TM4SF19 inhibitors and their uses

Abstract

The present invention relates to a composition for preventing or treating bone diseases, obesity or obesity-mediated metabolic diseases, cancer, and cancer metastasis, which contains an agent that inhibits the expression or activity of TM4SF19 (transmembrane 4 Lsix family member 19), and a method for screening for therapeutic drugs for the above diseases.

Description

[Technical Field]

[0001] The present invention relates to a composition for preventing or treating bone diseases, obesity or obesity-mediated metabolic diseases, cancer, and cancer metastasis, which comprises an inhibitor of the expression or activity of TM4SF19 (transmembrane 4L six family member 19), and a method for screening for therapeutic drugs for the above diseases. [Background technology]

[0002] Bones are dynamic tissues that change constantly throughout life. With the naked eye, bones are divided into the outer cortical bone (compact bone) and the inner trabecular bone (spongy bone). Cortical bone has great physical strength and plays a role in protecting and supporting the body, while trabecular bone plays a role in absorbing shock and maintaining a constant calcium balance.

[0003] Even after bone growth stops, old bone is broken down and lost (bone resorption), and new bone fills in the gaps (bone formation), a process that continues throughout life. This phenomenon is called bone remodeling.

[0004] Bone homeostasis is maintained through a balanced interaction between bone formation by osteoblasts and bone resorption by osteoclasts, and blood calcium levels are kept constant. However, bone metabolism imbalance can lead to bone-related diseases such as osteoporosis.

[0005] The interaction between osteoclasts and osteoblasts causes bone resorption and bone formation to occur sequentially, maintaining bone homeostasis. This phenomenon is called bone remodeling, and bone mass is determined by the balance of activity between osteoclasts and osteoblasts. The bone remodeling process occurs in a balanced manner, with osteoclasts promoting bone resorption followed by osteoblast-mediated bone formation. When this efficient balance is disrupted, bone loss occurs.

[0006] Osteoclasts undergo differentiation to become multinucleated osteoclasts, which then cause bone resorption. Osteoclast dysfunction leads to osteopetrosis, while increased bone resorption due to osteoclast overactivation leads to osteoporosis and inflammatory arthritis after menopause. To date, bisphosphonates and anti-RANKL antibodies have been developed to target osteoclast overactivation. However, general inhibition of osteoclasts affects the balance between osteoclasts and osteoblasts, significantly impacting bone formation and resulting in side effects. Therefore, the development of therapeutic agents that can more selectively inhibit osteoclasts is needed.

[0007] Osteoclasts are multinucleated cells derived from hematopoietic stem cells and differentiate from monocyte / macrophage lineages through stimulation with monocyte / macrophage colony-stimulating factor (M-CSF) and activation by receptor activator of nuclear factor κB (RANKL). Osteoclast differentiation occurs when osteoclast precursors differentiate into TRAP-positive mononuclear osteoclasts, which then mature into multinucleated osteoclasts through cell-cell fusion (see Figure 2c). These mature, multinucleated osteoclasts mediate bone resorption (Int J Mol Sci. 2020 Aug 8;21(16):5685).

[0008] Obesity, a biological phenomenon resulting from the complex interaction of genetic, metabolic, environmental, and behavioral factors, is defined as abnormal or excessive fat accumulation and can adversely affect health. Obesity, in particular, is known to be a significant risk factor for various adult diseases, including hypertension, type 2 diabetes, cancer, liver disease, hyperlipidemia, and atherosclerosis. Obesity is the result of a chronic imbalance between caloric intake and energy expenditure, resulting in impaired metabolic, endocrine, and immune functions in adipose tissue. This leads to metabolic abnormalities and reduced responsiveness (resistance) to insulin, the main fat storage signaling hormone. This leads to fat accumulation in metabolic organs other than adipose tissue, resulting in various forms of lipotoxicity. The most representative pathological manifestation of lipotoxicity is the inflammatory response, characterized by chronic low-grade inflammation. Before metabolic abnormalities and insulin resistance appear in obesity, macrophages (adipose tissue macrophages) are activated in adipose tissue, entering a pre-inflammatory phase. Thus, obesity induces a low-intensity chronic inflammatory state in the body, leading to various metabolic diseases. Thus, adipose tissue macrophages play an important role in the development of chronic inflammation and metabolic complications caused by obesity.

[0009] Fat stored in adipocytes is used as an important energy source in the body. Excessive adipocyte differentiation and an unbalanced energy supply can lead to obesity. Adipose tissue (adipose tissue) contains not only adipocytes but also various cells, such as adipose-derived stem cells (ASCs), immune cells, and endothelial cells, known as the stromal vascular fraction (SVF). Adipose tissue stores energy in the form of lipids but also functions as a heat insulator. It is divided into white adipose tissue (WAT), which stores nutrients, and brown adipose tissue (BAT), which consumes nutrients and generates heat. Adipose tissue also produces leptin, resistin, adiponectin, and tumor necrosis factor-α (TNFα). Adipocyte differentiation is a complex process that occurs through the interaction of various hormones and transcription factors. It is promoted by stimuli such as insulin, insulin-like growth factor-1, and growth hormone. During this process, increased expression of transcription factors, such as the CCAAT enhancer-binding protein (C / EBP) family and peroxisome proliferator-activated receptor (PPAR) gamma, is observed. These transcription factors, along with adipocyte regulatory factors, promote adipocyte differentiation, resulting in increased expression of enzymes such as fatty acid-binding protein aP2 and fatty acid biosynthesis enzymes. Furthermore, excessive triglyceride accumulation has been reported to be involved in the progression of fatty liver [J. Clin. Invest., 98, 1575–1584 (1996)].

[0010] Currently, the most commonly used obesity treatments are fat absorption inhibitors such as Xenical from Roche in Switzerland and appetite suppressants such as Meridia from Abbott in the United States, but these drugs have side effects such as headaches, elevated blood pressure, and diarrhea. Therefore, there is a need to develop new obesity treatments that do not cause side effects.

[0011] Cancer is one of the greatest threats to human health, and occurs when cells undergo a series of mutations, proliferating in an uncontrolled manner and becoming immortal. For cancers discovered in the early stages, treatments such as surgery, radiation therapy, and chemotherapy are available, but side effects are becoming a major problem, and in the case of terminal or metastatic cancer, patients have a limited lifespan without any specific treatment.

[0012] Recently, various biochemical mechanisms related to cancer have been elucidated and corresponding therapeutic agents have been developed, but a fundamental method for treating cancer has not yet been presented. As a result, research is actively underway to identify various cancer-related biomolecules and develop drugs that target them, and efforts are also being made to combine some of these drugs to enhance the effectiveness of cancer treatment.

[0013] Therefore, efforts to further discover cancer-related target molecules are of great importance.

[0014] TM4SF19 is a transmembrane 4L six family member (TM4SF19), and its use as a diagnostic marker for obesity has been disclosed in Korean Patent Registration No. 10-1781200. However, there have been no reports of TM4SF19 being used to treat bone-related diseases, obesity or obesity-related metabolic diseases, cancer, or to inhibit cancer metastasis. Summary of the Invention [Problem to be solved by the invention]

[0015] Therefore, the inventors conducted research into the relationship between various genes and bone diseases, obesity or obesity-mediated metabolic diseases, and cancer, in order to search for related genes with the aim of fundamentally treating bone diseases, obesity and various metabolic diseases mediated by obesity, cancer treatment, or suppressing cancer metastasis.As a result, they confirmed the various functions of TM4SF19, thereby completing the present invention. [Means for solving the problem]

[0016] An object of the present invention is to provide a pharmaceutical composition for preventing or treating bone diseases, which comprises an agent that inhibits the expression or activity of TM4SF19 as an active ingredient.

[0017] Another object of the present invention is to provide a method for screening for a drug for treating bone disease, which comprises the steps of treating a specimen suspected of having bone disease with a candidate substance for treating bone disease, and comparing the expression level of mRNA or protein of the TM4SF19 gene with that of a control group.

[0018] It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating obesity or obesity-mediated metabolic diseases, which comprises an inhibitor of TM4SF19 expression or activity as an active ingredient.

[0019] Yet another object of the present invention is to provide a method for screening for therapeutic agents for obesity or obesity-mediated metabolic diseases, which comprises the steps of treating a subject suspected of having obesity or an obesity-mediated metabolic disease with a candidate substance for treating obesity or an obesity-mediated metabolic disease, and comparing the expression level of mRNA or protein of the TM4SF19 gene with that of a control group.

[0020] It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating cancer or suppressing cancer metastasis, which comprises an inhibitor of TM4SF19 expression or activity as an active ingredient.

[0021] Another object of the present invention is to provide a method for screening drugs for treating cancer or cancer metastasis, which comprises treating a specimen suspected of cancer prevention, treatment or cancer metastasis with a candidate substance for cancer prevention, treatment or cancer metastasis suppression, and comparing the expression level of mRNA or protein of the TM4SF19 gene with that of a control group.

[0022] It is yet another object of the present invention to provide a fusion protein for inhibiting the expression or activity of TM4SF19.

[0023] Another object of the present invention is to provide a method for preventing or treating bone diseases, comprising administering to a subject a therapeutically effective amount of a composition for preventing or treating bone diseases, the composition comprising an inhibitor of TM4SF19 expression or activity.

[0024] Another object of the present invention is to provide a method for preventing or treating obesity or obesity-mediated metabolic diseases, comprising administering to a subject a composition for preventing or treating obesity or obesity-mediated metabolic diseases, comprising a therapeutically effective amount of a TM4SF19 expression or activity inhibitor.

[0025] Another object of the present invention is to provide a method for preventing or treating cancer or preventing or treating cancer metastasis, which comprises administering to a subject a composition for preventing, treating cancer or suppressing cancer metastasis, which comprises a therapeutically effective amount of an inhibitor of TM4SF19 expression or activity. [Effects of the Invention]

[0026] The agent for suppressing TM4SF19 expression or activity according to the present invention can be used to prevent or treat bone diseases, obesity, obesity-mediated metabolic diseases, cancer, and cancer metastasis.

[0027] Furthermore, a method for screening candidate substances for treating bone diseases, obesity, obesity-mediated metabolic diseases, cancer, and cancer metastasis that inhibit TM4SF19 can efficiently select candidate substances that can treat bone diseases, obesity, obesity-mediated metabolic diseases, cancer, and cancer metastasis. [Brief explanation of the drawings]

