KR20210056927A - Pharmaceutical composition for preventing or treating rheumatoid arthritis and bone diseases comprising recombinant stable galectin 9 protein - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating bone diseases comprising a recombinant stabilized galectin 9 protein. More specifically, the present invention relates to a pharmaceutical composition for preventing or treating bone diseases including rheumatoid arthritis, comprising recombinant stabilized galectin 9 protein as an active component in which an amino acid of a linker peptide is deleted and the amino acid of the C-terminal domain (CCRD) is deleted and substituted.

Description

Pharmaceutical composition for preventing or treating rheumatoid arthritis and bone diseases comprising recombinant stable galectin 9 protein}

The present invention relates to a recombinant stabilized galectin 9 protein and its use, and specifically relates to a pharmaceutical composition for preventing or treating bone diseases including rheumatoid arthritis comprising the protein as an active ingredient.

It was discovered that animal lectins that specifically recognize sugar chains having a β-galactoside structure exist in living organisms, and at least 14 kinds of genes have been identified so far. From its structure, galectins are classified into a prototype, chimera, and tandem repeat.

Galectin 9, one of the tandem repeat-type galectins, consists of two sugar chain recognition sites (sugar recognition domain, carbohydrate recognition domain: CRD) and a link peptide region connecting them, and the N-terminal domain (N -terminal Carbohydrate Recognition Domain; NCRD) and C-terminal Carbohydrate Recognition Domain (CCRD) are linked, and various activities have been reported to date. Regarding T cells, galectin 9 binds to Tim-3, induces apoptosis of Tim-3 positive Th1 cells, and suppresses autoimmune inflammation by inhibiting excessive Th1 response. In addition, it reduces Th17 cells, one of the causes or exacerbations of various intractable diseases such as autoimmune diseases, allergies and cancers expressed by Tim-3.

On the other hand, galectin 9 enhances immunity in some cases. It binds to Tim-3 on monocytes or dendritic cells to activate the cells and promote the production of inflammatory cytokines. In addition, the interaction of galectin 9 and Tim-3 in macrophages enhances immunity to exclude Mycobacterium tuberculosis.

In addition, evidence suggesting the existence of a genetic polymorphism was confirmed among the galectin 9 genes cloned from human cells or tissues, and recombinant galectin 9 produced by E. coli as a host acts directly on tumor cells (tumor The effect of inducing the metastasis and regression of cancer by the activity of inducing cell-cell adhesion and apoptosis, and the effect of inducing the metastasis and regression of cancer by the action through the immune system, and the effect of inducing the apoptosis of activated T cells, especially CD4-positive T cells, which are the cause of excessive immune response. In addition, the effect of treating human immunodeficiency virus (HIV) infection, the treatment of atopy and asthma, and the treatment of type 1 diabetes are known.

1) protease sensitivity of galectin 9 to use galectin 9 as an actual therapeutic agent; 2) low solubility; And 3) In order to solve the problem of a small yield, studies such as preparing a protease-resistant galectin 9 variant (G9Null; Non-Patent Document 1) by cleaving the linker peptide of galectin 9 are being continued. Effects such as KRAS mutant colon carcinoma antitumor activity (Non-Patent Document 2) are known.

On the other hand, bone is a dynamic tissue that undergoes continuous removal and reconstruction through bone resorption and formation. This process is carried out by two types of cells: osteoclast and osteoblast, respectively. Specifically, osteoblasts are responsible for bone formation, and osteoclasts are responsible for bone removal. In a normal environment, bone homeostasis is maintained by the balanced activity of the two cells. However, homeostasis can be stopped due to abnormal hyperactivity of these two types of cells, especially osteoclasts, resulting in arthritis, osteoporosis, periprosthetic osteolysis and Paget's disease. disease). Therefore, precise control of the differentiation and function of osteoclasts is important in the prevention and treatment of bone diseases.

For example, as a drug therapy for treating osteoporosis, it is a drug that promotes bone formation or inhibits bone resorption to prevent a decrease in the amount of bone or to increase the amount of bone. As a bone resorption inhibitor, calcium preparations, vitamin D preparations, bisphosphonate preparations (bisphosphonate), an estrogen agonist/antagonist, an estrogen agent, and an injection of teriparatide, a parathyroid hormone agent, as an stimulator of bone formation.

