TM4SF19 inhibitors and their uses

Abstract

To identify genes associated with bone disease, obesity, and various obesity-mediated metabolic disorders, and to investigate their association with cancer or cancer metastasis, we will study various genes and confirm the various functions of TM4SF19. [Solution] The present invention relates to compositions for the prevention or treatment of bone diseases, obesity or obesity-mediated metabolic diseases, cancer, and cancer metastasis, and to a method for screening therapeutic agents for the said diseases, comprising TM4SF19 (transmembrane 4L six family member 19) expression or activity inhibitors.

Description

[Technical Field]

[0001] The present invention relates to compositions for the prevention or treatment of bone diseases, obesity or obesity-mediated metabolic diseases, cancer, and cancer metastasis, and to a method for screening therapeutic agents for the said diseases, comprising an inhibitor of the expression or activity of TM4SF19 (transmembrane 4L6 family member 19). [Background technology]

[0002] Bones are active tissues that constantly change throughout a person's life. Visually, bones can be divided into the outer cortical bone (compact bone) and the inner trabecular bone (cancellous bone, spongy bone). The cortical bone has high physical strength and plays a role in protecting and supporting the body, while the trabecular bone absorbs shock or maintains a constant level of calcium.

[0003] Even after bone growth is interrupted, the old bone is broken down and disappears (bone resorption), and the area where new bone has been lost is filled in and fixed (bone formation). This process is repeated throughout life and is called bone remodeling.

[0004] Bone homeostasis is maintained by a balance between the interaction between osteoblasts (bone formation) and osteoclasts (bone resorption), which keeps blood calcium levels constant. However, bone metabolic imbalances can lead to bone-related diseases such as osteoporosis.

[0005] The process of bone resorption and bone formation occurs sequentially through the interaction of osteoclasts and osteoblasts, maintaining bone homeostasis. This phenomenon is called bone remodeling, and bone mass is determined by the balance of activity between osteoclasts and osteoblasts. In the process of bone remodeling, osteoclasts proceed with bone resorption, followed by bone formation by osteoblasts, which occurs in a balanced manner through coupling. When this efficient balance is disrupted, bone loss occurs.

[0006] Osteoclasts differentiate into multinucleated osteoclasts, which are responsible for bone resorption. Osteoclast dysfunction induces osteopetrosis, while increased bone resorption due to osteoclast hyperactivation induces postmenopausal osteoporosis and inflammatory arthritis. To date, bisphosphonates and anti-RANKL antibodies have been developed as drugs targeting osteoclast hyperactivation. However, general suppression of osteoclasts affects the balance between osteoclasts and osteoblasts, significantly impacting bone formation, and such general suppression is accompanied by side effects. Therefore, there is a need to develop therapeutic agents that can more selectively suppress osteoclasts.

[0007] Osteoclasts are multinucleated cells that originate from hematopoietic stem cells and differentiate from the mononuclear cell / macrophage system through M-CSF (monocyte / macrophage colony-stimulating factor) stimulation and activation by RANK (receptor activator of nuclear factor κB) ligand (RANKL). Osteoclast differentiation involves the differentiation of osteoclast precursors into TRAP-positive mononuclear osteoclasts, followed by cell-cell fusion to mature into multinucleated osteoclasts [see Figure 2c]. Such mature multinucleated osteoclasts cause bone resorption [Int J Mol Sci. 2020 Aug 8;21(16):5685.].

[0008] Obesity is a biological phenomenon resulting from the interaction of complex genetic, metabolic, environmental, and behavioral factors. Defined as the abnormal or excessive accumulation of fat, it can have adverse effects on health. In particular, obesity is known as a significant risk factor for various adult diseases such as hypertension, type 2 diabetes, cancer, liver disease, hyperlipidemia, and arteriosclerosis. Obesity is the result of a chronic imbalance between calorie intake and energy expenditure, accompanied by a loss of metabolic, endocrine, and immune function in adipose tissue. This leads to metabolic abnormalities and decreased responsiveness (resistance) to insulin, the main fat storage signaling hormone, causing fat accumulation in metabolic organs other than adipose tissue, resulting in various lipotoxicities. The most representative pathological phenomenon of lipotoxicity is an inflammatory response, characterized as a chronic low-grade inflammation. In obesity, before metabolic abnormalities and insulin resistance appear, activation of adipose tissue macrophages occurs in adipose tissue, leading to a pre-inflammatory stage. Thus, obesity induces a low-intensity chronic inflammatory state in the body, causing various metabolic diseases. Thus, macrophages in adipose tissue play a crucial role in the development of chronic inflammation and metabolic complications in the body caused by obesity.

[0009] Fat stored in adipocytes is used as an important energy source in the body. Obesity is induced by excessive differentiation of adipocytes and an unbalanced oversupply of energy. Adipose tissue contains not only adipocytes but also various other cells such as adipose-derived stem cells (ASCs), immune cells, and endothelial cells, and these are called stromal vascular fractions (SVFs). Adipose tissue stores energy in lipid form, but it can also have a warming effect on the body. It is divided into white adipose tissue (WAT), which stores nutrients, and brown adipose tissue (BAT), which consumes nutrients and generates heat. Adipose tissue can also produce leptin, resistin, adiponectin, and tumor necrosis factor-α (TNFα). Adipocyte differentiation is a highly complex process involving the interaction of various hormones and transcription factors. It is promoted by stimuli such as insulin, insulin-like growth factor-1, and growth hormone, and during this process, an increase in transcription factors such as the CCAA enhancer-binding protein (C / EBP) family and peroxisome proliferator-activated receptor (PPAR) gamma is observed. These transcription factors, along with adipocyte regulators, promote adipocyte differentiation and increase the expression levels of enzymes such as the fatty acid-binding protein aP2 and fatty acid synthase. On the other hand, it has been reported that excessive triglyceride accumulation is also 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 (Switzerland) and appetite suppressants such as Meridia from Abbott (USA). However, these drugs have the problem of causing side effects such as headaches, elevated blood pressure, and diarrhea. Therefore, there is a real need to develop new, targeted obesity treatments that do not have these side effects.

[0011] Cancer is one of the greatest diseases threatening human health, arising from a series of mutations that cause cells to proliferate uncontrollably and unregulatedly, becoming immortalized. While early-stage cancers can be treated with surgery, radiation therapy, and chemotherapy, their side effects have become a major problem. In the case of terminal or metastatic cancers, there is often no specific treatment available, leaving patients with a limited lifespan until their death.

[0012] Recently, various biochemical mechanisms related to cancer have been elucidated, and therapeutic agents have been developed accordingly. However, a fundamental cure for cancer has yet to be presented. As a result, research is actively underway to identify various biomolecules related to cancer and to develop drugs that target them. Efforts are also being made to enhance the effectiveness of cancer treatment by combining some of these drugs.

[0013] Therefore, further efforts to discover cancer-related target molecules are extremely important.

[0014] On the other hand, TM4SF19 is a transmembrane 4L six family member (TM4SF19), and its use as a marker for obesity diagnosis has been disclosed in Korean Patent No. 10-1781200. However, there have been no reports to date regarding TM4SF19's effects in treating bone-related diseases, obesity or obesity-related metabolic diseases, cancer, or cancer metastasis. [Overview of the project] [Problems that the invention aims to solve]

[0015] Therefore, the inventors of this invention conducted research on the association between various genes and bone diseases, obesity or obesity-mediated metabolic diseases, and cancer, with the aim of finding genes related to the fundamental treatment of bone diseases, obesity and various obesity-mediated metabolic diseases, cancer treatment, or suppression of cancer metastasis. As a result, the inventors completed this invention by confirming the various functions of TM4SF19. [Means for solving the problem]

[0016] The object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of bone disease, comprising a TM4SF19 expression or activity inhibitor as an active ingredient.

[0017] A further object of the present invention is to provide a screening method for therapeutic agents for bone disease, comprising the steps of treating a suspected bone disease specimen with a candidate substance for the treatment of bone disease, and comparing the expression levels of mRNA or protein of the TM4SF19 gene with those of a control group.

[0018] A further object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of obesity or obesity-mediated metabolic disease, comprising a TM4SF19 expression or activity inhibitor as an active ingredient.

[0019] A further object of the present invention is to provide a screening method for therapeutic agents for obesity or obesity-mediated metabolic diseases, comprising the steps of treating a suspected obese or obesity-mediated metabolic disease specimen with a candidate substance for the treatment of obesity or obesity-mediated metabolic disease, and comparing the expression levels of mRNA or protein of the TM4SF19 gene with those of a control group.

[0020] A further object of the present invention is to provide a pharmaceutical composition for cancer prevention, treatment, or suppression of cancer metastasis, comprising a TM4SF19 expression or activity inhibitor as an active ingredient.

[0021] Still another object of the present invention is to provide a method for screening a therapeutic agent for cancer treatment or cancer metastasis treatment, which includes treating a cancer prevention, treatment or cancer metastasis-suspected specimen with a candidate substance for cancer prevention, treatment or cancer metastasis suppression, and comparing the expression levels of the mRNA or protein of the TM4SF19 gene with a control group.

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

[0023] Still another object of the present invention is to provide a method for preventing or treating a bone disease, which includes administering to a subject a composition for preventing or treating a bone disease containing a therapeutically effective amount of an agent for suppressing TM4SF19 expression or activity.

[0024] Still another object of the present invention is to provide a method for preventing or treating obesity or obesity-mediated metabolic diseases, which includes administering to a subject a composition for preventing or treating obesity or obesity-mediated metabolic diseases containing a therapeutically effective amount of an agent for suppressing TM4SF19 expression or activity.

[0025] Still another object of the present invention is to provide a method for preventing or treating cancer prevention, treatment or cancer metastasis, which includes administering to a subject a composition for cancer prevention, treatment or cancer metastasis suppression containing a therapeutically effective amount of an agent for suppressing 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 for the prevention or treatment of bone diseases, obesity, obesity-mediated metabolic diseases, cancer, and cancer metastasis.

[0027] In addition, according to the method for screening a candidate substance for treating bone diseases, obesity, obesity-mediated metabolic diseases, cancer, and cancer metastasis that inhibits TM4SF19, candidate substances capable of treating bone diseases, obesity, obesity-mediated metabolic diseases, cancer, and cancer metastasis can be efficiently selected.