[0028] [Figure 1] Figure 1 shows the results of qPCR analysis of TM4SF19 gene expression after cells were extracted from mouse bone marrow and differentiated over time by treating them with M-CSF and RANKL (Receptor activator of nuclear factor kappa-B ligand). [Figure 2a] Figure 2a shows the results of TRAP staining after differentiation of mouse bone marrow-derived cells from wild-type (WT) mice and CRISPR knockout mice, TM4SF19KO, with M-CSF (25 ng / ml) + RANKL (100 ng / ml). [Figure 2b] Figure 2b shows the results of cells extracted from the bone marrow of TM4SF19KO, a CRISPR-induced TM4SF19 knockout mouse, and wild-type (WT) mice, differentiated under conditions where the RANKL concentration was fixed at 100 ng / ml and the M-CSF concentrations were varied at 25, 40, and 60 ng / ml, and then stained for TRAP (tartrate-resistant acid phosphatase), an osteoclast-related marker. [Figure 2c] FIG. 2c is a schematic diagram showing the differentiation process of osteoclasts. [Figure 3] Figure 3 shows the results of qPCR analysis of the expression of target genes involved in osteoclast (OC) differentiation, including Ctsk, Acp5, c-Fos, and Nfatc1, four days after cells were extracted from the bone marrow of wild-type (WT) and TM4SF19KO mice and differentiated by treatment with M-CSF or M-CSF + RANKL (M-CSF 25 ng / ml, RANKL 100 ng / ml). [Figure 4a] Figure 4a shows the results of F-actin staining to confirm the presence or absence of ectin belt formation during the differentiation of multinucleated osteoclasts after differentiation of wild-type and TM4SF19KO mouse bone marrow. [Figure 4b]Figure 4b shows pit formation confirmed by 1% toluidine blue staining after BMM cells were plated on a Dentin disc and differentiated. [Figure 5a] Figure 5a shows the results of microCT analysis of 8-week-old wild-type (WT) and TM4SF19KO female mice after either sham or ovariectomy (OVX), in which the femoral region was fixed 31 days after ovarian excision. [Figure 5b] Figure 5b shows the 3D microCT indices (bone microstructure indices) obtained by analyzing the microCT images using a 3D image analysis program, including total bone mineral density (BMD), bone volume (BV), the ratio of bone volume to tissue volume (%BV / TV), the mean number of bone trabeculae (Tb.N), the trabecular spacing (Tb.Sp), and the trabecular thickness (Tb.Th). [Figure 6] FIG. 6 shows the results of TRAP staining of femurs from 8-week-old wild-type (WT) mice and TM4SF19KO mice, which were fixed and decalcified. [Figure 7a] Figure 7a shows the results of TRAP staining after cells extracted from the bone marrow of wild-type (WT) mice and TM4SF19EC2Δ mice in which the TM4SF19 extracellular domain 2 (116–165) had been knocked out using CRISPR. The cells were differentiated by treatment with M-CSF 25 ng / ml or M-CSF + RANKL 100 ng / ml. [Figure 7b] Figure 7b shows the results of TRAP staining after cells extracted from the bone marrow of wild-type (WT) and TM4SF19EC2Δ mice were fixed at a RANKL concentration of 100 ng / ml and differentiated by treatment with M-CSF at a concentration of 25 ng / ml or 100 ng / ml. [Figure 7c] Figure 7c shows the results of F-actin staining to confirm the presence or absence of ectin belt formation, which occurs during the differentiation of multinucleated osteoclasts, after differentiation of wild-type and TM4SF19EC2Δ mouse bone marrow. [Figure 7d]Figure 7d shows wild-type and TM4SF19EC2Δ BMM cells plated on a Dentin disc and differentiated, followed by confirmation of pit formation using 1% toluidine blue staining. [Figure 8] FIG. 8 shows the results of qPCR confirmation of the expression of target genes (Ctsk, Acp5, c-Fos, Nfatc1) associated with osteoclast differentiation in wild-type (WT) mice and TM4SF19EC2Δ mice. [Figure 9a] Figure 9a shows the results of microCT analysis of 8-week-old WT and TM4SF19EC2Δ female mice, in which the ovaries were either sham-operated or ovariectomized (OVX), and the femurs were fixed 31 days later. [Figure 9b] Figure 9b shows the 3D microCT indices (bone microstructure indices) obtained by analyzing the microCT images using a 3D image analysis program, including total bone mineral density (BMD), bone volume (BV), the ratio of bone volume to tissue volume (%BV / TV), the average number of bone trabeculae (Tb.N), the trabecular spacing (Tb.Sp), and the trabecular thickness (Tb.Th). [Figure 10] Figure 10 shows the bone microarchitecture indexes obtained by microCT analysis of 8-week-old wild-type (WT), TM4SF19KO, and TM4SF19EC2Δ female mice after either sham or ovariectomy (OVX) of the ovaries, followed by fixation of the femurs 31 days later. [Figure 11] Figure 11 shows microCT images of 8-week-old wild-type (WT), TM4SF19KO, and TM4SF19EC2Δ female mice, in which the ovaries were either released without dissection (sham) or ovariectomized (OVX), and the femurs were fixed 31 days later. [Figure 12a] FIG. 12a is a schematic diagram showing the structures of hTm4sf19 mutants (hTM4SF19115-175Δ, hTm4sf19105-186Δ, hTm4sf105-196Δ, hTm4sf1994-186Δ, and hTm4sf1994-196Δ) in which a portion of the TM4SF19 sequence has been deleted. [Figure 12b] Figure 12b shows the results indicating that TM4SF19 binds to itself and is involved in cell-cell interactions. Among the mutants in which a partial sequence of TM4SF19 was deleted, the 94-196 deletion mutant did not bind to TM4SF19 itself. [Figure 12c] Figure 12c shows the results (bottom panel) of hTm4sf19, which is a deletion of the remaining sequences from hTm4sf19, leaving only the 94-196 sequence (top panel), demonstrating that hTm4sf1994-196 binds to itself and is involved in cell-cell interactions, just like the wild type (WT). [Figure 12d] Figure 12d shows the structures of the TM4SF19 fragments hTm4sf19 131-160, hTm4sf19 145-169, hTm4sf19 131-169, hTm4sf19 120-160, hTm4sf19 120-169, hTm4sf19 120-180, hTm4sf19 120-186, hTm4sf19 120-196, hTm4sf19 120-209, hTm4sf19 131-196, and hTm4sf19 145-196, and the results of immunoprecipitation confirming the mutual binding between N-terminally 3Flag-tagged TM4SF19 and hIgG1-Fc fusion proteins of the TM4SF19 fragments. [Figure 12e] Figure 12e, like Figure 12d, shows the TM4SF19 fragments hTm4sf19 131-160, hTm4sf19 145-169, hTm4sf19 131-169, hTm4sf19 120-160, hTm4sf19 120-169, hTm4sf19 120-180, hTm4sf19 120-186, hTm4sf19 120-196, hTm4sf19 120-209, hTm4sf19 131-196, and hTm4sf19 The figure shows the structure of 145 to 196 and the results of confirming the mutual binding between TM4SF19 tagged with 3Flag at the N-terminus and the hIgG1-Fc fusion protein of the TM4SF19 fragment by immunoprecipitation. [Figure 12f]FIG. 12f shows the results of immunoprecipitation confirming the mutual binding between integrin αv tagged with 3HA at the C-terminus and the hIgG1-Fc fusion protein of the TM4SF19 fragment. [Figure 12g] FIG. 12g shows the results of immunoprecipitation confirming the interaction between integrin β3 tagged with 3HA at the C-terminus and a hIgG1-Fc fusion protein of the TM4SF19 fragment. [Figure 12h] Figure 12h shows the interaction between TM4SF19 and integrin αv or β3, proteins that regulate osteoclast function and are involved in cytoskeletal rearrangement during multinucleated osteoclast formation, overexpressed in 293T cells or osteoclast precursor Raw264.7 cells. Additionally, the interaction between integrin αv or β3 tagged with 3HA at the C-terminus and TM4SF19 tagged with 3Flag at the N-terminus or a mutant lacking TM4SF19 EC2(115-175) was also examined. [Figure 12i] Figure 12i shows the results of immunoprecipitation confirming the interaction between TM4SF19 and siglec-15 with DC-Stamp, a membrane protein involved in regulating osteoclast function and involved in cytoskeletal rearrangement during multinucleated osteoclast formation. [Figure 13] Figure 13 shows the results of overexpressing TM4SF19 in mouse breast cancer cell line E0771 and examining cell proliferation, colony formation, and cell migration [LPCX: retroviral control vector name]. [Figure 14] FIG. 14 shows the results of confirming the suppression of lung metastasis after mouse-derived breast cancer cell E0771 was injected into the tail vein of wild-type (WT), TM4SF19KO, and TM4SF19EC2Δ mice. [Figure 15a]Figure 15a shows the results of qPCR analysis of target genes involved in cell migration (fibronectin, vimentin, CDH2, SNAI1, SNAI2) after bone marrow was collected from wild-type (WT) and TM4SF19KO mice and differentiated into bone marrow-derived macrophages, which were then co-cultured with breast cancer cells to induce cell migration (Figure 15a). [Figure 15b] Figure 15b shows the results of Western blotting to confirm the expression of metastasis-related proteins (vimentin, slug, snail, E-cadherin, and β-actin) [BMDM: bone marrow derived macrophages]. [Figure 16] Figure 16 shows the results of differentiating human adipose-derived mesenchymal stem cells (hADMSCs) into adipocytes, followed by confirming gene (C / EBPα, PPARγ, TM4SF19) expression by qPCR (top row) and protein (TM4SF19, PPARγ, FABP4) expression by Western blotting (bottom row). [Figure 17] FIG. 17 shows the results of confirming weight gain when 6-week-old wild-type (WT) mice and TM4SF19KO mice fed a normal diet were fed a high-fat diet (60% fat) for 16 weeks. [Figure 18] Figure 18 shows the results of 6-week-old wild-type (WT) mice and TM4SF19KO mice fed a normal diet and then a high-fat diet for 12 weeks, after which insulin resistance was confirmed by HOMA-IR (A), liver phenotype was confirmed, liver tissue was fixed and stained with H&E (B), and triglyceride levels in the liver were measured (C). [Figure 19a] FIG. 19a shows the weight gain observed in 6-week-old wild-type (WT) mice and TM4SF19KO mice fed a normal diet and a high-fat diet for 18 weeks. [Figure 19b] FIG. 19b shows the weight of each tissue. [Figure 19c] FIG. 19c is a graph confirming the weight of subcutaneous fat and visceral fat. [Figure 19d]FIG. 19d shows H&E staining of epididymal white adipose tissue (eWAT), confirming the adipose phenotype and macrophages in the adipose tissue surrounding the adipose tissue. [Figure 19e] Figure 19e shows the results of confirming the secretion of adiponectin, an anti-obesity cytokine, in serum [rtWAT: retroperitoneal white adipose tissue, sWAT: subcutaneous white adipose tissue, ingWAT: inguinal white adipose tissue, iWAT: interscapular white adipose tissue, iBAT: interscapular brown adipose tissue]. [Figure 20] Figure 20 shows the results of examining the expression of adipose differentiation markers (top row) and macrophages and M1-like macrophage markers (bottom row) in eWAT (epididymal white adipose tissue) and sWAT (subcutaneous white adipose tissue) of 6-week-old wild-type (WT) and TM4SF19KO mice fed a normal diet after 12 weeks of high-fat diet feeding. [Figure 21] Figure 21 shows the results of FACS analysis of epididymal white fat to determine the percentage of macrophages (A) and dendritic cells (B) in 6-week-old wild-type (WT) and TM4SF19KO mice fed a normal diet and then a high-fat diet for 12 weeks. [Figure 22-1] Figure 22-1 shows the results of examining the expression of macrophage marker genes MCP1 and F4 / 80, M1-like macrophage markers IL6 and TNFα, and M2-like macrophage marker IL10 in the stromal vascular fraction (SVF) of epididymal white fat in wild-type (WT) and TM4SF19KO mice fed a high-fat diet for 12 weeks. [Figure 22-2] This is a continuation of Figure 22-1. [Figure 23]FIG. 23 shows the results showing the role of TM4SF19 in inflammation, insulin resistance, and fatty liver in eWAT. [Figure 24a] Figure 24a shows the results of a high-fat diet-induced obesity mouse model in which 6-week-old mice were fed a normal diet and then started on a high-fat diet for 12 weeks, confirming the conversion of epididymal white fat to a beige or brown fat phenotype in TM4SF19KO mice. [Figure 24b] FIG. 24b shows the results confirming that Ucp1 expression is increased in the white fat of TM4SF19KO mice in a high-fat diet-induced obese mouse model. [Figure 24c] Figure 24c shows the process of creating an in vitro model of brown adipose differentiation. [Figure 24d] FIG. 24d shows the results confirming that Ucp1 was increased in TM4SF19KO in an in vitro model of brown adipose differentiation. [Figure 24e] Figure 24e shows the results confirming that TM4SF19 is involved in converting white adipocytes into beige adipocytes. [Figure 25a] Figure 25a shows a schematic diagram of the types of cells in each fraction of white fat, which was digested with collagen type I and then centrifuged to separate it into adipose tissue, infranatant, and SVF fractions. [Figure 25b] Figure 25b shows the results of confirming TM4SF19 expression in adipocytes and SVF separated from epididymal white fat and subcutaneous white fat of mice fed a high-fat diet (60% fat) for 24 weeks. [Figure 26] Figure 26 shows the results of extracting SVF from subcutaneous white fat of wild-type (WT) and TM4SF19KO mice, inducing differentiation into brown fat with isopropanol, and confirming the expression of thermogenic genes and beige fat marker genes. [Figure 27]Figure 27 shows the results of differentiating adipocytes in an in vitro model, contact co-culturing them with macrophages (right), and then checking marker genes for macrophages, inflammation, adiponectin, etc. (left) [Control group: Macrophages and adipocytes were grown separately, and then RNA was extracted and combined before use]. [Figure 28] Figure 28 shows the results of extracting SVF from wild-type (WT) mice, generating adipocytes, differentiating them into WT bone marrow macrophages, and then inducing inflammatory changes by contact co-culture or contact co-culture [control group (ctrl): macrophages and adipocytes were grown separately, and then RNA was extracted and combined for use]. [Figure 29] Figure 29 shows the results of extracting SVF from wild-type (WT) mice and TM4SF19KO mice, causing adipogenesis, collecting bone marrow from each mouse, differentiating it into macrophages, and then contact co-culture to induce inflammatory changes. [Figure 30] Figure 30 shows the results of extracting SVF from wild-type (WT) mice and TM4SF19KO mice and inducing adipogenesis, collecting bone marrow from each mouse and differentiating it into macrophages, and then inducing inflammatory changes by contact co-culture. [Control group (ctrl): Macrophages and adipocytes were grown separately, and then RNA was extracted and combined before use.] [Figure 31] Figure 31 shows the process of creating an in vitro insulin resistance model (top row), and the results of differentiating adipocytes for 12 days, treating them with TNFα for 24 hours, and then confirming the presence of C / EBPα, an adipocyte differentiation marker, and C / EBPβ, an insulin resistance in vitro model marker (center), as well as confirming an increase in TM4SF19 (bottom row). [Figure 32] FIG. 32 shows the results showing that insulin resistance is reduced in TM4SF19KO compared to the wild type in an in vitro insulin resistance model. [Figure 33]FIG. 33 shows the results of identifying markers involved in M1 polarization after differentiation of bone marrow from wild-type (WT) mice into macrophages. [Figure 34] Figure 34 shows the results (bottom) of bone marrow collected from wild-type (WT) mice and TM4SF19KO mice, differentiated into macrophages, and then performed M1 polarization. Markers involved in M1 polarization were confirmed after bone marrow collected from wild-type mice and TM4SF19KO mice and differentiated into macrophages. [Figure 35] Figure 35 shows the results of Western blotting performed on 293T cells after transiently overexpressing human TM4SF19 (hTM4SF19), human EC2Δ (hTM4SF19 115-175Δ), mouse TM4SF19 (mTM4SF19), and mouse EC2Δ (mTM4SF19 116-165Δ) to confirm expression using the polyclonal mouse TM4SF19 antibody previously prepared. [Figure 36] FIG. 36 shows the results of collecting bone marrow from wild-type mice, differentiating it into osteoclasts, treating it with TM4SF19 antibody, and confirming the results by TRAP staining. [Figure 37] Figure 37 shows the results of Coomassie staining (left and right) and Western blotting (center) after human TM4SF19120-169-Fc and mouse TM4SF19116-165-Fc were produced and purified. [Figure 38a] Figure 38a shows the results of TRAP staining of mM4SF19116-165-Fc during osteoclast differentiation, confirming the inhibition of multinuclear osteoclast formation [E1: sample eluted with elution buffer (20 mM glycine); E1-1: sample subjected to ultrafiltration / diafiltration (UF / DF) in Buffer A (50 mM phosphate, 50 mM NaCl, pH 7.0) (Fc may be more stable)]. [Figure 38b]FIG. 38b shows the inhibition of multinucleated osteoclast formation confirmed by TRAP staining after treatment of hTM4SF19120-169-Fc during osteoclast differentiation. [Figure 38c] Figure 38c shows the results of treating hIgG1-Fc, hTM4SF19120-169-Fc, and mTM4SF19116-165-Fc with the osteoclast differentiation process, and confirming by TRAP staining that hTM4SF19120-169-Fc and mTM4SF19116-165-Fc inhibit the formation of multinucleated osteoclasts. [Figure 38d] Figure 38d shows the results of F-actin staining to confirm the presence or absence of actin belt formation during differentiation of multinucleated osteoclasts after treatment of wild-type mouse bone marrow with 10 μg / ml of IgG-Fc and mTM4SF19-Fc. [Figure 38e] FIG. 38e shows that bone resorption was blocked by mTM4SF19-Fc, as confirmed by Toluidine blue staining. [Figure 39a] FIG. 39a is a micro-CT image showing the bone loss-inhibiting effect of mouse TM4SF19-Fc. [Figure 39b] Figure 39b shows 3D micro CT indices, including total bone mineral density (BMD), bone volume (BV), the ratio of bone volume to tissue volume (%BV / TV), mean number of trabeculae (Tb.N), and trabecular spacing (Tb.Sp), obtained by analyzing the micro CT image in Figure 39a using a 3D image analysis program. [Figure 39c] Figure 39c shows the histopathological analysis of mouse femur tissue through H&E staining. [Figure 39d] Figure 39d shows that bone loss was significantly suppressed after ovariectomy in 8-week-old female mice and injection of hIgG1-Fc, mouse TM4SF19-Fc(116-165), or human TM4SF19-Fc(131-169) into the tail vein. [Figure 40a]FIG. 40a is a graph showing the mRNA expression of Ctsk and Acp5 in cells differentiated under the conditions of treatment with 10 μg / ml of mouse TM4SF19-Fc while treating with M-CSF or M-CSF and RANKL. [Figure 40b] Figure 40b shows the results of examining the expression of osteoclast differentiation marker proteins after cells extracted from the bone marrow of wild-type (WT) mice were differentiated under conditions of treatment with M-CSF or M-CSF and RANKL (M-CSF 25 ng / ml, RANKL 100 ng / ml) and treated with mTM4SF19-Fc at 5 μg / ml. [Figure 41] FIG. 41 shows the results of FACS analysis of surface binding of Raw264.7 cells to mTM4SF19-Fc before and after differentiation. [Figure 42a] FIG. 42a is a schematic diagram of the experimental schedule for confirming the prevention and treatment of rheumatoid arthritis using TM4SF19-Fc. [Figure 42b] FIG. 42b shows the results of examining the state of the mouse joints 42 days after the induction of collagen-induced arthritis (CIA). [Figure 42c] Figure 42c shows the results of an analysis of the CIA score and the number of swollen joints examined during the experimental period. [Figure 42d] FIG. 42d is a microCT image showing that inflammation and bone damage that occurred in the paws of a collagen-induced arthritis mouse model were dose-dependently suppressed by mTM4SF19-Fc treatment. [Figure 42e] FIG. 42e shows the results showing that inflammation and bone damage that occurred in the paws of a mouse model of collagen-induced arthritis were dose-dependently suppressed by treatment with mTM4SF19-Fc. [Figure 42f]Figure 42f shows 3D microCT indices (bone microstructure indices) including total bone mineral density (BMD), bone volume (BV), the ratio of bone volume to tissue volume (%BV / TV), the average number of bone trabeculae (Tb.N), and the spacing between bone trabeculae (Tb.Sp) obtained by analyzing microCT images of the collagen-induced arthritis mouse model femur using a 3D image analysis program. [Figure 42g] Figure 42g shows the results of examining cartilage damage in a collagen-induced arthritis mouse model using toluidine blue, demonstrating that cartilage damage was suppressed in the mTM4SF19-Fc-treated group compared to the hIgG1-Fc-treated group. [Figure 42h] Figure 42h shows the semi-therapeutic effects of mTM4SF19-Fc and hTM4SF19-Fc(120-169) as confirmed by arthritis score in a mouse model of arthritis induced by LPS injection 3 days after collagen antibody treatment. The results confirmed that treatment with TM4SF19-Fc resulted in a lower arthritis disease score and reduced paw thickness compared to hIgG1-Fc. [Figure 42i] Figure 42i shows the therapeutic effect of mTM4SF19-Fc in a mouse model of arthritis induced by LPS injection 3 days after collagen antibody treatment, 8 days after the arthritis disease score reached its maximum. Compared to the untreated group, TM4SF19-Fc treatment reduced the arthritis disease score, reduced paw thickness, and inhibited cartilage damage with toludine blue, confirming the therapeutic effect. [Figure 43a] FIG. 43a is a schematic diagram of the experimental schedule for confirming the inhibitory effect of hTM4SF19-Fc on bone metastasis of breast cancer. [Figure 43b]FIG. 43b shows the results of confirming the state of cancer progression through bioluminescence image analysis. [Figure 43c] Figure 43c shows the results of a micro CT scan of a joint from a mouse with bone metastasis from breast cancer. [Figure 43d] Figure 43d shows the results of histological analysis of the joints of mice with bone metastasis of breast cancer through H&E staining. [Figure 44a] FIG. 44a is a schematic diagram of the experimental schedule for confirming the inhibitory effect of hTM4SF19-Fc(120-169) on bone metastasis of breast cancer. [Figure 44b] FIG. 44b shows the results of confirming the state of cancer progression through bioluminescence image analysis. [Figure 44c] Figure 44c shows the results of luminescence analysis of the joints of mice with bone metastasis of breast cancer. [Figure 44d] Figure 44d shows the results of a micro CT scan of a joint from a mouse with bone metastasis from breast cancer. [Figure 45] Figure 45 shows the results of injecting mouse-derived E0771 breast cancer cells into the tail vein of wild-type mice to induce lung metastasis, confirming that lung metastasis was significantly suppressed by mTM4SF19-Fc (116-165) and hTM4SF19-Fc (145-169). [Figure 46a] FIG. 46a shows that mTM4SF19-Fc treatment suppresses high-fat diet-induced obesity. [Figure 46b] FIG. 46b shows the effects of mTM4SF19-Fc treatment on reducing the weight of white fat in organs and suppressing fatty liver caused by a high-fat diet, as confirmed by organ weight relative to organ weight and body weight. [Figure 47] FIG. 47 shows the effects of mTM4SF19-Fc treatment on reducing fatty liver in liver tissue and neutral fat in plasma, as confirmed by H&E staining and Masson & trichrome staining. [Figure 48] Figure 48 shows that treatment with mTM4SF19-Fc (116-165) inhibited adipocyte differentiation in a dose-dependent manner, as confirmed by staining lipid droplets with Oil-red-O staining and by gene expression of adipogenesis markers C / EBPα and PPAR-γ. [Figure 49] FIG. 49 shows the results of staining with 0.05% crystal violet solution, confirming that TM4SF19-Fc inhibits cancer cell migration induced by osteoclast differentiation. [Figure 50a] Figure 50a shows the inhibition of cancer cell migration due to osteoclast differentiation in TM4SF19KO cells, confirmed by staining with 0.05% crystal violet solution. [Figure 50b] FIG. 50b shows the inhibition of cancer cell migration due to osteoclast differentiation in TM4SF19EC2Δ, confirmed by staining with 0.05% crystal violet solution. [Figure 51] FIG. 51 shows the results of stably overexpressing 3Flag-hTM4SF19 in MG63 osteosarcoma cell line, followed by MTT assay to confirm cell growth, colony formation, and migration. [Figure 52] FIG. 52 shows the results of stably overexpressing 3Flag-hTM4SF19 in HOS osteosarcoma cell line, followed by MTT assay to confirm cell growth, colony formation, and migration. [Figure 53] Figure 53 shows the results of knocking out TM4SF19 in the 143b osteosarcoma cell line using CRISPR, and then confirming cell growth in different clones (#4 and #6) using MTT analysis and colony formation to determine their swarming ability. [Figure 54a] FIG. 54a shows the results of MTT assay of cell growth while treating 143b osteosarcoma cells with hTM4SF19-Fc (120-169aa). [Figure 54b] Figure 54b shows the results of confirming swarming ability by colony formation. [Figure 54c]FIG. 54c shows the results of confirming cell growth by cell counting. [Figure 54d] FIG. 54d shows the results of confirming cell migration. [Figure 55] FIG. 55 shows that treatment with human TM4SF19-Fc (120-169aa) (10 μg / ml) inhibited the formation of U2OS and MG63 osteosarcoma colonies. [Figure 56] FIG. 56 shows that the cell migration ability of HOS osteosarcoma cells was inhibited by treatment with 10 μg / ml hTM4SF19-Fc(120-169aa) and hTM4SF19-Fc(145-169aa). [Figure 57a] FIG. 57a shows the results confirming the inhibition of pancreatic cancer cell colony formation by human TM4SF19-Fc (120-169aa). [Figure 57b] FIG. 57b shows the growth inhibition of pancreatic cancer cells by human TM4SF19-Fc (120-169aa) confirmed by cell counting. [Figure 58a] Figure 58a shows the sequence information of the TM4SF19 fragment and TM4SF19-Fc fusion protein used in the examples of this application (in each figure, the target sequence is shown in bold and is indicated as SEQ ID NOs: 8, 12, 15, and 18, and the enzyme site is the region between the target sequence and the immunoglobulin Fc region, and the immunoglobulin Fc region is shown in highlight and is indicated as SEQ ID NO: 9). [Figure 58b] Figure 58b, like Figure 58a, shows the sequence information of the TM4SF19 fragment and TM4SF19-Fc fusion protein used in the examples of this application. [Figure 58c] Figure 58c shows the sequence information of the TM4SF19 fragment and TM4SF19-Fc fusion protein used in the Examples of this application, similar to Figure 58a. [Figure 58d] Figure 58d shows the sequence information of the TM4SF19 fragment and TM4SF19-Fc fusion protein used in the Examples of this application, similar to Figure 58a. DETAILED DESCRIPTION OF THE INVENTION