In addition, among arthritis, rheumatoid arthritis is a typical chronic autoimmune disease, which is caused by inflammation of the synovial tissue surrounding the joint. It can occur in all joints where synovial membranes exist, and it is a chronic inflammatory disease that invades various organs throughout the body, causing severe joint disorders and premature death by destroying joint tissue. In some severe patients, tissues other than joints, such as the lungs, heart, eyes, gastrointestinal tract, skin and kidneys, can be affected, although rare. Moreover, inflammatory mediators, which are generally secreted in large amounts in the body, adversely affect bone metabolism, increasing the risk of osteoporosis and fracture. In fact, the incidence of osteoporosis in rheumatoid arthritis patients is confirmed to be about 15 to 20%.

Drug therapy to treat rheumatoid arthritis includes first-line drugs such as non-steroidal anti-inflammatory drugs, steroid drugs, which are hormones, and second-line drugs that inhibit rheumatoid arthritis itself by affecting the body's immune system. Need to be careful about. In particular, in the case of steroid preparations, when they are used, they feel good immediately and are abused, but the side effects are serious. It can also be a risk factor for osteoporosis when taken for a long time.

Accordingly, the present inventors have tried to develop a new therapeutic agent that has an effective therapeutic effect while minimizing the side effects of arthritis and/or osteoporosis therapeutic agents including rheumatoid arthritis. As a result, the C-terminal domain of the two sugar chain recognition sites of the existing wild-type galectin 9 (CCRD) and a recombinant stabilized galectin 9 protein in which amino acids of link peptides are deleted and substituted inhibit Th1 and Th17 differentiation, and induce apoptosis of fibroblast-like synoviocytes (FLS), The effect of inhibiting the differentiation of osteoclasts was confirmed, and the present invention was completed based on this.

Korean Patent Registration No. 10-1222281 Japanese Patent No. 5887661 U.S. Patent No. 9908921

Nishi, N et al, FEBS Lett. 2005 Apr 11;579(10):2058-64. Wiersma, VR et al, Autophagy. 2015;11(8):1373-88.

An object of the present invention is to provide a recombinant stabilized galectin 9 protein and a pharmaceutical composition for preventing or treating bone diseases comprising the same.

In order to achieve the object of the present invention, the present invention provides a recombinant stabilized galectin 9 protein having an amino acid sequence represented by SEQ ID NO: 1.

In addition, the present invention provides a recombinant vector comprising a gene encoding the protein.

In addition, the present invention provides a transformant into which the recombinant vector has been inserted.

In addition, the present invention provides a pharmaceutical composition for preventing or treating bone diseases, comprising a recombinant stabilized galectin 9 protein having an amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding the same as an active ingredient.

In addition, the present invention provides a health functional food for preventing or improving bone diseases, comprising a recombinant stabilized galectin 9 protein having an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

The recombinant stabilized galectin 9 protein of the present invention exhibits the effect of inhibiting Th1 and Th17 cell differentiation, killing synovial cells (FLS, Fibroblast-like synoviocytes), and inhibiting osteoclast differentiation, so that bone diseases including rheumatoid arthritis It can be usefully used as an active ingredient of a composition for prevention or treatment.

1 is a diagram confirming the degree of binding of Tim-3 of the recombinant stabilized galectin 9 protein (sGal-9) according to the present invention.
Figure 2 is a diagram confirming the effect of inhibiting Th1 and Th17 cell differentiation of sGal-9 according to the present invention.
3 is a diagram illustrating the apoptosis effect of sGal-9 according to the present invention on FLS (Fibroblast-like synoviocytes; synovial cells).
Figure 4 is a diagram confirming the effect of inhibiting osteoclast differentiation of sGal-9 according to the present invention.
5 is a diagram illustrating the effect of inhibiting bone resorption of sGal-9 according to the present invention.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily implement the present invention. Embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art. Accordingly, embodiments of the present invention may be modified into various other forms, and the scope of the present invention is not limited to the embodiments described below.

Throughout the specification of the present invention, when a certain part "includes" a certain constituent element, it means that other constituent elements may be further included rather than excluding other constituent elements unless otherwise specified.

The present invention provides a recombinant stabilized galectin 9 protein having an amino acid sequence represented by SEQ ID NO: 1.

In the present invention, the recombinant stabilized galectin 9 protein has a more stable molecular structure against proteases while maintaining the sugar chain recognition activity of wild type Galectin-9.