Brief Description of the Drawings

[0028] [Figure 1] Figure 1 shows the results of extracting cells from mouse bone marrow, treating them with M-CSF and RANKL (Receptor activator of nuclear factor kappa-β ligand), differentiating them at different time points, and then confirming TM4SF19 gene expression by qPCR. [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) by treatment with M-CSF (25 ng / ml) + RANKL (100 ng / ml). [Figure 2b] Figure 2b shows the results of extracting cells from the bone marrow of TM4SF19KO (a CRISPR-induced TM4SF19 knockout mouse) and wild-type (WT) mice, differentiating them under conditions where the RANKL concentration was fixed at 100 ng / ml and the M-CSF concentration varied at 25, 40, and 60 ng / ml, and then performing TRAP staining (Tartrate-resistant acid phosphatase staining), an osteoclast-related marker. [Figure 2c] Figure 2c is a schematic diagram illustrating the differentiation process of osteoclasts. [Figure 3] Figure 3 shows the results of qPCR testing four days after cells were extracted from the bone marrow of wild-type (WT) mice and TM4SF19KO mice, treated with M-CSF or M-CSF + RANKL (M-CSF 25 ng / ml, RANKL 100 ng / ml) for differentiation, and then tested for the expression of target genes related to osteoclast (OC) differentiation: Ctsk, Acp5, c-Fos, and Nfatc1. [Figure 4a] Figure 4a shows the presence or absence of ectin belt formation, which occurs during the differentiation of multinucleated osteoclasts, after differentiating bone marrow from wild-type and TM4SF19KO mice, as confirmed by F-actin staining. [Figure 4b]Figure 4b shows pit formation observed by 1% toluidine blue staining after BMM cells were plated onto a Dentin disc and differentiated. [Figure 5a] Figure 5a shows the results of microCT analysis performed on 8-week-old wild-type (WT) and TM4SF19KO female mice after either shaming (opening the ovary without detachment) or ovarian resection (OVX), followed by fixation of the thigh 31 days later. [Figure 5b] Figure 5b shows three-dimensional micro-CT indices (bone microstructure indices) obtained by analyzing micro-CT images using a three-dimensional image analysis program, including total bone mineral density (BMD), bone volume (BV), ratio of bone volume to tissue volume (%BV / TV), average number of trabeculae (Tb.N), inter-trabecular space (Tb.Sp), and trabecular thickness (Tb.Th). [Figure 6] Figure 6 shows the results of fixing the femurs of 8-week-old wild-type (WT) mice and TM4SF19KO mice, decalcifying them, and then staining them with TRAP. [Figure 7a] Figure 7a shows the results of extracting cells from the bone marrow of wild-type (WT) mice and TM4SF19EC2Δ mice in which TM4SF19 extracellular domain 2 (116-165) was knocked out using CRISPR, differentiating them by treatment with M-CSF 25 ng / ml or M-CSF + RANKL 100 ng / ml, and then staining them with TRAP. [Figure 7b] Figure 7b shows the results of extracting cells from the bone marrow of wild-type (WT) mice and TM4SF19EC2Δ mice, fixing them with RANKL at a concentration of 100 ng / ml, treating them with M-CSF at concentrations of 25 ng / ml or 100 ng / ml to induce differentiation, and then staining them with TRAP. [Figure 7c] Figure 7c shows the presence or absence of ectin belt formation, which occurs during the differentiation of multinucleated osteoclasts, after differentiating bone marrow from wild-type and TM4SF19EC2Δ mice, as confirmed by F-actin staining. [Figure 7d]Figure 7d shows pit formation observed by 1% toluidine blue staining after wild-type and TM4SF19EC2Δ BMM cells were plated onto a Dentin disc and differentiated. [Figure 8] Figure 8 shows the results of qPCR testing 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 performed 31 days after either shaming (opening the ovary without detachment) or ovarian resection (OVX) in 8-week-old wild-type (WT) and TM4SF19EC2Δ female mice, with the thigh fixed. [Figure 9b] Figure 9b shows three-dimensional micro-CT indices (bone microstructure indices) obtained by analyzing micro-CT images using a three-dimensional image analysis program, including total bone mineral density (BMD), bone volume (BV), ratio of bone volume to tissue volume (%BV / TV), average number of trabeculae (Tb.N), inter-trabecular space (Tb.Sp), and trabecular thickness (Tb.Th). [Figure 10] Figure 10 shows the results of micro-CT analysis of 8-week-old wild-type (WT), TM4SF19KO, and TM4SF19EC2Δ female mice after 31 days following either sham (opening the ovary without detachment) or ovariectomy (OVX), followed by fixation of the thigh, and displays the bone microstructure index. [Figure 11] Figure 11 shows images obtained by micro-CT imaging 31 days after 8-week-old wild-type (WT), TM4SF19KO, and TM4SF19EC2Δ female mice underwent either sham (opening the ovary without detachment) or ovarian resection (OVX) with the thigh fixed. [Figure 12a] Figure 12a is a schematic diagram of the structures of hTm4sf19 mutants (hTM4SF19115-175Δ, hTm4sf19105-186Δ, hTm4sf105-196Δ, hTm4sf1994-186Δ, and hTm4sf1994-196Δ) that have a partial sequence deletion in TM4SF19. [Figure 12b] Figure 12b shows results indicating that TM4SF19 binds to itself and is involved in intercellular interactions. Among the mutants in which a portion of the TM4SF19 sequence is deleted, the 94-196 deletion mutant shows that it does not bind to TM4SF19 itself. [Figure 12c] Figure 12c shows the results (bottom panel) indicating that hTm4sf1994-196 (top panel), in which only the 94-196 sequence of hTm4sf19 is retained and the remaining sequence is deleted, binds to self in the same way as the wild type (WT) and is involved in intercellular interactions. [Figure 12d] Figure 12d shows the structures of 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 used as TM4SF19 fragments, and the results of immunoprecipitation confirming the mutual binding between TM4SF19 tagged with 3Flag at the N-terminus and the hIgG1-Fc fusion protein of the aforementioned TM4SF19 fragments. [Figure 12e] Figure 12e, similar to Figure 12d, shows 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 used as TM4SF19 fragments. The structures of 145-196 and the results of immunoprecipitation confirming the mutual binding between TM4SF19 tagged with 3Flag at its N-terminus and the hIgG1-Fc fusion protein of the TM4SF19 fragment are shown. [Figure 12f]Figure 12f shows the results of immunoprecipitation confirming the mutual binding between integrin αv tagged with 3HA at its C-terminus and the hIgG1-Fc fusion protein of the TM4SF19 fragment. [Figure 12g] Figure 12g shows the results of immunoprecipitation confirming the interaction between integrin β3 tagged with 3HA at its C-terminus and the hIgG1-Fc fusion protein of the TM4SF19 fragment. [Figure 12h] Figure 12h shows the results of regulating the function of osteoclasts with TM4SF19 in 293T cells or osteoclast precursor Raw264.7 cells, and then overexpressing integrin αv or integrin β3, proteins involved in cytoskeletal rearrangement during multinucleated osteoclast formation, and confirming the presence or absence of interaction. It also shows the results of confirming the presence or absence of interaction between integrin αv or integrin β3 tagged with 3HA at the C-terminus, TM4SF19 tagged with 3Flag at the N-terminus, and mutants lacking TM4SF19 EC2(115-175). [Figure 12i] Figure 12i shows the results of immunoprecipitation confirming the interaction between TM4SF19 and DC-Stamp, a membrane protein involved in regulating osteoclast function and rearranging the cytoskeleton during multinucleated osteoclast formation, and siglec-15. [Figure 13] Figure 13 shows the results of overexpressing TM4SF19 in mouse-derived breast cancer cells E0771 to confirm cell proliferation, colony formation, and cell migration [LPCX: retroviral control vector name]. [Figure 14] Figure 14 shows the results of confirming the suppression of lung metastasis after injecting mouse-derived breast cancer cells E0771 into the tail veins of wild-type (WT), TM4SF19KO, and TM4SF19EC2Δ mice. [Figure 15a]Figure 15a shows the results of qPCR testing to confirm the expression of target genes involved in cell migration (fibronectin, vimentin, CDH2, SNAI1, SNAI2) after bone marrow was collected from wild-type (WT) mice and TM4SF19KO mice, differentiated into bone marrow-derived macrophages, and then co-cultured with breast cancer cells to induce cell migration (Figure 15a). [Figure 15b] Figure 15b shows the results of confirming the expression of translocation-related proteins (vimentin, slug, snail, E-cadherin, β-actin) by Western blotting [BMDM: bone marrow derived macrophages]. [Figure 16] Figure 16 shows the results of differentiating human adipose-derived mesenchymal stem cells (hADMSCs) into adipocytes, confirming gene expression (C / EBPα, PPARγ, TM4SF19) by qPCR (top panel), and confirming protein expression (TM4SF19, PPARγ, FABP4) by Western blotting (bottom panel). [Figure 17] Figure 17 shows the results of observing weight gain in 6-week-old wild-type (WT) mice and TM4SF19KO mice fed a normal diet, after being given a high-fat diet (60% fat) for 16 weeks. [Figure 18] Figure 18 shows the results of feeding 6-week-old wild-type (WT) mice and TM4SF19KO mice, which were fed a normal diet, with a high-fat diet for 12 weeks. Insulin resistance was assessed using HOMA-IR (A), liver phenotype was identified, liver tissue was fixed and stained with H&E (B), and triglyceride levels in the liver were measured (C). [Figure 19a] Figure 19a shows the results of observing weight gain in 6-week-old wild-type (WT) mice and TM4SF19KO mice fed a normal diet, after being fed a high-fat diet for 18 weeks. [Figure 19b] Figure 19b shows the weight of each tissue. [Figure 19c] Figure 19c shows the weight of subcutaneous fat and visceral fat. [Figure 19d]Figure 19d shows the phenotype of the adipose tissue and macrophages surrounding the epididymal white adipose tissue (eWAT) as confirmed by H&E staining. [Figure 19e] Figure 19e shows the results of confirming the secretion of adiponectin, an anti-obesity cytokine, in the serum [rtWAT: retroperitoenal 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 adipocyte differentiation markers (top panel), macrophages, and M1-like macrophage markers (bottom panel) in eWAT (epididymal white adipose tissue) and sWAT (subcutaneous white adipose tissue) after feeding 6-week-old wild-type (WT) mice and TM4SF19KO mice, which were fed a normal diet, on a high-fat diet for 12 weeks. [Figure 21] Figure 21 shows the results of FACS analysis of white adipose tissue in the epididymis of 6-week-old wild-type (WT) mice and TM4SF19KO mice that were fed a normal diet, followed by a 12-week high-fat diet, to identify macrophages (A)% and dendritic cells (B)%. [Figure 22-1] Figure 22-1 shows the results of feeding wild-type (WT) and TM4SF19KO mice a high-fat diet for 12 weeks, and then examining the expression of macrophage marker genes MCP1 and F4 / 80, as well as M1-like macrophage markers IL6 and TNFα, and M2-like macrophage marker IL10, in the stromal vascular fraction (SVF) of the white fat tissue of the epididymis. [Figure 22-2] This is a continuation of Figure 22-1. [Figure 23]Figure 23 shows the results illustrating 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 TM4SF19KO mice were fed a high-fat diet for 12 weeks after being introduced to a high-fat diet from 6-week-old mice that had been fed a normal diet. The change in epididymal white fat to beige or brown fat phenotype was confirmed. [Figure 24b] Figure 24b shows the results confirming that Ucp1 expression is increased in white adipose tissue of TM4SF19KO mice in a high-fat diet-induced obesity mouse model. [Figure 24c] Figure 24c shows the process for creating an in vitro model of brown adipose tissue differentiation. [Figure 24d] Figure 24d shows the results confirming that Ucp1 increased in TM4SF19KO in an in vitro model of brown adipose tissue 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 cell types in each fraction of white fat after digestion with collagen type I and subsequent centrifugation, separating it into adipose tissue, infranatant, and SVF fractions. [Figure 25b] Figure 25b shows the results of examining TM4SF19 expression in adipocytes and SVF separately in the white adipose tissue of the epididymis and subcutaneous white adipose tissue of mice fed a high-fat diet of 60% fat for 24 weeks. [Figure 26] Figure 26 shows the results of extracting SVF from subcutaneous white adipose tissue of wild-type (WT) and TM4SF19KO mice, differentiating it into brown adipose tissue with isopropanol, and confirming thermogenic gene expression and beige adipose tissue marker gene expression. [Figure 27]Figure 27 shows the results of identifying marker genes such as macrophages, inflammation, and adiponectin after contact co-culture with macrophages (right) following differentiation of adipocytes in an in vitro model [control group: used after culturing macrophages and adipocytes separately, extracting RNA, and combining them]. [Figure 28] Figure 28 shows the results of inducing inflammatory changes by extracting SVF from wild-type (WT) mice, generating adipocytes, differentiating them into WT bone marrow macrophages, and then either individually or by contact co-culture [control group (ctrl): macrophages and adipocytes were cultured separately, then RNA was extracted and combined before use]. [Figure 29] Figure 29 shows the results of extracting SVF from wild-type (WT) mice and TM4SF19KO mice, adipogenesis, bone marrow collection from each mouse, differentiation 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, inducing adipogenesis, collecting bone marrow from each mouse, differentiating it into macrophages, and then inducing inflammatory changes either individually or by contact co-culture [control group (ctrl): macrophages and adipocytes were cultured separately, then RNA was extracted and combined before use]. [Figure 31] Figure 31 shows the process of creating an in vitro insulin resistance model (top panel) and the results after differentiating adipocytes for 12 days and then treating them with TNFα for 24 hours, in which the adipocyte differentiation marker C / EBPα and the insulin resistance in vitro model marker C / EBPβ were confirmed (center), and an increase in TM4SF19 was also confirmed (bottom panel). [Figure 32] Figure 32 shows results demonstrating that insulin resistance is reduced in TM4SF19KO compared to wild-type individuals in an in vitro insulin resistance model. [Figure 33]Figure 33 shows the results of identifying markers involved in M1 polarization after differentiating macrophages from bone marrow extracted from wild-type (WT) mice. [Figure 34] Figure 34 shows the results (bottom panel) of M1 polarization performed after bone marrow was collected from wild-type (WT) mice and TM4SF19KO mice, differentiated into macrophages, and then markers involved in M1 polarization were identified. [Figure 35] Figure 35 shows the results of Western blotting performed to confirm expression using the previously prepared polyclonal mouse TM4SF19 antibody, after transiently overexpressing human TM4SF19 (hTM4SF19), human EC2Δ (hTM4SF19 115-175Δ), mouse TM4SF19 (mTM4SF19), and mouse EC2Δ (mTM4SF19 116-165Δ) in 293T cells. [Figure 36] Figure 36 shows the results of treating bone marrow collected from wild-type mice with TM4SF19 antibody and confirming the results by TRAP staining while differentiating it into osteoclasts. [Figure 37] Figure 37 shows the results of Coomassie staining (left and right) and Western blotting (center) after preparing and purifying human TM4SF19120-169-Fc and mouse TM4SF19116-165-Fc. [Figure 38a] Figure 38a shows the inhibition of multinucleated osteoclast formation by TRAP staining after treating mM4SF19116-165-Fc during the osteoclast differentiation process [E1; sample eluted with Elution buffer (20mM glycine). E1-1; sample subjected to Ultrafiltration / Diafiltration (UF / DF) with Buffer A (50mM phosphate, 50mM NaCl, pH 7.0) (Fc may be more stable)]. [Figure 38b]Figure 38b shows the inhibition of multinucleated osteoclast formation by TRAP staining after treating hTM4SF19120-169-Fc with a treatment that facilitates osteoclast differentiation. [Figure 38c] Figure 38c shows the results of treating hIgG1-Fc, hTM4SF19120-169-Fc, and mTM4SF19116-165-Fc with osteoclast differentiation processes, 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 treating wild-type mouse bone marrow with IgG-Fc and mTM4SF19-Fc at 10 μg / ml while differentiating it, and then confirming the presence or absence of actin belt formation during the differentiation of multinucleated osteoclasts by F-actin staining. [Figure 38e] Figure 38e shows confirmation by toluidine blue staining that bone resorption was blocked by mTM4SF19-Fc. [Figure 39a] Figure 39a is a micro CT scan image showing the bone loss suppression effect of mouse TM4SF19-Fc. [Figure 39b] Figure 39b shows the total bone mineral density (BMD), bone volume (BV), ratio of bone volume to tissue volume (%BV / TV), and average number of trabeculae (Tb.N), obtained by analyzing the micro CT images from Figure 39a using a 3D image analysis program, and represents 3D micro CT indices including the inter-trabecular space (Tb.Sp). [Figure 39c] Figure 39c shows the histopathological analysis of mouse femoral tissue via H&E staining. [Figure 39d] Figure 39d shows that bone loss was significantly suppressed after ovariectomy in 8-week-old female mice, followed by injection of hIgG1-Fc, mouse TM4SF19-Fc (116-165), and human TM4SF19-Fc (131-169) into the tail vein. [Figure 40a]Figure 40a shows a graph of Ctsk and Acp5 mRNA expression in cells differentiated under conditions of being treated with M-CSF or M-CSF and RANKL while being treated with 10 μg / ml of mouse TM4SF19-Fc. [Figure 40b] Figure 40b shows the results of extracting cells from the bone marrow of wild-type (WT) mice, differentiating them under conditions of M-CSF or M-CSF and RANKL (M-CSF 25 ng / ml, RANKL 100 ng / ml), and then treating them with mTM4SF19-Fc at 5 μg / ml, followed by confirmation of the expression of osteoclast differentiation marker proteins. [Figure 41] Figure 41 shows surface binding between Raw264.7 cells and mTM4SF19-Fc cells before and after differentiation, as confirmed by FACS analysis. [Figure 42a] Figure 42a is a schematic representation of the experimental schedule for confirming the prevention and treatment of rheumatoid arthritis using TM4SF19-Fc. [Figure 42b] Figure 42b shows the results of examining the joint condition of mice 42 days after the induction of collagen-induced arthritis (CIA). [Figure 42c] Figure 42c shows the results of an analysis of CIA scores and the number of swollen joints investigated during the experimental period. [Figure 42d] Figure 42d is a micro CT image showing that inflammation and bone damage in the foot of a collagen-induced arthritis mouse model were suppressed in a volume-dependent manner by mTM4SF19-Fc treatment. [Figure 42e] Figure 42e shows the results demonstrating that inflammation and bone damage occurring in the paw of a collagen-induced arthritis mouse model are suppressed in a dose-dependent manner by mTM4SF19-Fc treatment. [Figure 42f]Figure 42f shows the results of analyzing microCT images of the collagen-induced arthritis mouse model femur using a 3D image analysis program. The results include 3D microCT indices (bone microstructure indices) such as total bone mineral density (BMD), bone volume (BV), ratio of bone volume to tissue volume (%BV / TV), average number of trabeculae (Tb.N), and inter-trabecular space (Tb.Sp). [Figure 42g] Figure 42g shows the results of confirming cartilage damage in a collagen-induced arthritis mouse model using toluidine blue. It demonstrates 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 effect of mTM4SF19-Fc and hTM4SF19-Fc(120-169) observed in arthritis scores in a mouse model of arthritis induced by LPS injection three days after collagen antibody treatment. The results confirm that TM4SF19-Fc treatment resulted in a lower arthritis disease score and reduced paw thickness compared to hIgG1-Fc treatment. [Figure 42i] Figure 42i shows the therapeutic effect of mTM4SF19-Fc at 8 days after the arthritis disease score reached its maximum, in a mouse model of arthritis induced by LPS injection 3 days after collagen antibody treatment. Compared to the untreated group, treatment with TM4SF19-Fc resulted in a lower arthritis disease score, reduced paw thickness, and suppressed cartilage damage as measured by toludine blue, confirming the therapeutic effect. [Figure 43a] Figure 43a is a schematic representation of an experimental schedule for confirming the inhibitory effect of hTM4SF19-Fc on bone metastasis in breast cancer. [Figure 43b]Figure 43b shows the results of confirming the cancer progression status through bioluminescence imaging analysis. [Figure 43c] Figure 43c shows the results of a micro CT scan of the joints of mice with bone metastases from breast cancer. [Figure 43d] Figure 43d shows the results of histological analysis of the joints of mice with bone metastases from breast cancer, performed via H&E staining. [Figure 44a] Figure 44a is a schematic representation of an experimental schedule for confirming the inhibitory effect of hTM4SF19-Fc(120-169) on bone metastasis in breast cancer. [Figure 44b] Figure 44b shows the results of confirming the cancer progression status through bioluminescence imaging analysis. [Figure 44c] Figure 44c shows the results of luminescence analysis performed on the joints of mice with bone metastases from breast cancer. [Figure 44d] Figure 44d shows the results of a micro CT scan of the joints of mice with bone metastases from breast cancer. [Figure 45] Figure 45 shows the results of inducing lung metastasis by injecting mouse-derived E0771 breast cancer cells into the tail vein of wild-type mice, and confirming that lung metastasis was significantly suppressed by mTM4SF19-Fc(116-165) and hTM4SF19-Fc(145-169). [Figure 46a] Figure 46a shows that mTM4SF19-Fc treatment suppresses obesity caused by a high-fat diet. [Figure 46b] Figure 46b shows the effect 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 compared to body weight. [Figure 47] Figure 47 shows the effect of mTM4SF19-Fc treatment on reducing fatty liver in liver tissue and triglycerides in plasma, as confirmed by H&E staining and Masson & trichrome staining. [Figure 48] Figure 48 shows the results of confirming that adipocyte differentiation is suppressed in a dose-dependent manner by treatment with mTM4SF19-Fc(116~165) by staining lipid droplets with Oil-red-O staining, and by examining the gene expression of the adipogenesis markers C / EBPα and PPAR-γ. [Figure 49] Figure 49 shows the suppression of cancer cell migration induced by osteoclast differentiation with TM4SF19-Fc, confirmed by staining with 0.05% crystal violet solution. [Figure 50a] Figure 50a shows the suppression of cancer cell migration by osteoclast differentiation in TM4SF19KO, confirmed by staining with 0.05% crystal violet solution. [Figure 50b] Figure 50b shows the suppression of cancer cell migration by osteoclast differentiation in TM4SF19EC2Δ, confirmed by staining with 0.05% crystal violet solution. [Figure 51] Figure 51 shows the results of stably overexpressing 3Flag-hTM4SF19 in the MG63 osteosarcoma cell line, followed by confirmation of cell growth by MTT analysis, and observation of colony formation and migration. [Figure 52] Figure 52 shows the results of confirming colony formation and migration by MTT analysis after stably overexpressing 3Flag-hTM4SF19 in an HOS osteosarcoma cell line. [Figure 53] Figure 53 shows the results of CRISPR-mediated knockout of TM4SF19 in 143b osteosarcoma cell lines, followed by MTT analysis of cell growth in different clones (#4 and #6), and confirmation of colony formation as a measure of community building ability. [Figure 54a] Figure 54a shows the results of MTT analysis confirming cell growth in 143b osteosarcoma cells treated with hTM4SF19-Fc(120-169aa). [Figure 54b] Figure 54b shows the results of confirming community-building ability through colony formation. [Figure 54c]Figure 54c shows the results of confirming cell growth using cell counting. [Figure 54d] Figure 54d shows the results of confirming cell migration. [Figure 55] Figure 55 confirms that treatment with human TM4SF19-Fc(120-169aa)(10μg / ml) suppresses the formation of U2OS and MG63 osteosarcoma colonies. [Figure 56] Figure 56 confirms that the cell migration ability of HOS osteosarcoma cells is suppressed by treatment with 10 μg / ml hTM4SF19-Fc(120-169aa) and hTM4SF19-Fc(145-169aa). [Figure 57a] Figure 57a shows confirmation of the suppression of pancreatic cancer cell colony formation in human TM4SF19-Fc(120~169aa). [Figure 57b] Figure 57b shows the suppression of pancreatic cancer cell growth 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 embodiment of the present invention [In each figure, the target sequence is shown in bold and is indicated by SED ID NOs: 8, 12, 15, and 18; 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 by 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 embodiments of this application. [Figure 58c] Figure 58c, similar to Figure 58a, shows the sequence information of the TM4SF19 fragment and TM4SF19-Fc fusion protein used in the embodiments of this application. [Figure 58d] Figure 58d, like Figure 58a, shows the sequence information of the TM4SF19 fragment and TM4SF19-Fc fusion protein used in the embodiments of this application. [Modes for carrying out the invention]