[0029] The present invention will be described in detail below.

[0030] However, the 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. Furthermore, the embodiments of the present invention are provided to more completely explain the present invention to those having average knowledge in the art. Furthermore, throughout the specification, the term "comprise" a certain element does not exclude other elements, but means that other elements may also be included, unless otherwise specified.

[0031] The present invention relates to a pharmaceutical composition for preventing or treating bone diseases, which comprises an agent that inhibits the expression or activity of TM4SF19 as an active ingredient.

[0032] The present invention also relates to a pharmaceutical composition for preventing or treating obesity or obesity-mediated metabolic diseases, which comprises an agent that suppresses TM4SF19 expression or activity as an active ingredient.

[0033] The present invention also relates to a pharmaceutical composition for preventing or treating cancer or suppressing cancer metastasis, which comprises an inhibitor of TM4SF19 expression or activity as an active ingredient.

[0034] In the present invention, the term "TM4SF19" refers to transmembrane 4L six family member 19 (TM4SF19, OCTM4), which belongs to the transmembrane 4L six superfamily. These superfamily members are known to be involved in various cellular processes, including cell proliferation, motility, and adhesion, through interactions with integrins, and are associated with diseases such as liver fibrosis and cancer. However, the function of TM4SF19 has not been thoroughly studied.

[0035] The TM4SF19 protein is not only expressed in humans (Homo sapiens) or mice (Mus musculus), but the same protein may also be expressed in other mammals such as monkeys, cows, horses, dogs, and cats.

[0036] Human-derived TM4SF19 may be translated from mRNAs containing GenBank Accession Nos. NM_138461.4, NM_001204897.2, and NM_001204898.2, respectively, into peptides or proteins containing amino acid sequences represented by NP_612470.2, NP_001191826.1, and NP_001191827.1, respectively. Specifically, human TM4SF19 has three transcript variants (GenBank Accession Nos. NM_138461.4 (transcript variant 1), NM_001204897.2 (transcript variant 2), and NM_001204898.2 (transcript variant 3)) and three isoforms (GenBank Accession Nos. NP_612470.2 (isoform 1), NP_001191826.1 (isoform 2), and NP_001191827.1 (isoform 3). TM4SF19 used in the examples of the present invention is NP_612470.2 (isoform 1), derived from mRNA containing GenBank Accession No. NM_138461.4 (transcript variant 1). Isoform 1 is highly homologous to mouse TM4SF19.

[0037] Mouse TM4SF19 has GenBank Accession No. NP_001153874.1 and is represented by SEQ ID NO:7.

[0038] <GenBank Accession No.NP_612470.2(isoform 1)[SEQ ID NO:1]> MVSSPCTQAS SRTCSRILGL SLGTAALFAA GANVALLLPN WDVTYLLRGL LGRHAMLGTG 60 LWGGGLMVLT AAILISLMGW RYGCFSKSGL CRSVLTALLS GGLALLGALI CFVTSGVALK 120 DGPFCMFDVS SFNQTQAWKY GYPFKDLHSR NYLYDRSLWN SVCLEPSAAV VWHVSLFSAL 180 LCISLLQLLL VVVHVINSLL GLFCSLCEK

[0039] <GenBank Accession No.NP_001191826.1 (isoform 2) [SEQ ID NO: 2]> MVSSPCTQAS SRTCSRILGL SLGTAALFAA GANVALLLPN WDVTYLLRGL LGRHAMLGTG 60 LWGGGLMVLT AAILISLMGW RYGCFSKSGL CRSVLTALLS GGLALLGALI CFVTSGVALK 120 DGPFCMFDVS SFNQTQAWKY GYPFKDLHRI ICMTVRSGTP SAWSPLQLLS GTCPSSPPFC 180 ASACSSFSWW SFMSSTASWA FSAASARSDR QNLHLQAWVF SSSAVLNPFY KEWVRIINKL 240 PL

[0040] <GenBank Accession No.NP_001191827.1(isoform 3) [SEQ ID NO: 3]> MVSSPCTQAS SRTCSRILGL SLGTAALFAA GANVALLLPN WDVTYLLRGL LGRHAMLGTG 60 LWGGGLMVLT ALLSGGLALL GALICFVTSG VALKDGPFCM FDVSSFNQTQ AWKYGYPFKD 120 LHSRNYLYDR SLWNSVCLEP SAAVVWHVSL FSALLCISLL QLLLVVVHVI NSLLGLFCSL 180 CEK

[0041] <GenBank Accession No.NM_138461.4(transcript variant 1)[SEQ ID NO:4]> 1 61 ctctgaagac gcagcctttc tccaggttct gtctctccca ttctgattct tgacaccaga 121 tgcaggatgg tgtcctctcc ctgcacgcag gcaagctcac ggacttgctc ccgtatcctg 181 ggactgagcc ttgggactgc agccctgttt gctgctgggg ccaacgtggc actctccctt 241 cctaactggg atgtcaccta cctgttgagg ggcctccttg gcaggcatgc catgctggga 301 actgggctct ggggaggagg cctcatggta ctcactgcag ctatcctcat ctccttgatg 361 ggctggagat acggctgctt cattaagagt gggctctgtc gaagcgtgct tactgctctg 421 ttgtcaggtg gcctggcttt acttggagcc ctgatttgct ttgtcacttc tggagttgct 481 ctgaaagatg gtcctttttg catgtttgat gtttcatcct tcaatcagac acaagcttgg 541 aaatatggtt acccattcaa agacctgcat agtaggaatt atctgtatga ccgttcgctc 601 tggaactccg tctgcctgga gccctctgca gctgttgtct ggcacgtgtc cctcttctcc 661 gcccttctgt gcatcagcct gctccagctt ctcctggtgg tcgttcatgt catcaacagc 721 ctcctgggcc ttttctgcag cctctgcgag aagtgacagg cagaaccttc acttgcaagc 781 atgggtgttt tcatcatcgg ctgtcttgaa tcctttctac aagtagtggg tacgaattat 841 aaaaactt cccctttagg tatccctgga gtaataatga caaaaatt cactgcaggt 901 cggtggaatg atagaatgca ttttaaatca cattgtaaac ttccaggtga tccatggata 961 ggataataa ctaagttat ataattgttt aggaatttat agtccataaa atatcctcca 1021 gccagg

[0042] <GenBank Accession No. NM_001204897.2(transcript variant 2) [SEQ ID NO: 5]> 1 61 ctctgaagac gcagcctttc tccaggttct gtctctccca ttctgattct tgacaccaga 121 tgcaggatgg tgtcctctcc ctgcacgcag gcaagctcac ggacttgctc ccgtatcctg 181 ggactgagcc ttgggactgc agccctgttt gctgctgggg ccaacgtggc actctccctt 241 cctaactggg atgtcaccta cctgttgagg ggcctccttg gcaggcatgc catgctggga 301 actgggctct ggggaggagg cctcatggta ctcactgcag ctatcctcat ctccttgatg 361 ggctggagat acggctgctt cattaagagt gggctctgtc gaagcgtgct tactgctctg 421 ttgtcaggtg gcctggcttt acttggagcc ctgatttgct ttgtcacttc tggagttgct 481 ctgaaagatg gtcctttttg catgtttgat gtttcatcct tcaatcagac acaagcttgg 541 aaatatggtt acccattcaa agacctgcat agaattatct gtatgaccgt tcgctctgga 601 actccgtctg cctggagccc tctgcagctg ttgtctggca cgtgtccctc ttctccgcccc 661 ttctgtgcat cagcctgctc cagcttctcc tggtggtcgt tcatgtcatc aacagcctcc 721 tgggccttt ctgcagcctc tgcgagaagt gacaggcaga accttcactt gcaagcatgg 781 gtgttttcat catcggctgt cttgaatcct ttctacaagg agtgggtacg aattataaac 841 aaacttcccc tttaggtatc cctggagtaa taatgacaac aaaattcact gcaggtcggt 901 ggaatgatag aatgcatttt aaatcacatt gtaaacttcc aggtgatcca tggataggat 961 aaataactaa gttattataa ttgtttagga atttatagtc cataaaatat cctccagcca 1021 gg

[0043] <GenBank Accession No. NM_001204898.2(transcript variant 3) [SEQ ID NO: 6]> 1 aaagagtcct ggaaagacaa ccttcaggtc cagccctgga gctggaggag tggagcccca 61 ctctgaagac gcagcctttc tccaggttct gtctctccca ttctgattct tgacaccaga 121 tgcaggatgg tgtcctctcc ctgcacgcag gcaagctcac ggacttgctc ccgtatcctg 181 ggactgagcc ttgggactgc agccctgttt gctgctgggg ccaacgtggc actcctcctt 241 cctaactggg atgtcaccta cctgttgagg ggcctccttg gcaggcatgc catgctggga 301 actgggctct ggggaggagg cctcatggtg cttactgctc tgttgtcagg tggcctggct 361 ttacttggag ccctgatttg ctttgtcact tctggagttg ctctgaaaga tggtcctttt 421 tgcatgtttg atgtttcatc cttcaatcag acacaagctt ggaaatatgg ttacccattc 481 aaagacctgc atagtaggaa ttatctgtat gaccgttcgc tctggaactc cgtctgcctg 541 gagccctctg cagctgttgt ctggcacgtg tccctcttct ccgcccttct gtgcatcagc 601 ctgctccagc ttctcctggt ggtcgttcat gtcatcaaca gcctcctggg ccttttctgc 661 agcctctgcg agaagtgaca ggcagaacct tcacttgcaa gcatgggtgt tttcatcatc 721 ggctgtcttg aatcctttct acaaggagtg ggtacgaatt ataaacaaac ttccccttta 781 ggtatccctg gagtaataat gacaacaaaa ttcactgcag gtcggtggaa tgatagaatg 841 cattttaaat cacattgtaa acttccaggt gatccatgga taggataaat aactaagtta 901 ttataattgt ttaggaattt atagtccata aaatatcctc cagccagg

[0044] <GenBank Accession No.NP_001153874.1[SEQ ID NO:7]> MLSFSRVVNC SRTCSRFLGL SLGTASLCAA GANIALLFPN WDVTYLMRGL IGKHAMLGSG LWGGGLMVLL AATLISMTGS FSKSAPCLQV LIALLSSGLA LLGAVICFVT SGVALKDGPF CMFDVSSFNQ TQAWKFGYPF KDLHNRNYLY DRSLWTSVCL EPSKAVVWHV AFFSILLCIS LLQLLLVAIH LVNSILGLFC SFCEKH

[0045] The term "TM4SF19 expression or activity inhibitor" as used herein refers to a substance that reduces the expression of the TM4SF19 gene or the activity of the TM4SF19 protein. In a specific embodiment, the term "TM4SF19 gene expression inhibitor" refers to a substance that reduces the expression level or activity of the TM4SF19 gene by directly acting on the TM4SF19 gene or indirectly acting on upstream regulators of the TM4SF19 gene to reduce TM4SF19 gene expression at the transcriptional level, or by increasing the degradation of the expressed TM4SF19 gene or interfering with its activity. Specifically, the inhibitor may be at least one selected from the group consisting of, but not limited to, antisense nucleotides that bind complementary to the mRNA of the TM4SF19 gene, short hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), and ribozymes.

[0046] In the present invention, the term "antisense nucleotide" refers to a DNA or RNA sequence that is complementary to a specific gene and can bind to TM4SF19 mRNA. Because antisense nucleotides are long chains of monomer units, they can be easily synthesized against a target gene sequence.

