CN117460522A - TM4SF19 inhibitor and use thereof - Google Patents

Abstract

The present invention relates to compositions for preventing or treating bone diseases, obesity or obesity-mediated metabolic diseases, cancer and cancer metastasis, comprising inhibitors of transmembrane 4L six family member 19 (TM 4SF 19) expression or activity, and methods for screening for agents for treatment of such diseases.

Description

TM4SF19 inhibitor and use thereof

Technical Field

The present invention relates to a composition for preventing or treating bone diseases, obesity-mediated metabolic diseases, cancer or cancer metastasis, comprising an inhibitor of the expression or activity of transmembrane 4L six family member 19 (TM 4SF 19), and a method of screening for a medicament for treating the above diseases.

Background

Bones are active tissues that change continuously throughout a person's lifetime. Bones are visually classified into external cortical bone (cancellous bone) and internal trabecular bone (cancellous or spongy bone), wherein cortical bone is used to protect and support the body due to high physical strength and trabecular bone is used to absorb shock or sustain calcium changes continuously.

Even after the cessation of bone growth, a phenomenon in which old bone is destroyed and disappears (bone resorption) and new bone fills the place where old bone disappears (bone formation) is repeated throughout the life of a person, which is called bone remodeling.

When the interaction between bone formation by osteoblasts and bone resorption by osteoclasts reaches equilibrium, bone homeostasis is maintained and the calcium concentration in the blood is maintained continuously. Bone related diseases such as osteoporosis are caused by such imbalance in bone metabolism.

Through the interaction between osteoclasts and osteoblasts, a phenomenon in which bone homeostasis is maintained through bone resorption and bone formation that occur successively is called bone remodeling, and bone mass is determined by the balance of activities between osteoclasts and osteoblasts. The process of bone remodeling is balanced by the combination of bone resorption by osteoclasts and subsequent bone formation by osteoblasts. When this effective balance is broken, bone loss occurs.

Osteoclasts differentiate into multinucleated osteoclasts, which develop bone resorption. The dysfunction of osteoclasts leads to bone sclerosis and the overactivation of postmenopausal osteoclasts increases bone resorption, leading to postmenopausal osteoporosis and inflammatory arthritis. Bisphosphonates and anti-RANKL antibodies have been developed to date as drugs against overactivation of osteoclasts, but the overall inhibition of osteoclasts affects the balance between osteoclasts and osteoblasts, which greatly affects bone formation, but has side effects. Accordingly, there is a need to develop therapeutic agents capable of more selectively inhibiting osteoclasts.

Osteoclasts are derived from hematopoietic stem cells and are polynuclear cells differentiated from monocyte/macrophage lineage by stimulation of monocyte/macrophage colony stimulating factor (M-CSF) and activation of receptor activators of the nuclear factor κb (RANK) ligand (RANKL). Differentiation of osteoclasts is performed by differentiation of osteoclast precursors into TRAP positive single-core osteoclasts and maturation into multinucleated osteoclasts by cell-cell fusion [ see fig. 2C ]. These mature multinucleated osteoclasts lead to bone resorption [ Int J Mol sci.2020aug 8;21 (16):5685.].

Obesity is a biological phenomenon caused by complex interactions of genetic, metabolic, environmental and behavioral factors, defined as abnormal or excessive accumulation of fat, and can have adverse effects on health. In particular, obesity is considered as an important risk factor for various adult diseases such as hypertension, type II diabetes, cancer, liver disease, hyperlipidemia, arteriosclerosis, etc. Obesity is caused by a chronic imbalance between caloric intake and energy expenditure, accompanied by loss of metabolism, endocrine and immune functions of adipose tissue, and results in metabolic abnormality and reduced reactivity (resistance) to insulin, a major fat storage signaling hormone, which causes fat to accumulate in metabolic organs other than adipose tissue, resulting in several types of lipotoxicity. The most representative pathology of toxicity is an inflammatory response characterized by chronic low-degradation inflammation. In obesity, activation of macrophages occurs in adipose tissue before metabolic abnormalities and insulin resistance occur, and results in a pre-inflammatory step. Thus, obesity induces a low-intensity chronic inflammatory state in the body, causing various metabolic diseases. As described above, adipose tissue macrophages play an important role in the occurrence of chronic inflammation and metabolic complications in the body due to obesity.

Fat stored in adipocytes is used as an important energy source in vivo. Obesity is caused by excessive differentiation of adipocytes and imbalance of energy supply. In adipose tissue, there are various cells such as adipose-derived stem cells (ASC), immune cells, and endothelial cells, and adipocytes, which are called Stromal Vascular Fraction (SVF), adipose tissue stores energy in the form of lipids, but also plays a role in keeping the body warm. It is classified into White Adipose Tissue (WAT), which is fat that stores nutrients, and Brown Adipose Tissue (BAT), which consumes nutrients and generates heat. In addition, adipose tissue produces leptin, resistin, adiponectin, tumor necrosis factor-alpha (tnfa). The differentiation of adipocytes is very complex due to the interaction of various hormones and various transcription factors, and is stimulated by, for example, insulin-like growth factor-1, or growth hormone. In this process, an increase in transcription factors such as the CCAAT enhancer binding protein (C/EBP) family and peroxisome proliferator-activated receptor (PPAR) gamma is observed. These transcription factors, together with adipocyte-regulating factors, promote adipocyte differentiation, and the expression levels of enzymes such as fatty acid binding protein aP2 and fatty acid synthase are increased. On the other hand, excessive accumulation of triglycerides has been reported to be associated with the development of fatty liver [ j.clin.invest.,98,1575 1584 (1996) ].