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

[0030] On the other hand, 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, embodiments of the present invention are provided to further fully explain the present invention to a person with average skill in the art. Moreover, throughout the specification, "including" a component means that other components may be included, rather than excluding them, unless otherwise stated.

[0031] The present invention relates to a pharmaceutical composition for the prevention or treatment of bone disease, comprising a TM4SF19 expression or activity inhibitor as an active ingredient.

[0032] Furthermore, the present invention relates to a pharmaceutical composition for the prevention or treatment of obesity or obesity-mediated metabolic disease, comprising a TM4SF19 expression or activity inhibitor as an active ingredient.

[0033] Furthermore, the present invention relates to a pharmaceutical composition for cancer prevention, treatment, or suppression of cancer metastasis, comprising a TM4SF19 expression or activity inhibitor as an active ingredient.

[0034] In this 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, TM4SF19 has not been properly studied in relation to its function.

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

[0036] Human-derived TM4SF19 may be translated from mRNAs containing GenBank Accession No. 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. In other words, human-derived TM4SF19 has three transcription variants: GenBank Accession No. NM_138461.4 (transcript variant 1), NM_001204897.2 (transcript variant 2), and NM_001204898.2 (transcript variant 3), and three isoforms: GenBank Accession No. NP_612470.2 (isoform 1), NP_001191826.1 (isoform 2), and NP_001191827.1 (isoform 3). The TM4SF19 used in the examples of the present invention is NP_612470.2 (isoform 1), obtained from mRNA containing GenBank Accession No. NM_138461.4 (transcript variant 1). Isoform 1 has high homology to mouse TM4SF19.

[0037] The 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 TM4SF19 expression or activity inhibitor of the present invention means a substance that reduces the expression of the TM4SF19 gene or the activity of the TM4SF19 protein. In a specific form, the inhibitor of the TM4SF19 gene expression of the present invention means a substance that reduces the expression level or activity of the TM4SF19 gene by directly acting on the TM4SF19 gene or indirectly acting on the higher regulator of the TM4SF19 gene to reduce the expression of the TM4SF19 gene at the transcriptional level, or by increasing the degradation of the expressed TM4SF19 gene or interfering with its activity. Specifically, it may be, but is not limited to, at least one selected from the group consisting of antisense nucleotides that bind complementaryally to the mRNA of the TM4SF19 gene, small hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), and ribozyme.

[0046] In this 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. Since antisense nucleotides are long chains of monomer units, they may be readily synthesized for target gene sequences.

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

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

[0049] In this invention, the term "miRNA (microRNA)" refers to 21 to 23 non-coding RNAs that regulate gene expression after transcription by promoting the degradation of target RNA or suppressing its translation.

[0050] In this invention, the term "ribozyme" refers to an RNA molecule that has a function such as an enzyme that recognizes a specific base sequence and cleaves it itself. A ribozyme consists of a region that specifically binds to a complementary base sequence of the target messenger RNA strand and a region that cleaves the target RNA.

