Pharmaceutical composition for preventing or treating rheumatoid arthritis and bone disease comprising recombinant galectin-9 protein

Abstract

The present application relates to a pharmaceutical composition for preventing or treating bone diseases, comprising a recombinant galectin-9 protein, and in particular, to a pharmaceutical composition for preventing or treating bone diseases including rheumatoid arthritis, comprising a recombinant galectin-9 protein in which amino acids of a linker peptide are deleted and amino acids of a C-terminal domain (CCRD) are deleted and substituted, as an effective ingredient.

Description

Technical Field

[0001] This invention relates to recombinant stable galactolectin 9 protein and its uses, specifically to pharmaceutical compositions comprising the above-mentioned protein as an active ingredient for the prevention or treatment of bone diseases, including rheumatoid arthritis. Background Technology

[0002] Animal lectins that specifically recognize sugar chains with β-galactoside structures have been discovered in organisms, and at least 14 genes have been identified to date. Based on their structure, galactolectins are classified into prototype, chimera, and tandem repeat types.

[0003] Galactochondrin 9, one of the tandem repeat galactochondrins, consists of two glycan recognition sites (carbohydrate recognition domains, CRDs) and a linker peptide connecting them. This linker peptide connects the N-terminal Carbohydrate Recognition Domain (NCRD) to the C-terminal Carbohydrate Recognition Domain (CCRD), and its activities in various aspects have been reported. Regarding T cells, galactochondrin 9 binds to Tim-3 to induce apoptosis in Tim-3-positive Th1 cells, suppressing autoimmune inflammation by inhibiting excessive Th1 responses. Furthermore, it reduces Tim-3-expressing Th17 cells, which are a cause or aggravating factor in many intractable diseases, including autoimmune diseases, allergies, and cancer.

[0004] On the other hand, galactolectin 9 enhances immunity depending on the situation. It activates these cells by binding to Tim-3, which resembles monocytes or dendritic cells, thereby promoting the production of inflammatory cytokines. Furthermore, in macrophages, the interaction between galactolectin 9 and Tim-3 enhances the immunity that eliminates tuberculosis bacteria.

[0005] Meanwhile, suggestive evidence of genetic diversity has been confirmed among galactolectin 9 genes cloned from human cells or tissues. Recombinant galactolectin 9 produced using *E. coli* as a host has been shown to inhibit cancer metastasis and induce regression through direct action on tumor cells (intercellular adhesion and apoptosis-inducing activity) and through the immune system, as well as to induce and activate T cells, particularly inducing apoptosis of CD4-positive T cells, which are the cause of excessive immune responses. Furthermore, it is known to have therapeutic effects on human immunodeficiency virus (HIV) infection, allergies and asthma, and type 1 diabetes.

[0006] To utilize galectin 9 as a practical therapeutic agent, the following issues need to be addressed: 1) sensitivity to proteolytic enzymes; 2) low solubility; and 3) low availability. Research is ongoing to prepare proteolytic enzyme-resistant galectin 9 variants (G9Null; Non-Patent Literature 1) by cleaving the linker peptide of galectin 9. KRAS mutations are known to have antitumor activity against colon cancer (Non-Patent Literature 2).

[0007] On the other hand, bone is a dynamic tissue that is constantly being cleared and rebuilt through bone resorption and formation. This process is carried out by two types of cells: osteoclasts and osteoblasts. Specifically, osteoblasts are responsible for bone formation, while osteoclasts are responsible for bone removal. Under normal conditions, the permanence of bone is maintained through the balanced activity of these two cell types. However, permanence can be disrupted by abnormal overactivity of both cell types, especially osteoclasts, resulting in bone diseases such as arthritis, osteoporosis, periprosthetic osteolysis, and Paget's disease. Therefore, properly regulating osteoclast differentiation and function is crucial in the prevention and treatment of bone diseases.

[0008] For example, drug therapies for osteoporosis include medications that promote bone formation or inhibit bone resorption to prevent bone loss or increase bone mass. These include bone resorption inhibitors, such as calcium preparations, vitamin D preparations, bisphosphonates, estrogen agonists / antagonists, estrogen preparations, and bone formation promoters, such as teriparatide injections, which are parathyroid hormone preparations.

[0009] Furthermore, among arthritis, rheumatoid arthritis is a typical chronic autoimmune disease caused by inflammation of the synovial tissue surrounding the joints. It can occur in all joints with synovium, and as a disease affecting multiple organs throughout the body, it destroys joint tissue, causing severe joint dysfunction and is a chronic inflammatory disease leading to early death. Although rare, some severe cases can also affect tissues other than the joints, such as the lungs, heart, eyes, gastrointestinal tract, skin, and kidneys. Consequently, the large amounts of inflammatory mediators secreted in the body often adversely affect bone metabolism, increasing the risk of osteoporosis and fractures. It is confirmed that approximately 15%–20% of rheumatoid arthritis patients develop osteoporosis.