Specifically, the recombinant stabilized galectin 9 protein is a link region connecting two CRDs (Carbohydrate Recognition Domains) of wild-type galectin 9 having a structure of NCRD-linker-CCRD and a C-terminal Carbohydrate Recognition Domain (CCRD); C-terminal domain ) Is a recombinant protein produced by modifying. More specifically, the recombinant stabilized galectin 9 protein has all of the peptides of the linker region deleted, the amino acid sequence at positions 1 to 10 (SEQ ID NO: 3) in CCRD (SEQ ID NO: 2) is deleted, and Ala at position 13 (Alarine; A) is substituted with Pro (Proline; P) and may consist of an amino acid sequence represented by SEQ ID NO: 1, and 75% or more, preferably 80% or more, more preferably, with the amino acid sequence represented by SEQ ID NO: 1 May comprise an amino acid sequence having sequence homology of 90% or more, most preferably 95% or more, and prepared for a specific purpose to increase targeting sequence, tag, labeled residue, half-life or peptide stability. An amino acid sequence may additionally be included.

In addition, the recombinant protein of the present invention can be obtained by various methods well known in the art. As an example, it may be prepared using a polynucleotide recombination and protein expression system, or synthesized in vitro through chemical synthesis such as peptide synthesis, and a cell-free protein synthesis method.

As used herein, the term "polynucleotide" is a polymer to which nucleotides are bound, and serves to transmit genetic information. For the purposes of the present invention, encoding the recombinant protein of SEQ ID NO: 1, and 75% or more, preferably 85% or more, more preferably 90% or more, most preferably 95 with the polynucleotide sequence encoding the recombinant protein. It may include sequences having% or more sequence homology.

The term "homology" used in the present invention is intended to indicate a degree of similarity with a wild-type amino acid sequence or a polynucleotide sequence, and comparison of such homology can be performed using a comparison program well known in the art, 2 The homology between two or more sequences can be calculated as a percentage (%).

In addition, the present invention provides a recombinant vector comprising a gene encoding the protein.

In addition, the present invention provides a transformant into which the recombinant vector has been inserted.

The term "vector" used in the present invention refers to a plasmid, virus, or other medium known in the art into which a gene or a base sequence can be inserted or introduced, specifically, a linear DNA plasmid DNA, a recombinant non-viral vector, or a recombinant virus. It may be a sex vector or an inducible gene expression vector system, and the recombinant viral vector is a retrovirus, adenovirus, adeno-associated virus, helper-dependent adenovirus, herpes simplex virus, lentiviral vector, or It may be a vaccinia virus, but is not limited thereto. The nucleotide sequence according to the present invention may be operably linked to an expression control sequence, and the operably linked nucleotide sequence may be included in one expression vector including a selection marker and a replication origin. . The “operably linked” may be a gene and an expression control sequence linked in a manner that allows gene expression when an appropriate molecule is bound to the expression control sequence. "Expression control sequence" refers to a DNA sequence that controls the expression of a nucleotide sequence operably linked in a specific host cell. Such regulatory sequences include promoters for carrying out transcription, any operator sequences for regulating transcription, sequences encoding suitable mRNA ribosome binding sites, and sequences controlling termination of transcription and translation.

The term "transformation" used in the present invention means that the genetic properties of an organism are changed by a given DNA from the outside, that is, DNA, which is a type of nucleic acid extracted from a cell of a certain lineage, is introduced into living cells of another lineage. It means a phenomenon in which DNA enters the cell and changes its genotype. The cell may be a prokaryotic cell or a eukaryotic cell, but is not limited thereto.

In the present invention, the gene encoding the recombinant protein is prepared from a known sequence as described above, and a primer that can specifically recognize it is prepared, and using this, through a polymerase chain reaction (PCR). The gene can be amplified and introduced into a cell after introducing it into a vector as described above. Methods of introduction are known and include, for example, liposome-mediated transduction, calcium phosphate method, DEAE-dextran-mediated translocation, positively charged lipid mediated translocation, electroporation, transduction using a phage system, or infection using a virus. Including, but not limited to.

In addition, the present invention provides a pharmaceutical composition for preventing or treating bone diseases, comprising a recombinant stabilized galectin 9 protein having an amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding the same as an active ingredient.