[0047] As used herein, the term "small interfering RNA (siRNA)" refers to a short double-stranded RNA that can induce RNAi (RNA interference) through the cleavage of a specific mRNA. siRNA includes a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. Because siRNA can suppress the expression of a target gene, it is used in gene knockdown methods, gene therapy, and the like.

[0048] In the present invention, the term "shRNA (short hairpin RNA)" refers to a single-stranded RNA that is divided into a stem portion that forms a double-stranded portion through hydrogen bonds and a circular loop portion, and is processed by proteins such as Dicer to be converted into siRNA, which can perform the same function as siRNA.

[0049] As used herein, the term "miRNA (microRNA)" refers to 21 to 23 non-coding RNAs that regulate gene expression post-transcriptionally by promoting the degradation of target RNAs or by suppressing their translation.

[0050] In the present invention, the term "ribozyme" refers to an RNA molecule that has the function of an enzyme, recognizing and cleaving a specific base sequence. A ribozyme is composed of a region that specifically binds to a target messenger RNA strand, which has a base sequence complementary to the target messenger RNA strand, and a region that cleaves the target RNA.

[0051] In addition, in a specific embodiment, the inhibitor that suppresses the activity of the TM4SF19 protein of the present invention may be at least one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, fusion proteins, and antibodies that specifically bind to the TM4SF19 protein, but is not limited thereto.

[0052] In the present invention, the term "compound" includes any compound that can specifically bind to the TM4SF19 protein and inhibit its activity.

[0053] In the present invention, the term "peptide" refers to a peptide that has the advantage of having a strong binding affinity to target substances and is not denatured by heat or chemical treatment. Furthermore, due to its small molecular size, it can be attached to other proteins to form fusion proteins. Specifically, it can be attached to polymeric protein chains and used as a diagnostic kit or drug delivery substance.

[0054] In the present invention, the term "peptide mimetics" refers to compounds that inhibit the activity of the TM4SF19 protein by inhibiting its binding domain. Peptide mimetics may be peptides or non-peptides, and may be composed of amino acids linked by non-peptide bonds, such as psi bonds. They may also be "conformationally constrained" peptides, cyclic mimetics, or cyclic mimetics that include at least one exocyclic domain, a binding moiety (binding amino acids), and an active site. Peptide mimetics may be novel small molecules that are structured similarly to the secondary structural characteristics of the TM4SF19 protein, can mimic the inhibitory properties of large molecules such as antibodies or water-soluble receptors, and can act with effects comparable to those of natural antagonists.

[0055] In the present invention, the term "aptamer" refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and is capable of binding to a target molecule with high affinity and specificity.

[0056] In the present invention, the term "fusion protein" refers to a new protein created by combining two or more different proteins or proteins of the same kind, and is often called a chimera protein. Two or more heterologous proteins are combined part-to-part, part-to-whole, or whole-to-whole. In many cases, the sequence of one gene is joined to the sequence of another gene by matching codons as needed to create a hybrid gene, which is then expressed and produced as a protein.

[0057] In one embodiment, the fusion protein of the present invention may comprise a fragment of TM4SF19 that specifically binds to the TM4SF19 protein.

[0058] In addition to the above fragment, the antibody may further comprise an immunoglobulin Fc region.

[0059] The fragment may be the entire fragment or a portion of the fragment derived from extracellular loop 2 (EC2) of TM4SF19. The amino acid sequence portion corresponding to the entire EC2 may be the amino acid sequence portion from positions 120 to 169 of the human TM4SF19 protein or the amino acid sequence portion from positions 116 to 165 of the mouse TM4SF19 protein. The amino acid sequence portion corresponding to a portion of EC2 may include the amino acid sequence portion from positions 145 to 169 or the amino acid sequence portion from positions 131 to 169 of the human TM4SF19 protein.

[0060] Preferred fusion proteins according to the invention may comprise the amino acid sequence represented by SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, or SEQ ID NO:19.

[0061] In one embodiment, the present invention may include a fusion protein in which an immunoglobulin Fc region is linked to a fragment of TM4SF19, for example, a fusion protein in which an immunoglobulin Fc region is linked to all or part of an extracellular loop of TM4SF19, or a fusion protein in which an immunoglobulin Fc region is linked to all or part of an extracellular loop of TM4SF19 and all or part of a membrane protein. Here, a fusion protein in which an immunoglobulin Fc region is linked to a fragment of TM4SF19 includes not only one in which an immunoglobulin Fc region is directly linked to a fragment of TM4SF19, but also one in which an immunoglobulin Fc region is indirectly linked to a fragment of TM4SF19. When the immunoglobulin Fc region is indirectly bound to the TM4SF19 fragment, 1 to 10, for example, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, for example, 1 amino acid sequence corresponding to a linker, spacer, or enzyme site may further be present between the TM4SF19 fragment and the immunoglobulin Fc region.

[0062] Fragments of TM4SF19 that can be used in the present invention include, but are not limited to, various examples (hTm4sf19 94-196, hTm4sf19 131-160, hTm4sf19 145-169, hTm4sf19 131-169, hTm4sf19 120-160, hTm4sf19 120-169, hTm4sf19 120-180, hTm4sf19 120-186, hTm4sf19 120-196, hTm4sf19 120-209, hTm4sf19 131-196, and hTm4sf19 145-196). On the other hand, although the immunoglobulin Fc region is used as an example to stabilize the TM4SF19 protein, the fusion partner that can bind to the TM4SF19 protein is not limited to the immunoglobulin Fc region.

[0063] Although not limited thereto, the present invention further relates to a fusion protein for inhibiting the expression or activity of TM4SF19, which comprises a fragment derived from extracellular loop 2 of the TM4SF19 protein and an immunoglobulin Fc region.

[0064] The fragment derived from extracellular loop 2 (EC2) of the TM4SF19 protein may be an amino acid sequence portion corresponding to the whole or part of EC2.

[0065] The amino acid sequence portion corresponding to the entire EC2 may be the portion of amino acids 120 to 169 of the human TM4SF19 protein or the portion of amino acids 116 to 165 of the mouse TM4SF19 protein.

[0066] The amino acid sequence portion corresponding to a portion of EC2 may include the amino acid sequence from positions 145 to 169 of the human TM4SF19 protein.

[0067] The amino acid sequence portion corresponding to a portion of EC2 may include the amino acid sequence from positions 131 to 169 of the human TM4SF19 protein.

[0068] A preferred fusion protein according to the invention may comprise the amino acid sequence represented by SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16 or SEQ ID NO:19.

[0069] An enzyme site may be contained between the fragment derived from extracellular loop 2 (EC2) of the TM4SF19 protein and the immunoglobulin Fc region.

[0070] The portion of extracellular loop 2 of human TM4SF19 used as the fragment of TM4SF19 to generate the fusion protein is exemplary and not limiting.

[0071] In the present invention, the term "Fc region" refers to a protein comprising immunoglobulin heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3), but excluding the variable regions of the heavy and light chains of the immunoglobulin and light chain constant region 1 (CL1). It may further comprise the hinge region of the heavy chain constant region. The "Fc region" may be, but is not limited to, an Fc region derived from IgG, IgA, IgD, IgE, or IgM. In the following examples, human IgG1-Fc used to prepare the fusion protein is illustrative and not limiting.

[0072] The "Fc region" may also include an "altered immunoglobulin Fc region" or "Fc region mutant," which means an Fc region produced by substituting some amino acids or by combining different types of Fc regions. Preferably, the Fc region refers to an Fc region whose Fc receptor binding ability and / or complement binding ability have been altered, resulting in weakened antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) compared to a wild-type Fc region. In this case, the altered immunoglobulin Fc region may be selected from the group consisting of IgG1, IgG2, IgG3, IgD, IgG4, and combinations of these sequences.

[0073] In the present invention, the term "antibody" refers to a specific immunoglobulin directed against an antigenic site. "Antibody" includes monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, and human antibodies, and includes not only novel antibodies but also antibodies already known in the art or commercially available. The antibody includes not only full-length forms containing two heavy chains and two light chains, but also functional fragments of antibody molecules, so long as it specifically binds to TM4SF19. The functional fragment of the antibody molecule refers to a fragment that retains at least the antigen-binding function, and may include, but is not limited to, Fab, F(ab'), F(ab')2, Fv, etc.

[0074] The present invention relates to anti-TM4SF19 antibodies specific for all or part of extracellular loop 2 of human TM4SF19. In a specific embodiment, the present invention relates to anti-TM4SF19 antibodies specific for all or part of the extracellular loop 2 region of SEQ ID NOS: 115-175 of human TM4SF19 (GenBank Accession No. NP_612470.2). In another specific embodiment, the present invention relates to anti-TM4SF19 antibodies specific for all or part of the extracellular loop 2 region of human TM4SF19 (GenBank Accession No. 138461.4) (SEQ ID NOS: 120-169). The target sites in extracellular loop 2 of human TM4SF19 used to generate the antibodies are exemplary and not limiting.

[0075] According to the examples of the present invention, it has been revealed that the TM4SF19 protein is involved in cell-cell interactions through self-binding.

[0076] In particular, the inventors prepared antibodies against the TM4SF19 protein that target the extracellular loop region of the TM4SF19 protein and can inhibit the self-binding of the TM4SF19 protein, as well as fusion proteins that fuse Fc with fragments of TM4SF19 (e.g., the extracellular loop region, the extracellular loop region and membrane protein), and evaluated the efficacy of inhibitors of TM4SF19 protein activity.

[0077] In the present invention, the term "bone disease" may be at least one selected from the group consisting of metabolic bone disease, orthopedic bone disease, aplastic bone disease, degenerative bone disease, degenerative arthritis, rheumatoid arthritis, psoriatic arthritis, psoriatic spondylitis, age-related bone loss, osteoporosis, osteogenesis imperfecta, osteomalacia, osteopenia, fractures, bone defects and hip osteopenia, rickets, Paget's bone disease, periodontal disease, and bone damage caused by bone metastasis of cancer cells.

[0078] In the present invention, the term "obesity" refers to a state in which there is abnormal or excessive fat accumulation.

[0079] In the present invention, the term "obesity-mediated metabolic disease" includes diabetes, hypertension, hyperlipidemia, non-alcoholic steatohepatitis, and certain cancers, and more broadly includes hypertension, diabetes, insulin resistance syndrome, metabolic syndrome, obesity-related gastroesophageal reflux disease, arteriosclerosis, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, lipodystrophy, non-alcoholic steatohepatitis, cardiovascular disease, polycystic ovary syndrome, etc. When using the composition of the present invention, treatment of the above diseases can also be performed simultaneously. In addition, subjects to be treated for such obesity-related diseases also include subjects who wish to lose weight.

[0080] In the present invention, the term "cancer" is a general term for diseases caused by cells that have aggressive properties, such as dividing and growing despite normal growth limitations, invasive properties, such as infiltrating surrounding tissues, and metastatic properties, such as spreading to other parts of the body.

[0081] The type of cancer used in the present invention is not limited, and may be at least one selected from the group consisting of colorectal cancer, stomach cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma, uterine cancer, ovarian cancer, small intestine cancer, rectal cancer, cancer near the anus, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, lymphatic gland cancer, bladder cancer, gallbladder cancer, endocrine gland cancer, thyroid cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, spinal cord tumor, brainstem glioma, and pituitary adenoma.

[0082] The present invention includes uses of TM4SF19 related to the suppression of not only "cancer" but also "cancer metastasis."

[0083] Meanwhile, in the present invention, the term "prevention" refers to any action of administering the composition of the present invention to prevent the occurrence of a desired symptom or disease or to delay its occurrence or onset.

[0084] In the present invention, the term "treatment" means any action of administering the composition of the present invention to improve or eliminate the desired symptoms or disease.

[0085] On the other hand, the composition of the present invention may further comprise a pharmaceutically acceptable carrier or may be formulated together with a carrier.

[0086] In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate living organisms and does not inhibit the biological activity and properties of the administered compound. Pharmaceutical carriers acceptable for compositions formulated as liquid solutions include those that are sterile and biocompatible, such as physiological saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components. Other common additives, such as antioxidants, buffers, and bacteriostats, may also be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may also be added to formulate the compositions into injectable solutions, such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0087] The compositions of the present invention, comprising an inhibitor of TM4SF19 expression or activity and a pharmaceutically acceptable carrier, can be used in any dosage form containing the inhibitor as an active ingredient. They may be prepared as oral or parenteral dosage forms, and may be formulated in unit dosage forms for ease of administration and uniformity of dosage. Pharmaceutical dosage forms of the present invention include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal, or parenteral (including parenteral, intramuscular, subcutaneous, and intravenous) administration, or those suitable for administration by inhalation or injection. Oral dosage forms containing the compositions of the present invention as an active ingredient may be formulated as, for example, tablets, troches, lozenges, aqueous or oily suspensions, powders or granules, emulsions, hard or soft capsules, syrups, or elixirs.

[0088] The parenteral dosage form containing the composition of the present invention as an active ingredient may be formulated as an injectable form such as subcutaneous injection, intravenous injection, or intramuscular injection, or as a suppository injection, or as a spray such as an aerosol that can be inhaled through the respiratory tract. To formulate as an injectable dosage form, the composition of the present invention may be mixed with water together with a stabilizer or buffer to prepare a solution or suspension, which may then be formulated into a unit dose in an ampule or vial.

[0089] The compositions of the present invention are administered in a pharmaceutically effective amount, i.e., a therapeutically effective amount. In the present invention, a "therapeutically effective amount" refers to an amount sufficient to treat a disease. The effective dose level may be determined based on factors including the type and severity of the patient's disease, the activity and sensitivity of the drug, the administration time, administration route and excretion rate, the duration of treatment, concurrently used drugs, and other factors known in the medical field. The compositions of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, and in single or multiple administrations. That is, the total effective amount of the compositions of the present invention may be administered to a patient in a single dose or in a fractionated treatment protocol in which multiple doses are administered over a long period of time. Taking all of the above factors into consideration, it is important to administer an amount that provides maximum efficacy at a minimum dose without side effects, which can be easily determined by one skilled in the art.

[0090] The dosage of the pharmaceutical composition of the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage for parenteral administration is preferably 0.01 μg to 100 mg, more preferably 1 μg to 50 mg, per kg of body weight per day. However, since this may increase or decrease depending on the administration route, severity of obesity, sex, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0091] The compositions of the present invention may be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy and methods using biological response modifiers.

[0092] In another aspect, the present invention relates to a method for preventing or treating a bone disease, comprising administering to a subject a therapeutically effective amount of a composition for preventing or treating a bone disease, the composition comprising a substance that inhibits TM4SF19 expression or activity.

[0093] In another aspect, the present invention relates to a method for preventing or treating obesity or an obesity-mediated metabolic disease, comprising administering to a subject a composition for preventing or treating obesity or an obesity-mediated metabolic disease, the composition comprising a therapeutically effective amount of an inhibitor of TM4SF19 expression or activity as an active ingredient.

[0094] In another aspect, the present invention relates to a method for preventing or treating cancer or preventing or treating cancer metastasis, comprising administering to a subject a composition for preventing or treating cancer or suppressing cancer metastasis, the composition comprising a therapeutically effective amount of an inhibitor of TM4SF19 expression or activity as an active ingredient.

[0095] In the present invention, the term "administration" means introducing a predetermined substance into an individual by any appropriate method, and the route of administration of the preventive or therapeutic composition according to the present invention may be oral or parenteral administration via any common route as long as it can reach the target tissue. Furthermore, the preventive or therapeutic composition for cartilage-related diseases according to the present invention may be administered by any device that can transport the active ingredient to target cells.

[0096] The term "subject" as used herein includes mammals such as mice, rats, rabbits, cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, and the like, or humans, having an associated disease whose symptoms can be ameliorated by administration of a pharmaceutical composition according to the present invention.

[0097] The composition of the present invention may be administered via various oral or parenteral routes as long as it can reach the target tissue. Specifically, it may be administered by the oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, intraocular or intradermal route in a conventional manner.

[0098] The preventive or therapeutic methods of the present invention involve administering a therapeutically effective amount of the composition of the present invention. The therapeutically effective amount refers to an amount that promotes weight loss or a reduction in fat cell size. It is readily apparent to those skilled in the art that the appropriate total daily dose can be determined by a treating physician within the scope of sound medical judgment. The specific therapeutically effective amount for a particular patient will vary depending on the type and degree of response to be achieved, the specific composition, including whether other formulations are used, the patient's age, weight, general health, sex, and diet, the time and route of administration, the excretion rate of the composition, the duration of treatment, and various other factors, including similar factors known in the pharmaceutical arts. Therefore, the effective amount of the pharmaceutical composition suitable for the purpose of the present invention is preferably determined taking into account the above factors. In addition, the therapeutic effect can be enhanced by coadministering the composition of the present invention with a known therapeutic agent for a related disease.

