Composition for preventing or treating rheumatoid arthritis, comprising human monoclonal antibody specific to chi3l1

Abstract

The present invention relates to a composition for preventing or treating rheumatoid arthritis, comprising a human monoclonal antibody specific to CHI3L1. Specifically, the present invention relates to a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising an anti-CHI3L1 antibody as an active ingredient, and a health functional food for preventing or alleviating rheumatoid arthritis.

Description

Composition for preventing or treating rheumatoid arthritis comprising a human monoclonal antibody specific for HI3L1

[0001] The present invention relates to a composition for preventing or treating rheumatoid arthritis comprising a human monoclonal antibody specific for HI3L1.

[0002] Rheumatoid arthritis is a chronic, systemic, inflammatory autoimmune disease characterized by persistent inflammation of the joint lining, leading to swelling and pain, as well as destruction of joint cartilage and bone damage. Typical early symptoms include swelling and pain in the joints of the hands and feet, as well as morning stiffness and difficulty moving. Furthermore, it is known to be accompanied by symptoms beyond joints, such as osteoporosis, vasculitis, and organ involvement.

[0003] Although no cure for rheumatoid arthritis has been developed to date, nonsteroidal anti-inflammatory drugs, immunosuppressants, and antirheumatic drugs are used as medications for the treatment of rheumatoid arthritis.

[0004] However, nonsteroidal anti-inflammatory drugs have the effect of relieving pain, but they do not stop the progression of the disease and can cause gastrointestinal disorders when taken for a long time, and immunosuppressants such as TNF-α inhibitors can cause side effects such as latent tuberculosis.

[0005] Therefore, there is a need to develop new treatments that can effectively suppress rheumatoid arthritis while having fewer side effects compared to previously developed drugs.

[0006] 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.

[0007] Meanwhile, there has been no research on the development of a treatment for rheumatoid arthritis using a specific antibody against CHI3L1 that can suppress the pathogenic action of CHI3L1.

[0008] Accordingly, the inventors of the present invention prepared a monoclonal antibody specific to CHI3L1, and confirmed that rheumatoid arthritis 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.

[0009] Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising an anti-CHI3L1 antibody as an active ingredient.

[0010] Another object of the present invention is to provide a health functional food for preventing or improving rheumatoid arthritis, comprising an anti-CHI3L1 antibody as an active ingredient.

[0011] In order to achieve the above-described purpose of the present invention, the present invention provides a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising an anti-CHI3L1 antibody as an active ingredient.

[0012] 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.

[0013] 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.

[0014] 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.

[0015] In one embodiment of the present invention, the anti-CHI3L1 antibody may have an activity of inhibiting the expression of iNOS and COX-2; inhibiting the production of inflammatory cytokines TNF-α, IL-1β, and IL-18; or inhibiting the production of the chemokine CCL2.

[0016] In addition, the present invention provides a health functional food for preventing or improving rheumatoid arthritis, comprising an anti-CHI3L1 antibody as an active ingredient.

[0017] The monoclonal antibody specific to CHI3L1 according to the present invention can suppress the expression of inflammatory mediators involved in the development of rheumatoid arthritis, suppress the expression and production of inflammatory cytokines and chemokines, and effectively improve and treat the symptoms of rheumatoid arthritis. Therefore, the monoclonal antibody specific to CHI3L1 of the present invention can be usefully used in the manufacture of pharmaceuticals and health functional foods that can prevent, improve, and treat rheumatoid arthritis.

[0018] Figure 1 shows the results of analyzing the effect of improving rheumatoid arthritis according to treatment with the anti-CHI3L1 antibody of the present invention in mice induced with rheumatoid arthritis using clinical scores. Control: untreated control group, CIA: collagen-induced group, H1 Ab group: anti-CHI3L1 antibody administration group of the present invention (2.5 and 5 mg / kg), anti-TNF-α Ab group: experimental group administered anti-TNF-α antibody (5 mg / kg).

[0019] Figure 2 shows the results of observing the degree of improvement in symptoms according to treatment with the anti-CHI3L1 antibody of the present invention in mice induced with rheumatoid arthritis using visual observation, radiography, H&E staining, and Safranin O staining.

[0020] Figure 3 shows the results of measuring changes in the paw thickness of mice induced with rheumatoid arthritis following treatment with the anti-CHI3L1 antibody of the present invention.