[0010] Drug therapy for rheumatoid arthritis includes first-line medications such as nonsteroidal anti-inflammatory drugs (NSAIDs) and steroids (a type of hormone), as well as second-line medications that suppress rheumatoid arthritis by influencing the body's immune system. Most treatments require long-term use, thus requiring careful attention to side effects. Steroids, in particular, are prone to misuse due to their immediate effectiveness, leading to serious side effects. Furthermore, long-term use may be a risk factor for osteoporosis.

[0011] Therefore, as a result of the inventors' efforts to develop novel therapeutic agents that have effective therapeutic effects on arthritis, including rheumatoid arthritis, and / or osteoporosis with minimal side effects, it was confirmed that recombinant stable galactolectin 9 protein with the C-terminal domain (CCRD) and amino acids of the linker peptide in the two glycan recognition sites of existing wild-type galactolectin 9 has the effects of inhibiting Th1 and Th17 differentiation, inducing apoptosis of fibroblast-like synoviocytes (FLS), and inhibiting osteoclast differentiation. Based on this, the present invention was completed. Summary of the Invention

[0012] Technical issues

[0013] The purpose of this invention is to provide a pharmaceutical composition comprising recombinant stable galactolectin 9 protein and comprising thereof for the prevention or treatment of bone diseases.

[0014] Solution to the problem

[0015] To achieve the objectives of this invention, the present invention provides a recombinant stable galactolectin 9 protein having an amino acid sequence represented by sequence 1.

[0016] Furthermore, the present invention provides a recombinant vector containing a gene encoding the aforementioned protein.

[0017] Furthermore, the present invention provides a transformant having the aforementioned recombinant vector inserted therein.

[0018] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases, comprising a recombinant stable galactolectin 9 protein having an amino acid sequence represented by Sequence 1 or a polynucleotide encoding thereas as an active ingredient; a method for treating bone diseases, comprising administering to an individual a pharmaceutically effective amount of a recombinant stable galactolectin 9 protein having an amino acid sequence represented by Sequence 1 or a polynucleotide encoding thereas; a pharmaceutical composition for the prevention or treatment of bone diseases, comprising a recombinant stable galactolectin 9 protein having an amino acid sequence represented by Sequence 1 or a polynucleotide encoding thereas; and the use of a recombinant stable galactolectin 9 protein having an amino acid sequence represented by Sequence 1 or a polynucleotide encoding thereas for the preparation of a pharmaceutical composition for the prevention or treatment of bone diseases.

[0019] Meanwhile, the present invention provides a health food for preventing or improving bone diseases, comprising a recombinant stable galactolectin 9 protein having an amino acid sequence represented by Sequence 1 as an active ingredient; a method for preventing or improving bone diseases, comprising administering to an individual a pharmaceutically effective amount of a recombinant stable galactolectin 9 protein having an amino acid sequence represented by Sequence 1 or a polynucleotide encoding thereof; a health food for preventing or improving bone diseases, comprising a recombinant stable galactolectin 9 protein having an amino acid sequence represented by Sequence 1 or a polynucleotide encoding thereof; and the use of a recombinant stable galactolectin 9 protein having an amino acid sequence represented by Sequence 1 or a polynucleotide encoding thereof, for preparing a health food for preventing or improving bone diseases.

[0020] The effects of the invention

[0021] The recombinant stable galactosyllectin 9 protein of the present invention exhibits the effects of inhibiting the differentiation of Th1 and Th17 cells, inducing apoptosis of synovial cells (FLS, Fibroblast-like synoviocytes), and inhibiting osteoclast differentiation. It can be effectively used as an active ingredient in compositions for the prevention or treatment of bone diseases, including rheumatoid arthritis. Attached Figure Description

[0022] Figure 1 A graph to confirm the binding degree of Tim-3 to the recombinant stable galactolectin 9 protein (sGal-9) of the present invention.

[0023] Figure 2 The figure shows the effect of sGal-9 of the present invention in inhibiting the differentiation of Th1 and Th17 cells.

[0024] Figure 3 A figure illustrating the apoptosis effect of sGal-9 of the present invention on FLS (Fibroblast-like synoviocytes).

[0025] Figure 4 The figure shows the effect of sGal-9 in inhibiting osteoclast differentiation of the present invention.

[0026] Figure 5 A graph to confirm the effect of the sGal-9 of the present invention in inhibiting bone resorption. Detailed Implementation

[0027] The embodiments of the present invention will be further described in detail below to enable those skilled in the art to implement the present invention. These embodiments are provided to provide a more comprehensive explanation of the present invention to those skilled in the art. Therefore, the embodiments of the present invention can be modified in many other ways, and the scope of the present invention is not limited to the embodiments described below.

[0028] Throughout this specification, when a part is referred to as "containing" a structural element, unless the context clearly indicates a different meaning, it means that other structural elements may also be included, rather than excluding other structural elements.

[0029] The present invention provides a recombinant stable galactolectin 9 protein having an amino acid sequence represented by sequence 1.

