Fusion protein comprising a GLP-1 receptor agonist and an anti-oskar antibody and uses thereof

Abstract

The present invention relates to a fusion protein comprising a GLP-1 (glucagon-like peptide-1) receptor agonist and an anti-Osteoclast-associated receptor (OSCAR) antibody, a pharmaceutical composition, a food composition, and a functional food composition for preventing or treating arthritis, each comprising the fusion protein, and a method for preventing or treating arthritis, the method comprising administering the pharmaceutical composition.The fusion protein comprising a GLP-1 receptor agonist and an anti-Osteoclast-associated receptor (OSCAR) antibody of the present invention has excellent chondroprotective and pain-relieving effects and can therefore be widely used in the effective treatment of arthritis.

Description

[Technical Field]

[0001] The present invention relates to a fusion protein comprising a GLP-1 (Glucagon like peptide-1) receptor agonist and an anti-Osteoclast-associated receptor (OSCAR) antibody, a pharmaceutical composition, a food composition, and a functional food composition for preventing or treating arthritis, each comprising the fusion protein, and a method for preventing or treating arthritis, the method comprising administering the pharmaceutical composition. [Background technology]

[0002] Osteoarthritis is a disease characterized by the loss of cartilage without inflammatory changes associated with aging, resulting in joint deformation and localized degenerative changes. Osteoarthritis was previously thought to be a natural phenomenon resulting from the wear of cartilage as various bodily functions decline with age. However, it has recently become clear that cartilage changes and destruction are caused by various biological factors in addition to these mechanical factors. Unlike rheumatoid arthritis, osteoarthritis treatments involve the loss of cartilage and joint deformation without inflammatory changes. Therefore, the goal of osteoarthritis treatment is to alleviate joint pain and restore function by minimizing the causative factors that prevent the progression of cartilage degeneration.

[0003] Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly prescribed to treat osteoarthritis to relieve pain and control inflammation. Medications for osteoarthritis include conventional analgesics, nonsteroidal anti-inflammatory drugs, selective COX-2 inhibitors, narcotic and non-narcotic analgesics, intra-articular steroid injections, and disease-modifying osteoarthritis drugs (DMOARDs). Among the side effects of long-term NSAID treatment, the most common and sometimes emergency side effects are gastrointestinal side effects, ranging from mild dyspepsia to ulcers, bleeding, and perforation (Non-Patent Document 1). NSAIDs, the most commonly used drugs for osteoarthritis treatment, can cause serious cardiovascular side effects, with a high mortality rate of 5-10%. Since most osteoarthritis patients are elderly, NSAID-based osteoarthritis treatment is particularly risky. Therefore, to address these issues, there is a need for the rapid development of effective and safe therapeutic agents. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Best Practice & Research Clinical Gastroenterology 24 (2010) 121-132 Summary of the Invention [Problem to be solved by the invention]

[0005] Under these circumstances, the present inventors confirmed that a fusion protein comprising a GLP-1 receptor agonist and an anti-oscar antibody has excellent therapeutic effects for arthritis, and thus completed the present invention. [Means for solving the problem]

[0006] The present invention aims to provide a fusion protein comprising a glucagon-like peptide-1 (GLP-1) receptor agonist and an anti-oscar (osteoclast-associated receptor, OSCAR) antibody.

[0007] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating arthritis, which comprises the fusion protein as an active ingredient.

[0008] A further object of the present invention is to provide a food composition for preventing or ameliorating arthritis, which comprises the fusion protein as an active ingredient.

[0009] A further object of the present invention is to provide a functional food composition for preventing or ameliorating arthritis, which comprises the fusion protein as an active ingredient.

[0010] A further object of the present invention is to provide a method for preventing or treating arthritis, which comprises the step of administering the pharmaceutical composition to an individual.

[0011] A further object of the present invention is to provide use of the fusion protein for preventing, ameliorating, or treating arthritis.

[0012] Another object of the present invention is to provide a pharmaceutical composition containing the fusion protein for use in preventing or treating arthritis.

[0013] A further object of the present invention is to provide a food composition containing the fusion protein for use in preventing or ameliorating arthritis.