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

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

[0053] In this invention, the term "peptide" refers to a peptide that has the advantage of high binding affinity to target substances and does not undergo denaturation during heat / chemical treatment. Furthermore, due to its small molecular size, it can attach to other proteins and be used in fusion proteins. Specifically, because it can attach to high molecular weight protein chains, it can be used in diagnostic kits and drug delivery systems.

[0054] In this invention, the term "peptide mimetics" refers to molecules that inhibit the binding domain of the TM4SF19 protein and thereby suppress the activity of the TM4SF19 protein. Peptide mimetics may be peptides or non-peptides, and may consist of amino acids linked by non-peptide bonds such as psi bonds. They may also be "conformationally constrained" peptides, cyclic mimetics, or cyclic mimetics containing at least one exocyclic domain, a binding site (binding amino acid), and an active site. Peptide mimetics may be novel small molecules structured similarly to the secondary structural properties of the TM4SF19 protein, capable of mimicking the repressive properties of large molecules such as antibodies or water-soluble receptors, and capable of acting with effects equivalent 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 of its own and is characterized by its ability to bind to a target molecule with high affinity and specificity.

[0056] In this invention, the term "fusion protein" refers to a new protein created by the combination of two or more different proteins or the same type of protein, and is often called a chimeric protein. Two or more heterogeneous proteins are joined in parts, parts and wholes, or wholes and wholes. In many cases, the sequence of one gene and the sequence of another gene are joined together by matching codons as needed to create a hybrid gene, which is then expressed and produced as a protein.

[0057] As one example, the fusion protein according to the present invention may include a fragment of TM4SF19 that specifically binds to the TM4SF19 protein.

[0058] In addition to the aforementioned fragment, the immunoglobulin Fc region may be further included.

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

[0060] A preferred fusion protein according to the present invention may include an amino acid sequence represented by SEQ ID NO:10, SEQ ID NO:13, SEQ ID NO:16, or SEQ ID NO:19.

[0061] As an example, the present invention may include a fusion protein in which an immunoglobulin Fc region is bound to a fragment of TM4SF19, for example, a fusion protein in which an immunoglobulin Fc region is bound to all or part of the extracellular loop of TM4SF19, or a fusion protein in which an immunoglobulin Fc region is bound to all or part of the extracellular loop of TM4SF19 and all or part of a membrane protein. Here, the fusion protein in which an immunoglobulin Fc region is bound to a fragment of TM4SF19 includes not only those in which the immunoglobulin Fc region is directly bound to the fragment of TM4SF19, but also those in which the immunoglobulin Fc region is indirectly bound to the fragment of TM4SF19. When the immunoglobulin Fc region indirectly binds to the TM4SF19 fragment, there may be an additional 1 to 10 amino acid sequences, e.g., 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, e.g., 1 amino acid sequence, which may correspond to linkers, spacers, or enzyme sites, between the TM4SF19 fragment and the immunoglobulin Fc region.

[0062] The fragments of TM4SF19 that can be used in the present invention are not limited to, but include 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, while the immunoglobulin Fc region was used exemplarily for the stability of the TM4SF19 protein, the immunoglobulin Fc region is not the only fusion partner that can bind to the TM4SF19 protein.

[0063] The present invention further relates to a fusion protein for inhibiting TM4SF19 expression or activity, comprising a fragment derived from the extracellular loop 2 of the TM4SF19 protein and an immunoglobulin Fc region.

[0064] The fragment derived from the extracellular loop 2 (EC2) of the TM4SF19 protein may be an amino acid sequence site that corresponds to all or part of EC2.

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

[0066] The amino acid sequence region corresponding to part of EC2 may include the amino acid region between positions 145 and 169 of the human TM4SF19 protein.

[0067] The amino acid sequence region corresponding to part of EC2 may include the amino acid region between positions 131 and 169 of the human TM4SF19 protein.

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

[0069] The TM4SF19 protein may contain an enzyme site between the extracellular loop 2 (EC2)-derived fragment and the immunoglobulin Fc region. stomach.

[0070] The extracellular loop 2 region of human TM4SF19 used as a fragment for the construction of the fusion protein is illustrative and not limited to it.

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

[0072] Furthermore, the "Fc region" may include a "modified immunoglobulin Fc region" or an "Fc region variant," meaning that some amino acids in the Fc region are substituted, or that it is produced by combining different types of Fc regions. Preferably, it means an Fc region in which the binding affinity to the Fc receptor and / or the complement is modified, resulting in weakened antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) compared to a wild-type Fc region. In this case, the modified 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" means a specific immunoglobulin directed to an antigenic site. "Antibodies" include monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, and human antibodies, and include novel antibodies as well as antibodies already known or commercially available in the art. The antibody includes not only the full-length form containing two heavy chains and two light chains, but also functional fragments of the antibody molecule, insofar as it specifically binds to TM4SF19. The functional fragment of the antibody molecule means a fragment that possesses at least antigen-binding function, and this includes, but is not limited to, Fab, F(ab'), F(ab')2, Fv, etc.

[0074] This invention relates to anti-TM4SF19 antibodies specific to all or part of the extracellular loop 2 of human TM4SF19. Specifically, the invention relates to anti-TM4SF19 antibodies specific to all or part of the extracellular loop 2 region of human TM4SF19 SEQ ID NO. 115-175 from GenBank Accession No. NP_612470.2. In other specific forms, the invention relates to anti-TM4SF19 antibodies specific to all or part of the extracellular loop 2 region of human TM4SF19 SEQ ID NO. 120-169 from GenBank Accession No. 138461.4. The target sites of the extracellular loop 2 of human TM4SF19 used for antibody production are illustrative and not limited thereto.

[0075] According to the embodiments of the present invention, it was revealed that the TM4SF19 protein is involved in intercellular interactions through self-binding.

[0076] In particular, the inventors targeted the extracellular loop region of the TM4SF19 protein and produced an antibody against the TM4SF19 protein that can suppress the self-binding of the TM4SF19 protein, as well as a fusion protein in which fragments of TM4SF19 (e.g., the extracellular loop region, the extracellular loop region and membrane protein) are fused with Fc, and evaluated the efficacy of the TM4SF19 protein activity inhibitor.

[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, amorphous bone disease, degenerative bone disease, degenerative arthritis, rheumatoid arthritis, psoriatic arthritis, psoriatic spondylitis, age-related bone loss, osteoporosis, osteogenesis imperfecta, osteomalacia, osteopenia, fracture, bone defect and hip joint loss, rickets, Paget's bone disease, periodontal disease, and bone injury caused by bone metastasis of cancer cells.

[0078] In this invention, the term "obesity" means a condition in which there is abnormal or excessive accumulation of fat.

[0079] In this invention, the term "obesity-mediated metabolic disease" includes diabetes mellitus, hypertension, hyperlipidemia, non-alcoholic steatohepatitis, and certain cancers, and more broadly, hypertension, diabetes mellitus, insulin resistance syndrome, metabolic syndromes, obesity-related gastroesophageal reflux disease, arteriosclerosis, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, lipodystrophy, non-alcoholic steatohepatitis, cardiovascular disease, polycystic ovary syndrome, and the like. When using the compositions of this invention, the aforementioned diseases can be treated simultaneously. Furthermore, the target population for treatment of such obesity-related diseases also includes individuals who wish to lose weight.

[0080] In this invention, the term "cancer" refers collectively to diseases caused by cells that have aggressive characteristics, such as dividing and growing regardless of normal growth limits; invasive characteristics, such as invading surrounding tissues; and metastatic characteristics, such as spreading to other parts of the body.

[0081] There are no restrictions on the type of cancer used in the present invention, but it may be at least one selected from the group consisting of colorectal cancer, gastric 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, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic cancer, central nervous system (CNS) tumors, spinal cord tumors, brainstem glioma, and pituitary adenoma.

[0082] This invention includes applications of TM4SF19 related to the suppression of not only "cancer" but also "cancer metastasis."

[0083] On the other hand, in the present invention, the term "prevention" means all actions that involve administering the composition of the present invention to prevent the occurrence of a desired symptom or disease, or to delay its occurrence or manifestation.

[0084] In the present invention, the term "treatment" means all actions that involve administering the composition of the present invention to improve or eliminate a desired symptom or disease.

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

[0086] In this invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not irritate living organisms and does not inhibit the biological activity and properties of the administered compound. Acceptable pharmaceutically acceptable carriers in compositions formulated as liquid solutions are those suitable for sterilization and biological use, and may be physiological saline, sterile water, Ringer's solution, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components. Other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Diluents, dispersants, surfactants, binders, and lubricants may also be added to formulate the compositions into injectable preparations such as aqueous solutions, suspensions, emulsions, pills, capsules, granules, or tablets.

[0087] The composition comprising the TM4SF19 expression or activity inhibitor and a pharmaceutically acceptable carrier of the present invention is applicable to any dosage form containing it as an active ingredient and may be manufactured as an oral or parenteral dosage form, or as a unit dose form for ease of administration and uniformity of dosage. The pharmaceutically acceptable dosage forms of the present invention include forms suitable for oral, rectal, nasal, topical (including cheek and sublingual), subcutaneous, vaginal, or parenteral (including intramuscular, subcutaneous, and intravenous) administration, or forms suitable for inhalation or injection. Oral dosage forms containing the composition of the present invention as an active ingredient may be formulated as, for example, tablets, lozenges, water-soluble or oily suspensions, compounded powders or granules, emulsions, hard or soft capsules, syrups, or elixirs.

[0088] The composition of the present invention may be formulated as a parenteral dosage form containing the active ingredient in the form of injection, such as subcutaneous injection, intravenous injection, or intramuscular injection, as a suppository, or as a spray such as an aerosol that can be inhaled through a respirator. To formulate it as an injection dosage form, the composition of the present invention may be mixed with a stabilizer or buffer in water to produce a solution or suspension, which may then be formulated as a unit dose in ampoules or vials.

[0089] The composition of the present invention is administered in a pharmaceutically effective amount, i.e., a therapeutically effective amount. In the present invention, "therapeutically effective amount" means an amount sufficient to treat the disease, and the effective dose level may be determined according to factors including the type and severity of the patient's disease, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and elimination ratio, the duration of treatment, drugs used concurrently, and other factors known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple dose. That is, the total effective amount of the composition of the present invention may be administered to the patient as a single dose or by 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 the greatest effect with the minimum amount without side effects, which may be easily determined by those skilled in the art.

[0090] The dosage of the pharmaceutical composition of the present invention varies widely depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, and disease severity. The daily dose, when administered parenterally, is preferably 0.01 μg to 100 mg per kg of body weight, more preferably 1 μg to 50 mg per day. However, the dosage may increase or decrease depending on the administration route, severity of obesity, sex, weight, age, etc., and therefore, the above dosage does not limit the scope of the present invention by any means.

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

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

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

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

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

[0096] In the terminology of this invention, "subject" includes mammals such as mice, rats, rabbits, cattle, horses, sheep, pigs, goats, camels, antelopes, dogs, or humans, who have a related disease whose symptoms can be improved by administration of the pharmaceutical composition according to the present invention.

[0097] The compositions of the present invention may be administered via various routes, oral or parenteral, as long as they can reach the target tissue. Specifically, they may be administered in the usual manner via oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intra-arterial, transdermal, nasal, inhalation, intraocular, or intradermal routes.

[0098] The preventive or therapeutic method of the present invention comprises administering a therapeutically effective dose of the composition of the present invention. The therapeutically effective dose means an amount that enhances the effect of weight loss or reduction in the size of adipocytes. It is obvious to those skilled in the art that the appropriate total daily dose can be determined by a treating physician within the range of sound medical judgment. The specific therapeutically effective dose for a particular patient may 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 status, sex and diet, administration time, administration route and secretion rate of the composition, duration of treatment, and various other factors and similar factors known in the pharmaceutical field. Therefore, the effective dose of the pharmaceutical composition suitable for the purpose of the present invention is preferably determined by considering the matters described above. Furthermore, in some cases, the therapeutic effect can be enhanced by co-administering the composition of the present invention with known therapeutic agents for related diseases.

[0099] In another aspect, the present invention relates to the step of treating a suspected bone disease specimen with a candidate substance for the treatment of bone disease, and The present invention relates to a screening method for bone disease therapeutics, which includes a step of comparing the expression levels of mRNA or protein of the TM4SF19 gene with those of a control group.