As used herein, the term "prevention" refers to any action that suppresses the onset or delays the onset by administration of the composition.

As used herein, the term "treatment" refers to any action in which the symptoms of the disease are improved or beneficially changed by the administration of the composition.

In the present invention, the recombinant stabilized galectin 9 protein exhibits any one or more of the following properties, and thus may have an effect of preventing or treating bone diseases:

i) increased Tim-3 protein binding;

ii) inhibition of Th1 and Th17 cell differentiation;

iii) inhibition of T helper cells;

iv) death of fibroblast-like synoviocytes (FLS); And

v) Inhibition of osteoclast differentiation and bone resorption.

Specifically, the recombinant stabilized galectin 9 protein can inhibit helper T cells such as Th1 and Th17 through a mechanism of inducing apoptosis by binding with the Tim-3 protein expressed in T cells. In particular, the synovial cells are one of the important pathologies of rheumatoid arthritis along with inflammatory cytokines, and as the disease progresses, abnormal proliferation and activation of synovial cells is a problem, so that inflammation and destruction of joints may continue to occur.

In addition, the recombinant stabilized galectin 9 protein secretes MMP (matrix methalloproteinase) and cathepsin to destroy the matrix of articular cartilage, fibroblast-like synoviocytes. Can be destroyed.

In addition, the recombinant stabilized galectin 9 protein may inhibit osteoclast differentiation and bone resorption. In particular, the osteoclast is a multinuclear cell that destroys and absorbs bone tissue that is unnecessary in bone growth, and when the balance with osteoblasts involved in bone regeneration and regeneration is disrupted, osteoporosis may occur, causing bone loss. . In addition, the bone resorption (bone resorption) is a phenomenon in which bone is absorbed by the activity of bone-destructive cells, and bisphosphonate, a generally known osteoporosis drug, inhibits the bone resorption and prevents osteoporosis from worsening.

In the present invention, the bone diseases are arthritis, osteoporosis, periprosthetic osteolysis, osteolysis due to fatigue remnants of implants, Paget's disease, osteomalacia, rickets, osteopenia, calcium dysregulation, metastatic Bone cancer (metastatic bone cancer), inflammatory bone loss, endocrine disease or secondary bone loss (secondary bone loss) caused by drugs or periodontal disease accompanied by destruction of alveolar bone, but is not limited thereto.

In addition, the arthritis may be osteoarthritis, degenerative arthritis, rheumatoid arthritis, detachable osteochondritis, joint ligament damage, meniscus damage, joint misalignment, avascular necrosis, or juvenile idiopathic arthritis, but is not limited thereto.

In a specific embodiment of the present invention, the present inventors use a chromatography method after dissolving and purifying E. coli cells that induced the expression of a recombinant stabilized galectin 9 protein having an amino acid sequence represented by SEQ ID NO: 1 Thus, a recombinant protein was prepared. In addition, it was confirmed that the recombinant protein exhibited the effect of inhibiting Th1 and Th17 cell differentiation, the effect of killing fibroblast-like synoviocytes (FLS), and the inhibitory effect of osteoclast differentiation. Therefore, the recombinant protein and the polynucleotide encoding the same can be usefully used as an active ingredient in a composition for preventing or treating bone diseases.

On the other hand, the recombinant protein of the present invention or the polynucleotide encoding the same can be transported in a pharmaceutically acceptable carrier such as colloidal suspension, powder, saline, lipids, liposomes, microspheres, or nano-spherical particles. . They can be complexed or associated with the vehicle and are known in the art such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids. It can be delivered in vivo using known delivery systems.

In addition, pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, which are commonly used in formulation. Polyvinyl pyrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. In addition, a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like may be additionally included in addition to the above components.

The pharmaceutical composition of the present invention can be administered orally or parenterally (for example, intramuscular, intravenous, intraperitoneal, subcutaneous, intradermal, or topically applied) according to a desired method, and the dosage is It depends on the condition and weight, the degree of the disease, the form of the drug, the route of administration and the time, but may be appropriately selected by a person skilled in the art.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.

As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is the type, severity, and drug of the patient's disease. Activity, sensitivity to drugs, time of administration, route of administration and rate of excretion, duration of treatment, factors including drugs used concurrently, and other factors well known in the medical field. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent, or may be used in combination with surgery, hormone therapy, drug treatment, and biological response modifier, and may be administered simultaneously, separately or sequentially with the agent, single or It can be administered multiple times. It is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects in consideration of all of the above factors, and this can be easily determined by a person skilled in the art.

Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient in the body, inactivation rate, excretion rate, type of disease, and drugs used in combination, and the route of administration, Obesity may increase or decrease depending on the severity, sex, weight, and age.

In addition, the present invention provides a health functional food for preventing or improving bone diseases, comprising a recombinant stabilized galectin 9 protein having an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

In the present invention, the contents of the recombinant stabilized galectin 9 protein and bone disease are the same as those described above, and the detailed descriptions refer to the above.

As used herein, the term "improvement" refers to any action that at least reduces the severity of a parameter related to the condition being treated, for example, symptoms. In this case, the health functional food composition may be used before or after the onset stage of the disease in order to prevent or improve bone disease, and simultaneously with or separately from the drug for treatment.

Meanwhile, in the present invention, the recombinant stabilized galectin 9 protein having the amino acid sequence represented by SEQ ID NO: 1 inhibits Th1 and Th17 cell differentiation, fibroblast-like-synoviocytes (FLS) killing effect and osteoclasts. Since it was confirmed that the (osteoclast) differentiation inhibitory effect was shown, the recombinant protein can be usefully used as an active ingredient of a health functional food for preventing or improving bone disease.

In the health functional food of the present invention, the active ingredient may be added to the food as it is or may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use (for prevention or improvement). In general, when preparing food or beverage, the health functional food of the present invention may be added in an amount of preferably 15% by weight or less, preferably 10% by weight or less based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of health control, the amount may be less than the above range.

The health functional food of the present invention may contain other ingredients as essential ingredients without particular limitation in addition to containing the active ingredient. For example, as in ordinary beverages, various flavoring agents or natural carbohydrates may be included as additional ingredients. Examples of the above-described natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, common sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. I can. The proportion of the natural carbohydrate can be appropriately determined by the choice of a person skilled in the art.

In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents and heavy weight agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid. And salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like. These components may be used independently or in combination, and the proportion of these additives may also be appropriately selected by those skilled in the art.

Hereinafter, the present invention will be described in more detail through Preparation Examples and Examples. However, the following Preparation Examples and Examples are intended to aid understanding of the present invention, and are not intended to limit the scope of the present invention thereto.

<Preparation Example 1> Preparation of recombinant stabilized galectin 9 protein

An expression vector containing a gene encoding a recombinant stabilized galectin 9 protein having an amino acid sequence of SEQ ID NO: 1 was prepared, and the expression vector was introduced into E. coli by a heat shock method. Expression of the recombinant protein was carried out by culturing E. coli in an LB medium containing 50 µg/ml kanamycin, adding arabinose when the absorbance at 600 nm reached 0.7 to induce expression of the recombinant protein. Then, the cells inducing the expression of the recombinant protein were lysed and filtered, and the target protein was captured using a cation exchange method and an affinity column to obtain a highly purified recombinant stabilized galectin 9 protein in high yield.

<Example 1> Recombinant stable Galectin 9 protein (Recombinant stable Galectin 9 protein, sGal-9) Tim-3 protein binding ability confirmation

Recombinant human Tim-3 (R&D systems, Cat. No. 10241-TI-050) at a concentration of 0.5 μg/ml was added 100 μl to each well of a 96-well ELISA plate (Nunc, Cat. No. 44-2404-21). After adding and reacting overnight at room temperature, it was washed three times with DPBS (Dulbecco's phosphate-buffered saline; Welgene, Cat. No. LB011-02) containing 0.05% of Tween 20 (Sigma Cat. No. P9416). After washing, 200 µl of DPBS containing 1% bovine serum albumin (BSA) was added to each well, incubated for 1 hour at room temperature, and washed three times with a wash buffer. After washing three times, the recombinant stabilized galectin 9 protein (sGal-9) obtained in <Preparation Example 1> was diluted to various concentrations (0, 5, 10 and 20 μg/ml), and 100 μl each was added to each well at room temperature. After incubation for 2 hours at, it was washed 3 times with a washing buffer.

As an experimental group, Gal-9 antibody (R&D systems, Cat. No. 1015238) was diluted 1:1000 with 1% BSA/DPBS, added 100 µl to each well, and incubated for 1 hour at room temperature, and then used as a washing buffer. Washed twice. As a control, anti-mouse HRP antibody (Invitrogen, Cat. No. 31430) was diluted 1:1000 with 1% BSA/DPBS, added to each well, and incubated for 1 hour at room temperature for 1 hour. Washed twice.