Currently, fat absorption inhibitors such as Xenical (Roche, switzerland) and anorectics such as Meridia (Abbott us) are widely used as weight-loss drugs, but these drugs have problems of side effects such as headache, blood pressure elevation and diarrhea. Therefore, there is a need to develop a weight loss drug without side effects against new targets.

Cancer is one of the biggest diseases threatening human health, caused by cells undergoing a series of mutations, proliferating in an unlimited, uncontrolled manner, and becoming endless. In the case of early detection of cancer, there are treatments such as surgery, radiation therapy and chemotherapy, but side effects are becoming a big problem, and in the case of advanced or metastatic cancer, life ends with limited life without special treatment.

Recently, although various biochemical mechanisms associated with cancer have been identified and related therapeutic agents have been developed, basic methods of treating cancer have not been proposed. Accordingly, research is actively being conducted to identify various cancer-related molecules in vivo and develop drugs targeting them, and attempts are also being made to enhance cancer therapeutic effects by combining some of these drugs.

Thus, efforts to additionally find cancer-related target molecules are very important.

Meanwhile, the use of a transmembrane 4L six family member (TM 4SF 19) as a marker for diagnosis of obesity has been disclosed in Korean patent No. 10-1781200. However, to date, there has been no report on the role of TM4SF19 in the treatment of bone related diseases, the treatment of obesity or obesity related metabolic diseases, and the treatment of cancer or the inhibition of cancer metastasis.

Disclosure of Invention

[ technical problem ]

Accordingly, in order to find genes involved in basic treatments for bone diseases, obesity, various metabolic diseases mediated by obesity, cancer treatment or inhibition of cancer metastasis, the present inventors studied the correlation of various genes with bone diseases, obesity or obesity-mediated metabolic diseases and cancers, and as a result confirmed various functions of TM4SF 19. Thus, the present invention has been completed.

[ technical solution ]

The present invention relates to providing a pharmaceutical composition for preventing or treating bone diseases, which comprises an inhibitor of TM4SF19 expression or activity as an active ingredient.

The present invention is also directed to a method of screening for a drug for treating bone disease, the method comprising: treating a sample suspected of being a bone disease with a candidate for treating the bone disease; and comparing the mRNA or protein expression level of the TM4SF19 gene with a control.

The present invention also relates to providing a pharmaceutical composition for preventing or treating obesity or obesity-mediated metabolic diseases, comprising an inhibitor of TM4SF19 expression or activity as an active ingredient.

The present invention is also directed to a method of screening for a drug for treating obesity or an obesity-mediated metabolic disease, the method comprising: samples suspected of being obesity or an obesity-mediated metabolic disease are treated with a candidate substance for treating obesity or an obesity-mediated metabolic disease, and the mRNA or protein expression level of the TM4SF19 gene is compared to a control.

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

The present invention is also directed to a method of screening for a drug for treating cancer or inhibiting metastasis of cancer, the method comprising: treating a sample suspected of being cancer or inhibiting cancer metastasis with a candidate for preventing or treating cancer or inhibiting cancer metastasis; and comparing the mRNA or protein expression level of the TM4SF19 gene with a control.

The invention also relates to the provision of fusion proteins useful for inhibiting TM4SF19 expression or activity.

The invention also relates to a method for providing a prophylactic or therapeutic treatment of a bone disease, the method comprising: a therapeutically effective amount of a composition for preventing or treating a bone disease comprising an inhibitor of TM4SF19 expression or activity is administered to a subject.

The present invention also relates to a method for preventing or treating obesity or an obesity-mediated metabolic disease comprising administering to a subject a therapeutically effective amount of a composition for preventing or treating obesity or an obesity-mediated metabolic disease comprising an inhibitor of TM4SF19 expression or activity.

The invention also relates to a method of providing prophylaxis or treatment of cancer or cancer metastasis, the method comprising administering to a subject a therapeutically effective amount of a composition for prophylaxis or treatment of cancer or inhibition of cancer metastasis, the composition comprising an inhibitor of TM4SF19 expression or activity.

[ advantageous effects ]

Inhibitors of TM4SF19 expression or activity according to the invention may be used for the prevention or treatment of bone diseases, obesity-mediated metabolic diseases, cancer or cancer metastasis.

In addition, by screening a method for inhibiting TM4SF19 for a candidate for treating bone disease, obesity-mediated metabolic disease, cancer, or cancer metastasis, a candidate capable of treating bone disease, obesity-mediated metabolic disease, cancer, or cancer metastasis can be effectively selected.