[0014] A further object of the present invention is to provide a functional food composition containing the fusion protein for use in preventing or improving arthritis. [Effects of the Invention]

[0015] The fusion protein of the present invention comprising a GLP-1 receptor agonist and an anti-oscar antibody has excellent chondroprotective and pain-relieving effects, and can therefore be widely used in the effective treatment of arthritis. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic diagram showing the structure of the fusion protein PF1803. [Figure 2] FIG. 1 shows the results of SDS-PAGE and Western blotting performed to confirm the PF1803 protein. [Figure 3] FIG. 1 shows the results of ELISA performed to confirm the structure of the PF1803 protein. [Figure 4] FIG. 1 shows the cell death inhibitory effect of PF1803 in chondrocytes in which cell death was induced. [Figure 5] FIG. 1 shows that PF1803 reduces the expression of caspase 3 and caspase 8 in chondrocytes in which cell death has been induced. [Figure 6] FIG. 1 shows that PF1803 reduces the expression level of MMP3 and increases the expression level of Aggrecan in chondrocytes in which cell death has been induced. [Figure 7] FIG. 1 shows histological analysis confirming the chondroprotective effect of PF1803 in an arthritis animal model. [Figure 8] FIG. 1 shows weight bearing confirming the pain-relieving effect of PF1803 in an arthritis animal model. DETAILED DESCRIPTION OF THE INVENTION

[0017] These will be described in detail below. Note that each description and embodiment disclosed in the present invention also applies to other descriptions and embodiments. In other words, all combinations of various elements disclosed in the present invention are included in the present invention. Furthermore, the present invention is not limited to the following specific description.

[0018] Additionally, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the present invention.

[0019] To achieve the above object, one aspect of the present invention provides a fusion protein comprising a GLP-1 (glucagon-like peptide-1) receptor agonist and an anti-OSCAR (osteoclast-associated receptor, OSCAR) antibody.

[0020] In the present invention, the term "GLP-1 receptor agonist" refers to a protein that binds to the receptor for GLP-1 (glucagon-like peptide-1), a gastrointestinal hormone derived from the transcription product of the glucagon gene, and plays a role in lowering blood glucose levels. The GLP-1 receptor agonist is not limited to a specific substance as long as it selectively stimulates the GLP-1 receptor and has a signal transduction pathway similar to that of GLP-1. Specific examples include GLP-1 and its derivatives. The GLP-1 derivative can be prepared from GLP-1 by any of the following methods: substitution, addition, deletion, and modification of some amino acids, or a combination thereof. Such GLP-1 derivatives are substances well known in the art, and include, for example, liraglutide, exendin-4, lixisenatide, dulaglutide, albiglutide, and the like.

[0021] In the present invention, "osteoclast-associated receptor (OSCAR)" refers to a cell surface receptor belonging to the leukocyte receptor complex and having two immunoglobulin (Ig) domains. The oskar protein or a fragment thereof of the present invention may be derived from human or mouse. Genetic information such as the amino acid sequence of the human or mouse oskar protein and the nucleotide sequence encoding it can be obtained from publicly known databases, such as GenBank of the National Center for Biotechnology Information (NCBI), but is not limited thereto.

[0022] In the present invention, the term "antibody" refers to a proteinaceous molecule that specifically binds to an antigenic site of a protein or peptide molecule. Such antibodies can be produced by cloning each gene into an expression vector using conventional methods, obtaining a protein encoded by the marker gene, and then using conventional methods from the obtained protein. The antibody is composed of two light chains and two heavy chains, each of which consists of a variable domain with a variable amino acid sequence and a constant domain with a constant sequence. The antigen-binding site is located at the terminus of the three-dimensional structure of the variable domain, and this site is formed by the aggregation of three complementarity-determining regions (CDRs) present in each of the light chain and the heavy chain. The CDRs are particularly variable in amino acid sequence among the variable domains, and this high variability allows the production of antibodies specific to various antigens. The present invention encompasses not only intact antibodies but also antigen-binding fragments of the antibody molecules.