[0099] In another aspect, the present invention provides a method for treating a subject suspected of having a bone disease with a candidate substance for treating a bone disease; The present invention relates to a method for screening for therapeutic agents for bone diseases, which comprises the step of comparing the expression level of mRNA or protein of the TM4SF19 gene with that of a control group.

[0100] In another aspect, the present invention provides a method for treating a subject suspected of having obesity or an obesity-mediated metabolic disorder with a candidate substance for treating obesity or an obesity-mediated metabolic disorder; The present invention relates to a method for screening for a therapeutic drug for obesity or an obesity-mediated metabolic disease, which comprises the step of comparing the expression level of mRNA or protein of the TM4SF19 gene with that of a control group.

[0101] In another aspect, the present invention provides a method for treating a specimen suspected of cancer prevention, treatment, or cancer metastasis with a candidate substance for cancer prevention, treatment, or suppression of cancer metastasis; The present invention relates to a method for screening for a drug for treating cancer or cancer metastasis, which comprises the step of comparing the expression level of mRNA or protein of the TM4SF19 gene with that of a control group.

[0102] In the present invention, the term "specimen" refers to an individual or sample used to screen candidate substances for treating relevant diseases, and includes, without limitation, mammals including dogs, cows, pigs, rabbits, chickens, mice, and humans, and includes samples such as whole blood, serum, blood, plasma, saliva, urine, sputum, lymph, cells, and tissues separated from the individual.

[0103] As used herein, the term "control group" refers to a sample that has not been treated with a candidate substance. Furthermore, the term "candidate substance" as used herein refers to a drug that is tested for changes in TM4SF19 expression or activity. Targets for measuring the ability to prevent or treat related diseases by directly or indirectly altering TM4SF19 expression levels or activity include any molecule, such as a protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, or a wide range of chemical compounds. Such candidate substances include both natural and synthetic substances.

[0104] As used herein, the term "therapeutic drug" refers to a substance that prevents or treats the associated disease.

[0105] The screening method of the present invention may be performed by treating an individual suspected of having a related disease with a candidate therapeutic substance, comparing the expression levels of TM4SF19 gene mRNA or protein with those of a control group not treated with the candidate substance, and determining a substance that reduces the expression of TM4SF19 gene mRNA or protein compared to the control group as a therapeutic agent for the related disease. Analytical methods for measuring mRNA levels include, but are not limited to, reverse transcriptase-polymerase reaction, competitive reverse transcriptase-polymerase reaction, real-time reverse transcriptase-polymerase reaction, RNase protection assay, Northern blotting, DNA chip, etc.

[0106] Analytical methods for measuring protein levels include, but are not limited to, Western blot, ELISA, radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chips.

[0107] Furthermore, according to one embodiment of the present invention, we confirmed that TM4SF19 expression increases during osteoclast differentiation. In TM4SF19-deficient models (TM4SF19 knockout mice, TM4SF19KO, and TM4SF19 extracellular domain 2 (116-165) knockout mice, TM4SF19EC2△), we confirmed that mononuclear osteoclasts are formed during osteoclast differentiation, while multinucleated osteoclast formation is suppressed. Osteoclasts that fail to differentiate into multinucleated osteoclasts retain the osteoclast phenotype and maintain low levels of bone resorption activity. In other words, the TM4SF19 inhibitors of the present invention, which can selectively suppress the formation of multinucleated osteoclasts, do not completely abolish bone resorption function but can more selectively regulate bone resorption, resulting in significantly reduced side effects compared to therapeutic agents such as anti-RANKL antibodies that inhibit the formation of mononuclear osteoclasts. We also confirmed that a TM4SF19-deficient model prevents bone loss following ovariectomy. Furthermore, we investigated the osteoclast differentiation process by treating the osteoclasts with anti-mouse TM4SF19 polyclonal antibody, mouse TM4SF19 EC2 (GenBank Accession No. NP_001153874.1, 116-165)-Fc fusion protein, and human TM4SF19 EC2 (GenBank Accession No. NP_612470.2 (isoform 1), 120-169)-Fc fusion protein. We confirmed that these inhibitors promote the formation of mononuclear osteoclasts but suppress the formation of multinuclear osteoclasts during osteoclast differentiation. Therefore, we conclude that substances that inhibit TM4SF19 expression or activity are effective in preventing or treating bone diseases.

[0108] Furthermore, according to one embodiment of the present invention, TM4SF19 expression was increased during the adipocyte differentiation of hADMSCs (human adipose-derived mesenchymal stem cells). In TM4SF19 knockout mice, weight gain was suppressed in a high-fat diet-induced obesity model, and the amount of adipose tissue-associated macrophages caused by obesity was reduced, confirming that TM4SF19 acts on adipocyte differentiation and inflammatory responses involving macrophages. As a result, TM4SF19KO mice were confirmed to reduce insulin resistance and suppress fatty liver in a high-fat diet-induced obesity mouse model. Therefore, it can be concluded that substances that inhibit TM4SF19 expression or activity are effective in preventing or treating obesity and obesity-mediated metabolic diseases.

[0109] Furthermore, according to one embodiment of the present invention, overexpression of TM4SF19 increased the proliferation, migration, and colony formation of mouse-derived breast cancer and human osteosarcoma cells, and TM4SF19 deficiency inhibited the lung metastasis of breast cancer cells and the proliferation, migration, and colony formation of osteosarcoma cells, confirming the inhibitory effect of bone metastasis of breast cancer cells. Therefore, it is clear that substances that inhibit the expression or activity of TM4SF19 are effective in preventing and treating cancer and cancer metastasis.

[0110] Furthermore, according to one embodiment of the present invention, treatment with human TM4SF19 EC2 (GenBank Accession No. NP_612470.2 (isoform 1) 120-169)-Fc fusion protein inhibited the growth and aggregation ability of pancreatic cancer cells, and was confirmed to have an inhibitory effect on osteosarcoma cell proliferation, migration, and colony formation. Therefore, it is clear that substances that inhibit TM4SF19 expression or activity are effective in preventing and treating cancer and cancer metastasis. [Example]

[0111] The advantages and features of the present invention, as well as methods for achieving them, will become clearer with reference to the following detailed experimental and manufacturing examples. However, the present invention is not limited to the following experimental and manufacturing examples, and may be embodied in various different forms, and the following examples are provided solely for the purpose of complete disclosure of the present invention and are intended to be illustrative and not restrictive of the present invention. The present invention is provided to fully convey the scope of the invention.

[0112] [Example] Example 1: Role of TM4SF19 in osteoclastogenesis Figure 1 shows the results of qPCR analysis of TM4SF19 mRNA expression over time after cells extracted from mouse bone marrow were treated with M-CSF and RANKL (Receptor activator of nuclear factor kappa-B ligand) to induce differentiation into osteoclasts (OCs). As shown in Figure 1, TM4SF19 expression increased with the progression of osteoclast differentiation. Expression of ACP5 and CTSK, genes related to osteoclast differentiation, also increased with the progression of osteoclast differentiation.

[0113] Example 2: Confirmation of the role of TM4SF19 using TM4SF19 knockout mice (1) TM4SF19 knockout and suppression of multinucleated osteoclast formation Cells were extracted from the bone marrow of TM4SF19KO, a CRISPR-induced TM4SF19 knockout mouse, and wild-type (WT) mice, and differentiated by treatment with M-CSF 25ng / ml + RANKL 100ng / ml. They were then stained for TRAP (tartrate-resistant acid phosphatase), an osteoclast-related marker.

[0114] As shown in Figure 2a, when bone marrow cells from TM4SF19KO mice were induced to differentiate into osteoclasts, they differentiated into mononuclear osteoclasts but did not differentiate into multinucleated osteoclasts.

[0115] Using the same method, cells were extracted from the bone marrow of TM4SF19KO (a CRISPR-induced TM4SF19 knockout mouse) and wild-type (WT) mice, and differentiated under conditions where the RANKL concentration was fixed at 100ng / ml and the M-CSF concentration was varied at 25, 40, and 60ng / ml. Then, they were stained for TRAP (tartrate-resistant acid phosphatase), an osteoclast-related marker.

[0116] As shown in Figure 2b, when bone marrow cells from TM4SF19KO mice were induced to differentiate into osteoclasts, it was confirmed that the impaired multinucleated cell formation was not restored even by treatment with high concentrations of M-CSF.

[0117] Figure 2c is a schematic diagram showing the differentiation process of osteoclasts. As shown in Figure 2c, osteoclast precursors first differentiate into TRAP-positive mononuclear osteoclasts, which then mature into giant multinucleated cells through cell-cell fusion and incomplete cytokinesis. Mature, multinucleated osteoclasts have high bone-resorbing activity, whereas osteoclasts that do not differentiate into multinucleated osteoclasts retain the osteoclast phenotype and express osteoclast-related markers such as TRAP and cadepsin K, but maintain low levels of bone-resorbing activity.

[0118] Therefore, the fact that TM4SF19 knockout prevents the differentiation and maturation of multinucleated osteoclasts during osteoclast differentiation suggests that it may be possible to attempt to prevent or treat bone loss-related diseases through the inhibition of TM4SF19. In particular, TM4SF19 inhibitors can selectively suppress the formation of multinucleated osteoclasts, which does not completely abolish the bone resorption function of osteoclasts but can more selectively regulate bone resorption and has significantly reduced side effects compared to therapeutic agents such as anti-RANKL antibodies that inhibit the formation of mononuclear osteoclasts.

[0119] (2) Confirmation of suppression of gene expression involved in osteoclast differentiation in TM4SF19KO mice Cells were extracted from the bone marrow of wild-type (WT) mice and TM4SF19KO mice, which were knocked out using CRISPR. After differentiation with or without M-CSF and RANKL treatment, the expression of genes involved in osteoclast differentiation, Ctsk, Acp5, c-Fos, and Nfatc1, was confirmed using qPCR.

[0120] FIG. 3 is a graph showing the mRNA expression of Ctsk, Acp5, c-Fos, and Nfatc1 in cells differentiated with or without M-CSF and RANKL treatment.

[0121] As shown in Figure 3, the expression of genes involved in osteoclast differentiation was significantly suppressed during differentiation of bone marrow cells derived from TM4SF19KO mice into osteoclasts compared to the control group (WT).

[0122] (3) Confirmation of actin belt formation and bone resorption inhibition After differentiation of wild-type (WT) and TM4SF19KO mouse bone marrow, the presence or absence of actin belt formation, which occurs during the differentiation of multinucleated osteoclasts, was confirmed by F-actin staining. While the wild-type (WT) mice formed normal actin belts, the TM4SF19KO mice did not form actin belts (Figure 4a).

[0123] After plating BMM cells on dentin discs and differentiation, pit formation was observed. Bone resorption was confirmed in wild-type (WT) cells, but was blocked in TM4SF19KO cells (Figure 4b).

[0124] (4) Confirmation of bone loss suppression by OVX Eight-week-old wild-type (WT) and TM4SF19KO female mice underwent either sham or ovariectomy (OVX), and femurs were fixed on day 31 and analyzed by microCT.

[0125] Furthermore, the micro-CT images were analyzed using a 3D image analysis program to obtain 3D micro-CT indices (bone microstructure indices) including bone volume (BV), the ratio of bone volume to tissue volume (%BV / TV), the average number of bone trabeculae (Tb.N), and the trabecular spacing (Tb.Sp).

[0126] We confirmed that ovariectomy-induced bone loss was suppressed in TM4SF19KO mice (Figure 5).

[0127] Regardless of ovariectomy, bone mineral density is higher in TM4SF19KO mice compared with wild-type (WT) mice.

[0128] (5) Confirmation of osteoclast formation by TRAP staining of femur The femurs of 8-week-old wild-type (WT) and TM4SF19KO mice were fixed, decalcified, and then TRAP stained.

[0129] As a result, as shown in Figure 6, TRAP staining of the femurs of TM4SF19KO mice confirmed the lack of multinuclear osteoclast formation.

[0130] Example 3-1: Confirmation of impaired multinucleated osteoclast formation in TM4SF19 extracellular loop 2 (EC2)-deficient mice (1) Confirmation of impairment of multinucleated osteoclast formation Cells were extracted from the bone marrow of wild-type (WT) mice and mice with TM4SF19EC2 Del (also referred to as TM4SF19EC2Δ), in which the TM4SF19 extracellular domain 2 (116-165) had been knocked out using CRISPR. They were differentiated with or without treatment with 25 ng / ml M-CSF and 100 ng / ml RANKL, and then TRAP staining was performed.

[0131] As a result, as shown in FIG. 7a, when the bone marrow of TM4SF19EC2Δ mice was differentiated into osteoclasts, it was confirmed that they differentiated into mononuclear osteoclasts but not into multinuclear osteoclasts.

[0132] Figure 7b shows the results of TRAP staining of cells extracted from the bone marrow of wild-type (WT) mice and TM4SF19EC2Δ mice in which the TM4SF19 extracellular domain 2 (116-165) had been knocked out using CRISPR. The cells were differentiated in a fixed RANKL concentration of 100 ng / ml and treated with M-CSF at 25 ng / ml or 100 ng / ml. When bone marrow from TM4SF19EC2Δ mice was differentiated into osteoclasts, the impaired formation of multinucleated osteoclasts was not restored even with high concentrations of M-CSF.

[0133] Example 3-2: Effect of cytoskeletal rearrangement by TM4SF19EC2Δ (1) Confirmation of actin belt formation and inhibition of bone resorption During the differentiation process of osteoclasts, multinucleated osteoclasts (mature osteoclasts) are formed, and actin belts consisting of podosomes are formed. The podosomes then adhere to the bone, causing bone resorption.

[0134] Cells were extracted from the bone marrow of wild-type and TM4SF19EC2Δ mice and differentiated in MCSF 60 ng / ml and RANKL 100 ng / ml. The cells were then fixed, stained with phalloidin FITC, and examined for F-actin belt formation using a confocal microscope (400x magnification).

[0135] We confirmed that TM4SF19EC2Δ inhibits the formation of multinucleated osteoclasts and affects integrin signaling-related cytoskeleton rearrangement. As shown in Figure 7c, actin belts are formed upon differentiation of multinucleated osteoclasts in wild-type cells, but not in TM4SF19EC2Δ cells.

[0136] After plating BMM cells on Dentin discs and differentiation, pit formation was confirmed by 1% toluidine blue staining. The results showed that bone resorption was observed in wild-type (WT) cells, but was blocked in TM4SF19EC2Δ cells (Figure 7d).

[0137] (2) Confirmation of suppression of osteoclast-related gene expression qPCR was performed to confirm the expression of target genes (Ctsk, Acp5, c-Fos, Nfatc1) related to osteoclast differentiation in wild-type (WT) mice and TM4SF19EC2Δ mice in which the TM4SF19 extracellular domain 2 (116-165) was knocked out using CRISPR.

[0138] As shown in Figure 8 , the expression of genes involved in osteoclast differentiation (Ctsk, Acp5, c-Fos, Nfatc1) was significantly suppressed during osteoclast differentiation in TM4SF19EC2Δ mice compared to the control group.

[0139] (3) Confirmation of bone loss inhibition Eight-week-old wild-type (WT) and TM4SF19EC2Δ female mice underwent either sham or ovariectomy (OVX), and femurs were fixed on day 31 and analyzed by microCT.

[0140] Furthermore, the micro-CT images were analyzed using a 3D image analysis program to obtain 3D micro-CT indices (bone microstructure indices) including bone volume (BV), the ratio of bone volume to tissue volume (%BV / TV), the average number of bone trabeculae (Tb.N), and the trabecular spacing (Tb.Sp).

[0141] We confirmed that ovariectomy-induced bone loss was suppressed in TM4SF19EC2Δ mice (Figure 9).

[0142] Eight-week-old wild-type (WT), TM4SF19KO, and TM4SF19EC2Δ mice (all female) were either released without ovary dissection (sham) or ovariectomized (OVX). Thirty-one days later, the femurs were fixed and subjected to microCT (μCT) analysis.