Drawings

FIG. 1 shows the expression of the TM4SF19 gene over time after treatment of cells isolated from mouse bone marrow with M-CSF and nuclear factor kappa-beta receptor activator ligand (RANKL) for differentiation, confirmed by qPCR.

FIG. 2A is the TRAP staining results of bone marrow derived cells of wild type mice and CRISPR knockout mice, TM4SF19KO mice, which have been treated with M-CSF (25 ng/ml) +RANKL (100 ng/ml) for differentiation. FIG. 2B shows the results of bone marrow derived macrophages (BMM) of TM4SF19 knockout mice (TM 4SF19 KO) generated using CRISPR and wild-type (WT) mice after differentiation with anti-tartrate acid phosphatase (TRAP), an osteoclast-associated marker, fixed at a RANKL concentration of 100ng/ml at an M-CSF concentration of 25, 40 or 60ng/ml. Fig. 2C is a schematic diagram illustrating an osteoclast differentiation process.

FIG. 3 shows the expression of target genes Ctsk, acp5, c-Fos and Nfatc1 associated with Osteoclast (OC) differentiation, confirmed by qPCR, 4 days after treatment of bone marrow cells of wild-type (WT) and TM4SF19KO mice with M-CSF or M-CSF+RANKL (M-CSF: 25ng/ml, RANKL:100 ng/ml).

FIG. 4A shows that after differentiating BMM of wild type and TM4SF19KO mice, whether actin bands are formed in osteoclast polynucleization was confirmed by F-actin staining.

Fig. 4B shows pit formation confirmed by 1% toluidine blue staining after spreading the BMM on dentin discs and allowing them to differentiate.

Fig. 5A shows the results of microscopic CT analysis of 8 week old Wild Type (WT) and TM4SF19KO female mice, which were dissected without ovariectomy (sham surgery) or Ovariectomy (OVX), and then their femur was fixed on day 31.

Fig. 5B shows 3D micro CT parameters (bone micro structural parameters), including Bone Volume (BV), bone volume to tissue volume ratio (% BV/TV), average trabecular number (tb.n), trabecular separation (tb.sp) and trabecular thickness (tb.th), obtained by analyzing micro CT images using a 3D imaging procedure.

FIG. 6 is TRAP staining results of the femur of 8 week old wild-type (WT) and TM4SF19KO mice that have been fixed and decalcified.

FIG. 7A shows TRAP staining results obtained after treatment with M-CSF (25 ng/ml) or M-CSF+RANKL (100 ng/ml) and differentiation of cells isolated from bone marrow of Wild Type (WT) mice and TM4SF19EC 2.DELTA.mice in which TM4SF19 extracellular domain 2 (116-165) was knocked out by CRISPR. FIG. 7B shows the result of TRAP staining after treatment of cells isolated from bone marrow of wild-type (WT) mice and TM4SF19EC 2.DELTA.mice by fixing the RANKL concentration to 100ng/ml and changing the M-CSF concentration to 25ng/ml or 100ng/ml and differentiating. FIG. 7C shows actin band formation during multinucleated osteoclast differentiation confirmed by F-actin staining after differentiation of bone marrow from Wild Type (WT) and TM4SF19EC 2. Delta. Mice. FIG. 7D shows pit formation confirmed by 1% toluidine blue staining after plating Wild Type (WT) and TM4SF19EC 2. DELTA. BMM on dentinal discs and differentiating them.

FIG. 8 shows the expression of target genes (Ctsk, acp5, c-Fos and nfatt 1) associated with osteoclast differentiation in wild-type (WT) mice and TM4SF19EC 2. Delta. Mice analyzed by qPCR.

Fig. 9A shows the results of microscopic CT analysis of 8 week old Wild Type (WT) and TM4SF19EC2 delta female mice, which were dissected without ovariectomy (sham surgery) or Ovariectomy (OVX), and then their femur was fixed after day 31.

Fig. 9B shows 3D micro CT parameters (bone micro structural parameters), including Bone Volume (BV), bone volume to tissue volume ratio (% BV/TV), average trabecular number (tb.n), trabecular separation (tb.sp) and trabecular thickness (tb.th), obtained by analyzing micro CT images using a 3D imaging procedure.

Fig. 10 is the results of microscopic CT analysis of 8 week old Wild Type (WT), TM4SF19KO and TM4SF19EC2 delta female mice, showing skeletal microstructure parameters, which were dissected without ovariectomy (sham) or Ovariectomy (OVX), and then their femur was fixed after day 31.

Fig. 11 is a microscopic CT image of a group of 8 week old Wild Type (WT), TM4SF19KO and TM4SF19EC2 delta female mice that were dissected without ovariectomy (sham) or Ovariectomy (OVX) and then their femur was fixed after day 31.