[0023] In the present invention, the term "antibody fragment" refers to any portion of an antibody, and includes, but is not limited to, scFv, dsFv, Fab, Fab', F(ab')2, sdAb, nanobody, and combinations thereof. These antibody fragments contain an antigen-recognition site. The Fab has a structure comprising light-chain and heavy-chain variable regions, a light-chain constant region, and the first heavy-chain constant region (CH1 domain), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing at least one cysteine ​​residue at the C-terminus of the heavy-chain CH1 domain. F(ab')2 antibodies are generated by disulfide bonding of cysteine ​​residues in the hinge region of Fab'. Fv (variable fragment) refers to the minimum antibody fragment comprising only the heavy-chain variable region and the light-chain variable region. Disulfide-stabilized Fvs (dsFvs) have a heavy chain variable region linked to a light chain variable region by a disulfide bond, while single-chain Fvs (scFvs) generally have a heavy chain variable region linked to a light chain variable region by a covalent bond via a peptide linker. Such antibody fragments may be obtained using proteases or produced by genetic recombination techniques. Furthermore, the sdAbs and nanobodies are single variable domain antibody fragments, including, but not limited to, antibody fragments produced by proteolysis or genetic recombination techniques of the variable domains of heavy-chain antibodies containing a naturally occurring single variable domain (VH) and two constant domains (CH2 and CH3), and single domain antibody fragments produced by artificially modifying the antibody light or heavy chain variable domains.

[0024] The anti-oscar antibodies of the present invention are antibodies that act on the oscar protein, and include antibodies that inhibit the binding of oscar to collagen.

[0025] In one embodiment of the present invention, the anti-OSCAR antibody comprises a heavy chain variable region and a light chain variable region, and is at least one selected from the group consisting of, but not limited to: 1) an anti-OSCAR antibody or a fragment thereof comprising a heavy chain variable region represented by SEQ ID NO: 1 and a light chain variable region represented by SEQ ID NO: 2; 2) an anti-OSCAR antibody or a fragment thereof comprising a heavy chain variable region represented by SEQ ID NO: 3 and a light chain variable region represented by SEQ ID NO: 4; and 3) an anti-OSCAR antibody or a fragment thereof comprising a heavy chain variable region represented by SEQ ID NO: 5 and a light chain variable region represented by SEQ ID NO: 6.

[0026] The fusion protein of the present invention is an artificially synthesized protein in which a GLP-1 receptor agonist and an anti-oscar antibody are bound, but is not limited thereto.

[0027] The fusion proteins of the present invention may comprise a GLP-1 receptor agonist and an anti-oscar antibody linked directly, via a linker, or may further comprise another protein moiety, but are not limited thereto. The linking method for the fusion proteins of the present invention may be any method known in the art, as long as it does not alter the structure or activity of the linked proteins. The linker may be, but is not limited to, a peptidic or non-peptidic linker consisting of 1 to 20 amino acids.

[0028] The fusion proteins of the present invention can be produced by conjugating a substance that extends the half-life of a protein (e.g., a GLP-1 receptor agonist and / or an anti-oscar antibody) or by introducing a mutation to prevent degradation in vivo. Any method known in the art may be used as long as it acts on a protein to improve its durability. The substance that extends the half-life is selected from the group consisting of high molecular weight polymers, fatty acids, cholesterol, albumin and its fragments, albumin-binding substances, antibodies, antibody fragments, FcRn-binding substances, in vivo connective tissue, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin. The FcRn-binding substance is an immunoglobulin Fc region, but is not particularly limited thereto. It goes without saying that the immunoglobulin Fc region may be a protein having a partial amino acid sequence with deletion, modification, substitution, conservative substitution, or addition, as long as it has the same or equivalent activity as the above protein, even if it has such a partial amino acid sequence.

[0029] The immunoglobulin Fc region may be composed of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 domains, and may include a hinge region. The immunoglobulin Fc region may be selected from the group consisting of IgG, IgA, IgD, IgE, IgM, combinations thereof, and hybrids thereof, but is not limited to these.

[0030] Furthermore, the immunoglobulin Fc region of the present invention includes not only naturally occurring amino acid sequences but also sequence variants thereof. An amino acid sequence variant refers to an amino acid sequence that differs from the naturally occurring amino acid sequence by deletion, insertion, non-conservative or conservative substitution of at least one amino acid residue, or a combination thereof.

[0031] Specifically, the immunoglobulin Fc region includes CH2 and CH3 regions and is a monomer or dimer, but is not limited thereto. Alternatively, the immunoglobulin Fc region may be an immunoglobulin Fc region of human IgG1 (SEQ ID NO: 8) in which the 297th amino acid is substituted from asparagine to alanine (SEQ ID NO: 9), but is not limited thereto. In one example, the immunoglobulin Fc region has the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 9, but is not limited thereto.

