermatitis induced by PA, the mRNA and protein levels of CHI3L1, IL-1β, IL-4, IL-6, CXCL1, and TSLP were measured in the skin tissue of the mice.

[0146]

[0147] As a result, the expression levels of these inflammatory mediators were found to be significantly increased in the group in which atopic dermatitis was induced by PA treatment compared to the normal control group. Meanwhile, the group treated with the anti-CHI3L1 antibody of the present invention showed a decrease in both the mRNA expression levels and protein levels of inflammatory cytokines in a dose-dependent manner, and the effect of reducing the inflammatory cytokine levels by the anti-CHI3L1 antibody treatment was found to be more superior than that of the IL-4R antibody treatment group (Figs. 3a and 3b).

[0148]

[0149] <Example 4>

[0150] Analysis of anti-inflammatory activity of anti-CHI3L1 antibody treatment in atopic dermatitis-like reconstituted human skin model (RHS) and HaCaT cells.

[0151] To confirm the anti-inflammatory effect of anti-CHI3L1 antibodies on atopic dermatitis, an atopic dermatitis-like human skin model was used. First, changes in epidermal thickness were analyzed in response to anti-CHI3L1 antibody treatment in a reconstituted human skin model (RHS) and HaCaT cell lines.

[0152]

[0153] As a result, when the anti-CHI3L1 antibody of the present invention was treated in an atopic dermatitis-like reconstructed human skin model (RHS), the epidermal thickness increased by atopic dermatitis was significantly reduced (Fig. 4a), and the increased expression and production of inflammatory cytokines IL-1β, IL-6, CXCL8, TSLP, and CHI3L1 induced by the atopic dermatitis cocktail were also inhibited by the anti-CHI3L1 antibody treatment (Figs. 4b and 4c).

[0154] In addition, the anti-inflammatory effect of anti-CHI3L1 antibody treatment on human keratinocyte HaCaT cells was investigated, and the results showed that the release and expression levels of inflammatory cytokines were reduced by anti-CHI3L1 antibody treatment (Figures 5a and 5b), and the anti-inflammatory and atopic dermatitis prevention effects of anti-CHI3L1 antibody treatment were found to be superior to those of the group treated with IL-4R antibody.

[0155]

[0156] <Example 5>

[0157] Analysis of the effect of anti-CHI3L1 antibodies on STAT3 signaling

[0158] STAT3 is known to be a transcription factor associated with inflammatory responses in atopic dermatitis models, and STAT3 activation is known to induce transcription of inflammatory cytokines and chemokines, including IL-1, IL-6, IL-23, and TNF-α, contributing to the persistence of skin inflammatory responses. Furthermore, STAT3 activation is known to promote keratinocyte hyperproliferation and epidermal proliferation, contributing to the increased skin thickness observed in atopic dermatitis. Therefore, the present inventors determined whether the anti-CHI3L1 antibody of the present invention could modulate STAT3 signaling in relation to the therapeutic efficacy of atopic dermatitis.

[0159]

[0160] To this end, first, to investigate whether the anti-CHI3L1 antibody of the present invention can inhibit STAT3 phosphorylation in an atopic dermatitis model treated with PA, changes in the expression levels of phosphorylated STAT3 and CHI3L1 due to anti-CHI3L1 antibody treatment were analyzed using Western blot and immunohistochemical analysis.

[0161] As a result, the PA-treated mouse tissues showed increased expression of CHI3L1 and phosphorylated STAT3, but when treated with the anti-CHI3L1 antibody of the present invention, the expression of CHI3L1 and phosphorylated STAT3 was suppressed in a treatment dose-dependent manner (Figs. 6a and 6b). In addition, the RHS tissues treated with the atopic dermatitis-inducing cocktail showed a significant increase in the phosphorylation of STAT3 and the expression of CHI3L1 compared to the control group, whereas the increased phosphorylation of STAT3 and the expression of CHI3L1 were significantly reduced by the anti-CHI3L1 antibody treatment of the present invention (Fig. 6c). These results were also observed in the HaCaT cell line treated with an atopic dermatitis-inducing cocktail. It was confirmed that the anti-CHI3L1 antibody-treated group significantly suppressed the increase in the expression of p-STAT3 and CHI3L1 induced by the atopic dermatitis-inducing cocktail treatment (Fig. 6d). Furthermore, it was confirmed that the inhibitory effect of the anti-CHI3L1 antibody on the expression of p-STAT3 and CHI3L1 was superior to that of the IL-4R antibody.