FIG. 12A is a hTm SF19 variant illustrating a partial sequence deleted for TM4SF19 (hTM 4SF19 _115-175Δ 、hTm4sf19 _105-186Δ 、hTm4sf19 _105-196Δ 、hTm4sf19 _94-186Δ And hTm sf19 _94-196Δ ) Is a schematic diagram of the structure of (a). FIG. 12B is a graph showing the results of TM4SF19 binding to itself and participating in intercellular interactions. It was shown that, among the variants deleted for part of the sequence of TM4SF19, the 94-196 deleted variants did not bind to TM4SF19 itself. FIG. 12C shows hTm sf19 _94-196 (top) binds to Wild Type (WT) and participates in the results of interactions (bottom) in which other sequences are deleted from hTm sf19 in addition to sequences 94-196. FIGS. 12D and 12E are results showing the structure of hTm SF19 131-160, hTm SF19 145-169, hTm4SF19 131-169, hTm SF19 120-160, hTm4SF19 120-169, hTm4SF19 120-180, hTm4SF19 120-186, hTm4SF19 120-196, hTm4SF19 120-209, hTm SF19 131-196 and hTm4SF19 145-196 used as Tm4SF19 fragments, and the interaction between TM4SF19 and hIgG1-Fc fusion proteins, wherein the N-terminus of the TM4SF19 or each TM4SF19 fragment is labeled with 3Flag, by immunoprecipitation. FIG. 12F is a display screenThe result of interaction between integrin αv (wherein 3HA is labeled at the C-terminus) and each TM4SF19 fragment fused to hIgG1 was confirmed by hyperimmune precipitation. FIG. 12G is a graph showing the results of confirming the interaction between integrin beta 3 (wherein 3HA is labeled at the C-terminus) and each TM4SF19 fragment fused to hIgG1 by immunoprecipitation. Fig. 12H is a result of confirming interaction after overexpression of integrin αv or integrin β3 (a protein that regulates osteoclast function and participates in cytoskeletal rearrangement during formation of polynuclear osteoclasts) and TM4SF19 in 293T cells or osteoclast precursor Raw 264.7 cells. It also shows the interaction between integrin αv or integrin β3 (wherein 3HA is tagged at the C-terminus) and TM4SF19 or TM4SF19 EC2 (115-175) deleted variants (wherein 3Flag is tagged at the N-terminus). FIG. 12I shows confirmation of interactions between TM4SF19 and DC-Stamp or siglec-15, which are membrane proteins involved in regulation of osteoclast function and cytoskeletal rearrangement during multinucleated osteoclast formation by immunoprecipitation.

FIG. 13 is the result of confirming cell proliferation, colony formation and cell migration after overexpressing TM4SF19 in mouse breast cancer cells E0771[ LPCX: retrovirus control vector ].

FIG. 14 is a result of confirming inhibition of lung metastasis after injection of murine breast cancer cells E0771 into the tail veins of Wild Type (WT), TM4SF19KO mice and TM4SF19EC 2. Delta. Mice.

FIG. 15 shows the expression of target genes involved in cell migration (CDH 2, SNAI1 and SNAI 2) confirmed by qPCR (FIG. 15A) and the expression of metastasis-associated proteins (vimentin, slug, bnail, E-cadherin, β -actin) confirmed by Western blotting (FIG. 15B) after isolating cells from bone marrow of wild-type (WT) mice and TM4SF19KO mice and differentiating into bone marrow-derived macrophages and co-culturing with breast cancer cells to allow cell migration [ BMDM: bone marrow-derived macrophages ].

FIG. 16 shows the expression of genes (C/EBP. Alpha., PPARgamma.) confirmed by qPCR (upper panel) and the expression of proteins (TM 4SF19, PPARgamma, and FABP 4) confirmed by Western blotting after differentiation of human adipose-derived mesenchymal stem cells (hADMSCs) (lower panel).

FIG. 17 is a result of confirming weight gain in 6 weeks old Wild Type (WT) mice and TM4SF19KO mice, which had been fed a normal diet while being fed a high fat diet (60% fat) for 16 weeks.

FIG. 18 shows (A) insulin resistance confirmed by HOMA-IR, (B) liver phenotype obtained by fixing liver tissue and stained with H & E, and (C) triglyceride levels in liver after feeding 6 weeks old Wild Type (WT) mice and TM4SF19KO mice, which have been fed a normal diet, for 12 weeks on a high fat diet.

FIG. 19 shows (a) weight gain, (b) weight of each tissue, and (c) weight of subcutaneous fat and visceral fat, measured when 6 weeks old Wild Type (WT) mice and TM4SF19KO mice (which had been fed with normal diet) were fed with high fat diet for 18 weeks, (d) fat phenotype and adipose tissue macrophages around fat confirmed by H & E staining of epididymal white adipose tissue (eWAT), and (E) secretion of anti-obesity cytokine adiponectin in serum [ rtWAT: retroperitoneal white adipose tissue, sWAT: subcutaneous white adipose tissue, ingWAT: inguinal white adipose tissue, iWAT: inter-scapular white adipose tissue, iBAT: inter-scapular brown adipose tissue ].