[0021] Figure 4 shows the results of Western blot analysis to confirm changes in the expression of inflammatory markers iNOS and COX-2 when treating mice with rheumatoid arthritis induced with the anti-CHI3L1 antibody of the present invention.

[0022] Figure 5 shows the results of confirming the effect of suppressing the expression of inflammatory cytokines when treating mice with rheumatoid arthritis with the anti-CHI3L1 antibody of the present invention.

[0023] The present invention is characterized by providing a composition for preventing or treating rheumatoid arthritis, which comprises a monoclonal antibody specific for CHI3L1 (Chitinase-3-like 1) as an active ingredient.

[0024] Chitinase-3-like 1 (CHI3L1) is a secreted heparin-binding glycoprotein expressed in various cell types, including 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. Furthermore, CHI3L1 is also known to be crucially involved in neuroinflammatory diseases such as atherosclerosis, liver damage, stroke, and Alzheimer's disease.

[0025] 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 rheumatoid arthritis.

[0026] 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.

[0027] 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 for 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 sera of patients with metastatic cancer or chronic inflammatory diseases.

[0028] Meanwhile, the inventors of the present invention have for the first time discovered that the monoclonal antibody of the present invention specific for CHI3L1 has excellent effects in preventing, improving, and treating rheumatoid arthritis.

[0029] According to one embodiment of the present invention, in a group treated with a monoclonal antibody specific to CHI3L1 of the present invention, i.e., an anti-CHI3L1 antibody, in mice in which rheumatoid arthritis was induced by collagen administration, the clinical score of rheumatoid arthritis was effectively reduced compared to a group that was not treated with the antibody, and the swelling and thickness of the feet increased by rheumatoid arthritis were also improved, and it was confirmed that the invasion of pannus tissue into the bone space and the destroyed joint structure were also improved, and the reduction of bone destruction and fibrosis was effectively suppressed. In addition, it was confirmed that the rheumatoid arthritis improvement effect according to the treatment with the monoclonal antibody specific to CHI3L1 of the present invention was almost similar to or superior to the effect of a group treated with an anti-TNF-α antibody known as a conventional rheumatoid arthritis treatment.

[0030] Furthermore, in another embodiment of the present invention, it was analyzed whether the monoclonal antibody specific to CHI3L1 of the present invention affects the expression of iNOS and COX-2, which are inflammatory markers associated with the onset of rheumatoid arthritis. As a result, it was confirmed that the antibody of the present invention can effectively reduce the expression levels of iNOS and COX-2 increased by the onset of rheumatoid arthritis. In addition, it was confirmed that the expression levels of inflammatory cytokines TNF-α, IL-1β, IL-18, and chemokine CCL2 were also effectively suppressed.

[0031] Therefore, through these results, the inventors were able to confirm that the monoclonal antibody specific to CHI3L1 of the present invention can be used as a new treatment agent for rheumatoid arthritis.

[0032] Therefore, the present invention can provide a pharmaceutical composition for preventing or treating rheumatoid arthritis, comprising an anti-CHI3L1 antibody as an active ingredient.

[0033] 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).

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] Additionally, equivalent or similar biological activity can be achieved by substitution between amino acids having similar hydrophilicity values.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] In the following examples of the present invention, experiments were performed using the 3A10-F1 (H1) antibody, which is a humanized monoclonal antibody that specifically binds to CHI3L1, as an anti-CHI3L1 antibody according to the present invention.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] In addition, the present invention can provide a food composition for preventing or improving rheumatoid arthritis, which comprises an anti-CHI3L1 antibody as an active ingredient, and can also provide a health functional food for preventing or improving rheumatoid arthritis, which comprises the composition.

[0052] The food composition of the present invention can be applied to, for example, various foods, beverages, gum, tea, vitamin complexes, health functional foods, etc., and can also be added to foods or beverages to produce a desired effect.

[0053] The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. The health beverage composition of the present invention has no particular limitations on other ingredients other than containing the above-mentioned effective ingredient as an essential ingredient in the indicated ratio, and may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, conventional sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g, per 100 ml of the composition of the present invention.

[0054] In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), 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, it may contain fruit pulp for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

[0055] 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.