A solution of 1:1 mixture of Substrate Reagent (R&D systems, Cat. No. DY999) A and B was added to each washed well and incubated at room temperature for 20 minutes. After incubation, 50 µl of a stop solution (2 N H2SO4) was added to each well and measured at 450 nm using a microplate reader to check the optical density (O.D) value.

As a result, as shown in FIG. 1, it was confirmed that the optical density increased as the concentration of sGal-9 increased, and it was confirmed that the degree of bonding between sGal-9 and Tim-3 was excellent in a concentration-dependent manner (FIG. 1).

<Example 2> Confirmation of the inhibitory effect of sGal-9 on T helper cell differentiation

Anti-CD3 (eBioscience, Cat. No. 16-0031-82) with a concentration of 2 µg/ml diluted with PBS (Welgene, Cat. No. LB 001-02) was added 100 µl each to a 96-well plate or 24 wells. After dispensing 500 µl of each plate and reacting overnight at 4° C., it was washed once with PBS and used for cell culture. Dispensing into a 96-well plate or 24-well plate coated with CD4, CD62L and T cells and incubating for 4 days at 37°C and 5% CO2 conditions, anti-IL-4, anti-IL-2 to induce differentiation into Th1 cells And anti-IL-12 cytokines and anti-IFN-γ, anti-IL-4, anti-IL-2 and anti-IL-6 cytokines were added to induce differentiation into Th17 cells. After the cytokine was added, sGal-9 having a concentration of 1 μg/ml was added and incubated for 4 days at 37°C and 5% CO2.

After cultivation, IFN-γ cells of Th1 cells and IL-17 cytokines of Th17 cells were measured according to the manufacturer's procedure using an ELISA assay kit (Mouse DuoSet, R&D Systems).

As a result, as shown in FIG. 2, it was confirmed that the differentiation of the helper T cells Th1 and Th17 cells was inhibited as the concentration of sGal-9 increased, and it was confirmed that the effect of inhibiting the differentiation of the helper T cells of sGal-9 was excellent ( Fig. 2).

<Example 3> Fibroblast-like-synoviocytes (FLS, Fibroblast-like synoviocytes) killing effect of sGal-9 was confirmed

FLS cells (cell passage: RA_FLS 2-92#P7) were dispensed into each well of a 48-well plate (SPL, Cat. 100 µl of sGal-9 at concentrations (2.5, 5, and 10 µg/ml) was added to each well. After 16 hours, the supernatant was collected and stored in a deep freezer.

Cells for fluorescence-activated cell sorting (FACS) are washed in a 15 ml tube, and 200 μl of FACS buffer is added, and then stored at 4° C. for 1 hour and Annexin V-FITC/7-AAD (Biolegend, Cat. No. 640922) was performed. Stained cells were filtered with a 70 μm cell strainer (BD Falcon, Cat. No. 352350) in a tube for FACS.

As a result, as shown in Fig. 3, it was confirmed that the fibroblast-like-synovial cell killing effect increased as the concentration of sGal-9 increased, and it was confirmed that the synovial cell killing effect of sGal-9 is excellent (Fig. 3). ).

<Example 4> Confirmation of the inhibitory effect of sGal-9 on osteoclast differentiation

Osteoclasts were passaged 1 ml each at a cell density of 2.5 x 105 cells/ml in each well of a 12-well plate (Corning, Cat. No. 3513), and after 24 hours, various concentrations (0.1, 0.5, 1, 2, 4, and 10 μg/ml) of sGal-9 and 1 ml of PBS were added to each well as a control group.

After completion of differentiation, the stained osteoclasts of the experimental group and the control group were stained with TRAP using a TRAP Staining Kit (K-ASSAY, Cat. No. KT-008) and counted using an optical microscope, and then three or more nuclei were observed. The resulting multinuclear cells (TRAP(+) MNCs) were judged to have differentiated into osteoclasts.

As a result, as shown in FIG. 4, it was confirmed that the number of stained osteoclasts decreased as the concentration of sGal-9 increased, and it was confirmed that the effect of inhibiting osteoclast differentiation of sGal-9 was excellent (FIG. 4 ).