FIG. 20 shows the expression of adipogenic differentiation markers (upper panel), macrophage/M1-like macrophage markers (lower panel) in epididymal white adipose tissue (eWAT) and subcutaneous white adipose tissue (sWAT) after 6 weeks of age wild-type (WT) mice and TM4SF19KO mice (which have been fed a normal diet) were fed a high fat diet for 12 weeks.

Fig. 21 shows the percentage of macrophages (a) and dendritic cells (B) confirmed by FACS analysis of epididymal white adipose tissue (eWAT) after 6 weeks of age wild-type (WT) mice and TM4SF19KO mice (which have been fed a normal diet) were fed a high fat diet for 12 weeks.

FIG. 22 shows the results of confirming the expression of macrophage marker genes MCP1 and F4/80, M1-like macrophage marker IL6 and TNFα, and M2-like macrophage marker IL10 in Stromal Vascular Fraction (SVF) derived from epididymal white adipose tissue after wild-type (WT) and TM4SF19KO mice were fed a high fat diet for 12 weeks.

FIG. 23 shows the effect of TM4SF19 on inflammation, insulin resistance and fatty liver in eWAT.

Fig. 24A is a result of confirming the conversion of eeat of TM4SF19KO mice to beige or brown adipocyte phenotype in a high fat diet-induced obese mouse model that was fed a high fat diet for 12 weeks after starting the high fat diet for 6 week old mice fed a normal diet.

FIG. 24B is a result of confirming Ucp1 expression in white fat of TM4SF19KO mice in a high fat diet-induced obese mouse model.

Figure 24C shows the in vitro process of the brown fat differentiation model.

Fig. 24D is a result of confirming that Ucp1 was increased in TM4SF19KO in an in vitro model of brown fat differentiation.

Fig. 24E is a result of confirming that TM4SF19 participates in changing white adipocytes into beige adipocytes.

FIG. 25A is a schematic representation of the cell types in each fraction after digestion of white fat with type I collagen and separation by centrifugation into adipose tissue, lower fat and SVF fractions. This is a schematic representation of the cell types in each fraction.

Fig. 25B is a result of confirming expression of TM4SF19 after epididymal white fat and subcutaneous white fat of mice fed a high fat diet (60% fat) for 24 weeks were separated into adipocytes and SVF.

FIG. 26 shows thermogenic gene expression and beige fat marker gene expression after SVF extraction from subcutaneous white fat and induced differentiation into brown fat using isopropanol in wild-type (WT) and TM4SF19KO mice.

FIG. 27 is the results of co-culture of adipocytes with macrophages (right) after differentiation using an in vitro model, followed by identification of marker genes for macrophages, inflammation, and adiponectin (left) [ control: after the growth of macrophages and adipocytes, RNA is extracted, combined and used ].

FIG. 28 is a graph showing SVF extraction from wild-type (WT) mice to allow adipogenesis, differentiation into WT bone marrow macrophages, followed by culture alone or co-culture with contact [ control (ctrl): results of inflammatory changes were induced after macrophages and adipocytes were cultured alone, extracted, combined, and RNA was used.

FIG. 29 is the result of inducing inflammatory changes after SVF was extracted from wild-type (WT) mice and TM4SF19KO mice to allow adipogenesis, differentiation into WT bone marrow macrophages, and then contact co-culture.

FIG. 30 is a graph showing SVF extraction from wild-type (WT) mice and TM4SF19KO mice to allow adipogenesis, differentiation into macrophages, followed by culture alone or co-culture with contact [ control (ctrl): results of inflammatory changes were induced after macrophages and adipocytes were cultured alone, extracted, combined, and RNA was used.

FIG. 31 is a graph showing the results of confirming the in vitro insulin resistance model production process (up) and the in vitro adipogenic differentiation marker C/EBP alpha and insulin resistance model marker C/EBP beta (in) and TM4SF19 increase (down) after 12 days of adipocyte differentiation followed by treatment with TNF alpha for 24 hours.

FIG. 32 shows an in vitro insulin resistance model with reduced TM4SF19KO compared to the wild type.

FIG. 33 is a result of confirming markers involved in M1 polarization after bone marrow extraction from Wild Type (WT) mice and differentiation into macrophages.

FIG. 34 is a result of confirming M1 polarization after bone marrow was extracted from wild-type (WT) mice and TM4SF19KO mice and differentiated into macrophages, and markers involved in M1 polarization after bone marrow was extracted from wild-type mice and TM4SF19KO mice and differentiated into macrophages (lower panel).

FIG. 35 is a graph showing the results of transient overexpression of human TM4SF19 (hTM 4SF 19), human EC 2. Delta (hTM 4SF19 115-175. Delta.), mouse TM4SF19 (mTM SF 19), and mouse EC 2. Delta (mTM 4SF19 116-165. Delta.) in 293T cells, as confirmed by Western blotting using previously manufactured polyclonal mouse TM4SF19 antibodies.