[0100] In another embodiment, the present invention relates to the step of treating a specimen suspected of being obese or having an obesity-mediated metabolic disease with a candidate substance for the treatment of obesity or an obesity-mediated metabolic disease, and The present invention relates to a screening method for drugs that treat obesity or obesity-mediated metabolic disorders, which includes a step of comparing the expression levels of mRNA or protein of the TM4SF19 gene with those of a control group.

[0101] In another aspect, the present invention relates to the step of treating a specimen suspected of being used for cancer prevention, treatment, or cancer metastasis with a candidate substance for cancer prevention, treatment, or cancer metastasis suppression, and The present invention relates to a screening method for cancer or cancer metastasis therapies, which includes a step of comparing the expression levels of mRNA or protein of the TM4SF19 gene with those of a control group.

[0102] In the present invention, the term "specimen" means an individual or sample used to screen for candidate substances for the treatment of related diseases, and this includes, without limitation, mammals including dogs, cattle, pigs, rabbits, chickens, mice, and humans, and includes samples such as whole blood, serum, blood, plasma, saliva, urine, sputum, lymph, cells, and tissues isolated from such individuals.

[0103] In this invention, the term "control group" means a sample that has not been treated with a candidate substance. Furthermore, the term "candidate substance" as used in this invention means a substance used to test for TM4SF19 expression or activity changes, and includes any molecule, such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and a wide range of compounds, as a target for measuring the ability to prevent or treat related diseases by directly or indirectly altering TM4SF19 expression levels or activity. Such candidate substances include all synthetic substances as well as natural substances.

[0104] In this invention, the term "therapeutic drug" means a substance that prevents or treats a related disease.

[0105] The screening method of the present invention may be performed by treating individuals suspected of having the related disease with a candidate therapeutic substance, and then comparing the expression levels of TM4SF19 gene mRNA or protein with a control group that has not been treated with the candidate substance. Substances that reduce the expression of TM4SF19 gene mRNA or protein compared to the control group may be identified as therapeutic agents for the related disease. Analytical methods for measuring mRNA levels include, but are not limited to, reverse transcriptase polymerization, competitive reverse transcriptase polymerization, real-time reverse transcriptase polymerization, RNase protection assays, Northern blotting, and DNA chips.

[0106] Analytical methods for measuring protein levels include, but are not limited to, Western blotting, ELISA, radioimmunoanalysis, radioimmunodiffusion, Octarony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation analysis, complement fixation analysis, FACS, and protein chips.

[0107] Furthermore, according to one embodiment of the present invention, it was confirmed that TM4SF19 expression increases during the osteoclast differentiation process. In TM4SF19 deficiency models (TM4SF19 knockout mouse, TM4SF19KO, and TM4SF19 extracellular domain 2 (116~165) knockout mouse, TM4SF19EC2△), it was confirmed that mononuclear osteoclasts were formed during the osteoclast differentiation process, while the formation of multinucleated osteoclasts was suppressed. Osteoclasts that failed to differentiate into multinucleated osteoclasts retained their osteoclast phenotype and maintained low levels of bone resorption activity. In other words, the TM4SF19 inhibitor of the present invention, which can selectively suppress the formation of multinucleated osteoclasts, does not completely eliminate bone resorption function, but rather can more selectively regulate bone resorption, thus significantly reducing side effects compared to therapeutic agents that suppress mononuclear osteoclast formation from the stage, such as anti-RANKL antibodies. Furthermore, we confirmed that the TM4SF19 deficiency model prevents bone loss due to ovariectomy. In addition, when osteoclast differentiation was treated 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 during the osteoclast differentiation process, while suppressing the formation of multinuclear osteoclasts. Therefore, it can be seen that substances that inhibit TM4SF19 expression or TM4SF19 activity are effective in preventing or treating bone diseases.

[0108] Furthermore, according to one embodiment of the present invention, TM4SF19 expression increased during the adipocyte differentiation process of hADMSCs (human adipose-derived mesenchymal stem cells). In TM4SF19 knockout mice, it was confirmed that weight gain was suppressed in a high-fat diet-induced obesity model, and the amount of adipose tissue-associated macrophages caused by obesity decreased, confirming that TM4SF19 acts on adipocyte differentiation and inflammatory responses including macrophages. As a result, it was confirmed that TM4SF19 KO mice reduced insulin resistance and suppressed fatty liver in a high-fat diet-induced obesity mouse model. Therefore, it can be seen that substances that suppress 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, TM4SF19 overexpression increased the proliferation, migration, and colonization of mouse-derived breast cancer and human osteosarcoma cells, while TM4SF19 deficiency suppressed lung metastasis of breast cancer cells and inhibited the proliferation, migration, and colonization of osteosarcoma cells, confirming an inhibitory effect on bone metastasis of breast cancer cells. Therefore, it can be seen that substances that inhibit TM4SF19 expression or activity 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 suppressed the growth and colonization ability of pancreatic cancer cells, and confirmed an inhibitory effect on osteosarcoma cell proliferation, migration, and colony formation. Therefore, it can be seen that substances that inhibit TM4SF19 expression or substances that inhibit TM4SF19 activity are effective in preventing and treating cancer and cancer metastasis. [Examples]

[0111] The advantages and features of the present invention, as well as methods for achieving them, will become clear with reference to the experimental and manufacturing examples described in detail below. However, the present invention is not limited to the experimental and manufacturing examples disclosed below, but can be embodied in various different forms, provided merely to complete the disclosure of the present invention and to fully inform those skilled in the art of which the invention pertains.

[0112] [Examples] Example 1: The role of TM4SF19 in osteoclast formation. Figure 1 shows the results of qPCR testing of TM4SF19 mRNA expression as differentiation progressed, after cells were extracted from mouse bone marrow and treated with M-CSF and RANKL (Receptor activator of nuclear factor kappa-β ligand) to induce differentiation into osteoclasts (OCs). As shown in Figure 1, it was confirmed that TM4SF19 expression increased with the progression of osteoclast differentiation. ACP5 CTSK expression, a gene 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) Confirmation of TM4SF19 knockout and suppression of multinucleated osteoclast formation Cells were extracted from the bone marrow of TM4SF19KO (TM4SF19 knockout mice) and wild-type (WT) mice using CRISPR. These cells were differentiated by treatment with M-CSF 25 ng / ml + RANKL 100 ng / ml, and then stained with TRAP (Tartrate-resistant acid phosphatase staining), an osteoclast-related marker.

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

[0115] Using the same method, cells were extracted from the bone marrow of TM4SF19KO (TM4SF19 knockout mice) and wild-type (WT) mice using CRISPR. The cells were then 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. Finally, TRAP staining (Tartrate-resistant acid phosphatase staining), an osteoclast-related marker, was performed.

[0116] As shown in Figure 2b, when TM4SF19KO mouse bone marrow cells were differentiated into osteoclasts, it was confirmed that the impairment of multinucleated cell formation could not be reversed even with high-concentration M-CSF treatment.

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

[0118] Therefore, the fact that TM4SF19 knockout prevents differentiation and maturation into multinucleated osteoclasts during the osteoclast differentiation process 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 allows for more selective regulation of bone resorption rather than completely eliminating the bone resorption function of osteoclasts, and can significantly reduce side effects compared to therapeutic agents that suppress mononucleated osteoclast formation, such as anti-RANKL antibodies.

[0119] (2) Confirmation of suppression of gene expression involved in osteoclast differentiation in TM4SF19KO mice Cells were extracted from the bone marrow of TM4SF19KO (TM4SF19 knockout mice) and wild-type (WT) mice using CRISPR. After differentiation under conditions of M-CSF and RANKL treatment or untreated conditions, the expression of genes involved in osteoclast differentiation, namely Ctsk, Acp5, c-Fos, and Nfatc1, was confirmed by qPCR four days later.

[0120] Figure 3 shows a graph of mRNA expression levels of Ctsk, Acp5, c-Fos, and Nfatc1 in cells differentiated under conditions of M-CSF and RANKL treatment, or without treatment.

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

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

[0123] BMM cells were plated onto a dentin disc and differentiated, after which pit formation was confirmed. Bone resorption was observed in wild-type (WT) cells, but bone resorption was blocked in TM4SF19KO cells (Figure 4b).

[0124] (4) Confirmation of bone loss suppression by OVX After 8-week-old wild-type (WT) and TM4SF19KO female mice underwent either sham (opening the ovary without detachment) or ovarian resection (OVX), the femur was fixed 31 days later and then analyzed by micro-CT.

[0125] Furthermore, by analyzing the micro-CT images using a 3D image analysis program, we obtained 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 trabeculae (Tb.N), and the inter-trabecular space (Tb.Sp).

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

[0127] Regardless of ovariectomy, TM4SF19KO mice have higher bone mineral density than wild-type (WT) mice.

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

[0129] As a result, as shown in Figure 6, a deficiency in multinucleated osteoclast formation was confirmed by TRAP staining of the femur of TM4SF19KO mice.

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

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

[0132] Figure 7b shows the results of extracting cells from the bone marrow of wild-type (WT) mice and TM4SF19EC2Δ mice in which TM4SF19 extracellular domain 2 (116-165) was knocked out using CRISPR. These cells were then differentiated after fixing the RANKL concentration at 100 ng / ml and treating them with M-CSF concentrations of 25 ng / ml or 100 ng / ml, followed by TRAP staining. When the bone marrow of TM4SF19EC2Δ mice was differentiated into osteoclasts, the damage to multinucleated osteoclast formation was not restored even with high-concentration M-CSF treatment.

[0133] Example 3-2: Effects of TM4SF19EC2Δ on cytoskeletal rearrangement (1) Confirmation of actin belt formation and suppression of bone resorption During the differentiation process of osteoclasts, when multinucleated osteoclasts (mature osteoclasts) are formed, an actin belt composed of podosomes is formed, and the podosomes adhere to the bone, leading to bone resorption.

[0134] Cells were extracted from the bone marrow of wild-type mice and TM4SF19EC2Δ mice, differentiated with MCSF 60 ng / ml and RANKL 100 ng / ml, then fixed, stained with phalloidin FITC, and examined under a confocal microscope (400x) to confirm the presence or absence of F-actin belt formation.

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

[0136] BMM cells were plated onto a dentin disc and differentiated, after which pit formation was confirmed by 1% toluidine blue staining. As a result, bone resorption was observed in wild-type (WT) cells, but bone resorption was confirmed to be blocked in TM4SF19EC2Δ cells (Figure 7d).

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

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

[0139] (3) Confirmation of bone loss suppression After 8-week-old wild-type (WT) and TM4SF19EC2Δ female mice underwent either sham (opening the ovary without detachment) or ovarian resection (OVX), the femur was fixed 31 days later and then analyzed by micro-CT.

[0140] Furthermore, by analyzing the micro-CT images using a 3D image analysis program, we obtained 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 trabeculae (Tb.N), and the inter-trabecular space (Tb.Sp).

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

[0142] Micro CT (μCT) analysis was performed 31 days after either shaming (opening the ovaries without detachment) or ovariectomy (OVX) was performed on 8-week-old wild-type (WT) mice, TM4SF19KO mice, and TM4SF19EC2Δ mice (all female) by fixing the thigh.

[0143] Figure 10 shows three-dimensional micro-CT indices (bone microstructure indices) obtained by analyzing micro-CT images using a three-dimensional image analysis program, including bone volume (BV), the ratio of bone volume to tissue volume (%BV / TV), the average number of trabeculae (Tb.N), the gaps between trabeculae (Tb.Sp), and the thickness of trabeculae (Tb.Th).

[0144] Figure 11 shows an image of a micro CT scan.

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

[0146] Example 4: Confirmation of self-binding and inter-integrin bonding of TM4SF19 (1) Confirmation of self interaction between TM4SF19 and TM4SF19 To confirm whether TM4SF19 is involved in cell-to-cell interaction, TM4SF19 was created with HA and flag tagging, respectively. Transient overexpression was performed on 293T cells with TM4SF19 tagged with 3HA to the N-terminus, wild-type cells tagged with 3Flag to the N-terminus, and a TM4SF19 deletion mutant to check for binding via immunoprecipitation. The TM4SF19 deletion mutant was created as shown in Figure 12a. The hTm4sf19 mutant (hTM4SF19) in which a portion of the TM4SF19 sequence was deleted (as shown in Figure 12a) was created. 115-175 Δ, hTm4sf 105-186 Δ, hTm4sf19 105-196 Δ, hTm4sf19 94-186 Δ and hTm4sf19 94-196 For Δ), after tagging each of the three flags, the intercellular connections were analyzed by immunoprecipitation.