[0143] Figure 10 shows the 3D microCT indices (bone microstructure indices) obtained by analyzing the microCT images using a 3D image analysis program, including bone volume (BV), the ratio of bone volume to tissue volume (%BV / TV), the average number of bone trabeculae (Tb.N), the trabecular spacing (Tb.Sp), and the trabecular thickness (Tb.Th).

[0144] Figure 11 shows the micro-CT image.

[0145] As a result, as shown in Figures 10 and 11, we confirmed that TM4SF19KO mice and TM4SF19EC2Δ mice suppressed bone loss induced by ovariectomy, and that even in the sham group that did not undergo ovariectomy (OVX), TM4SF19KO mice and TM4SF19EC2Δ mice had higher bone density than wild-type (WT) mice.

[0146] Example 4: Confirmation of TM4SF19 self-binding and mutual binding with integrins (1) Confirmation of self-interaction between TM4SF19 and TM4SF19 To confirm whether TM4SF19 is involved in cell-to-cell interaction, we generated TM4SF19 tagged with HA and flag. 293T cells were transiently overexpressed with TM4SF19 tagged with 3HA at the N-terminus, wild-type TM4SF19 tagged with 3Flag at the N-terminus, and a TM4SF19 deletion mutant, and we examined their binding by immunoprecipitation. The TM4SF19 deletion mutant was generated as shown in Figure 12a. The hTM4SF19 mutant (hTM4SF19), in which a partial sequence of TM4SF19 shown in Figure 12a was deleted, was used. 115-175 Δ, hTm4sf 105-186 Δ, hTm4sf19 105-196 Δ, hTm4sf19 94-186 Δ and hTm4sf19 94-196 Δ) was also tagged with 3Flag, and the intercellular binding was analyzed by immunoprecipitation.

[0147] As a result, we confirmed that TM4SF19 is involved in cell-to-cell interactions and binds to itself (Fig. 12b). However, a mutant (hTm4sf19) lacking transmembrane 3 (TM3), extracellular region 2 (EC2), and transmembrane 4 (TM4) of TM4SF19 was isolated. 94-196These results confirmed that the transmembrane 3-extracellular region 2-transmembrane 4 region of TM4SF19 is important for TM4SF19 self-interaction, and that positions 94-196 of TM4SF19 are particularly important for TM4SF19-TM4SF19 self-interaction.

[0148] (2) Identification of the region involved in the TM4SF19-TM4SF19 self-interaction To verify that the region between positions 94 and 196 of TM4SF19 is important for the self-interaction between TM4SF19 and TM4SF19, we used 3Flag-hTm4sf19. 94-196Δ hTm4sf19, which is different from the above, retains only the 94-196 sequence of hTm4sf19 and deletes the remaining sequence. 94-196 We generated a TM4SF19 deletion mutant (Figure 12c, upper panel). Similar to the method described above, we transiently overexpressed TM4SF19 tagged with 3HA at the N-terminus, wild-type TM4SF19 tagged with 3Flag at the N-terminus, and the TM4SF19 deletion mutant in 293T cells, and examined their binding by immunoprecipitation. The results showed that a mutant (hTm4sf19) expressing transmembrane 3, EC2 (extracellular region 2), and transmembrane 4 of TM4SF19 was able to bind to the TM4SF19 transmembrane. 94-196 ) self-bound similarly to the wild-type (WT) (Fig. 12c, bottom panel). These results reaffirmed that the 94-196 aa region of TM4SF19 is an important site for the TM4SF19-TM4SF19 self-interaction.

[0149] (3) Interaction between TM4SF19 and TM4SF19-Fc The mutual binding between TM4SF19 tagged with 3Flag at the N-terminus and the hIgG1-Fc fusion protein of the TM4SF19 fragment was confirmed by immunoprecipitation using the same method as above in 293T cells. The TM4SF19 fragments used were the hTm4sf19 131-160, hTm4sf19 145-169, hTm4sf19 131-169, hTm4sf19 120-160, hTm4sf19 120-169, hTm4sf19 120-180, hTm4sf19 120-186, hTm4sf19 120-196, hTm4sf19 120-209, hTm4sf19 131-196, and hTm4sf19 145-196 regions (upper panel of Figure 12d). As a result, as shown at the bottom of Figure 12d and Figure 12e, it was confirmed that the 160-169aa region of hTm4sf19 plays an important role in binding, and that the 145-196 region of hTM4SF19-Fc plays an important role in increasing binding to TM4SF19.

[0150] (4) Confirmation of the interaction between integrin αv and TM4SF19 We confirmed the interaction between integrin αv, which plays an important role in osteoclast differentiation, and TM4SF19. To identify the key binding region of TM4SF19-Fc binding to integrin αv-TM4SF19, we performed immunoprecipitation in 293T cells to confirm the interaction between integrin αv tagged with 3HA at the C-terminus and a hIgG1-Fc fusion protein of the TM4SF19 fragment (Fig. 12f).

[0151] (5) Confirmation of the interaction between integrin β3 and TM4SF19 We confirmed the interaction between integrin β3 and TM4SF19, which plays an important role in osteoclast differentiation. To identify the key binding region of TM4SF19-Fc binding to integrin β3-TM4SF19, we performed immunoprecipitation in 293T cells to confirm the interaction between integrin β3 tagged with 3HA at the C-terminus and a hIgG1-Fc fusion protein of the TM4SF19 fragment (Fig. 12g).

[0152] Figure 12h shows the results of overexpressing integrin αv or integrin β3, a protein involved in cytoskeletal rearrangement during multinucleated osteoclast formation, in 293T cells or osteoclast precursor Raw264.7 cells, which regulate osteoclast function with TM4SF19. TM4SF19 binds to both integrin αv and integrin β3.

[0153] TM4SF19 is a member of the Tetraspanins family, and the extracellular region 2 (large extracellular loop) of this family is known to play an important role in binding with other binding partners. To confirm whether the extracellular region of TM4SF19 is essential for interacting with partners, we performed immunoprecipitation to confirm the mutual binding of TM4SF19 wt or proteins with deleted extracellular region 2 (115-175) with integrin αv or integrin β3. While TM4SF19 wt was confirmed to interact with them, hTM4SF19 115~175Δ This is the result of confirming that no bonding occurs.

[0154] Figure 12i shows the results of immunoprecipitation analysis of TM4SF19 and siglec-15, a membrane protein involved in regulating osteoclast function and cytoskeletal rearrangement during multinucleated osteoclast formation. TM4SF19 was N-terminally 3HA-tagged, DC-Stamp was N-terminus-tagged with 3Flag, and Siglec-15 was C-terminus-tagged with 3Flag. These proteins were then expressed in 293T cells, and their interactions were confirmed by immunoprecipitation.

[0155] Example 5: Confirmation of increased proliferation, colony formation, and migration of cancer cells due to overexpression of TM4SF19 After overexpressing TM4SF19 (hTM4SF19) in mouse breast cancer cells E0771, we performed time-dependent cell proliferation, colony formation assays, and cell migration assays.

[0156] Figure 13a shows a graph demonstrating that TM4SF19 overexpression significantly increased cell proliferation in E0771 breast cancer cells at 24, 48, and 72 hours. Figure 13b shows colony formation after plating 1,000 TM4SF19-overexpressing and control cells onto E0771 breast cancer cells, demonstrating that TM4SF19 overexpression increases colony formation. Figure 13c shows that TM4SF19-overexpressing and control cells onto E0771 breast cancer cells were plated in a migration chamber and cell migration was assessed over time, demonstrating that TM4SF19 overexpression increases cell migration.

[0157] Example 6: Confirmation of suppression of cancer cell metastasis by TM4SF19 deficiency (1) Confirmation that TM4SF19 deficiency suppresses lung metastasis of breast cancer cells Breast cancer cells E0771 were injected into the tail vein of wild-type (WT), TM4SF19KO, and TM4SF19EC2Δ mice. The mice were sacrificed 12 days later, and lung metastasis was confirmed by staining the lungs with Indian ink.

[0158] The lung metastasis phenotype of breast cancer cells in wild-type (WT) mice, TM4SF19KO mice, and TM4SF19EC2Δ mice was confirmed, and the number of nodules formed in the lungs was measured.

[0159] As a result, we confirmed that lung metastasis of breast cancer cells was significantly suppressed in TM4SF19KO mice and TM4SF19EC2Δ mice compared to wild-type (WT) mice (Figure 14).

[0160] (2) Confirmation of suppression of breast cancer cell metastasis-related gene and protein expression in TM4SF19 deficiency The surrounding environment of cancer cells plays an important role in cancer metastasis, and the extent of metastasis can be influenced by anti-tumor factors released by macrophages. Bone marrow was obtained from TM4SF19KO mice (TMKO) and wild-type (WT) mice, differentiated into bone marrow-derived macrophages (BMDMs), and then placed in the lower chamber of a migration chamber. Breast cancer cells were co-cultured in the upper chamber. The expression of target genes involved in cell migration (fibronectin, vimentin, CDH2, SNAI1, and SNAI2) was confirmed by qPCR, and the expression of metastasis-related proteins (vimentin, slug, snail, E-cadherin, and β-actin) was confirmed by Western blotting.

[0161] As a result, we confirmed that co-culture with TM4SF19KO mouse macrophages suppressed the expression of target genes involved in cell migration (fibronectin, vimentin, CDH2, SNAI1, SNAI2) (Fig. 15a) and metastasis-related proteins (vimentin, slug, snail, E-cadherin, β-actin) (Fig. 15b), indicating that TM4SF19 deficiency suppresses the expression of cancer cell metastasis-related genes and proteins.

[0162] Example 7: Confirmation of the involvement of TM4SF19 in human ADMSC adipogenesis Human adipose-derived mesenchymal stem cells (hADMSCs) were differentiated into adipocytes by growing them in DMEM / F12 medium containing 10 μg / ml insulin, 1 nM 3,3',5-Triiodo-L-thyronine, 1 μM dexamethasone, and 1 μM rosiglitazone. Gene expression (C / EBPα, PPARγ) was confirmed by qPCR on days 0, 14, and 21, and protein expression (PPARγ, FABP4) was confirmed by Western blotting.

[0163] As a result, it was confirmed that the expression level of TM4SF19 increased due to the adipogenic differentiation of human ADMSCs (FIG. 16).

[0164] Example 8: Confirmation of the obesity and metabolic disease-related role of TM4SF19 (1) Feed TM4SF19KO mice a high-fat diet to confirm weight gain Six-week-old wild-type (WT) mice and TM4SF19KO mice fed a normal diet were fed a high-fat diet (60% fat) for 16 weeks to confirm weight gain.

[0165] As a result, obesity was suppressed in TM4SF19KO mice despite being on a high-fat diet (Figure 17).

[0166] (2) Confirmation of insulin resistance and fatty liver resistance After feeding wild-type (WT) and TM4SF19KO mice a high-fat diet, insulin resistance was confirmed by HOMA-IR (A), liver phenotype was confirmed, liver tissue was fixed and stained with H&E (B), and intrahepatic triglyceride levels were measured (C).

[0167] As a result, TM4SF19KO mice reduced insulin resistance and suppressed fatty liver formation in a high-fat diet-induced obese mouse model. Therefore, we confirmed that TM4SF19KO mice (6-week-old mice fed a normal diet were fed a high-fat diet for 12 weeks) are resistant to high-fat diet-induced insulin resistance and fatty liver (Figure 18).

[0168] (3) Confirmation of resistance to high-fat-induced obesity Six-week-old wild-type (WT) and TM4SF19KO mice were fed a normal diet and then a high-fat diet for 18 weeks to confirm weight gain (Fig. 19a). After 18 weeks, the mice were sacrificed and the weights of each tissue (Fig. 19b), including subcutaneous and visceral fat (Fig. 19c), were measured. H&E staining of epididymal white adipose tissue (eWAT) confirmed the adipose phenotype and adipose tissue macrophages surrounding the adipose tissue (Fig. 19d). Serum secretion of the anti-obesity cytokine adiponectin was also confirmed (Fig. 19e) [rtWAT: retroperitoneal white adipose tissue, sWAT: subcutaneous white adipose tissue, ingWAT: inguinal white adipose tissue, iWAT: interscapular white adipose tissue, iBAT: interscapular brown adipose tissue].

[0169] As a result, TM4SF19KO mice exhibited suppressed obesity and increased serum adiponectin secretion even in a high-fat diet-induced obesity mouse model, confirming that TM4SF19KO mice are resistant to high-fat-induced obesity (18 weeks) (Figure 19).

[0170] (4) Confirmation of adipogenesis and expression of macrophage markers, M1-like macrophage markers Six-week-old wild-type (WT) and TM4SF19KO mice fed a normal diet were then fed a high-fat diet for 12 weeks. The expression of adipocyte differentiation markers (top), macrophage markers, and M1-like macrophage markers (bottom) was confirmed by qPCR in eWAT (white adipose tissue in the epididymis) and sWAT (subcutaneous white adipose tissue).

[0171] As a result, despite being on a high-fat diet, the expression of adipocyte differentiation markers, macrophage markers, and M1-like macrophage markers was reduced in the epididymal and subcutaneous white fat of TM4SF19KO mice (Figure 20).

[0172] (5) Confirmation of the amount of adipose tissue-associated macrophages Six-week-old wild-type (WT) and TM4SF19KO mice were fed a normal diet and then a high-fat diet for 12 weeks. After this, the percentage of macrophages (A) and dendritic cells (B) in the white fat of the epididymis was determined by FACS analysis.

[0173] As a result, despite a high-fat diet, % macrophages and dendritic cells were reduced in the white fat of the epididymis of TM4SF19KO mice, and the amount of adipose tissue-associated macrophages caused by obesity was reduced in TM4SF19KO (Figure 21).

[0174] (6) Expression of marker genes in the stromal vascular cell population of white adipose tissue After 12 weeks of high-fat diet feeding in 6-week-old wild-type (WT) and TM4SF19KO mice fed a normal diet, qPCR was performed to confirm the expression of phage marker genes in the stromal vascular fraction (SVF) in the white fat of the epididymis.

[0175] As a result, we isolated stromal vascular fraction (SVF) from the epididymal white fat of TM4SF19KO mice and confirmed that the expression of macrophage markers MCP1 and F4 / 80 (Fig. 22a) and M1-like macrophage markers IL6 and TNFα was reduced (Fig. 22b), and the expression of M2-like macrophage marker IL10 was increased (Fig. 22c).

[0176] (7) Confirmation of the role of TM4SF19 in inflammation, insulin resistance, and hepatic steatosis in eWAT To confirm the role of TM4SF19 in eWAT on inflammation, insulin resistance, and hepatic steatosis, we performed a high-fat diet-induced obese mouse model experiment.

[0177] As a result, we confirmed that TM4SF19 acts on adipocyte differentiation and inflammatory responses including macrophages, and that TM4SF19 deficiency reduces insulin resistance and suppresses fatty liver (Figure 23).

[0178] (8) TM4SF19 deficiency induces conversion of white adipocytes to beige adipocytes In a high-fat diet-induced obesity mouse model (6-week-old mice fed a normal diet were fed a high-fat diet for 12 weeks after the start of the diet), we confirmed the conversion of epididymal white fat to beige or brown fat phenotype in TM4SF19KO mice. Furthermore, we confirmed increased Ucp1 expression in the white fat of TM4SF19KO mice in a 12-week high-fat diet-induced obesity mouse model. Furthermore, we confirmed that TM4SF19 is involved in the conversion of white adipocytes to beige adipocytes by confirming increased Ucp1 expression in TM4SF19KO mice in an in vitro model of brown fat differentiation (Figure 24).

[0179] Figure 24a shows the formation of beige or brown fat phenotype and fatty liver in the epididymal white fat of high-fat diet-induced obese WT or TM4SF19KO mice, confirmed by H&E staining.

[0180] Figure 24b shows the expression of Ucp1, a brown fat marker, in the epididymal white fat and subcutaneous fat of high-fat diet-induced obese WT and TM4SF19KO mice. Ucp1 expression was confirmed to be increased in the white fat of TM4SF19KO mice.

[0181] Figure 24c is a schematic diagram showing the beige / brown adipocyte differentiation method in white fat. Adipocyte progenitor cells extracted from white fat of WT and TM4SF19KO mice were grown in DMEM / F-12 media supplemented with T3 and insulin. Two days after reaching confluence, differentiation was initiated by treatment with DMI and indometacin. From day 2, the cells were treated with rosiglitazone and cultured from day 7 to day 8 to induce beige / brown adipocyte differentiation.