FIG. 36 shows the results of bone marrow extraction and differentiation into osteoclasts from wild-type mice, treatment with TM4SF19 antibody, and staining with TRAP.

FIG. 37 shows human TM4SF19 after concentration of the medium _120-169 -Fc and mouse TM4SF19 _116-165 Coomassie staining of Fc (left, right) and western blotting (medium).

FIG. 38A shows the confirmation by TRAP staining from mM4SF19 _116-165 Inhibition of osteoclast differentiation-induced multinucleated osteoclast generation by Fc [ E1; samples eluted with elution buffer (20 mM glycine). E1-1; samples (Fc was probably more stable) were subjected to ultrafiltration/diafiltration (UF/DF) using buffer A (50 mM phosphate, 50mM NaCl, pH 7.0)]。

FIG. 38B shows hTM4SF19 confirmed by TRAP staining _120-169 -inhibition of polynuclear osteoclast formation by Fc treatment.

FIG. 38C shows confirmation by TRAP staining, hTM4SF19 _120-169 -Fc and mTM SF19 4 _116-165 Fc was obtained by hIgG1-Fc, hTM4SF19 _120-169 -Fc and mTM SF19 4 _116-165 Fc treatment inhibits multi-core osteoclast formation.

FIG. 38D shows confirmation of actin band formation by F-actin staining during multinucleated osteoclast formation of wild-type mouse BMM and treatment with 10. Mu.g/ml each of IgG-Fc and mTM4SF 19-Fc.

FIG. 38E shows that bone resorption is confirmed by blue-toluidine staining with mTM SF 19-Fc.

FIG. 39A is a set of microscopic CT images showing bone loss inhibition by mouse TM4SF 19-Fc.

Fig. 39B shows 3D micro-CT parameters including Bone Volume (BV), bone volume to tissue volume ratio (%bv/TV), average trabecular number (tb.n) and trabecular separation (tb.sp), obtained by analyzing the micro-CT images of fig. 39A using a 3D imaging procedure.

Fig. 39C shows the results of histopathological analysis of mouse femoral tissue by H & E staining.

FIG. 39D shows that bone loss was significantly inhibited after ovariectomy in 8 week old female mice, after tail vein injection of hIgG1-Fc, mouse TM4SF19-Fc (116-165), and human TM4SF19-Fc (131-169).

FIG. 40A is a set of graphs showing mRNA expression of Ctsk and Acp5 in differentiated cells under treatment with M-CSF or M-CSF and RANKL and treatment with 10. Mu.g/ml mouse TM4SF 19-Fc.

FIG. 40B shows the results of confirming the expression of osteoclast differentiation marker protein after treatment with 5. Mu.g/ml of mTM SF19-Fc while differentiating under the conditions of treating cells extracted from WT mouse bone marrow with M-CSF or M-CSF and RANKL (M-CSF 25ng/ml, RANKL 100 ng/ml).

FIG. 41 shows the surface binding of mTM SF19-Fc to Raw264.7 cells before and after differentiation confirmed by FACS analysis.

FIG. 42A schematically shows an experimental protocol for confirming the prevention and treatment of rheumatoid arthritis using TM4SF 19-Fc. Fig. 42B shows the confirmation of joint status in mice 42 days after collagen-induced arthritis (CIA) induction. Fig. 42C shows the results of the analysis of CIA scores and swollen joint numbers investigated during the experiment. Fig. 42D is a set of microscopic CT images showing that inflammation and bone damage occurring in the paw of a collagen-induced arthritis mouse model was dose-dependently inhibited by mTM SF19-Fc treatment. Fig. 42E is a graph showing the results of dose-dependent inhibition of inflammation and bone injury by mTM SF19-Fc therapy occurring in the paw of a collagen-induced arthritis mouse model. Fig. 42F shows 3D micro-CT parameters (bone micro-structural parameters), including Bone Volume (BV), bone volume to tissue volume ratio (% BV/TV), average trabecular number (tb.n), and trabecular separation (tb.sp), by analyzing micro-CT images of the femur of collagen-induced arthritic mice using a 3D imaging procedure. Fig. 42G is the result of confirming cartilage damage in collagen-induced arthritic mice using toluidine blue. Cartilage damage was shown to be inhibited in the mTM4SF19-Fc treated group compared to the hIgG1-Fc treated group. FIG. 42H shows the semi-therapeutic effect of mTM SF19-Fc and hTM4SF19-Fc (120-169) in an LPS-injected arthritis-induced mouse model assessed by arthritis scoring three days after collagen antibody treatment. It was confirmed that treatment with TM4SF19-Fc reduced the arthritic disease score and reduced paw thickness compared to hIgG 1-Fc. Fig. 42I shows the therapeutic effect of mTM SF19-Fc in an arthritis-induced mouse model that received LPS injection 3 days after collagen antibody treatment, with highest arthritic disease score 8 days after LPS injection. It was confirmed by toluidine blue that TM4SF19-Fc treatment resulted in a decrease in arthritic disease score and paw thickness and inhibited cartilage damage compared to the untreated group.