[0056]

[0057] <Example 1>

[0058] Preparation of anti-CHI3L1 antibodies that specifically bind to CHI3L1: Phage display library panning.

[0059] Antibodies to Chi3L1 that showed significant binding affinity to Chi3L1 were selected using phage display technology. Binding affinity to hChi3L1 was confirmed by 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) before 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 reacted with Anti-M13-HRP (1:5,000). 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.

[0060] 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.

[0061] 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).

[0062] The 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 1] below.

[0063] Antibody sequence information SEQ ID NO: 3A10-F1 (H1) Amino acid sequence of the heavy chain variable region of the antibody EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSGISGSGGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAGVGTFDVWGQGTLVTVSS14E3-F2 (M3) Amino acid sequence of the heavy chain variable region of the antibody QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYDIHWVRQAPGQGLEWMGIISPYLGITIYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARRYFYYQSEAFDYWGQGTLVTVSS23A10-F1 (H1) Amino acid sequence of the light chain variable region of the antibody Amino acid sequence of the light chain variable region of the antibody DIQMTQSPSSLSASVGDRVTITCRASQTISSWLNWYQQKPGKAPKLLIYAASRLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPLTFGQGTKVEIK34E3-F2 (M3) 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

[0064]

[0065] <Example 2>

[0066] Confirmation of improvement and therapeutic effect of anti-CHI3L1 antibody treatment of rheumatoid arthritis in a mouse animal model of arthritis

[0067] All animal experiments conducted in the examples of the present invention were conducted in accordance with the guidelines (CBNUA-754-14-01) of the Institutional Animal Care and Use Committee (IACUC) of the Laboratory Animal Research Center of Chungbuk National University. Every effort was made to minimize animal suffering and reduce the number of animals used. The mice used in this experiment were male C57BL / 6 (7 weeks old) mice obtained from Taconic Korea (Daehan Biolink Co., Ltd., Umsung, Chungbuk, Korea). They were housed in a sterile cage with automatic temperature control (21-25°C), relative humidity (45-65%), and a 12-hour light-dark cycle, and food and water were available ad libitum.

[0068]

[0069] <2-1> Clinical score analysis

[0070] To determine whether the anti-CHI3L1 antibody of the present invention is effective in improving the symptoms of rheumatoid arthritis, the inventors produced mice in which rheumatoid arthritis was induced by administration of CIA (collagen), a well-known method in the art. Specifically, 7-week-old male C57BL / 6 mice were intravenously injected with 5 mg of a collagen II antibody cocktail (CII-Ab, Chondrex, Redmond, WA, USA) on day 0, and 50 μg (ip) was administered on day 3. The mice were visually inspected daily for the appearance of peripheral arthritis to produce mice that developed rheumatoid arthritis.

[0071] Thereafter, the anti-CHI3L1 antibody (3A10-F1(H1) antibody) of the present invention was administered to rheumatoid arthritis mice at 2.5 mg / kg and 5.0 mg / kg, respectively, and the clinical scores for rheumatoid arthritis symptoms over time were evaluated. At this time, a group administered with an anti-TNF-α antibody (5.0 mg / kg), known as a rheumatoid arthritis treatment, was used as a positive control group.

[0072]

[0073] As a result, as shown in Fig. 1, in the case of mice induced with rheumatoid arthritis, the clinical score of rheumatoid arthritis was found to increase significantly over time, whereas in the group treated with the anti-CHI3L1 antibody of the present invention, the symptoms of rheumatoid arthritis were alleviated and the clinical score for the disease was found to decrease, and this phenomenon was found to be almost similar to the group treated with the anti-TNF-α antibody.

[0074]

[0075] <2-2> Photographic analysis and tissue staining analysis

[0076] For the mouse group used in the above Example <2-1>, the hind paws of each mouse were analyzed by visual observation, X-ray photography, H&E staining, and Safranin O staining.

[0077]

[0078] As a result, as shown in Fig. 2, in mice induced with rheumatoid arthritis, swelling of the ankles and feet was significantly increased compared to the normal group, whereas in the group treated with the anti-CHI3L1 antibody of the present invention, the increased swelling of the ankles and feet was significantly reduced, and it was confirmed that the improvement in symptoms of rheumatoid arthritis due to the anti-CHI3L1 antibody treatment was at a similar level to the group treated with the anti-TNF-α antibody.