[0030] In this invention, the recombinant stable galectin-9 protein retains the glycan recognition activity of wild-type galectin-9 while having a more stable molecular structure for proteases.

[0031] Specifically, the aforementioned recombinant stable galactocelenin 9 protein is a recombinant protein prepared by altering the linker regions of the two CRD (Carbohydrate Recognition Domain) and the CCRD (C-terminal Carbohydrate Recognition Domain) of wild-type galactocelenin 9 with an NCRD-adaptor-CCRD structure. More specifically, the recombinant stable galactosyllectrin 9 protein can be formed by completely deleting the peptide in the linker region, deleting the amino acid sequence at positions 1 to 10 in CCRD (Sequence 2) (Sequence 3), and replacing the alanine (Ala; A) at position 13 with proline (Pro; P). It can include amino acid sequences with more than 75% sequence homology to the amino acid sequence represented by Sequence 1, preferably, it can include amino acid sequences with more than 80% sequence homology to the amino acid sequence represented by Sequence 1, more preferably, it can include amino acid sequences with more than 90% sequence homology to the amino acid sequence represented by Sequence 1, and most preferably, it can include amino acid sequences with more than 95% sequence homology to the amino acid sequence represented by Sequence 1. It can also include a targeting sequence, a tag, a labeled residue, or an amino acid sequence prepared for a specific purpose of increasing half-life or peptide stability.

[0032] Furthermore, the recombinant protein of the present invention can be obtained by various methods known in the art to which this invention pertains. For example, it can be prepared using polynucleotide recombination and protein expression systems, or by in vitro synthesis methods such as peptide synthesis, or by cell-free protein synthesis methods.

[0033] As used in this invention, the term "polynucleotide" refers to a polymer of bound nucleotides that serves to transmit genetic information. For the purposes of this invention, the invention may include a sequence encoding a recombinant protein of sequence 1 and having at least 75% sequence homology with the polynucleotide sequence encoding the recombinant protein; preferably, it may include a sequence having at least 85% sequence homology with the polynucleotide sequence encoding the recombinant protein; more preferably, it may include a sequence having at least 90% sequence homology with the polynucleotide sequence encoding the recombinant protein; and most preferably, it may include a sequence having at least 95% sequence homology with the polynucleotide sequence encoding the recombinant protein.

[0034] The term “homology” as used in this invention is used to indicate the degree of similarity to wild-type amino acid sequences or polynucleotide sequences. Such comparison of homology can be performed using procedures well known in the art to which this invention pertains. Homology between two or more sequences can be calculated as a percentage (%).

[0035] Furthermore, the present invention provides a recombinant vector containing a gene encoding the aforementioned protein.

[0036] Furthermore, the present invention provides a transformant having the aforementioned recombinant vector inserted therein.

[0037] As used in this invention, the term "vector" can refer to plasmids, viruses, or other media known in the technical field to which this invention pertains, into which genes or base sequences are inserted or introduced. Specifically, it can be linear deoxyribonucleic acid (DNA), plasmid DNA, recombinant nonviral vectors, recombinant viral vectors, or inducible gene expression vector systems. The recombinant viral vectors can be retroviruses, adenoviruses, adeno-associated viruses, helper virus-dependent adenoviruses, herpes simplex viruses, lentiviral vectors, or vaccinia viruses, but are not limited thereto. The base sequences of this invention can be operably linked to expression regulatory sequences, and these operably linked base sequences can be contained within an expression vector that simultaneously includes a selection marker and a replication origin. The term "operably linked" can refer to a gene and an expression regulatory sequence linked in a manner capable of gene expression when a suitable molecule binds to the expression regulatory sequence. An "expression control sequence" refers to a DNA sequence that regulates the expression of the operably linked base sequence in a specific host cell. Such regulatory sequences may include promoters for carrying out transcription, arbitrary operon sequences for regulating transcription, sequences encoding appropriate messenger ribonucleic acid (mRNA) ribosome binding sites, and sequences for terminating transcription and interpretation.

[0038] The term "transformation" as used in this invention refers to altering the genetic properties of an organism by injecting DNA from an external source. Specifically, it refers to the phenomenon where, when DNA, one of the nucleic acids extracted from cells of one system of an organism, is introduced into living cells of another system, the DNA enters the cell and causes a change in genetic traits. The cells mentioned can be prokaryotic or eukaryotic cells, but are not limited to these.

[0039] In this invention, primers capable of specifically recognizing the gene encoding the recombinant protein are prepared from the known sequences described above. These primers can be used to amplify the gene via polymerase chain reaction (PCR) and then introduced into the vector described above before being introduced into cells. The introduction methods are known, and include, but are not limited to, liposome-mediated translocation, calcium phosphate translocation, DEAE-glucan-mediated translocation, cationic lipid-mediated translocation, electroporation, transduction using phage systems, or viral infection methods.

[0040] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases, comprising a recombinant stable galactolectin 9 protein having an amino acid sequence represented by Sequence 1 or a polynucleotide encoding therethe as an active ingredient.