[0147] As a result, we confirmed that TM4SF19 is involved in cell-to-cell interaction and binds to itself (Figure 12b). However, a mutant (hTm4sf19) lacking transmembrane 3 (TM3), extracellular region 2 (EC2), and transmembrane 4 (TM4) of TM4SF19 was also found. 94-196Δ) was confirmed not to undergo self-binding. From these results, it was confirmed that the transmembrane 3 - extracellular region 2 - transmembrane 4 region of TM4SF19 is important for TM4SF19 self-interaction, and the positions 94 - 196 of TM4SF19 are particularly important regions for self-interaction between TM4SF19 - TM4SF19.

[0148] (2) Confirmation of the region involved in self-interaction between TM4SF19 - TM4SF19 To verify that the positions 94 - 196 of TM4SF19 are important regions for self-interaction between TM4SF19 - TM4SF19, this time, 3Flag-hTm4sf19 94-196Δ different from it, hTm4sf19 with only the 94 - 196 sequence remaining and the remaining sequences deleted 94-196 was prepared as a TM4SF19 deletion mutant (upper part of Figure 12c). Similar to the method described above, 293T cells were transiently overexpressed with TM4SF19 tagged with 3HA at the N-terminus, wild-type tagged with 3Flag at the N-terminus, and the TM4SF19 deletion mutant, and it was confirmed whether they bind by immunoprecipitation. As a result, it was confirmed that the mutant (hTm4sf19 94-196 ) expressing transmembrane 3, EC2 (extracellular region 2), and transmembrane 4 of TM4SF19 self-binds in the same way as the wild-type (WT) (lower part of Figure 12c). From these results, it was reconfirmed that the 94 - 196aa region of TM4SF19 is an important site for TM4SF19 - TM4SF19 self-interaction.

[0149] (3) TM4SF19 and TM4SF19 - Fc mutual binding 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 method described above in 293T cells. The TM4SF19 fragments used were the regions 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 (upper panel of Figure 12d). As a result, as shown at the bottom of Figure 12d and in 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 with TM4SF19.

[0150] (4) Confirmation of interaction between integrin αv and TM4SF19 We confirmed the interaction between integrin αv and TM4SF19, which play a crucial role in osteoclast differentiation. To identify the key binding region where TM4SF19-Fc binds to integrin αv-TM4SF19, we examined the interaction between hIgG1-Fc fusion proteins of integrin αv tagged with 3HA at the C-terminus and TM4SF19 fragments in 293T cells by immunoprecipitation (Figure 12f).

[0151] (5) Confirmation of interaction between integrin β3 and TM4SF19 We confirmed the interaction between integrin β3 and TM4SF19, which play a crucial role in osteoclast differentiation. To identify the important binding region where TM4SF19-Fc binds to integrin β3-TM4SF19, we examined the interaction between hIgG1-Fc fusion proteins of integrin β3 tagged with 3HA at its C-terminus and TM4SF19 fragments in 293T cells by immunoprecipitation (Figure 12g).

[0152] Figure 12h shows the results of regulating the function of osteoclasts with TM4SF19 in 293T cells or osteoclast precursor Raw264.7 cells, and then overexpressing integrin αv or integrin β3, proteins involved in cytoskeletal rearrangement during multinucleated osteoclast formation, and confirming the presence or absence of interaction. The results confirmed that TM4SF19 binds to both integrin αv and integrin β3.

[0153] TM4SF19 belongs to the Tetraspanins family, and it is known that the extracellular region 2 (large extracellular loop) of this family plays a crucial role in binding with other binding partners. To confirm whether the extracellular region of TM4SF19 is essential for interaction with partners, the mutual binding of TM4SF19 wt or a protein with extracellular region 2 (115-175) deleted with integrin αv or integrin β3 was confirmed by immunoprecipitation. While interaction with them was confirmed for TM4SF19 wt, hTM4SF19 115~175Δ This is the result of confirming that no coupling occurs.

[0154] Figure 12i shows the results of immunoprecipitation testing the interaction between TM4SF19 and DC-Stamp and siglec-15, membrane proteins involved in the regulation of osteoclast function and cytoskeletal rearrangement during multinucleated osteoclast formation. TM4SF19 was expressed with N-terminal 3HA tagging, DC-Stamp with 3 Flags tagged to the N-terminus, and Siglec-15 with 3 Flags tagged to the C-terminus. The interactions were then confirmed by immunoprecipitation after expression in 293T cells.

[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-derived breast cancer cells E0771, we performed time-dependent cell proliferation and colony formation assays, as well as cell migration assays.

[0156] Figure 13a is a graph showing that cell proliferation of breast cancer cells E0771 significantly increased after 24, 48, and 72 hours following TM4SF19 overexpression. Figure 13b shows the colony formation ability after plating 1000 breast cancer cells E0771 with TM4SF19 overexpression and 1000 control cells, demonstrating that TM4SF19 overexpression increases colony formation. Figure 13c shows the evaluation of cell migration ability over time after plating breast cancer cells E0771 with TM4SF19 overexpression and control cells in a migration chamber, demonstrating that TM4SF19 overexpression increases cell migration.

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

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

[0159] As a result, we confirmed that TM4SF19KO mice and TM4SF19EC2Δ mice showed significantly suppressed lung metastasis of breast cancer cells 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 environment surrounding cancer cells acts as a crucial factor in cancer metastasis, and the degree of metastasis can be altered by antitumor factors released by macrophages. Bone marrow was obtained from TM4SF19KO mice (TMKO) and wild-type (WT) mice and differentiated into bone marrow-derived macrophages (BMDM). These were then placed in the lower part of a migration chamber, and breast cancer cells were placed in the upper chamber for co-culture. After co-culture, the expression of target genes involved in cell migration (fibronectin, vimentin, CDH2, SNAI1, SNAI2) in the migrated cells was confirmed by qPCR, and Western blotting was performed to confirm the expression of metastasis-related proteins (vimentin, slug, snail, E-cadherin, β-actin).

[0161] As a result, co-culturing with TM4SF19KO mouse macrophages suppressed the expression of target genes involved in cell migration (fibronectin, vimentin, CDH2, SNAI1, SNAI2) (Figure 15a), and suppressed the expression of metastasis-related proteins (vimentin, slug, snail, E-cadherin, β-actin) (Figure 15b). This indicates 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 adipocyte differentiation. 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 TM4SF19 expression levels increased due to human ADMSC adipogenic differentiation (Figure 16).

[0164] Example 8: Confirmation of the role of TM4SF19 in obesity and metabolic diseases. (1) Feed TM4SF19KO a high-fat diet and observe weight gain. We observed weight gain in 6-week-old wild-type (WT) mice and TM4SF19KO mice that had been fed a normal diet, after they were given a high-fat diet (60% fat) for 16 weeks.

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

[0166] (2) Confirmation of insulin and fatty liver tolerance Wild-type (WT) mice and TM4SF19KO mice were fed a high-fat diet, then insulin resistance was assessed using HOMA-IR (A), liver phenotype was determined, liver tissue was fixed, stained with H&E (B), and intrahepatic triglyceride levels were measured (C).

[0167] As a result, TM4SF19KO mice exhibited reduced insulin resistance and suppressed fatty liver formation in a high-fat diet-induced obese mouse model. Therefore, it was confirmed that TM4SF19KO mice (6-week-old mice fed a normal diet and then given 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 tolerance to high-fat-induced obesity Six-week-old wild-type (WT) mice and TM4SF19KO mice fed a normal diet were given a high-fat diet for 18 weeks to observe weight gain (Figure 19a). After 18 weeks, the weights of various tissues were examined by sacrificing the mice (Figure 19b), and the weights of subcutaneous and visceral fat were examined (Figure 19c). The fat phenotype and adipose tissue macrophages surrounding the fat were confirmed by H&E staining of epididymal white adipose tissue (eWAT) (Figure 19d), and the secretion of the anti-obesity cytokine adiponectin in the serum was also confirmed (Figure 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 showed suppressed obesity in a high-fat diet-induced obesity mouse model, and serum adiponectin secretion also increased. Therefore, it was confirmed that TM4SF19KO mice are resistant to high-fat-induced obesity (18 weeks) (Figure 19).

[0170] (4) Confirmation of adipocyte differentiation and expression of macrophage markers and M1-like macrophage markers. After feeding 6-week-old wild-type (WT) mice and TM4SF19KO mice a normal diet, and then feeding them a high-fat diet for 12 weeks, the expression of adipocyte differentiation markers (top) and macrophages and M1-like macrophage markers (bottom) in eWAT (epididymal white adipose tissue) and sWAT (subcutaneous white adipose tissue) was confirmed by qPCR.

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

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

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

[0174] (6) Expression of marker genes in interstitial vascular cells of white adipose tissue Six-week-old wild-type (WT) mice and TM4SF19KO mice fed a normal diet were then given a high-fat diet for 12 weeks. qPCR was then performed to confirm the expression of lophage marker genes in stromal vascular fractions (SVF) of the white adipose tissue of the epididymis.

[0175] As a result, we isolated stromal vascular fraction (SVF) from the white adipose tissue of the epididymis of TM4SF19KO mice and confirmed that the expression of macrophage markers MCP1 and F4 / 80 (Figure 22a), as well as M1-like macrophage markers IL6 and TNFa, was decreased (Figure 22b), while the expression of M2-like macrophage marker IL10 was increased (Figure 22c).

[0176] (7) Confirmation of the role of TM4SF19 in inflammation, insulin resistance, and fatty liver in eWAT To confirm the role of TM4SF19 in inflammation, insulin resistance, and fatty liver in eWAT, a high-fat diet-induced obesity mouse model experiment was conducted.

[0177] As a result, it was confirmed that TM4SF19 acts on adipocyte differentiation and the inflammatory response including macrophages, and that the lack of TM4SF19 reduces insulin resistance and suppresses fatty liver (Figure 23).

[0178] (8) Confirmation of the conversion from white adipocytes to beige adipocytes due to TM4SF19 deficiency (Starting from 6-week-old mice fed a normal diet and given a high-fat diet for 12 weeks) In a high-fat diet-induced obese mouse model, the conversion of white fat in the epididymis of TM4SF19KO mice to a beige or brown fat phenotype was confirmed. Also, it was confirmed that the expression of Ucp1 in the white fat of TM4SF19KO mice increased in a 12-week high-fat diet-induced obese mouse model, and by confirming that Ucp1 increased in TM4SF19KO in a brown fat differentiation in vitro model, it was confirmed that TM4SF19 is involved in changing white adipocytes into beige adipocytes (Figure 24).

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

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

[0181] Figure 24c is a schematic diagram illustrating the method for beige / brown adipocyte differentiation in white adipose tissue. Adipocyte progenitor cells extracted from white adipose tissue of WT and TM4SF19KO mice were cultured in DMEM / F-12 media with T3 and insulin. Two days after becoming confluent, they were treated with DMI and indometacin to initiate differentiation. From day 2, rosiglitazone was treated, and the cells were cultured until day 7-8 to induce beige / brown adipocyte differentiation.

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

[0183] (9) Confirmation of TM4SF19 expression in adipocytes and vascular cells (SVF) of white adipose tissue. In mice fed a high-fat diet for 24 weeks, TM4SF19 expression was confirmed by separating adipocytes and SVF in the white adipose tissue of the epididymis and subcutaneous white adipose tissue.

[0184] By confirming TM4SF19 expression in both adipose tissue and SVF containing macrophages in eWAT and sWAT of mice induced by a high-fat diet (6-week-old mice fed a normal diet were given a high-fat diet for 24 weeks), it is evident that TM4SF19 has a function not only in adipocytes but also in SVF containing macrophages and mononuclear cells (monocytes) (Figure 25).

[0185] Figure 25a shows a schematic diagram of the cell types in each fraction of white fat after digestion with collagen type I and subsequent centrifugation to separate it into adipose tissue, infranatant, and SVF fractions.

[0186] Figure 25b shows the results of examining TM4SF19 expression in adipocytes and SVF separately in the white adipose tissue of the epididymis and subcutaneous white adipose tissue of mice fed a high-fat diet of 60% fat for 24 weeks.

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

[0188] Increased expression of thermogenic genes and beige fat markers in TM4SF19KO mouse SVF suggested that TM4SF19KO mice may be involved in the browning or beigeing of white fat (Figure 26).

[0189] (11) TM4SF19 expression in co-culture of macrophages and 3T3-L1 adipocytes In obesity caused by a high-fat diet, the interaction between 3T3-L1 adipose tissue and macrophages plays a crucial role.

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

[0191] Co-culture of 3T3-L1 adipocytes and 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 subjected to adipogenesis. After differentiating wild-type (WT) bone marrow into macrophages, inflammatory changes were induced by co-culturing each cell individually or in contact with the other cells. For the control group, macrophages and adipocytes were cultured separately, then their RNA was extracted and combined before use.