FIG. 43A schematically shows an experimental protocol for confirming the inhibition of breast cancer bone metastasis using mTM4SF 19-Fc. Fig. 43B shows the result of confirming the state of progression of cancer by bioluminescence image analysis. Fig. 43C shows a micro CT scan of the joints of mice with breast cancer bone metastases. Fig. 43D shows the results of histological analysis of joints of mice whose breast cancer has metastasized to bone by H & E staining.

FIG. 44A schematically shows an experimental protocol for confirming the inhibition of breast cancer bone metastasis using hTM4SF19-Fc (145-169). Fig. 44B shows the result of confirming the state of progression of cancer by bioluminescence image analysis. Fig. 44C shows the results of luminescence analysis of joints of mice with bone metastases of breast cancer. Fig. 44D shows a micro CT scan of a mouse joint with bone metastasis from breast cancer.

FIG. 45 is a result of confirming that lung metastasis is significantly inhibited by mTM SF19-Fc (116-165) and hTM4SF19-Fc (145-169) after lung metastasis is caused by injection of murine E0771 breast cancer cells into the tail vein of wild-type mice.

Figure 46A shows that mTM4SF19-Fc treatment inhibited high fat diet-induced obesity.

Fig. 46B shows that mTM SF19-Fc treatment induced a decrease in white fat weight and an inhibitory effect of fatty liver in organs by high fat diet, which was confirmed by organ weight and the ratio of organ weight to body weight.

FIG. 47 shows the effect of reducing fatty liver and triglyceride in plasma as confirmed by H & E staining and Masson trichromatic staining with respect to mTM SF19-Fc treatment.

FIG. 48 shows that treatment of lipid droplets by oil red-O staining and treatment with mTM SF19-Fc (116-165) inhibited adipocyte differentiation dose-dependently as confirmed by gene expression of adipogenic markers C/EBP alpha and PPAR-gamma.

FIG. 49 shows confirmation of the inhibition of osteoclast differentiation-induced cancer cell migration by TM4SF19-Fc by staining with 0.05% crystal violet solution.

FIG. 50A shows that the inhibition of tumor cell migration by osteoclast differentiation in TM4SF19KO was confirmed by staining with 0.05% crystal violet solution.

FIG. 50B shows that osteoclast differentiation in TM4SF19EC 2. Delta. Was confirmed to inhibit tumor cell migration by staining with 0.05% crystal violet solution.

FIG. 51 is the results of confirming cell growth and confirming colony formation and migration after stable overexpression of 3Flag-hTM4SF19 in MG63 osteosarcoma cell line by MTT assay.

FIG. 52 is the results of confirmation of cell growth and confirmation of colony formation and migration after stable overexpression of 3Flag-hTM4SF19 in HOS osteosarcoma cell lines by MTT assay.

FIG. 53 is a result of MTT assay confirming cell growth in 143b osteosarcoma cell line using different TM4SF19 clones (# 4 and # 6) that had been knocked out by CRISPR, and confirming reproductive ability by colony formation.

FIG. 54 is a graph showing the results of confirmation of cell growth, confirmation of colony ability by colony formation (FIG. 54B) and confirmation of cell migration (FIG. 54D) of the cells of osteosarcoma 143B treated with hTM4SF19-Fc (aa 120-169) by MTT assay (FIG. 54A) and cell number (FIG. 54C).

FIG. 55 shows that treatment with human TM4SF19-Fc (aa 120-169) (10. Mu.g/ml) inhibited U2OS and MG63 osteosarcoma colony formation.

FIG. 56 shows that treatment with 10. Mu.g/ml of hTM4SF19-Fc (aa 120-169) and hTM4SF19-Fc (aa 145-169) inhibited the cell migration ability of HOS osteosarcoma cells.

FIG. 57A shows inhibition of pancreatic cancer cell colony formation by human TM4SF19-Fc (aa 120-169).

FIG. 57B shows confirmation of inhibition of pancreatic cancer cell growth by human TM4SF19-Fc (aa 120-169) by cell counting.

FIGS. 58A to 58D show information on the sequences of the TM4SF19 fragment and the TM4SF19-Fc fusion protein used in the examples of the present invention [ in each figure, the corresponding target sequences are shown in bold and represented by SEQ ID NO:8, 12, 15 or 18; the enzyme site is a site between the target sequence and the immunoglobulin Fc region; the Fc region of the immunoglobulin is the highlighted portion, indicated by SEQ ID NO:9 ].

Detailed Description

Hereinafter, the present invention will be described in detail.

Meanwhile, the embodiments of the present invention may be modified in various different forms, and should not be construed as limiting the scope of the present invention to the following embodiments. Furthermore, embodiments of the present invention are provided to more fully explain the present invention to those skilled in the art. Furthermore, throughout the specification, when a portion "comprises" an element means that it can also include other elements, not exclude elements, unless specifically stated.

The present invention relates to a pharmaceutical composition for preventing or treating bone diseases, which comprises an inhibitor of TM4SF19 expression or activity as an active ingredient.