[0032] In a specific embodiment, the fusion protein of the present invention consists of the amino acid sequence of SEQ ID NO: 7 and is used in combination with PF1803, but is not limited thereto.

[0033] The fusion protein of the present invention is characterized by containing a GLP-1 receptor agonist and an anti-oscar antibody, thereby allowing the GLP-1 receptor and the anti-oscar antibody to act simultaneously. Furthermore, by containing a half-life extender, the fusion protein exhibits long-lasting efficacy in the body. Therefore, the fusion protein of the present invention is an excellent therapeutic agent for target diseases, particularly arthritis, based on the simultaneous action of the GLP-1 receptor and the anti-oscar antibody and the extended half-life.

[0034] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating arthritis, which comprises the fusion protein as an active ingredient.

[0035] The fusion protein is as described above.

[0036] "Arthritis" as used herein refers to a disease characterized by inflammation and pain caused by damage to the bones and ligaments that form the joint due to damage or degenerative changes in the cartilage that protects the joint. This term is also used to refer to osteoarthritis. Arthritis in the present invention includes degenerative arthritis, osteochondritis dissecans, articular ligament injury, meniscus injury, joint malalignment, avascular necrosis, rheumatoid arthritis, pediatric idiopathic arthritis, trauma, inflammatory arthritis, and arthritis caused by infection, specifically, but not limited to, degenerative arthritis and rheumatoid arthritis. The pharmaceutical composition of the present invention exhibits the effect of delaying cartilage destruction or alleviating pain, and is effective in preventing or treating arthritis, but is not limited to these.

[0037] In the present invention, "prevention" means any action that prevents or delays arthritis by administering a composition of the present invention, and "treatment" means any action that improves or favorably changes the symptoms of arthritis by administering a composition of the present invention.

[0038] The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, excipient, or diluent. Such pharmaceutically acceptable carrier, excipient, or diluent may be non-naturally occurring. Specifically, the composition may be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, topical preparations, suppositories, and sterile injection solutions by conventional methods. Examples of the carrier, excipient, and diluent contained in the pharmaceutical composition of the present invention include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, they are prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Oral solid formulations include tablets, pills, powders, granules, and capsules. These solid formulations are prepared by mixing the extract and its fractions with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to commonly used excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid formulations include suspensions, oral solutions, emulsions, and syrups. In addition to commonly used diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavoring agents, and preservatives, are used. Parenteral formulations include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions 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, cocoa butter, laurin butter, and glycerol gelatin.

[0039] Yet another aspect of the present invention provides a food composition for preventing or ameliorating arthritis, which comprises the fusion protein as an active ingredient.

[0040] Yet another aspect of the present invention provides a functional food composition for preventing or ameliorating arthritis, which comprises the fusion protein as an active ingredient.

[0041] The fusion protein, arthritis and prevention are as described above.

[0042] In the present invention, "improvement" refers to any action of using the composition to improve or favorably change the symptoms of individuals who have developed a disease or who are suspected of having the disease.

[0043] The term "food" as used herein refers to any food in the conventional sense, such as meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, soft drinks, tea, supplemental drinks, alcoholic beverages, vitamin complexes, and functional foods, as long as it contains the fusion protein of the present invention. The food composition may be prepared by adding raw materials and ingredients commonly used in the art, and the types of ingredients are not particularly limited. For example, similar to conventional foods, additional ingredients may include, but are not limited to, various herbal extracts, nutritive and acceptable food additives, and natural carbohydrates. The amount of active ingredient to be added may be determined appropriately depending on the intended use.

[0044] The term "functional food" as used herein is synonymous with food for special health use (FoSHU) and refers to a food manufactured and processed using specific ingredients for the purpose of health supplementation, or by extracting, concentrating, purifying, or mixing specific ingredients contained in food ingredients, and designed and processed so that the ingredients fully exert their biological regulatory functions in the body, such as biodefense, regulation of biological rhythms, and disease prevention and recovery. The functional food composition has functions related to disease prevention, disease recovery, etc. The functional food of the present invention is used interchangeably with terms known in the art, such as functional health food.

[0045] Yet another aspect of the present invention provides a method for preventing or treating arthritis, comprising the step of administering the pharmaceutical composition to an individual.

[0046] In the present invention, an "individual" may be any mammal, including mice, livestock, humans, or farmed fish, that has developed or is at risk of developing arthritis.