[0193] TM4SF19 levels increased after co-culture of SVF adipocyte differentiation and myeloid macrophage differentiation (Figure 28).

[0194] SVF was extracted from wild-type (WT) mice and TM4SF19KO mice, and fat was produced. Bone marrow was then collected from each mouse and differentiated into macrophages, which were then co-cultured in contact to induce inflammatory changes.

[0195] As a result, as shown in Figure 29, MCP1 and IL6 levels 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) mice and TM4SF19KO mice, and adipocytes were induced to produce fat. Bone marrow was then collected from each mouse and differentiated into macrophages, followed by ctrl culture or co-culture to induce inflammatory changes. For the control group (ctrl culture), macrophages and adipocytes were cultured separately, then RNA was extracted and combined before use.

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

[0198] When differentiated adipocytes and BMDM were co - cultured, the inflammatory signal increased.

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

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

[0201] TNFα is a model that mimics the increase in the surrounding inflammation signal when obesity is induced in vivo by inducing inflammation.

[0202] SVF was extracted from wild - type (WT) mice and TM4SF19KO mice for adipogenic differentiation and treated with TNFα for 24 hours. Then, C / EBPα, which is an adipogenic marker, C / EBPβ, which is an in vitro insulin resistance model marker, and TM4SF19 were confirmed by qPCR. As a result, it was confirmed that TM4SF19 increased in the in vitro insulin resistance model (Figures 31, 32). It was confirmed that TM4SF19KO decreased insulin resistance (C / EBPβ is an insulin resistance marker).

[0203] (14) The role of TM4SF19 in macrophage differentiation and polarization Bone marrow was collected from wild-type (WT) mice and TM4SF19KO mice, differentiated into macrophages, and then markers involved in M1 polarization were identified. M1 macrophages are involved in global inflammatory signaling.

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

[0205] Manufacturing Example 1: Production and validation of TM4SF19 antibody (1) Production of polyclonal mouse TM4SF19 antibody After predicting antibody immunogenicity and determining possible targets, polyclonal mouse TM4SF19 antibodies were constructed targeting the 141-159aa region of the mouse TM4SF19 extracellular loop 2. (See SEQ ID NO:7 for details on the mouse TM4SF19 protein.)

[0206] (2) Verification 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. Western blotting was then performed to confirm expression using the previously prepared polyclonal mouse TM4SF19 antibody.

[0207] The polyclonal mouse TM4SF19 antibody recognized both human TM4SF19 and mouse TM4SF19. However, it could not recognize the extracellular loop 2 deletion mutant (Figure 35).

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

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

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

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

[0212] The mouse TM4SF19-Fc(116-165) showed good secretion, so it was purified and subjected to in vitro testing.

[0213] Human TM4SF19-Fc (115-175) is not secreted well, so we created Fc for a new region (120-169).

[0214] The mouse TM4SF19-Fc was purified because it secretes well.

[0215] Mouse strains TM4SF19EC2-Fc(116-165) were prepared, purified, and used in subsequent experiments.

[0216] Human TM4SF19-EC2-Fc (115-175) did not express well, so it was regenerated using a conserved sequence with mouse TM4SF19-EC2-Fc and expression was confirmed. Human TM4SF19-Fc (120-169) showed more secretion in the media compared to extracellular loop 2 (115-175), and this was used in subsequent experiments (Figure 37).

[0217] The fusion protein used in the examples of the present invention is shown in Figure 58.

[0218] Hereafter, "hTM4SF19-Fc" refers to the hTM4SF19-Fc(120-169aa) fusion protein, and "mTM4SF19-Fc" refers to the mouse TM4SF19-Fc(116-165aa) 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 used to treat the osteoclast differentiation process. Bone marrow-derived cells were treated with MCSF 60 ng / ml and RANKL 100 ng / ml for osteoclast differentiation, followed by TRAP staining.

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

[0221] After differentiating wild-type mouse bone marrow, the cells were treated with IgG-Fc and mTM4SF19-Fc at 10 μg / ml, and the presence or absence of actin belt formation during the differentiation of multinucleated osteoclasts was confirmed by F-actin staining. It was confirmed that mTM4SF19-Fc suppressed actin belt formation, and that TM4SF19 was localized to the actin belt by the mouse TM4SF19 antibody (Figure 38d).

[0222] After plating BMM cells onto a dentin disc, they were treated with IgG-Fc and mTM4SF19-Fc at 10 μg / ml while differentiating, and pit formation was confirmed by 1% toluidine blue staining. As shown in Figure 38e, it was confirmed that bone resorption was blocked by mTM4SF19-Fc.

[0223] Example 11: Confirmation of bone loss recovery using TM4SF19-Fc Eight-week-old female mice underwent ovariectomy, and then, starting one week later, were injected with mouse TM4SF19-Fc once a week via the tail vein for three weeks. One week after the final injection, the mice were sacrificed, their femurs were fixed, and micro-CT scans were performed. Mouse TM4SF19-Fc concentrations of 2.5 mg / kg and 5 mg / kg were used.

[0224] Figure 39a is a micro CT scan image showing bone loss recovery using the 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), ratio of bone volume to tissue volume (%BV / TV), average number of trabeculae (Tb.N), and inter-trabecular space (Tb.Sp) (Figure 39b).

[0226] As shown in Figure 39b, we confirmed that total BMD (Bone mineral density), %BV / TV (bone volume per tissue volume), and Tb.N (trabecular number) decreased after ovariectomy and increased in a dose-dependent manner with mTM4SF19-Fc. On the other hand, we confirmed that Tb.Sp (trabecular separation) decreased with mTM4SF19-Fc. These results confirm that mouse TM4SF19-Fc rescued ovariectomy-induced bone loss.

[0227] Furthermore, mouse femoral tissue was histopathologically analyzed through H&E staining. As a result, as shown in Figure 39c, it was confirmed that ovariectomy reduced the number of trabecular bones, and that TM4SF19-Fc treatment restored the reduction in the number of trabecular bones in a volume-dependent manner.

[0228] Furthermore, after ovariectomy of 8-week-old female mice, mouse TM4SF19-Fc (116-165) and human TM4SF19-Fc (131-169) were administered via IV injection. Bone loss was significantly suppressed by human and mouse TM4SF19-Fc compared to the untreated group (Figure 39d). Mouse TM4SF19-Fc (116-165) was administered at a dose of 10 mg / kg, and human TM4SF19-Fc (131-169) at a dose of 25 mg / kg.

[0229] The human TM4SF19 region, specifically the area including cells 131-169, can rescue bone loss caused by oophorectomy.

[0230] These results suggest the potential for developing TM4SF19-Fc as a treatment for osteoporosis.

[0231] Example 12: Verification of the role of TM4SF19 using TM4SF19-Fc (1) Osteoclast formation 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 10 μg / ml of mTM4SF19-Fc (the control group was treated with a buffer), 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 M-CSF treatment or M-CSF and RANKL treatment (WT1, 2, 3, 4, and 5 represent the results obtained by differentiating bone marrow cells from five wild-type mice into osteoclasts; the group treated with M-CSF alone represents the group that did not differentiate).

[0233] As shown in Figure 40a, mTM4SF19-Fc treatment significantly suppressed the expression of genes involved in osteoclast differentiation 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 mTM4SF19-Fc at a concentration of 5 μg / ml, the expression of osteoclast differentiation marker proteins was examined. 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 the multinucleation of osteoclasts because its ability to bind to the surface increases during differentiation. Therefore, we performed surface binding with mTM4SF19-Fc before and after differentiation of Raw264.7 cells using FACS analysis. As shown in Figure 41, there was almost no difference in surface binding between the control IgG1 (light gray graph) and mouseTM4SF19-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 with mTM4SF19-Fc actually increased after differentiation.

[0236] As shown in Figure 41, it was confirmed that the 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 the collagen-induced arthritis mouse model according to the experimental schedule shown in Figure 42a.

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

[0239] The CIA score is assessed in the following ways: Score 0 (normal paw), Score 1 (one or two toes inflamed and swollen), Score 2 (3+ toes inflamed 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] After inducing CIA (Collagen-Induced Arthritis), the joint condition of mice was examined 42 days later. As shown in Figure 42b, compared to the untreated Sham group, the control group treated with IgG1 showed swelling in all of the toes and the tops of the feet. However, in the group treated with 10 mg / kg of mTM4SF19-Fc, edema in the toes and tops of the feet was somewhat suppressed, and at 25 mg / kg, the edema phenomenon was clearly suppressed.

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

[0242] Furthermore, we performed micro-CT scans on the feet of the collagen-induced arthritis mouse model femur and analyzed the images.

[0243] Figure 42d is a micro CT image showing that inflammation and bone damage in the foot of a collagen-induced arthritis mouse model were suppressed in a dose-dependent manner by treatment with mTM4SF19-Fc. Collagen-induced arthritis caused toe curvature and inflammatory bone damage in the toe joints and ankles, but inflammation and bone damage in the joints were suppressed in a dose-dependent manner in mice treated with mTM4SF19-Fc compared to the control group of mice treated with hIgG1-Fc.

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

[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), ratio of bone volume to tissue volume (%BV / TV), average number of trabeculae (Tb.N), and inter-trabecular space (Tb.Sp).

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

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

[0248] Furthermore, in a collagen antibody-induced arthritis mouse model, the semi-therapeutic effect of mTM4SF19-Fc and hTM4SF19-Fc(120-169) was confirmed by arthritis score. Three days after administering collagen antibodies to Balbc mice, LPS was injected to induce arthritis. Compared to the control group, mTM4SF19-Fc 25 mg / kg and hTM4SF19-Fc(120-169) 25 mg / kg showed an arthritis-suppressing effect (Figure 42h). It was confirmed that TM4SF19-Fc treatment resulted in lower arthritis disease scores and reduced foot thickness compared to hIgG1-Fc treatment.

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

[0250] Three days after administering collagen antibodies, LPS was injected to induce arthritis. To confirm the therapeutic effect of mTM4SF19-Fc, patients were treated with 50 mg / kg of mTM4SF19-Fc starting from day 8, when the arthritis disease score was highest. From day 9 to day 19, the arthritis disease score was checked every two days.

[0251] Compared to the untreated group, treatment with mTM4SF19-Fc resulted in lower arthritis scores, reduced foot thickness, and suppressed cartilage damage as confirmed by 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 in Figure 43a, MDA-MB231-luc breast cancer cells (a cell line expressing luciparaze in MDA-MB231 for imaging purposes) were injected into the tail of 6-week-old female NOD-SCID mice via caudal artery (CA) injection to induce bone metastasis. After induction of bone metastasis, hIgG1 or hTM4SF19-Fc was administered intravenously. Intravenous administration of TM4SF19-Fc was started 2 days after cancer cell injection, and 10 mpk or 25 mpk was administered twice a week. Cancer progression was confirmed through bioluminescence imaging analysis.

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

[0254] Figure 43c shows the results of micro-CT scans of the joints of mice with bone metastases from breast cancer. Micro-CT analysis confirmed that bone damage occurs due to bone metastases from breast cancer, and that this bone damage is repaired in a dose-dependent manner by hTM4SF19-Fc treatment.

[0255] Furthermore, histological analysis was performed on the joints of mice with bone metastases from breast cancer using H&E staining. As a result, as shown in Figure 43d, it was confirmed that breast cancer bone metastases and tumor growth were suppressed in a dose-dependent manner by hTM4SF19-Fc treatment.

[0256] According to the experimental schedule in Figure 44a, bone metastases were induced in 6-week-old female NOD-SCID mice by caudal artery (CA) injection of MDA-MB231-luc breast cancer cells (a cell line expressing luciferase in MDA-MB231 for imaging purposes) into the tail. After induction, intravenous administration of control hIgG1 or hTM4SF19-Fc at a dose of 50 mg / kg was started 30 days later.

[0257] Bioluminescence imaging analysis was performed at 7-day intervals until the 21st after injection, confirming that bone metastases were suppressed by hTM4SF19-Fc(120-169) (Figure 44b). Luminescence analysis of sacrificial limbs removed after luciferin injection confirmed that bone metastases were suppressed by TM4SF19-Fc treatment (Figure 44c). Micro CT confirmed that bone destruction occurs due to bone metastases from breast cancer and is rescued by hTM4SF19-Fc treatment (Figure 44d).