<Example 5> Confirmation of the inhibitory effect of sGal-9 on bone resorption

In each well of the Osteo Assay Surface multi-well plate (Corning, Cat. No. 3987) coated with an inorganic 3-dimensional crystalline resembling a bone structure in vivo, osteoclast cells are placed in each well. Passage 1 ml each at a cell density of 2.5 x 105 cells/ml, and after 24 hours, each well with sGal-9 and PBS as a control group as an experimental group of various concentrations (0.1, 0.5, 1, 2, 4 and 10 μg/ml) 1 ml was added to each. The experimental group (sGal-9), the control group (PBS), and the cell culture solution (MEM-alpha, gibco, Cat. No. 12561-056) were replaced daily until the differentiation was completed.

After completion of differentiation, cells were removed with 20% SDS (Bleaching solution), and the Osteo Assay Surface multi-well plate was observed under a microscope.

As a result, as shown in Fig. 5, it was confirmed that the effect of inhibiting bone resorption of sGal-9 is excellent (Fig. 5).

In conclusion, through the results of <Example 1> to <Example 5>, sGal-9 increased the Tim-3 protein binding ability in a concentration-dependent manner, inhibited Th1 and Th17 cell differentiation, and increased synovial cell death effect, and osteoclast differentiation. It was confirmed that bone diseases can be prevented or treated by increasing the inhibitory effect and bone resorption inhibitory effect.

<110> GBiologics <120> Pharmaceutical composition for preventing or treating rheumatoid arthritis and bone diseases comprising recombinant stable galectin 9 protein <130> PB2020-166 <150> KR 10-2019-0143752 <151> 2019-11-11 <160> 3 <170> KoPatentIn 3.0 <210> 1 <211> 284 <212> PRT <213> Artificial Sequence <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 Sequence <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 Sequence <220> <223> CCRD 1-10 <400> 3 Thr Pro Ala Ile Pro Pro Met Met Tyr Pro 1 5 10

Claims (12)

Recombinant stabilized galectin 9 protein having an amino acid sequence represented by SEQ ID NO: 1.
The recombinant stabilized galectin 9 protein according to claim 1, wherein the recombinant stabilized galectin 9 protein maintains the sugar chain recognition activity of wild-type galectin 9.
A recombinant vector comprising a gene encoding the protein of claim 1.
A transformant into which the recombinant vector of claim 3 has been inserted.
A pharmaceutical composition for preventing or treating bone disease, comprising a recombinant stabilized galectin 9 protein having an amino acid sequence represented by SEQ ID NO: 1 or a polynucleotide encoding the same as an active ingredient.
The pharmaceutical composition for preventing or treating bone disease according to claim 5, wherein the recombinant stabilized galectin 9 protein exhibits any one or more of the following characteristics:
i) increased Tim-3 protein binding;
ii) inhibition of Th1 and Th17 cell differentiation;
iii) inhibition of T helper cells;
iv) death of fibroblast-like synoviocytes (FLS); And
v) Inhibition of osteoclast differentiation and bone resorption.
The method of claim 5, wherein the bone disease is arthritis, osteoporosis, periprosthetic osteolysis, osteolysis due to fatigue remnants of an implant, Paget's disease, osteomalacia, rickets, osteopenia, and calcium dysregulation. , Metastatic bone cancer (metastatic bone cancer), inflammatory bone loss, endocrine disease or secondary bone loss caused by drugs (secondary bone loss) and periodontal disease accompanied by destruction of the alveolar bone, characterized in that any one or more selected from the group consisting of, A pharmaceutical composition for preventing or treating bone disease.
8. Characterized in, a pharmaceutical composition for preventing or treating bone disease.
The pharmaceutical composition for preventing or treating bone disease according to claim 5, wherein the bone disease is rheumatoid arthritis or osteoporosis.
The pharmaceutical composition for preventing or treating bone disease according to claim 5, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
The method of claim 5, wherein the pharmaceutical composition is formulated for oral administration, intramuscular administration, intravenous administration, intraperitoneal administration, subcutaneous administration, intradermal administration, or topical administration, A pharmaceutical composition for preventing or treating bone disease.
Health functional food for preventing or improving bone disease, comprising a recombinant stabilized galectin 9 protein having an amino acid sequence represented by SEQ ID NO: 1 as an active ingredient.

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