[0182] Figures 24d and 24e show the results of confirming gene expression and protein expression of Ucp1 after beige / brown adipocyte differentiation.

[0183] (9) TM4SF19 expression was confirmed in white fat adipocytes and stromal vascular fraction (SVF). TM4SF19 expression was confirmed in adipocytes and SVF in the epididymal and subcutaneous white fat of mice fed a high-fat diet for 24 weeks.

[0184] The confirmation of TM4SF19 expression in both adipose tissue and SVF containing macrophages in eWAT and sWAT of high-fat diet-induced mice (6-week-old mice fed a normal diet were fed a high-fat diet for 24 weeks after starting the diet) indicates that TM4SF19 functions not only in adipocytes but also in SVF containing macrophages and monocytes (Figure 25).

[0185] Figure 25a shows a schematic diagram of the types of cells in each fraction of white fat, which was digested with collagen type I and then centrifuged to separate it into adipose tissue, infranatant, and SVF fractions.

[0186] Figure 25b shows the results of confirming TM4SF19 expression in adipocytes and SVF separated from epididymal white fat and subcutaneous white fat of mice fed a high-fat diet (60% fat) for 24 weeks.

[0187] (10) Isoproterenol treatment of stromal vascular fraction (SVF) isolated from sWAT of TM4SF19KO and wild-type mice induced brown adipose differentiation. SVF was extracted from subcutaneous white fat cells of wild-type (WT) and TM4SF19KO mice, and differentiation into brown fat cells was induced with isoproterenol. qPCR was performed to confirm the expression of thermogenic genes and beige fat marker genes.

[0188] The expression of thermogenic genes and beige fat markers was increased in the SVF of TM4SF19KO mice, suggesting that TM4SF19KO mice can contribute to the transformation of white fat into brown or beige fat (Figure 26).

[0189] (11) TM4SF19 expression in co-cultures of macrophages and 3T3-L1 adipocytes Interactions between 3T3-L1 adipose tissue and macrophages play an important role in high-fat diet-induced obesity.

[0190] As shown in the right panel of Figure 27, adipocytes were differentiated in an in vitro model and then contact co-cultured with macrophages. The expression of marker genes for macrophages, inflammation, and anti-obesity (macrophage marker; MCP-1, inflammation marker; TNFα, IL6, MMP3, anti-obesity marker; adiponectin) was then confirmed by qPCR. As a control, macrophages and adipocytes were grown separately, and the RNA was extracted and combined for use.

[0191] Co-culture of 3T3-L1 adipocytes with macrophages increased the expression of MMP3, an inflammation-related gene, and also increased TM4SF19.

[0192] (12) Induction of inflammatory changes by co-culture of adipocytes and macrophages in a contact system SVF was extracted from wild-type (WT) mice, and adipogenesis was induced. WT bone marrow was differentiated into macrophages, which were then cultured separately or in contact with each other to induce inflammatory changes. As a control, macrophages and adipocytes were cultured separately, and RNA was extracted and combined.

[0193] TM4SF19 was increased by co-culture of SVF adipocyte differentiation and bone marrow macrophage differentiation (Figure 28).

[0194] SVF was extracted from wild-type (WT) and TM4SF19KO mice, adipocytes were allowed to form, and bone marrow was collected from each mouse and differentiated into macrophages. The mice were then co-cultured to induce inflammatory changes.

[0195] As a result, as shown in Figure 29, MCP1 and IL6 were reduced in the differentiation of TM4SF19KO differentiated adipocytes + macrophages compared to the differentiation co-culture of WT differentiated adipocytes + macrophages.

[0196] SVF was extracted from wild-type (WT) and TM4SF19KO mice, and adipocytes were allowed to form. Bone marrow was collected from each mouse and differentiated into macrophages. These were then cultured in ctrl or co-culture to induce inflammatory changes. As a control (ctrl culture), macrophages and adipocytes were cultured separately, and RNA was extracted and combined for use.

[0197] In a co-culture model following differentiation of SVF adipocytes and bone marrow macrophages, TM4SF19KO BMDMs reduced the expression of inflammatory response markers, and the inflammatory response was reduced in TM4SF19KO adipocyte-TM4SF19KO macrophage co-cultures compared with wt adipocyte-wt macrophages (Figure 30).

[0198] When differentiated adipocytes were co-cultured with BMDMs, inflammatory signals were increased.

[0199] (13) Confirmation of TM4SF19 in an in vitro insulin resistance model An in vitro insulin resistance model was created.

[0200] 3T3-L1 adipocytes were differentiated for 12 days and treated with TNFα for 24 hours. Then, the adipogenic marker C / EBPα, and the in vitro insulin resistance model markers C / EBPβ and TM4SF19 were confirmed by qPCR.

[0201] TNFα induces inflammation, and this model mimics the increased inflammation signaling that occurs in the surrounding area when obesity is induced in vivo.

[0202] SVF was extracted from wild-type (WT) and TM4SF19KO mice and subjected to adipogenesis. After 24 hours of treatment with TNFα, the adipogenesis marker C / EBPα and the in vitro insulin resistance model markers C / EBPβ and TM4SF19 were analyzed by qPCR. The results confirmed that TM4SF19 expression was increased in the in vitro insulin resistance model (Figures 31 and 32). TM4SF19KO mice were also shown to have reduced insulin resistance (C / EBPβ is an insulin resistance marker).

[0203] (14) Role of TM4SF19 in macrophage differentiation and polarization We harvested bone marrow from wild-type (WT) and TM4SF19KO mice, differentiated them into macrophages, and identified markers involved in M1 polarization, which is involved in pan-inflammatory signaling.

[0204] We confirmed that inflammatory markers and TM4SF19 increased with differentiation of wild-type (WT) mouse bone marrow-derived M1 macrophages (Figure 33), and that bone marrow-derived M1 macrophage differentiation markers decreased in TM4SF19KO mice (Figure 34).

[0205] Production Example 1: Production and verification of TM4SF19 antibody (1) Generation of polyclonal mouse TM4SF19 antibody After predicting antibody immunogenicity and determining potential targets, we generated a polyclonal mouse TM4SF19 antibody targeting the 141-159aa region of mouse TM4SF19 extracellular loop 2. (See SEQ ID NO: 7 for the mouse TM4SF19 protein.)

[0206] (2) Validation of TM4SF19 antibody Human TM4SF19 (hTM4SF19), human EC2Δ (hTM4SF19 115-175Δ), mouse TM4SF19 (mTM4SF19), and mouse EC2Δ (mTM4SF19 116-165Δ) were transiently overexpressed in 293T cells, and Western blotting was performed to confirm expression using the polyclonal mouse TM4SF19 antibody prepared previously.

[0207] The polyclonal mouse TM4SF19 antibody recognized both human and mouse TM4SF19, but not the extracellular loop 2 deletion mutant (Fig. 35).

[0208] Example 9: Verification of the role of TM4SF19 using TM4SF19 antibodies (1) Confirmation of the inhibition of osteoclast differentiation by the TM4SF19 antibody Bone marrow was collected from wild-type (WT) mice, and differentiated into osteoclasts. The cells were treated with TM4SF19 antibody and then confirmed by TRAP staining.

[0209] As a result, the TM4SF19 antibody inhibited osteoclast multinucleation in a dose-dependent manner (FIG. 36).

[0210] Production Example 2: Preparation of TM4SF19-Fc Using sequence information and phylogenetic tree information for the TM4SF family, we constructed TM4SF19-Fc fusion proteins against human TM4SF19 and mouse TM4SF19 extracellular loop2.

[0211] TM4SF19-Fc is a form in which human IgG1-Fc is fused to the c-terminus of human TM4SF19 or mouse TM4SF19 extracellular loop 2.

[0212] Since mouse TM4SF19-Fc(116-165) is well secreted, it was purified and subjected to in vitro testing.

[0213] Since human TM4SF19-Fc (115-175) is not well secreted, a new Fc was prepared against the region (120-169).

[0214] Mouse TM4SF19-Fc was purified because it is well secreted.

[0215] Mouse TM4SF19EC2-Fc(116-165) was generated and purified and used in subsequent experiments.

[0216] Because human TM4SF19-EC2-Fc (115-175) showed poor expression, we recreated it with a sequence conserved with mouse TM4SF19-EC2-Fc and confirmed its expression. Human TM4SF19-Fc (120-169) showed higher secretion in the median region than extracellular loop 2 (115-175), and this was used in subsequent experiments (Figure 37).

[0217] The fusion proteins used in the examples according to the present invention are shown in FIG.

[0218] Hereinafter, "hTM4SF19-Fc" refers to the hTM4SF19-Fc (120-169 aa) fusion protein, and "mTM4SF19-Fc" refers to the mouse TM4SF19-Fc (116-165 aa) fusion protein.

[0219] Example 10: Verification of the role of TM4SF19 using TM4SF19-Fc hIgG1-Fc, hTM4SF19 120-169 -Fc and mTM4SF19 116-165 -Fc was treated during osteoclast differentiation. For osteoclast differentiation, bone marrow-derived cells were treated with MCSF 60 ng / ml and RANKL 100 ng / ml, and then TRAP staining was performed.

[0220] The results are shown in Figures 38a to 38c. Purified mTM4SF19 EC2-Fc was treated during osteoclast differentiation to confirm the inhibition of multinucleated osteoclast formation [E1: Sample eluted with Elution buffer (20 mM glycine); E1-1: Sample subjected to UF / DF (Ultrafiltration / Diafiltration) with Buffer A (50 mM phosphate, 50 mM NaCl, pH 7.0) (Fc may be more stable)]. Furthermore, purified hTM4SF19 120-169 -Fc treatment inhibited multinucleated osteoclast formation.

[0221] After treatment of wild-type mouse bone marrow with 10 μg / ml of IgG-Fc or mTM4SF19-Fc, the formation of actin belts during the differentiation of multinucleated osteoclasts was confirmed by F-actin staining. mTM4SF19-Fc inhibited actin belt formation, and the mouse TM4SF19 antibody localized TM4SF19 to the actin belt (Fig. 38d).

[0222] BMM cells were plated on dentin discs and then treated with 10 μg / ml of IgG-Fc and mTM4SF19-Fc during differentiation. Pit formation was confirmed by 1% toluidine blue staining. As shown in Figure 38e, bone resorption was blocked by mTM4SF19-Fc.

[0223] Example 11: Confirmation of bone loss recovery using TM4SF19-Fc Eight-week-old female mice were ovariectomized and injected with mouse TM4SF19-Fc via the tail vein once a week for three weeks, starting one week after the last injection. One week after the final injection, the mice were sacrificed, and their femurs were fixed and subjected to microCT analysis. Mouse TM4SF19-Fc concentrations of 2.5 mg / kg and 5 mg / kg were used.

[0224] FIG. 39a is a micro-CT image showing bone loss recovery by mouse TM4SF19-Fc.

[0225] The micro CT images in Figure 39a were analyzed using a 3D image analysis program to obtain 3D micro CT indices (bone microstructure indices) including total bone mineral density (BMD), bone volume (BV), the ratio of bone volume to tissue volume (%BV / TV), the mean number of bone trabeculae (Tb.N), and the trabecular spacing (Tb.Sp) (Figure 39b).

[0226] As shown in Figure 39b, total BMD (bone mineral density), %BV / TV (bone volume per tissue volume), and Tb.N (trabecular number) were decreased by ovariectomy and increased by mTM4SF19-Fc in a dose-dependent manner. Meanwhile, Tb.Sp (trabecular separation) was decreased by mTM4SF19-Fc. These results confirm that mouse TM4SF19-Fc rescued ovariectomy-induced bone loss.

[0227] Furthermore, mouse femoral tissues were histopathologically analyzed by H&E staining. As shown in Figure 39c, the number of trabeculae was reduced by ovariectomy, and TM4SF19-Fc treatment restored this reduction in trabecular number in a dose-dependent manner.

[0228] In addition, 8-week-old female mice underwent ovariectomy and were then intravenously injected with mouse TM4SF19-Fc (116–165) or human TM4SF19-Fc (131–169). Compared to the untreated group, human and mouse TM4SF19-Fc significantly suppressed bone loss (Figure 39d). Mouse TM4SF19-Fc (116–165) was administered at 10 mg / kg, and human TM4SF19-Fc (131–169) was administered at 25 mg / kg.

[0229] The human TM4SF19 region encompassing 131-169 can rescue bone loss following ovariectomy.

[0230] These results suggest the possibility of developing TM4SF19-Fc as a therapeutic agent for osteoporosis.

[0231] Example 12: Verification of the role of TM4SF19 using TM4SF19-Fc (1) Osteoclastogenesis Cells were extracted from the bone marrow of wild-type (WT) mice and differentiated under conditions of M-CSF or M-CSF and RANKL treatment. Then, the cells were treated with 10 μg / ml of mTM4SF19-Fc (the control group was buffer-treated). The expression of Ctsk and Acp5, genes involved in osteoclast differentiation, was confirmed by qPCR.

[0232] Figure 40 is a graph showing the mRNA expression of Acp5 and Ctsk in cells differentiated under conditions of treatment with M-CSF or M-CSF and RANKL (WT1, 2, 3, 4, and 5 represent the results obtained by differentiating bone marrow cells obtained from each of five wild-type mice into osteoclasts; the group treated with M-CSF only represents the undifferentiated group).

[0233] As shown in FIG. 40a, the expression of genes involved in osteoclast differentiation was significantly suppressed by mTM4SF19-Fc treatment compared to the control group (WT).

[0234] Similarly, cells were extracted from the bone marrow of wild-type (WT) mice and differentiated under conditions of M-CSF or M-CSF and RANKL treatment. After treatment with 5 μg / ml of mTM4SF19-Fc, the expression of osteoclast differentiation marker proteins was confirmed. As shown in Figure 40b, it was confirmed that treatment with mTM4SF19-Fc significantly suppressed the expression of osteoclast differentiation marker proteins compared to the control group (WT).

[0235] (2) Surface binding with mTM4SF19-Fc We hypothesized that TM4SF19-Fc blocks osteoclast multinucleation due to its increased surface binding ability during differentiation, and performed FACS analysis to measure surface binding of mTM4SF19-Fc before and after differentiation of Raw264.7 cells. As shown in Figure 41, there was little difference in surface binding between the control IgG1 (light gray graph) and mouse TM4SF19-Fc (dark gray graph) before differentiation. However, after differentiation, mTM4SF19-Fc (dark gray graph) shifted to the right compared to the control IgG1 (light gray graph), confirming that surface binding of mTM4SF19-Fc indeed increased after differentiation.

[0236] As shown in Figure 41, it was confirmed that binding to mTM4SF19-Fc increased after differentiation.

[0237] Example 13: Prevention and treatment of rheumatoid arthritis using TM4SF19-Fc The semi-therapeutic effect of mTM4SF19-Fc was confirmed in a collagen-induced arthritis mouse model according to the experimental schedule in Figure 42a.

[0238] DBA / 1 mice were purified for 14 days and then injected with chicken collagen II mixed with Complete Freund's Adjuvant (CFA), followed by a boost injection 18 days later with chicken collagen II mixed with Incomplete Freund's Adjuvant (IFA). mTM4SF19-Fc was administered intravenously at 10 mg / kg or 25 mg / kg twice weekly starting on day 15 after the initial injection. CIA scores were monitored twice weekly until day 63, when the mice were sacrificed.

[0239] The CIA score is rated as follows: Score 0 (normal paw), Score 1 (one or two toes inflamed and swollen), Score 2 (three or more toes inflamed with or without paw swelling, or mild swelling of the entire paw), Score 3 (entire paw inflamed and swollen), Score 4 (severely swollen paw and all toes, or ankylosed paw and toes).

[0240] The condition of the mouse joints was examined 42 days after the induction of CIA (Collagen-Induced Arthritis). As shown in Figure 42b, the control group treated with IgG1 showed swelling of the toes and instep compared to the untreated sham group. However, in the group treated with 10 mg / kg mTM4SF19-Fc, swelling of the toes and instep was somewhat suppressed, and at 25 mg / kg, the swelling phenomenon was clearly suppressed.