[0041] As used in this invention, the term "prevention" refers to all behaviors that suppress or delay the onset of disease by administering a composition.

[0042] The term "treatment" as used in this invention refers to all actions that improve or cure the symptoms of the aforementioned disease by administering a composition.

[0043] In this invention, the recombinant stable galactose lectin 9 protein described above can have the effect of preventing or treating bone diseases by exhibiting one or more of the following properties:

[0044] i) Increased binding affinity to Tim-3 protein;

[0045] ii) Inhibit the differentiation of Th1 and Th17 cells;

[0046] iii) Inhibit helper T cells; iv) Induce apoptosis in fibroblast-like synovial cells; and

[0047] v) Inhibit osteoclast differentiation and bone resorption.

[0048] Specifically, the aforementioned recombinant stable galactolectin 9 protein can inhibit helper T cells such as Th1 and Th17 by inducing apoptosis through binding to the Tim-3 protein expressed by T cells. In particular, synovial cells, along with inflammatory cytokines, are one of the important pathological factors in rheumatoid arthritis. As the disease progresses, the abnormal proliferation and activation of synovial cells become problematic, leading to persistent joint inflammation and destruction.

[0049] Furthermore, the aforementioned recombinant stable galactolectin 9 protein can induce apoptosis in fibroblast-like synoviocytes that destroy the matrix of articular cartilage by secreting matrix metalloproteinases (MMPs) and cathepsins.

[0050] Furthermore, the aforementioned recombinant stable galactolectin 9 protein can inhibit osteoclast differentiation and bone resorption. In particular, osteoclasts, as multinucleated cells that destroy and absorb unnecessary bone tissue during bone growth, can cause osteoporosis, leading to bone loss, when their balance with osteoblasts involved in bone formation and regeneration is disrupted. Moreover, since bone resorption occurs through osteoclast activity, bisphosphonates, commonly known osteoporosis medications, prevent further deterioration of osteoporosis by inhibiting this bone resorption.

[0051] In this invention, the aforementioned bone diseases may include, but are not limited to, arthritis, osteoporosis, periprosthetic osteolysis, osteolysis caused by implant debris, Paget's disease, osteomalacia, rickets, osteopenia, calcium metabolism disorder, metastatic bone cancer, inflammatory bone destruction, secondary bone loss caused by endocrine diseases or drugs, or periodontal diseases accompanied by alveolar bone destruction.

[0052] Furthermore, the aforementioned arthritis can be osteoarthritis, degenerative osteoarthritis, rheumatoid arthritis, osteochondritis dissecans, joint ligament injury, meniscus injury, joint dislocation, ischemic necrosis, or juvenile idiopathic arthritis, but is not limited to these.

[0053] In a specific embodiment of the present invention, the inventors prepared the recombinant protein by lysing and purifying *E. coli* cells that expressed a recombinant stable galactolectin 9 protein having the amino acid sequence represented by Sequence 1, and then using chromatography. Furthermore, it was confirmed that the recombinant protein inhibits the differentiation of Th1 and Th17 cells, induces apoptosis in fibroblast-like synovial cells, and inhibits osteoclast differentiation. Therefore, the recombinant protein and the polynucleotide encoding it can be effectively used as active ingredients in compositions for the prevention or treatment of bone diseases.

[0054] On the other hand, the recombinant protein of the present invention or the polynucleotide encoding it can be transported by pharmaceutically acceptable carriers such as colloidal suspensions, powders, physiological saline, lipids, liposomes, microspheres, or nanospheres. This can be related to the means of transport and the formation of the complex, and can be transported into the organism using transport systems known in the art to which this invention pertains, such as lipids, liposomes, microparticles, gold nanoparticles, polymers, condensation reactants, polysaccharides, polyamino acids, dendritic molecules, saponins, adsorption enhancers, or fatty acids.

[0055] In addition, pharmaceutically acceptable carriers may include, but are not limited to, lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil, etc., commonly used in formulations. Furthermore, in addition to the above-mentioned components, lubricants, humectants, sweeteners, flavorings, emulsifiers, suspending agents, preservatives, etc., may also be included.

[0056] The pharmaceutical composition of the present invention can be administered orally or parenterally according to the desired method (e.g., intramuscular, intravenous, intraperitoneal, subcutaneous, intradermal, or local). The dosage can vary depending on the patient's condition and weight, the severity of the disease, the form of the drug, the route of administration, and the timing, and can be appropriately selected by those skilled in the art to which this invention pertains.

[0057] The pharmaceutical compositions of the present invention are administered in a pharmaceutically effective amount.

[0058] The term "pharmaceutically effective amount" as used in this invention refers to an adequate amount that can treat a disease in a reasonable benefit / risk ratio suitable for medical treatment. The level of the effective dose can be determined based on factors including the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the timing of administration, the route of administration and metabolic rate, the duration of treatment, and other factors well-known in the medical field. The pharmaceutical compositions of this invention can be administered as a single therapeutic agent, or in combination with surgery, hormone therapy, drug therapy, and biological response modifiers. They can be administered simultaneously, alone, or sequentially with the aforementioned preparations, and can be administered once or multiple times. Importantly, considering all the above factors, the dosage that achieves the maximum effect with the minimum amount without side effects can be easily determined by those skilled in the art to which this invention pertains.