[0047] The pharmaceutical compositions of the present invention can be administered orally or parenterally, including, but not limited to, cutaneous, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, gastrointestinal, intra-articular, topical, sublingual, intravaginal or rectal routes, using administration methods commonly used in the art.

[0048] The appropriate dosage of the pharmaceutical composition of the present invention can be determined by the attending physician within the scope of sound medical judgment and can be administered in single or divided doses. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will preferably vary depending on the type and degree of response to be achieved, whether other formulations are used, the specific composition, the patient's age, weight, general health, sex, diet, time of administration, route of administration, excretion rate of the composition, duration of treatment, etc.

[0049] Yet another aspect of the present invention provides use of the fusion protein for preventing, ameliorating or treating arthritis.

[0050] Yet another aspect of the present invention provides a pharmaceutical composition comprising the fusion protein for use in preventing or treating arthritis.

[0051] Yet another aspect of the present invention provides a food composition containing the fusion protein for preventing or ameliorating arthritis.

[0052] Yet another aspect of the present invention provides a use of a functional food composition containing the fusion protein for preventing or ameliorating arthritis.

[0053] The fusion protein, pharmaceutical composition, food composition, functional food composition, arthritis, prevention, amelioration, treatment, etc. are as described above. [Example]

[0054] The present invention will be described in more detail below with reference to examples. These examples are intended to explain the present invention more specifically, but the present invention is not limited to these examples. [Example]

[0055] Fusion protein production and validation A fusion protein (PF1803; SEQ ID NO: 7) containing a GLP-1 receptor agonist and an anti-oskar antibody was synthesized to form a GLP-1-Fc-VH-VL fusion protein (Figure 1). The synthesized PF1803 was cloned into the pcDNA3.1 vector using the Nhe1 / HindIII site. The cloned vector was transfected into CHO-S cells. On day 3, the supernatant was collected, centrifuged, and filtered to remove the cells and supernatant. The supernatant was then purified using protein A beads (Cytiva, Hitraprene Preotain A).

[0056] The production of PF1803 was confirmed by SDS-PAGE gel under non-reducing and reducing conditions and Western blot analysis using anti-GLP1 Ab (Abcam) (Figure 2).

[0057] Human OSCAR protein was dispensed into an immuno-96 microwell plate at 300 ng per well in coating buffer and surface-immobilized overnight at 4°C. 20 μl of 5% skim milk in PBST was added per well, followed by incubation at 37°C for 1 hour to block nonspecific binding. PF1803 was then diluted 3-fold from a maximum concentration of 1000 nM to 0.017 nM, and 100 μl of the diluted solution was dispensed into each well and incubated at 37°C for 1 hour. After washing three times with 200 μl of PBST, mouse anti-GLP1 antibody (Abcam) was diluted 1:2000 and dispensed into each well at 100 μl. The plate was then incubated at 37°C for 1 hour. After washing three more times with PBST, 100 μl of anti-mouse IgG-HRP (Millipore) diluted 1:3000 was added to each well and incubated at 37°C for 1 hour. The wells were then washed three times with PBST. Next, 100 μl of TMB (BD) solution was added to each well and incubated at room temperature for 5 minutes. The reaction was then stopped with 100 μl of 1N HCl. Finally, the absorbance was measured at 450 nm using a spectrophotometer (Spectrmax iD3, Molecular Devices). The EC value was 7.494 nM. 50 The values ​​were confirmed (Fig. 3), confirming that the produced PF1803 protein bound to both OSCAR and GLP-1 antibodies. [Example]

[0058] MIA-induced arthritis model The animal model of arthritis used in the present invention is a model in which osteoarthritis is induced by injecting monosodium iodoacetate (MIA) into the joint cavity. MIA activates matrix metalloproteinase (MMP), inhibits proteoglycan synthesis in cartilage, and causes necrosis of chondrocytes, making it an animal model similar to the symptoms of degenerative arthritis in patients.

[0059] Specifically, monosodium iodoacetate (MIA, I2512, Sigma, Poole, UK) was dissolved in injectable saline at concentrations of 20 mg / mL and 60 mg / mL and prepared on the day of the experiment (day 0). After separating the animals into groups, on the day of the experiment, the animals were placed in an anesthesia chamber and anesthetized with isoflurane via inhalation. Subsequently, 50 μL of MIA (MIA 1.3 mg / body) was injected into the right knee joint via the infrapatellar ligament using a 26.5-gauge, 1-cc syringe. The experimental groups and administered substances are shown in Table 1.