[0079]

[0080] In addition, histopathological analysis of the ankle joint was performed using hematoxylene and eosin staining and safranin O staining. As a result, the group induced with rheumatoid arthritis showed that pannus tissue invaded the bone space and the joint structure was destroyed compared to the normal group, and a decrease in bone destruction and fibrosis was confirmed. On the other hand, in the group treated with the anti-CHI3L1 antibody of the present invention, the invasion of pannus tissue into the bone space and the destruction of the joint structure were improved, and the decrease in bone destruction and fibrosis was effectively suppressed.

[0081]

[0082] <2-3> Foot thickness analysis

[0083] The paw thickness was measured and analyzed in a group of mice induced with rheumatoid arthritis and a group of mice administered the anti-CHI3L1 antibody of the present invention.

[0084]

[0085] As a result, as shown in Fig. 3, the group of mice induced with rheumatoid arthritis showed a significantly increased thickness of the paws compared to the normal group, whereas the group treated with the anti-CHI3L1 antibody of the present invention showed an improvement in the thickness of the paws thickened by rheumatoid arthritis to almost a normal level.

[0086]

[0087] <2-4> Analysis of expression levels of inflammatory markers

[0088] It is known that the expression levels of inflammatory markers increase with the onset of rheumatoid arthritis. Therefore, the inventors of the present invention analyzed, through Western blot, whether the anti-CHI3L1 antibody of the present invention could modulate the expression levels of iNOS and COX-2, known inflammatory markers.

[0089]

[0090] As a result, as shown in Fig. 4, in the group of mice in which rheumatoid arthritis was induced, compared to the normal control group, the protein expression levels of the inflammatory markers iNOS and COX-2 were found to have increased, whereas in the group treated with the anti-CHI3L1 antibody of the present invention, the increased expression levels of iNOS and COX-2 were found to have decreased, and the level of decrease in the expression of these inflammatory markers was found to be almost similar to that of the anti-TNF-α antibody treated group.

[0091]

[0092] <2-5> Analysis of inflammatory cytokine levels

[0093] Furthermore, in order to confirm whether the anti-CHI3L1 antibody of the present invention can regulate the expression of inflammatory cytokines, the inventors analyzed the mRNA levels of inflammatory cytokines TNF-α, IL-1β, IL-18 and chemokine CCL2 using qRT-PCR in the mouse groups used in the <2-1> experiment.

[0094]

[0095] As a result, as shown in Fig. 5, in the group with rheumatoid arthritis, the expression levels of TNF-α, IL-1β, IL-18, and CCL2 were found to be significantly increased compared to the normal group, whereas in the group treated with the anti-CHI3L1 antibody of the present invention, the increased expression levels of TNF-α, IL-1β, IL-18, and CCL2 were found to be decreased.

[0096]

[0097] Through the above results, the inventors of the present invention were able to find that the monoclonal antibody (anti-CHI3L1 antibody) that specifically binds to CHI3L1 of the present invention can effectively prevent, improve, and treat rheumatoid arthritis.

[0098]

[0099] 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 rheumatoid arthritis, 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 rheumatoid arthritis, characterized in that it comprises 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.

3. In paragraph 1, A pharmaceutical composition for preventing or treating rheumatoid arthritis, 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 rheumatoid arthritis, 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, Inhibits the expression of iNOS and COX-2; Inhibits the production of inflammatory cytokines TNF-α, IL-1β, and IL-18; or A pharmaceutical composition for the prevention or treatment of rheumatoid arthritis, characterized by having an activity of inhibiting the production of chemokine CCL2.

6. A health functional food for preventing or improving rheumatoid arthritis, containing anti-CHI3L1 antibody as an active ingredient.

7. In paragraph 6, 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 rheumatoid arthritis, characterized in that it comprises 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.

8. In paragraph 6, A health functional food for preventing or improving rheumatoid arthritis, 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.

9. In paragraph 6, A health functional food for preventing or improving rheumatoid arthritis, 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.

10. In paragraph 6, The above anti-CHI3L1 antibody, Inhibits the expression of iNOS and COX-2; Inhibits the production of inflammatory cytokines TNF-α, IL-1β, and IL-18; or A health functional food for preventing or improving rheumatoid arthritis, characterized by having an activity of inhibiting the production of chemokine CCL2.

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