[0059] Specifically, the effective amount of the pharmaceutical composition of the present invention can vary depending on the patient's age, gender, condition, weight, absorption rate, inactivation rate, excretion rate of the active ingredient in the body, type of disease, and concurrent drugs, and can be increased or decreased according to the route of administration, severity of obesity, gender, weight, age, etc.

[0060] Meanwhile, the present invention provides a health food for preventing or improving bone diseases, comprising a recombinant stable galactoglucan 9 protein having an amino acid sequence represented by sequence 1 as an active ingredient.

[0061] In this invention, the content concerning the above-mentioned recombinant stable galactolectin 9 protein and bone disease is the same as described above, and the specific description will refer to the above content.

[0062] As used in this invention, the term "improvement" refers to all behaviors that at least reduce parameters related to the treated state, such as the severity of symptoms. In this context, the aforementioned health food composition can be used, simultaneously with or alone, with a therapeutic agent to prevent or improve bone diseases, either before or after the onset of the relevant disease.

[0063] On the other hand, this invention has confirmed that the recombinant stable galactolectin 9 protein having the amino acid sequence represented by Sequence 1 exhibits effects such as inhibiting Th1 and Th17 cell differentiation, inducing apoptosis in fibroblast-like synovial cells, and inhibiting osteoclast differentiation. Therefore, the above-mentioned recombinant protein can be effectively used as an active ingredient in health-promoting functional foods for the prevention or improvement of bone diseases.

[0064] In the health functional food of the present invention, the active ingredients can be directly added to the food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of active ingredients mixed can be appropriately determined according to the purpose of its use (prevention or improvement). Generally, when preparing it as a food or beverage, preferably, less than 15% by weight of the health functional food of the present invention can be added relative to the raw materials, more preferably, less than 10% by weight of the health functional food of the present invention can be added. However, in cases where it is consumed for health and hygiene purposes or for long-term health regulation purposes, the above amounts can be below the above range.

[0065] In addition to the aforementioned effective ingredients, the health-functional food of the present invention may also contain other ingredients as essential components without limitation. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, similar to ordinary beverages. Examples of the aforementioned natural carbohydrates may be monosaccharides, such as glucose and fructose; disaccharides, such as maltose and sucrose; and polysaccharides, such as common sugars like dextrin and cyclodextrin, as well as sugar alcohols such as xylitol, sorbitol, and erythritol. In addition to the aforementioned components, natural flavoring agents (such as sematrandrine and stevia leaf extract (e.g., lebodiin A and glycyrrhizic acid)) and synthetic flavoring agents (such as saccharin and aspartame) may be advantageously used as flavoring agents. The proportion of the aforementioned natural carbohydrates can be appropriately determined by those skilled in the art to which this invention pertains.

[0066] In addition to the above-mentioned ingredients, the health functional food of the present invention may also contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. These ingredients may be used alone or in combination, and the proportions of these additives may be appropriately selected by those skilled in the art to which this invention pertains.

[0067] Furthermore, the present invention provides a method for preventing or treating bone diseases, comprising the step of administering to an individual a pharmaceutically effective amount of a recombinant stable galactolectin 9 protein having an amino acid sequence represented by Sequence 1 or a polynucleotide encoding thereus.

[0068] Furthermore, the present invention provides a method for preventing or improving bone diseases, comprising the step of administering to an individual a pharmaceutically effective amount of a recombinant stable galactolectin 9 protein having an amino acid sequence represented by Sequence 1 or a polynucleotide encoding therethe.

[0069] The recombinant stable galactosyllectin 9 protein or the polynucleotide encoding it, having the amino acid sequence represented by Sequence 1 of the present invention, exhibits the effects of increasing Tim-3 protein binding, inhibiting Th1 and Th17 cell differentiation, inhibiting helper T cells, inducing apoptosis of fibroblast-like synovial cells, inhibiting osteoclast differentiation, and inhibiting bone resorption. Therefore, it can be effectively used in the treatment of bone diseases.

[0070] Furthermore, the present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases, comprising a recombinant stable galactolectin 9 protein having an amino acid sequence represented by Sequence 1 or a polynucleotide encoding thereus.

[0071] Furthermore, the present invention provides a health-promoting functional food for the prevention or improvement of bone diseases, comprising a recombinant stable galactolectin 9 protein having an amino acid sequence represented by Sequence 1 or a polynucleotide encoding therethe.

[0072] Furthermore, the present invention provides the use of a recombinant stable galactolectin 9 protein having the amino acid sequence represented by Sequence 1 or the polynucleotide encoding thereus, for the preparation of pharmaceutical compositions for the prevention or treatment of bone diseases.