[0060] [Table 1]

[0061] Osteoarthritis was induced by MIA, and then the test substance groups (G3 to G6) received a predetermined dose of the test substance homogenized in a vehicle, which was then administered orally to the knee joint in a volume of 1 ml once every seven days. The normal control group and vehicle control group received the same amount of vehicle alone in the knee joint once every seven days on the same schedule as the test substance administration. The positive control group received a predetermined dose of celecoxib homogenized in a vehicle, which was then administered orally once daily. [Example]

[0062] Inhibitory effect on chondrocyte cell death Example 3-1. Inhibition of chondrocyte death To induce chondrocyte death, mouse chondrocytes were treated with 10 ng / ml of IL-1β. To evaluate the effect of anti-OSCAR antibodies, they were treated with OSC (Oscar-binding triple-helical peptide) (SEQ ID NO: 10), which induces signaling from primary cells expressing OSCAR.

[0063] After treatment with IL-1β and OSCAR, the cell viability of each chondrocyte was measured after treatment with GLP1-Fc (a fusion protein combining GLP-1 and the Fc region of an antibody), anti-OSCAR antibody, or PF1803.

[0064] As a result, it was confirmed that the death of chondrocytes treated with PF1803 was significantly inhibited in a concentration-dependent manner compared to the anti-OSCAR antibody and GLP-1 groups (Fig. 4).

[0065] Example 3-2. Cell death pathway (decreased activity of caspase 3 and caspase 8) In Example 3-1, the activities of caspase 3 and caspase 8 were measured in chondrocytes treated with GLP1-Fc, anti-OSCAR antibody, or PF1803 after IL-1β and OSCAR treatment. Because caspase 3 and caspase 8 are proteins that indicate cell death, their levels were measured and compared.

[0066] The cleavage activity of caspase 3 and caspase 8 was measured using a colorimetric assay kit for caspase 3 or caspase 8 (caspase 3 kit (Biovision K106) and caspase 8 kit (Biovision K113)).

[0067] The results confirmed that the activity of caspase 3 and caspase 8 was significantly reduced in chondrocytes treated with PF1803 compared to the anti-OSCAR antibody and GLP-1 groups (Figure 5). This suggests that PF1803 has an excellent effect in delaying cartilage destruction and regenerating cartilage.

[0068] Example 3-3. Cell Death Transduction Pathway (Decreased Expression of MMP3 and Increased Expression of Aggrecan) In Example 3-1, the expression levels of MMP3 and Aggrecan in each type of chondrocyte treated with IL-1β and OSC, followed by GLP1-Fc, anti-OSCAR antibody, or PF1803 were measured by Western blotting.

[0069] Specifically, MMP3 (matrix metalloproteinase-3) is a catabolic marker, and an increase in MMP3 is an indicator of chondrocyte destruction, while Aggrecan is an anabolic marker, and an increase in Aggrecan is an indicator of chondrocyte regeneration, so the expression levels of MMP3 and Aggrecan were measured.

[0070] As a result, in the chondrocytes treated with PF1803, compared with the anti-OSCAR antibody and GLP-1 groups, The expression level of MMP3 was significantly decreased, and the expression level of Aggrecan was significantly increased. (Figure 6) This suggests that PF1803 has excellent effects in delaying cartilage destruction and regenerating cartilage. [Example]

[0071] PF1803's effect on delaying cartilage destruction Joints from each animal model group were harvested and paraffin blocks were prepared. H&E and Safranin O staining were performed on all samples to observe the extent of cartilage destruction and inflammatory cell proliferation. OARSI and Mankin scores were assigned based on the results of Safranin O staining, and differences from the control group were statistically analyzed.