[0241] Meanwhile, the graph in Figure 42c shows the results of analyzing the CIA score and the number of swollen joints examined during the experimental period. Compared to the control group treated with IgG1, the mTM4SF19-Fc treatment group demonstrated a dose-dependent reduction in CIA score and edema.

[0242] Furthermore, micro-CT images of the paws of the collagen-induced arthritis mouse model femur were taken and the images were analyzed.

[0243] Figure 42d shows micro-CT images demonstrating that mTM4SF19-Fc treatment dose-dependently suppressed inflammation and bone damage in the paws of a mouse model of collagen-induced arthritis. Collagen-induced arthritis causes toes to curl and bone damage due to inflammation in the joints and ankles. Compared to mice treated with hIgG1-Fc (control group), inflammation and bone damage in the joints of mice treated with mTM4SF19-Fc were dose-dependently suppressed.

[0244] Figure 42e shows that inflammation and bone damage in the paws of a mouse model of collagen-induced arthritis were dose-dependently suppressed by mTM4SF19-Fc treatment, and when mice with the same RA score were compared, bone damage was suppressed in mice treated with mTM4SF19-Fc compared to mice treated with hIgG1-Fc (control group).

[0245] Furthermore, micro-CT images of the collagen-induced arthritis mouse model femur were analyzed using a 3D image analysis program to obtain 3D micro-CT indices (bone microstructure indices) including BMD, bone volume (BV), the ratio of bone volume to tissue volume (%BV / TV), the mean number of bone trabeculae (Tb.N), and the trabecular spacing (Tb.Sp).

[0246] FIG. 42f shows that collagen-induced arthritis reduces BMD (bone mineral density) and Tb.N (trabecular number) compared to the sham group, but TM4SF19-Fc suppresses bone loss.

[0247] Figure 42g shows the results of examining cartilage damage in a collagen-induced arthritis mouse model using toluidine blue, demonstrating that cartilage damage was suppressed in the mTM4SF19-Fc-treated group compared to the hIgG1-Fc-treated group.

[0248] Furthermore, the semi-therapeutic effects of mTM4SF19-Fc and hTM4SF19-Fc(120-169) were confirmed by arthritis score in a mouse model of collagen antibody-induced arthritis. Balbc mice were administered collagen antibody and then injected with LPS three days later to induce arthritis. Compared to the control group, mTM4SF19-Fc 25 mg / kg and hTM4SF19-Fc(120-169) 25 mg / kg exhibited arthritis-suppressing effects (Figure 4h). Compared to hIgG1-Fc, TM4SF19-Fc treatment resulted in lower arthritis scores and reduced paw thickness.

[0249] Furthermore, in a collagen-induced arthritis mouse model, arthritis was induced by injection of LPS 3 days after administration of the collagen antibody, and the therapeutic effect of mTM4SF19-Fc was confirmed by arthritis score.

[0250] Three days after administration of the collagen antibody, arthritis was induced by injection of LPS. To confirm the therapeutic effect of mTM4SF19-Fc, 50 mg / kg of mTM4SF19-Fc was administered from the 8th day, when the arthritis disease score was highest, and the arthritis disease score was checked every two days from the 9th to the 19th day.

[0251] Compared to the untreated group, mTM4SF19-Fc treatment reduced arthritis scores, paw thickness, and cartilage damage confirmed with toluidine blue (Figure 42i). In other words, mTM4SF19-Fc demonstrated a therapeutic effect on arthritis.

[0252] Example 14: Suppression of bone metastasis of breast cancer using hTM4SF19-Fc According to the experimental schedule shown in Figure 43a, 6-week-old female NOD-SCID mice were injected with MDA-MB231-luc breast cancer cells (a cell line in which luciparum was expressed in MDA-MB231 for imaging purposes) into the caudal artery (CA) to induce bone metastasis. After bone metastasis induction, hIgG1 or hTM4SF19-Fc was intravenously injected. TM4SF19-Fc was administered intravenously starting two days after cancer cell injection, at a dose of 10 mpk or 25 mpk twice a week. Cancer progression was monitored using bioluminescence imaging analysis.

[0253] On day 45 after cancer cell injection, the presence or absence of bone metastasis was confirmed through bioluminescence imaging analysis. As shown in Figure 43b (left), bone metastasis was suppressed in the hTM4SF19-Fc-treated group compared to the control hIgG1-treated group. On day 45, after luciferin injection, the mice were sacrificed, and their legs were removed for luminescence analysis. As shown in Figure 43b (right), it was confirmed that hTM4SF19-Fc treatment suppressed bone metastasis.

[0254] Figure 43c shows the results of micro-CT scans of the joints of mice with bone metastasis from breast cancer. Micro-CT analysis confirmed that bone damage was caused by bone metastasis from breast cancer and that treatment with hTM4SF19-Fc dose-dependently repaired the bone damage.

[0255] Furthermore, histological analysis of the joints of mice with bone metastasis of breast cancer was performed using H&E staining. As shown in Figure 43d, it was confirmed that treatment with hTM4SF19-Fc dose-dependently suppressed breast cancer bone metastasis and tumor growth.

[0256] According to the experimental schedule shown in Figure 44a, 6-week-old female NOD-SCID mice were injected with MDA-MB231-luc breast cancer cells (a luciferase-expressing MDA-MB231 cell line for imaging purposes) into the caudal artery (CA) to induce bone metastasis. Thirty days after induction, control hIgG1 or hTM4SF19-Fc was administered intravenously at 50 mg / kg.

[0257] Bioluminescence imaging analysis was performed every 7 days up to the 21st day after injection, confirming that bone metastasis was suppressed by hTM4SF19-Fc(120-169) (Figure 44b). After luciferin injection, the mice were sacrificed and their legs were excised for luminescence analysis, confirming that TM4SF19-Fc treatment suppressed bone metastasis (Figure 44c). MicroCT analysis confirmed that bone destruction caused by breast cancer bone metastasis was rescued by hTM4SF19-Fc treatment (Figure 44d).

[0258] Example 15: Inhibition of lung metastasis of breast cancer using TM4SF19-Fc Mouse-derived E0771 breast cancer cells were injected into the tail vein of wild-type mice to induce lung metastasis. hIgG1-Fc, mouse TM4SF19-Fc (116-165), and human TM4SF19-Fc (145-169) were each injected intravenously at 10 mg / kg. The inhibition of lung metastasis of breast cancer cells was confirmed by counting the number of nodules that formed in the lungs after staining with Indian ink. As shown in Figure 45, mouse TM4SF19-Fc (116-165) and human TM4SF19-Fc (145-169) significantly inhibited lung metastasis.

[0259] Example 16: Suppression of high-fat diet-induced obesity and fatty liver by mTM4SF19-Fc treatment Six-week-old mice were purchased and fed a high-fat diet for seven weeks. After one week of high-fat diet purification, the mice were fed a high-fat diet for nine weeks and administered mouse TM4SF19-Fc twice a week via subcutaneous or intravenous injection. As a result, as shown in Figure 46a, administration of 10 mg / kg of mTM4SF19-Fc suppressed high-fat diet-induced obesity.

[0260] The mice were sacrificed and the distribution of white fat in the organs was analyzed. As shown in Figure 46b, it was confirmed that mTM4SF19-Fc treatment reduced the weight of white fat, and also demonstrated a clear inhibitory effect on fatty liver.

[0261] Furthermore, liver tissue was observed using H&E staining and Masson and trichrome staining. As shown in Figure 47 (left), the effect of reducing fatty liver was clear compared to the control group treated with hIgG1-Fc. In addition, a reduction in plasma triglycerides was confirmed (Figure 47 (right)).

[0262] Example 17: Inhibitory effect of TM4SF19-Fc treatment on adipocyte differentiation Purified mouse TM4SF19-Fc (115-165) was treated in C3H10T1 / 2 cells, and it was confirmed that adipocyte differentiation was inhibited in a dose-dependent manner (FIG. 48).

[0263] The control group was not treated with differentiation inducers, while the experimental group was treated with differentiation inducers (dexamethasone 1 μM, IBMX 500 μM, insulin 4 μg / ml (DMI), and rosiglitazone 5 μM during adipocyte differentiation) and mouse TM4SF19 cells. 115-165 Adipocyte differentiation was observed after treatment with -Fc at 0 ng / ml, 1.25 ng / ml, 2.5 ng / ml, 5 ng / ml, and 10 ng / ml.

[0264] Furthermore, we confirmed the expression of C / EBP alpha and PPAR-gamma, which are major markers of adipocyte differentiation. We found that expression increased in the DMI-treated group compared to the group without differentiation induction (no DMI), and that expression was suppressed by treatment with mouse TM4SF19-Fc(116-165).

[0265] Example 18: Cancer cell migration inhibitory effect induced by osteoclast differentiation (1) TM4SF19-Fc Cancer cell migration induced by osteoclast differentiation was inhibited by mTM4SF19-Fc or hTM4SF19-Fc(120-169) and stained with 0.05% crystal violet solution. Bone marrow-derived macrophages were plated in 12-well plates and induced to differentiate into MCSF and RNAKL. They were then treated with 10 μg / ml of mTM4SF19-Fc or hTM4SF19-Fc, or no RNAKL was added. MDA-MB231 or PC3M cancer cells were then placed on the migration chamber and migration was assessed. Migrated cancer cells were stained with crystal violet.

[0266] As a result, as shown in Figure 49, it was confirmed that TM4SF19-Fc inhibited the migration of cancer cells induced by osteoclast differentiation.

[0267] (2)TM4SF19KO We confirmed the inhibition of cancer cell migration due to osteoclast differentiation in TM4SF19KO. Bone marrow-derived macrophages extracted from wt and TM4SF19KO mice were added with MCSF, and differentiated with or without RANKL. MDA-MB231 or PC3M cancer cells were then placed on the migration chamber and migration was monitored. Migrated cancer cells were stained with crystal violet.

[0268] The results, shown in Figure 50a, showed that cancer cell migration was suppressed by osteoclast differentiation in TM4SF19KO.

[0269] (3)TM4SF19EC2Δ We confirmed the inhibition of cancer cell migration by osteoclast differentiation in TM4SF19EC2Δ. Bone marrow-derived macrophages extracted from wt and TM4SF19EC2Δ mice were treated with MCSF and differentiated with or without RANKL. MDA-MB231 or PC3M cancer cells were then placed on the migration chamber and migration was monitored. Migrated cancer cells were stained with crystal violet.

[0270] The results shown in Figure 50b show that TM4SF19EC2Δ inhibited cancer cell migration through osteoclast differentiation.

[0271] Example 19: Confirmation of osteosarcoma cell proliferation, migration and colony formation (1) TM4SF19 overexpression After stably overexpressing 3Flag-hTM4SF19 in MG63 and HOS osteosarcoma cell lines, cell growth was assessed by MTT assay to confirm colony formation and migration. The results showed that TM4SF19 overexpression increased osteosarcoma cell proliferation, migration, and colony formation (Figures 51 and 52).

[0272] In addition, after knocking out TM4SF19 in the 143b osteosarcoma cell line using CRISPR, cell growth was confirmed in different clones (#4 and #6) using MTT assay, and colony formation was confirmed.

[0273] As a result, it was confirmed that suppression of TM4SF19 expression inhibited the proliferation and colony formation of osteosarcoma cell 143b (FIG. 53).

[0274] Overexpression of TM4SF19 increases osteosarcoma cell proliferation and migration, whereas suppression of TM4SF19 expression suppresses osteosarcoma cell proliferation and colony formation.

[0275] (2) TM4SF19-Fc treatment While treating the 143b osteosarcoma cell line hTM4SF19-Fc(120-169), cell growth was confirmed by MTT assay and cell counting, swarming ability was confirmed by colony formation, and cell migration was confirmed.

[0276] Treatment with hTM4SF19-Fc inhibited the growth of 143b osteosarcoma cells as confirmed by MTT assay (Fig. 54a) and cell counting (Fig. 54c), as well as the ability to swarm by colony formation (Fig. 54b) and cell migration (Fig. 54d).

[0277] It was confirmed that treatment with human TM4SF19-Fc (120-169aa) (10 μg / ml) inhibited colony formation in U2OS and MG63 osteosarcoma cells (FIG. 55).

[0278] It was confirmed that the cell migration ability of HOS osteosarcoma cells was inhibited by treatment with 10 μg / ml hTM4SF19-Fc(120-169aa) and hTM4SF19-Fc(145-169aa) (FIG. 56).

[0279] This suggests that hTM4SF19-Fc containing at least 145 to 169 aa exhibits activity.

[0280] Example 20: Confirmation of inhibition of pancreatic cancer cell proliferation and colony formation Pancreatic cancer cell lines Panc-1 and MIA-PaCa2 were treated with 100 μg / ml hTM4SF19-Fc(120-169), and the cell aggregation ability was confirmed by colony formation assay. As shown in Figure 57a, hTM4SF19-Fc(120-169) inhibited the aggregation ability of pancreatic cancer cell lines. Furthermore, pancreatic cancer cell lines Panc-1, MIA-PaCa2, and AsPC-1 were treated with 62.5 μg / ml or 125 μg / ml hTM4SF19-Fc(120-169), and cell growth inhibition was confirmed by cell counting (Figure 57b). The results confirmed that TM4SF19-Fc treatment inhibited the growth and aggregation ability of pancreatic cancer cells.

Claims

1. A pharmaceutical composition for preventing or treating a bone disease, comprising as an active ingredient a fusion protein having activity of suppressing the expression or activity of TM4SF19 (transmembrane 4L six family member 19) protein, the fusion protein comprising a fragment derived from extracellular loop 2 of the protein and an immunoglobulin Fc region, the fragment derived from extracellular loop 2 of TM4SF19 corresponds to the whole or part of EC2 of human or mouse TM4SF19 protein; the amino acid sequence portion corresponding to the entire EC2 is the 120th to 169th amino acids of the human TM4SF19 protein, and the fragment derived from extracellular loop 2 of TM4SF19 necessarily contains the 145th to 169th amino acids of the human TM4SF19 protein, or the amino acid sequence portion corresponding to the entire EC2 is the 116th to 165th amino acids of the mouse TM4SF19 protein; and The human TM4SF19 protein is represented by the amino acid sequence of SEQ ID NO: 1, and the mouse TM4SF19 protein is represented by the amino acid sequence of SEQ ID NO: 7; The bone disease is one or more selected from the group consisting of osteoclast dysfunction, osteopetrosis, arthritis, osteopenia, and bone damage. A pharmaceutical composition for preventing or treating bone diseases.

2. The fragment derived from extracellular loop 2 of human TM4SF19 is represented by the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 18; The fragment derived from extracellular loop 2 of the mouse TM4SF19 protein is represented by the amino acid sequence of SEQ ID NO: 8; The pharmaceutical composition of claim 1.

3. 2. The pharmaceutical composition of claim 1, wherein the fusion protein is represented by the amino acid sequence of SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16 or SEQ ID NO:

19.

4. A fusion protein having an activity of suppressing the expression or activity of TM4SF19 (transmembrane 4L six family member 19) protein, comprising a fragment derived from extracellular loop 2 of the protein and an immunoglobulin Fc region, the fragment derived from extracellular loop 2 of TM4SF19 corresponds to the whole or part of EC2 of human or mouse TM4SF19 protein; the amino acid sequence portion corresponding to the entire EC2 is the amino acid region of 120 to 169 of the human TM4SF19 protein, and the fragment derived from extracellular loop 2 of TM4SF19 necessarily contains the amino acid region of 145 to 169 of the human TM4SF19 protein, or the amino acid sequence portion corresponding to the entire EC2 is the amino acid region of 116 to 165 of the mouse TM4SF19 protein; The human TM4SF19 protein is represented by the amino acid sequence of SEQ ID NO: 1, and the mouse TM4SF19 protein is represented by the amino acid sequence of SEQ ID NO:

7. A fusion protein having the activity of suppressing the expression or activity of TM4SF19, comprising a fragment derived from extracellular loop 2 of the TM4SF19 protein and an immunoglobulin Fc region.

5. The fragment derived from extracellular loop 2 of human TM4SF19 is represented by the amino acid sequence of SEQ ID NO: 12, SEQ ID NO: 15 or SEQ ID NO: 18; The fragment derived from extracellular loop 2 of the mouse TM4SF19 protein is represented by the amino acid sequence of SEQ ID NO: 8; The fusion protein of claim 4.

6. The fusion protein of claim 4, comprising an amino acid sequence represented by SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, or SEQ ID NO: 19.

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