[0073] Furthermore, the present invention provides the use of a recombinant stable galactolectin 9 protein having the amino acid sequence represented by Sequence 1 or the polynucleotide encoding thereus, for the preparation of health functional foods for the prevention or improvement of bone diseases.

[0074] The present invention will now be described in more detail through preparation examples and embodiments. However, these preparation examples and embodiments are only for the purpose of helping to understand the present invention and are not intended to limit the scope of protection of the present invention.

[0075] Preparation Example 1. Preparation of recombinant stable galactolectin 9 protein

[0076] An expression vector containing a gene encoding a recombinant stable galactolectin 9 protein with the amino acid sequence of sequence 1 was prepared and introduced into *E. coli* via heat shock. Expression of the recombinant protein was induced as follows: *E. coli* were cultured in LB medium containing 50 μg / ml kanamycin, and arabinose was added at 600 nm when the absorbance reached 0.7 to induce recombinant protein expression. The cells inducing recombinant protein expression were then lysed and filtered, and the target protein was captured using cation exchange chromatography and an affinity column, resulting in a high-purity recombinant stable galactolectin 9 protein with a high yield.

[0077] Example 1. Confirmation of the Tim-3 protein binding ability of recombinant stable galectin 9 protein (sGal-9).

[0078] Add 100 μl of 0.5 μg / ml recombinant human Tim-3 (R&Dsystems, Cat. No. 10241-TI-050) to each well of a 96-well enzyme-linked immunosorbent assay (ELISA) plate (Nunc, Cat. No. 44-2404-21) and incubate overnight at room temperature. Then wash three times with Dulbecco's phosphate-buffered saline (DPBS; Welgene, Cat. No. LB011-02) containing 0.05% Tween 20 (Sigma, Cat. No. P9416). After washing, add 200 μl of Dulbecco's phosphate-buffered saline containing 1% bovine serum albumin (BSA) to each well and incubate for 1 hour at room temperature. Finally, wash three times with wash buffer. After washing three times, the recombinant stable galactose lectin 9 protein (sGal-9) obtained in Preparation Example 1 was diluted to multiple concentrations (0 μg / ml, 5 μg / ml, 10 μg / ml and 20 μg / ml), and 100 μl was added to each well. After incubation at room temperature for 2 hours, the wells were washed three times with washing buffer.

[0079] Gal-9 antibody (R&Dsystems, Cat. No. 1015238) was diluted 1:1000 with 1% bovine serum albumin / DuPont phosphate buffer. 100 μl of the diluted solution was added to each well, and the mixture was incubated at room temperature for 1 hour. The wells were then washed three times with washing buffer to form the experimental group. Anti-mouse HRP antibody (Invitrogen, Cat. No. 31430) was diluted 1:1000 with 1% bovine serum albumin / DuPont phosphate buffer. 100 μl of the diluted solution was added to each well, and the wells were incubated at room temperature for 1 hour. The wells were then washed three times with washing buffer to form the control group.

[0080] A solution of Substrate Reagent (R&D Systems, Cat. No. DY999) A and B, mixed in a 1:1 ratio, was added to each washed well (100 μl). The wells were then incubated at room temperature under light-protected conditions for 20 minutes. After incubation, 50 μl of stop solution (2N H2SO4) was added to each well, and the optical density (OD) value was confirmed by measuring at a wavelength of 450 nm using a microplate reader.

[0081] The results are as follows Figure 1 As shown, it was confirmed that the optical density increased with increasing sGal-9 concentration, confirming that sGal-9 exhibited excellent binding to Tim-3 in a concentration-dependent manner.Figure 1 ).

[0082] Example 2. Confirmation of the effect of sGal-9 in inhibiting T helper cell differentiation.

[0083] Anti-CD3 (eBioscience, Cat. No. 16-0031-82), diluted with phosphate-buffered saline (PBS; Welgene, Cat. No. LB 001-02) at a concentration of 2 μg / ml, was added to each well of a 96-well plate (100 μl of the isolate) or to each well of a 24-well plate (500 μl of the isolate). After incubation overnight at 4°C, the isolates were washed once with phosphate-buffered saline before use in cell culture. After culturing the isolates in 96-well or 24-well plates coated with CD4, CD62L, and T cells at 37°C and 5% CO2 for 4 days, anti-IL-4, anti-IL-2, and anti-IL-12 cytokines were added to induce differentiation into Th1 cells. Anti-IFN-γ, anti-IL-4, anti-IL-2, and anti-IL-6 cytokines were added to induce differentiation into Th17 cells. After adding cytokines, sGal-9 at a concentration of 1 μg / ml was added and the mixture was cultured at 37°C and 5% CO2 for 4 days.

[0084] After culture, the IFN-γ cytokine of Th1 cells and the IL-17 cytokine of Th1 cells were detected using an enzyme-linked immunosorbent assay kit (Mouse DuoSet, R&D Systems) according to the manufacturer's instructions.