[0162] Based on this, the inventors of the present invention were able to determine that the anti-CHI3L1 antibody of the present invention can regulate the expression levels of p-STAT3 and CHI3L1, and thus can prevent, improve, and treat atopic dermatitis by inhibiting the signal transduction of STAT3 involved in atopic dermatitis.

[0163]

[0164] <Example 6>

[0165] Analysis of the Relationship Between CHI3L1-Mediated Atopic Dermatitis and CXCL8

[0166] The results of the previously confirmed examples confirmed that CHI3L1 is related to skin inflammation of atopic dermatitis. Therefore, the present inventors performed a HumanBase protein-protein interaction network analysis to determine which factors are involved in the inflammation induced by CHI3L1 in the development of atopic dermatitis. As a result, it was found that CHI3L1 was associated with TGF-β1, NCF2, CTSS, CXCL8, and CD14. In particular, among these, CXCL8 showed the highest increase in expression level by recombinant CHI3L1 treatment (Fig. 7). In addition, the present inventors were able to confirm that the mRNA levels of CHI3L1 and CXCL8 were also increased in CHI3L1-overexpressing HaCaT cells (Fig. 8b), and conversely, it was confirmed that CHI3L1 knockdown decreased the mRNA levels of CHI3L1 and CXCL8 (Fig. 8c).

[0167] In addition, the present inventors verified whether CXCL8 is involved in the CHI3L1-mediated skin inflammatory response. Treatment with CXCL8 was shown to increase the mRNA levels of atopic dermatitis-related cytokines IL-1β, IL-6, CXCL8, TSLP, and CHI3L1, and the group treated with an atopic dermatitis-inducing cocktail showed a further increase in the expression levels of these cytokines (Fig. 8d). Meanwhile, the group in which CXCL8 was knocked down (si-CXCL treatment group) showed a decrease in the mRNA levels of cytokines increased by the atopic dermatitis-inducing cocktail (Fig. 8e).

[0168] Furthermore, to verify whether the above results were confirmed in an analysis performed on serum samples from atopic dermatitis patients, the inventors investigated the levels of CHI3L1 and CXCL8 in serum samples from atopic dermatitis patients.

[0169] As a result, it was observed that the concentration of CHI3L1 was increased in the serum of atopic dermatitis patients compared to the healthy control group (Fig. 9a), and the ROC curve analysis results showed that CHI3L1 had a sensitivity of 86.7%, a specificity of 80.0%, a cut-off value of 31.33 ng / ml, and an area under the curve of 0.873 (Fig. 9a). In addition, the serum level of CXCL8 was found to be increased in the patient group compared to the healthy control group, and the ROC curve analysis results of serum CXCL8 showed that the sensitivity, specificity, cut-off value, and area under the curve were 73.33%, 93.33%, 278.57 pg / ml, and 0.902, respectively (Fig. 9b). In addition, Pearson's correlation analysis confirmed that there was a correlation between the serum levels of CHI3L1 and CXCL8 in patients (Fig. 9c).

[0170] Through these results, the inventors of the present invention were able to find that CHI3L1 and CXCL8 are significantly increased in expression when atopic dermatitis occurs, and that this increase in expression has high sensitivity and specificity, so that they can be usefully used as biomarkers for the diagnosis of atopic dermatitis.

[0171]

[0172] The present invention has been described above, focusing on preferred embodiments thereof. Those skilled in the art will appreciate that the present invention can be implemented in modified forms without departing from its essential characteristics. Therefore, the disclosed embodiments should be considered illustrative rather than limiting. The scope of the present invention is set forth in the claims, not the foregoing description, and all differences within the scope equivalent thereto should be construed as being encompassed by the present invention.

Claims

1. A pharmaceutical composition for preventing or treating atopic dermatitis, comprising an anti-CHI3L1 antibody as an active ingredient.

2. In paragraph 1, The above anti-CHI3L1 antibody is a monoclonal antibody that specifically binds to CHI3L1. (1) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 5; a CDR2 consisting of the amino acid sequence of SEQ ID NO: 6; and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 7; and A light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 11; a CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13, or (2) A pharmaceutical composition for preventing or treating atopic dermatitis, characterized by comprising a heavy chain variable region comprising a CDR1 consisting of an amino acid sequence of SEQ ID NO: 8; a CDR2 consisting of an amino acid sequence of SEQ ID NO: 9; and a CDR3 consisting of an amino acid sequence of SEQ ID NO: 10; and a light chain variable region comprising a CDR1 consisting of an amino acid sequence of SEQ ID NO: 14; a CDR2 consisting of an amino acid sequence of SEQ ID NO: 15; and a CDR3 consisting of an amino acid sequence of SEQ ID NO:

16.