[0258] Example 15: Suppression of lung metastasis of breast cancer using TM4SF19-Fc E0771 breast cancer cells derived from mice were injected into the tail vein of wild-type mice to induce lung metastasis. After intravenous injection of 10 mg / kg each of hIgG1-Fc, mouse TM4SF19-Fc (116-165), and human TM4SF19-Fc (145-169), the presence or absence of suppression of lung metastasis of breast cancer cells was confirmed by staining with Indian ink and counting the number of nodules that formed in the lungs. As shown in Figure 45, lung metastasis was significantly suppressed by mouse TM4SF19-Fc (116-165) and human TM4SF19-Fc (145-169).

[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 purifying them with a high-fat diet for one week, they were administered mouse TM4SF19-Fc twice a week via subcutaneous or intravenous injection while continuing to be fed a high-fat diet for nine weeks. As shown in Figure 46a, it was confirmed that administration of mTM4SF19-Fc at a dose of 10 mg / kg suppressed obesity caused by the high-fat diet.

[0260] Analysis of the distribution of white fat within organs using sacrificial mice revealed, as shown in Figure 46b, that mTM4SF19-Fc treatment reduced the weight of white fat, demonstrating a clear inhibitory effect on fatty liver.

[0261] Furthermore, observation of liver tissue through H&E staining and Masson and trichrome staining revealed a clear reduction in fatty liver compared to the control group treated with hIgG1-Fc, as shown in Figure 47 (left). A decrease in triglycerides in plasma was also confirmed (Figure 47 (right)).

[0262] Example 17: Inhibitory effect of TM4SF19-Fc treatment on adipocyte differentiation When C3H10T1 / 2 cells were treated with purified mouse TM4SF19-Fc(115-165), adipocyte differentiation was confirmed to be suppressed in a dose-dependent manner (Figure 48).

[0263] The control group received no differentiation-inducing factors, while the experimental group received differentiation-inducing factors (dexamethasone 1 μM, IBMX 500 μM, insulin 4 μg / ml (DMI), rosiglitazone 5 μM) along with mouse TM4SF19. 115-165 Adipocyte differentiation was observed after treating with -Fc at 0 ng / ml, 1.25 ng / ml, 2.5 ng / ml, 5 ng / ml, and 10 ng / ml.

[0264] Furthermore, we examined the expression of C / EBP alpha and PPAR-gamma, the main markers of adipocyte differentiation. The results showed that the expression of these cells increased in the DMI-treated group compared to the group that did not undergo differentiation induction (no DMI), and that their expression was suppressed by treatment with mouse TM4SF19-Fc(116~165).

[0265] Example 18: Inhibitory effect of cancer cell migration induced by osteoclast differentiation (1) TM4SF19-Fc The suppression of cancer cell migration induced by osteoclast differentiation by mTM4SF19-Fc or hTM4SF19-Fc(120-169) was confirmed by staining with 0.05% crystal violet solution. After plating bone marrow-derived macrophages in 12 wells, they were treated with 10 μg / ml of mTM4SF19-Fc or hTM4SF19-Fc while differentiating them into MCSF and RNAKL, or they were not differentiated by not adding RNAKL, and cancer cells MDA-MB231 or PC3M were placed on top of the migration chamber and migrated. The migrated cancer cells were stained with crystal violet.

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

[0267] (2) TM4SF19KO This study confirmed the suppression of cancer cell migration by osteoclast differentiation in TM4SF19KO. Bone marrow-derived macrophages extracted from wt and TM4SF19KO were inoculated with MCSF, and either differentiated with RANKL or not differentiated without RANKL. Cancer cells MDA-MB231 or PC3M were placed on top of the migration chamber and migrated. The migrated cancer cells were stained with crystal violet.

[0268] The results, shown in Figure 50a, demonstrate the suppression of cancer cell migration by osteoclast differentiation in TM4SF19KO.

[0269] (3) TM4SF19EC2Δ This study confirmed the suppression of cancer cell migration by osteoclast differentiation in TM4SF19EC2Δ. Bone marrow-derived macrophages extracted from wt and TM4SF19EC2Δ were inoculated with MCSF, and either differentiated with RANKL or not differentiated without RANKL. Cancer cells MDA-MB231 or PC3M were placed on top of the migration chamber and migrated. The migrated cancer cells were stained with crystal violet.

[0270] The results, shown in Figure 50b, demonstrate that in TM4SF19EC2Δ, the differentiation of osteoclasts suppressed cancer cell migration.

[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 analyzed by MTT analysis to confirm colony formation and migration. As a result, it was confirmed that TM4SF19 overexpression increased the proliferation, migration, and colony formation of osteosarcoma cells (Figures 51 and 52).

[0272] Furthermore, after knocking out TM4SF19 in 143b osteosarcoma cell lines using CRISPR, cell growth was confirmed by MTT analysis in different clones (#4 and #6), and colony formation was observed.

[0273] As a result, we confirmed that suppressing TM4SF19 expression inhibits the proliferation and colony formation of osteosarcoma cell 143b (Figure 53).

[0274] Overexpression of TM4SF19 increases osteosarcoma cell proliferation and migration. Suppression of TM4SF19 expression inhibits 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 analysis and cell counting, community formation ability was confirmed by colony formation, and cell migration was observed.

[0276] We confirmed that the growth of 143b osteosarcoma cells was suppressed by hTM4SF19-Fc treatment using MTT analysis (Figure 54a) and cell counting (Figure 54c), confirmed the ability to colonize by colony formation (Figure 54b), and confirmed cell migration (Figure 54d).

[0277] Treatment with human TM4SF19-Fc(120-169aa) (10 μg / ml) was confirmed to suppress the formation of U2OS and MG63 osteosarcoma cell colonies (Figure 55).

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

[0279] This suggests that hTM4SF19-Fc containing at least 145-169 aa is active.

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

Claims

1. A pharmaceutical composition for the prevention or treatment of bone disease, comprising a TM4SF19 (transmember 4L six family member 19) expression or activity inhibitor as an active ingredient.

2. The pharmaceutical composition according to claim 1, wherein the TM4SF19 (transmember 4L six family member 19) expression or activity inhibitor is at least one selected from the group consisting of antisense nucleotides that bind complementarily to the mRNA of the TM4SF19 gene, short hairpin RNA (shRNA), short interfering RNA (siRNA), microRNA (miRNA), and ribozyme.

3. The pharmaceutical composition according to claim 1, wherein the TM4SF19 (transmember 4L six family member 19) expression or activity inhibitor is at least one selected from the group consisting of compounds, peptides, peptide mimes, aptamers, fusion proteins, and antibodies that specifically bind to the TM4SF19 protein.

4. The pharmaceutical composition according to claim 3, wherein the fusion protein that specifically binds to the TM4SF19 protein comprises a fragment of TM4SF19 and an immunoglobulin Fc region.

5. The pharmaceutical composition according to claim 4, wherein the fragment of TM4SF19 comprises all or part of the extracellular loop 2 of TM4SF19.

6. The pharmaceutical composition according to claim 1, wherein the bone disease is at least one selected from the group consisting of metabolic bone disease, orthopedic bone disease, amorphous bone disease, degenerative bone disease, degenerative arthritis, rheumatoid arthritis, psoriatic arthritis, psoriatic spondylitis, age-related bone loss, osteoporosis, osteogenesis imperfecta, osteomalacia, osteopenia, fracture, bone defect and hip joint loss, rickets, Paget's bone disease, periodontal disease, and bone injury caused by bone metastasis of cancer cells.

7. The step of treating a suspected bone disease specimen with a candidate substance for bone disease treatment, A method for screening bone disease therapeutics, comprising the step of comparing the expression levels of mRNA or protein of the TM4SF19 gene with those of a control group.

8. A pharmaceutical composition for the prevention or treatment of obesity or obesity-mediated metabolic disease, comprising a TM4SF19 expression or activity inhibitor as an active ingredient.

9. The pharmaceutical composition according to claim 8, wherein the TM4SF19 (transmember 4L six family member 19) expression or activity inhibitor is at least one selected from the group consisting of antisense nucleotides that bind complementarily to the mRNA of the TM4SF19 gene, short hairpin RNA (shRNA), short interfering RNA (siRNA), microRNA (miRNA), and ribozyme.

10. The pharmaceutical composition according to claim 8, wherein the TM4SF19 (transmember 4L six family member 19) expression or activity inhibitor is at least one selected from the group consisting of compounds, peptides, peptide mimes, aptamers, fusion proteins, and antibodies that specifically bind to the TM4SF19 protein.

11. The pharmaceutical composition according to claim 10, wherein the fusion protein that specifically binds to the TM4SF19 protein comprises a fragment of TM4SF19 and an immunoglobulin Fc region.

12. The pharmaceutical composition according to claim 11, wherein the fragment of TM4SF19 comprises all or part of the extracellular loop 2 of TM4SF19.

13. The method according to claim 8. The pharmaceutical composition according to claim 8, wherein the obesity-mediated metabolic disease is at least one selected from the group consisting of hypertension, diabetes mellitus, insulin resistance syndrome, metabolic syndrome, obesity-related gastroesophageal reflux disease, arteriosclerosis, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, lipodystrophy, non-alcoholic steatohepatitis, cardiovascular disease, and polycystic ovary syndrome.

14. The steps include treating a specimen suspected of being obese or having an obesity-mediated metabolic disease with a candidate substance for the treatment of obesity or an obesity-mediated metabolic disease, A method for screening therapeutic drugs for obesity or obesity-mediated metabolic diseases, comprising the step of comparing the expression levels of mRNA or protein of the TM4SF19 gene with those of a control group.

15. A pharmaceutical composition for cancer prevention, treatment, or suppression of cancer metastasis, comprising a TM4SF19 expression or activity inhibitor as an active ingredient.

16. The pharmaceutical composition according to claim 15, wherein the TM4SF19 (transmember 4L six family member 19) expression or activity inhibitor is at least one selected from the group consisting of antisense nucleotides that bind complementary to the mRNA of the TM4SF19 gene, short hairpin RNA (shRNA), short interfering RNA (siRNA), microRNA (miRNA), and ribozyme.

17. The pharmaceutical composition according to claim 15, wherein the TM4SF19 (transmember 4L six family member 19) expression or activity inhibitor is at least one selected from the group consisting of compounds, peptides, peptide mimes, aptamers, fusion proteins, and antibodies that specifically bind to the TM4SF19 protein.

18. The pharmaceutical composition according to claim 17, wherein the fusion protein that specifically binds to the TM4SF19 protein comprises a fragment of TM4SF19 and an immunoglobulin Fc region.

19. The pharmaceutical composition according to claim 18, wherein the fragment of TM4SF19 comprises all or part of the extracellular loop 2 of TM4SF19.

20. The pharmaceutical composition according to claim 15, wherein the cancer is at least one selected from the group consisting of colorectal cancer, gastric 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, perianal cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, renal cell carcinoma, renal pelvic cancer, central nervous system (CNS) tumor, spinal cord tumor, brainstem glioma, and pituitary adenoma.

21. A step of treating a specimen suspected of being used for cancer prevention, treatment, or cancer metastasis with a candidate substance for cancer prevention, treatment, or suppression of cancer metastasis, A method for screening cancer or cancer metastasis therapeutic agents, comprising the step of comparing the expression levels of mRNA or protein of the TM4SF19 gene with those of a control group.

22. A fusion protein for inhibiting TM4SF19 expression or activity, containing a fragment derived from the extracellular loop 2 of the TM4SF19 protein and an immunoglobulin Fc region.

23. The fusion protein according to claim 22, wherein the fragment derived from extracellular loop 2 (EC2) of the TM4SF19 protein is an amino acid sequence region corresponding to all or part of EC2.

24. The fusion protein according to claim 22, wherein the amino acid sequence region corresponding to the entire EC2 is the 120th to 169th amino acid region of the human TM4SF19 protein or the 116th to 165th amino acid region of the mouse TM4SF19 protein.

25. The fusion protein according to claim 22, wherein the amino acid sequence region corresponding to a part of EC2 includes the amino acid region between positions 145 and 169 of the human TM4SF19 protein.

26. The fusion protein according to claim 22, comprising an amino acid sequence represented by SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, or SEQ ID NO:

19.

27. A method for preventing or treating a bone disease, comprising the step of administering a composition for the prevention or treatment of a bone disease containing a therapeutically effective amount of a TM4SF19 expression or activity inhibitor to a subject.

28. A method for preventing or treating obesity or obesity-mediated metabolic disease, comprising the step of administering to a subject a composition for the prevention or treatment of obesity or obesity-mediated metabolic disease comprising a therapeutically effective amount of a TM4SF19 expression or activity inhibitor.

29. A method for cancer prevention, treatment, or prevention or treatment of cancer metastasis, comprising the step of administering a composition for cancer prevention, treatment, or inhibition of cancer metastasis containing a therapeutically effective amount of a TM4SF19 expression or activity inhibitor to a subject.

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