[0085] The results are as follows Figure 2 As shown, the differentiation of Th1 and Th17 cells, which act as helper T cells, was confirmed to be inhibited with increasing sGal-9 concentration, thus confirming the excellent inhibitory effect of sGal-9 on helper T cell differentiation. Figure 2 ).

[0086] Example 3. Confirmation of the effect of sGal-9 on inducing apoptosis in fibroblast-like synoviocytes (FLS).

[0087] With 5×10 4200 μl of FLS cells (cell passage: RA_FLS 2-92#P7) were aliquoted into each well of a 48-well plate (SPL, Cat. No. 30048) at a cell density of 200 cells / ml. After 24 hours, 100 μl of sGal-9 at different concentrations (2.5 μg / ml, 5 μg / ml, and 10 μg / ml) were added to each well. The supernatant was collected after 16 hours and stored in a deep freezer.

[0088] Cells intended for Fluorescence Activated Cell Sorting (FACS) were loaded into 15 ml tubes and washed. 200 μl of FACS buffer was added, and the tubes were incubated at 4°C for 1 hour. Cells were then stained with Annexin V-FITC / 7-AAD (Biolegend, Cat. No. 640922). The stained cells were filtered through a 70 μm cell filter (BD Falcon, Cat. No. 352350) in FACS tubes.

[0089] The results are as follows Figure 3 As shown, the effect of sGal-9 in inducing apoptosis in fibroblast-like synovial cells increased with increasing sGal-9 concentration, thus confirming the excellent apoptosis-inducing effect of sGal-9 on synovial cells. Figure 3 ).

[0090] Example 4. Confirmation of the effect of sGal-9 in inhibiting osteoclast differentiation

[0091] Osteoclasts were injected at a concentration of 2.5 × 10⁻⁶. 5 Cells were passaged by adding 1 ml of the cell density to each well of a 12-well culture plate (Corning, Cat. No. 3513). After 24 hours, 1 ml of sGal-9 at different concentrations (0.1 μg / ml, 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml and 10 μg / ml) as the experimental group was added to each well, and 1 ml of phosphate buffer solution was added to each well as the control group.

[0092] After differentiation, osteoclasts in the experimental and control groups were stained with TRAP using a TRAP Staining Kit (K-ASSAY, Cat. No. KT-008). After counting under an optical microscope, the observed multinucleated cells with more than three nuclei (TRAP(+)MNCs) were identified as osteoclasts.

[0093] The results are as followsFigure 4 As shown, the number of stained osteoclasts decreased with increasing sGal-9 concentration, thus confirming the excellent inhibitory effect of sGal-9 on osteoclast differentiation. Figure 4 ).

[0094] Example 5. Confirmation of the effect of sGal-9 in inhibiting bone resorption

[0095] Osteoclasts were injected at a concentration of 2.5 × 10⁻⁶. 5 Cells at a density of 1 ml / ml were added to each well of an OsteoAssay Surface multi-well plate (Corning, Cat. No. 3987) coated with inorganic 3-dimensional crystalline material similar to in vivo bone tissue for passage. After 24 hours, 1 ml of sGal-9 at different concentrations (0.1 μg / ml, 0.5 μg / ml, 1 μg / ml, 2 μg / ml, 4 μg / ml, and 10 μg / ml) as the experimental group was added to each well, and 1 ml of phosphate-buffered saline (PBS) as the control group was added to each well. The experimental group (sGal-9), control group (PBS), and cell culture medium (MEM-alpha, Gibco, Cat. No. 12561-056) were changed daily until differentiation was completed.

[0096] After differentiation, the cells were washed with 20% sodium dodecyl sulfate (SDS) (bleaching solution) and then observed under a microscope in Osteo Assay Surface multi-well culture plates.

[0097] The results are as follows Figure 5 As shown, sGal-9 has been confirmed to have excellent effects in inhibiting bone resorption. Figure 5 ).