3. In paragraph 1, A pharmaceutical composition for preventing or treating atopic dermatitis, characterized in that the anti-CHI3L1 antibody comprises (a) a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 1; and (b) a light chain variable region consisting of an amino acid sequence of SEQ ID NO:

3.

4. In paragraph 1, A pharmaceutical composition for preventing or treating atopic dermatitis, characterized in that the anti-CHI3L1 antibody comprises (a) a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 2; and (b) a light chain variable region consisting of an amino acid sequence of SEQ ID NO:

4.

5. In paragraph 1, The above anti-CHI3L1 antibody, Reduces the thickness of the skin epidermis increased by atopic dermatitis; Inhibits mast cell infiltration; Reduce the weight of the lymph nodes; Inhibits the expression and production of inflammatory cytokines; and A pharmaceutical composition for preventing or treating atopic dermatitis, characterized in that it can prevent, improve or treat atopic dermatitis by inhibiting the expression of p-STAT3 and CHI3L1.

6. In paragraph 5, A pharmaceutical composition for preventing or treating atopic dermatitis, characterized in that the inflammatory cytokine is IL-1β, IL-4, CXCL8 or TSLP.

7. A cosmetic composition for preventing or improving atopic dermatitis, comprising an anti-CHI3L1 antibody as an active ingredient.

8. In paragraph 7, The above anti-CHI3L1 antibody is a monoclonal antibody that specifically binds to CHI3L1. (1) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 5; a CDR2 consisting of the amino acid sequence of SEQ ID NO: 6; and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 7; and A light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 11; a CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13, or (2) A cosmetic composition for preventing or improving atopic dermatitis, characterized by comprising a heavy chain variable region comprising a CDR1 consisting of an amino acid sequence of SEQ ID NO: 8; a CDR2 consisting of an amino acid sequence of SEQ ID NO: 9; and a CDR3 consisting of an amino acid sequence of SEQ ID NO: 10; and a light chain variable region comprising a CDR1 consisting of an amino acid sequence of SEQ ID NO: 14; a CDR2 consisting of an amino acid sequence of SEQ ID NO: 15; and a CDR3 consisting of an amino acid sequence of SEQ ID NO:

16.

9. In paragraph 7, A cosmetic composition for preventing or improving atopic dermatitis, characterized in that the anti-CHI3L1 antibody comprises (a) a heavy chain variable region consisting of an amino acid sequence of sequence number 1; and (b) a light chain variable region consisting of an amino acid sequence of sequence number 3.

10. In paragraph 7, A cosmetic composition for preventing or improving atopic dermatitis, characterized in that the anti-CHI3L1 antibody comprises (a) a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 2; and (b) a light chain variable region consisting of an amino acid sequence of SEQ ID NO:

4.

11. A health functional food for preventing or improving atopic dermatitis, containing anti-CHI3L1 antibody as an active ingredient.

12. In paragraph 11, The above anti-CHI3L1 antibody is a monoclonal antibody that specifically binds to CHI3L1. (1) a heavy chain variable region comprising a CDR1 consisting of the amino acid sequence of SEQ ID NO: 5; a CDR2 consisting of the amino acid sequence of SEQ ID NO: 6; and a CDR3 consisting of the amino acid sequence of SEQ ID NO: 7; and A light chain variable region comprising a CDR1 comprising the amino acid sequence of SEQ ID NO: 11; a CDR2 comprising the amino acid sequence of SEQ ID NO: 12; and a CDR3 comprising the amino acid sequence of SEQ ID NO: 13, or (2) A health functional food for preventing or improving atopic dermatitis, characterized by comprising a heavy chain variable region comprising a CDR1 consisting of an amino acid sequence of SEQ ID NO: 8; a CDR2 consisting of an amino acid sequence of SEQ ID NO: 9; and a CDR3 consisting of an amino acid sequence of SEQ ID NO: 10; and a light chain variable region comprising a CDR1 consisting of an amino acid sequence of SEQ ID NO: 14; a CDR2 consisting of an amino acid sequence of SEQ ID NO: 15; and a CDR3 consisting of an amino acid sequence of SEQ ID NO:

16.

13. In paragraph 11, A health functional food for preventing or improving atopic dermatitis, characterized in that the anti-CHI3L1 antibody comprises (a) a heavy chain variable region consisting of an amino acid sequence of sequence number 1; and (b) a light chain variable region consisting of an amino acid sequence of sequence number 3.

14. In paragraph 11, A health functional food for preventing or improving atopic dermatitis, characterized in that the anti-CHI3L1 antibody comprises (a) a heavy chain variable region consisting of an amino acid sequence of sequence number 2; and (b) a light chain variable region consisting of an amino acid sequence of sequence number 4.

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