[0072] As a result, cartilage destruction was completely observed in animals treated with the positive control group. Compared to the group treated with anti-OSCAR antibody and GLP-1, animals treated with PF1803 were confirmed to have a superior effect in delaying cartilage destruction (Figure 7). [Example]

[0073] Pain relief effect of PF1803 The weight bearing measurement test measured changes in weight bearing (or weight distribution) between the normal hind leg (left) and the arthritic hind leg (right) due to knee pain in one hind leg after arthritis was induced. The weight bearing of each hind leg was measured using an incapacitance meter (Model 600, IITC, USA). Because the weight bearing varied depending on the animal's leg position, the animal was accurately positioned in the holder to ensure both legs were positioned symmetrically. Only one person was required to hold the animal in place to minimize error. The machine was activated when each animal was accurately positioned in the holder. Two measurements were taken, each with a 5-second measurement time. The average of these measurements was used as the weight bearing (g) value for each leg. Regarding the timing of measurements, the vehicle control, experimental, and positive control groups were measured before arthritis induction by MIA administration (day 0) to obtain baseline values, and then again before test substance administration (day 3) to separate the groups. Subsequently, measurements were taken twice weekly at the designated times. The results of the weight bearing measurement test were converted into a weight bearing ratio using the following formula and analyzed.

[0074]

number

[0075] As a result, it was confirmed that the PF1803 administration group had a superior pain-reducing effect compared to the positive control group or the group administered with anti-OSCAR antibody and GLP-1 in combination (FIG. 8).

[0076] That is, it was suggested that the fusion protein of the present invention comprising a GLP-1 receptor agonist and an anti-oscar antibody is useful for preventing or treating arthritis.

[0077] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential features thereof. It should be understood that the above examples are merely illustrative and not limiting. The present invention should be construed as including all modifications and variations derived from the meaning and scope of the claims, rather than the specification, and their equivalents.

Claims

1. A fusion protein comprising a glucagon-like peptide-1 (GLP-1) receptor agonist and an anti-osteoclast-associated receptor (OSCAR) antibody, wherein the anti-oscar antibody comprises a heavy chain variable region and a light chain variable region: 1) an anti-oskar antibody or an antigen-binding fragment thereof comprising a heavy chain variable region represented by SEQ ID NO: 1 and a light chain variable region represented by SEQ ID NO: 2; 2) an anti-oskar antibody or an antigen-binding fragment thereof comprising a heavy chain variable region represented by SEQ ID NO: 3 and a light chain variable region represented by SEQ ID NO: 4; and 3) An anti-oskar antibody or an antigen-binding fragment thereof comprising a heavy chain variable region represented by SEQ ID NO: 5 and a light chain variable region represented by SEQ ID NO:

6. A fusion protein comprising at least one selected from the group consisting of:

2. The fusion protein according to claim 1, wherein the GLP-1 receptor agonist is GLP-1 or a derivative thereof.

3. The fusion protein of claim 1 , wherein the fusion protein further comprises a half-life extender.

4. The fusion protein of claim 3 , wherein the half-life extending substance is an immunoglobulin Fc region.

5. The fusion protein according to claim 4 , wherein the immunoglobulin Fc region has an asparagine to alanine substitution at amino acid position 297.

6. The fusion protein of claim 1 , wherein the fusion protein is represented by SEQ ID NO:

7.

7. A pharmaceutical composition for preventing or treating arthritis, comprising the fusion protein according to any one of claims 1 to 6 as an active ingredient.

8. The pharmaceutical composition of claim 7, wherein the arthritis is degenerative arthritis or rheumatoid arthritis.

9. The pharmaceutical composition according to claim 7, characterized in that the pharmaceutical composition slows cartilage destruction or reduces pain.

10. A food composition for preventing or ameliorating arthritis, comprising the fusion protein according to any one of claims 1 to 6 as an active ingredient.

11. A functional food composition for preventing or ameliorating arthritis, comprising the fusion protein according to any one of claims 1 to 6 as an active ingredient.

12. 10. A method for preventing or treating arthritis, comprising administering the pharmaceutical composition of claim 7 to a non-human individual.

13. 1) an anti-oskar antibody or an antigen-binding fragment thereof comprising a heavy chain variable region represented by SEQ ID NO: 1 and a light chain variable region represented by SEQ ID NO: 2; 2) an anti-oskar antibody or an antigen-binding fragment thereof comprising a heavy chain variable region represented by SEQ ID NO: 3 and a light chain variable region represented by SEQ ID NO: 4; and 3) An anti-oskar antibody or an antigen-binding fragment thereof comprising a heavy chain variable region represented by SEQ ID NO: 5 and a light chain variable region represented by SEQ ID NO:

6. An anti-oskar antibody comprising a heavy chain variable region and a light chain variable region comprising at least one selected from the group consisting of:

14. A pharmaceutical composition for preventing or treating arthritis, comprising the anti-oscar antibody of claim 13 as an active ingredient.

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