[0098] In summary, the results of Examples 1 to 5 confirm that sGal-9 enhances the binding capacity of Tim-3 protein in a concentration-dependent manner, improves the inhibition of Th1 and Th17 cell differentiation and the effect of inducing synovial cell apoptosis, improves the inhibition of osteoclast differentiation and the effect of inhibiting bone resorption, and confirms that it can prevent or treat bone diseases. SEQUENCE LISTING <110> G Biotech Co., Ltd. <120> Pharmaceutical compositions containing recombinant stable galactolectin 9 protein for the prevention or treatment of rheumatoid arthritis and bone diseases. <130> OPB2021-017CN <150> KR 10‑2019‑0143752 <151> 2019‑11‑11 <150> PCT / KR2020 / 015782 <151> 2020‑11‑11 <160> 3 <170> PatentIn version 3.2 <210> 1 <211> 284 <212> PRT <213> Artificial <220> <223> sGal‑9 <400> 1 Met Ala Phe Ser Gly Ser Gln Ala Pro Tyr Leu Ser Pro Ala Val Pro 1 5 10 15 Phe Ser Gly Thr Ile Gln Gly Gly Leu Gln Asp Gly Leu Gln Ile Thr 20 25 30 Val Asn Gly Thr Val Leu Ser Ser Ser Gly Thr Arg Phe Ala Val Asn 35 40 45 Phe Gln Thr Gly Phe Ser Gly Asn Asp Ile Ala Phe His Phe Asn Pro 50 55 60 Arg Phe Glu Asp Gly Gly Tyr Val Val Cys Asn Thr Arg Gln Asn Gly 65 70 75 80 Ser Trp Gly Pro Glu Glu Arg Lys Thr His Met Pro Phe Gln Lys Gly 85 90 95 Met Pro Phe Asp Leu Cys Phe Leu Val Gln Ser Ser Asp Phe Lys Val 100 105 110 Met Val Asn Gly Ile Leu Phe Val Gln Tyr Phe His Arg Val Pro Phe 115 120 125 His Arg Val Asp Thr Ile Ser Val Asn Gly Ser Val Gln Leu Ser Tyr 130 135 140 Ile Ser Phe Gln His Pro Pro Tyr Pro Met Pro Phe Ile Thr Thr Ile 145 150 155 160 Leu Gly Gly Leu Tyr Pro Ser Lys Ser Ile Leu Leu Ser Gly Thr Val 165 170 175 Leu Pro Ser Ala Gln Arg Phe His Ile Asn Leu Cys Ser Gly Asn His 180 185 190 Ile Ala Phe His Leu Asn Pro Arg Phe Asp Glu Asn Ala Val Val Arg 195 200 205 Asn Thr Gln Ile Asp Asn Ser Trp Gly Ser Glu Glu Arg Ser Leu Pro 210 215 220 Arg Lys Met Pro Phe Val Arg Gly Gln Ser Phe Ser Val Trp Ile Leu 225 230 235 240 Cys Glu Ala His Cys Leu Lys Val Ala Val Asp Gly Gln His Leu Phe 245 250 255 Glu Tyr Tyr His Arg Leu Arg Asn Leu Pro Thr Ile Asn Arg Leu Glu 260 265 270 Val Gly Gly Asp Ile Gln Leu Thr His Val Gln Thr 275 280 <210> 2 <211> 146 <212> PRT <213> Artificial <220> <223> CCRD <400> 2 Thr Pro Ala Ile Pro Pro Met Met Tyr Pro His Pro Ala Tyr Pro Met 1 5 10 15 Pro Phe Ile Thr Thr Ile Leu Gly Gly Leu Tyr Pro Ser Lys Ser Ile 20 25 30 Leu Leu Ser Gly Thr Val Leu Pro Ser Ala Gln Arg Phe His Ile Asn 35 40 45 Leu Cys Ser Gly Asn His Ile Ala Phe His Leu Asn Pro Arg Phe Asp 50 55 60 Glu Asn Ala Val Val Arg Asn Thr Gln Ile Asp Asn Ser Trp Gly Ser 65 70 75 80 Glu Glu Arg Ser Leu Pro Arg Lys Met Pro Phe Val Arg Gly Gln Ser 85 90 95 Phe Ser Val Trp Ile Leu Cys Glu Ala His Cys Leu Lys Val Ala Val 100 105 110 Asp Gly Gln His Leu Phe Glu Tyr Tyr His Arg Leu Arg Asn Leu Pro 115 120 125 Thr Ile Asn Arg Leu Glu Val Gly Gly Asp Ile Gln Leu Thr His Val 130 135 140 Gln Thr 145 <210> 3 <211> 10 <212> PRT <213> Artificial <220> <223> CCRD 1-10 <400> 3 Thr Pro Ala Ile Pro Pro Met Met Tyr Pro 1 5 10

Claims

1. Use of a recombinant stable galactolectin 9 protein or a polynucleotide encoding the amino acid sequence represented by sequence 1, characterized in that, A pharmaceutical composition for the preparation of a drug for the prevention or treatment of bone diseases, wherein the bone diseases are selected from one or more of the following groups: arthritis, osteoporosis, periprosthetic osteolysis, osteolysis caused by implant debris, Paget's disease, osteomalacia, rickets, osteopenia, calcium metabolism disorder, bone metastases, inflammatory bone destruction, secondary bone destruction caused by endocrine diseases or drugs, and periodontal diseases accompanied by alveolar bone destruction.

2. The use according to claim 1, characterized in that, The arthritis mentioned above was selected from the group consisting of osteoarthritis, rheumatoid arthritis, osteochondritis dissecans, and juvenile idiopathic arthritis.

3. The use according to claim 1, characterized in that, The aforementioned bone diseases are rheumatoid arthritis or osteoporosis.

4. The use according to claim 1, characterized in that, The above-mentioned pharmaceutical composition also contains a pharmaceutically acceptable carrier.

5. The use according to claim 1, characterized in that, The above-mentioned pharmaceutical composition is formulated for oral, intramuscular, intravenous, intraperitoneal, subcutaneous, intradermal, or local administration.

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