The present invention also relates to a pharmaceutical composition for preventing or treating obesity or obesity-mediated metabolic diseases, comprising an inhibitor of TM4SF19 expression or activity as an active ingredient.

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

The term "TM4SF19" as used herein refers to transmembrane 4L six family member 19 (TM 4SF19, OCTM 4), which belongs to the transmembrane 4L 6 superfamily. Such superfamily members are known to be involved in a variety of cellular processes, including cell proliferation, movement and adhesion, through interactions with integrins, and are associated with diseases such as liver fibrosis and cancer. However, the relationship of TM4SF19 to its function has not been properly studied.

In the case of the TM4SF19 protein, the same protein can be expressed not only in humans (homo sapiens) or mice (mice), but also in other mammals such as monkeys, cows, horses, dogs, or cats.

Human TM4SF19 can be translated into peptides or proteins that include the corresponding amino acid sequences represented by np_612470.2, np_001191826.1 and np_001191827.1 from the corresponding mrnas of GenBank accession numbers nm_138461.4, nm_001204897.2 and nm_ 001204898.2. That is, human TM4SF19 includes three types of transcriptional variants (GenBank accession No. nm_138461.4 (transcriptional variant 1), nm_001204897.2 (transcriptional variant 2), nm_001204898.2 (transcriptional variant 3)) and three types of isoforms (GenBank accession nos. np_612470.2 (isoform 1), np_001191826.1 (isoform 2), np_001191827.1 (isoform 3)). The TM4SF19 used in embodiments of the present invention is np_612470.2 (isoform 1) obtained from mRNA including GenBank accession No. nm_138461.4 (transcriptional variant 1). Isoform 1 has a high degree of homology with mouse TM4SF 19.

Mouse TM4SF19 is GenBank accession number NP-001153874.1, represented by SEQ ID NO: 7.

< GenBank accession number NP-612470.2 (isoform 1) [ SEQ ID NO:1] >)

< GenBank accession number NP-001191826.1 (isoform 2) [ SEQ ID NO:2] >)

< GenBank accession number NP-001191827.1 (isoform 3) [ SEQ ID NO:3] >)

< GenBank accession No. NM-138461.4 (transcriptional variant 1) [ SEQ ID NO:4] >

< GenBank accession No. NM-001204897.2 (transcriptional variant 2) [ SEQ ID NO:5] >)

< GenBank accession No. NM-001204898.2 (transcriptional variant 3) [ SEQ ID NO:6] >

< GenBank accession number NP-001153874.1[SEQ ID NO:7] >

The inhibitor of TM4SF19 expression or activity of the present invention means a substance which reduces TM4SF19 gene expression or TM4SF19 protein activity. In one embodiment, the inhibitor for inhibiting the expression of the TM4SF19 gene of the present invention refers to a substance that acts directly on the TM4SF19 gene or indirectly on an up-regulating factor of the TM4SF19 gene to reduce the expression of the TM4SF19 gene at the transcription level, increase the degradation of the expressed TM4SF19 gene, or inhibit the activity thereof, thereby reducing the expression level of the TM4SF19 gene or the activity thereof. Specifically, the inhibitor for inhibiting the expression of the TM4SF19 gene may include one or more selected from the group consisting of antisense nucleotides, small hairpin RNAs (shrnas), small interfering RNAs (sirnas), micrornas (mirnas), and ribozymes, which complementarily bind to mRNA of the TM4SF19 gene, but the present invention is not limited thereto.

The term "antisense nucleotide" as used herein refers to a DNA or RNA sequence that is complementary to a particular gene and is capable of binding to TM4SF19 mRNA. Since antisense nucleotides are long chains of monomeric units, they can readily synthesize target gene sequences.

The term "small interfering RNA (siRNA)" refers to a short double-stranded RNA capable of inducing RNA Interference (iRNA) by cleavage of a specific mRNA. The siRNA includes a sense RNA strand having a sequence homologous to the target gene mRNA and an antisense RNA strand having a sequence complementary thereto. Because siRNA can inhibit expression of a target gene, it is used in gene knockout methods or gene therapies.

The term "short hairpin RNA (shRNA)" as used herein is a single-stranded RNA, which is divided into a stem portion forming a double-stranded portion by hydrogen bonding and a loop portion forming a loop, and is processed by a protein such as dicer to become siRNA, and thus can exert the same function as siRNA.

The term "microRNA (mRNA)" as used herein refers to non-coding RNA of 21 to 23-nt nucleotides that modulates post-transcriptional gene expression by promoting degradation or inhibiting translation of the target RNA.

The term "ribozyme" as used herein refers to an RNA molecule that recognizes a particular base sequence and has an enzyme-like function of cleaving the sequence itself. Ribozymes consist of a region that specifically binds to the complementary base sequence of the target messenger RNA strand and a region that cleaves the target RNA.

Further, in one embodiment, the inhibitor for inhibiting the activity of TM4SF19 protein according to the present invention may be one or more selected from the group consisting of a compound, a peptide, a peptidomimetic, an aptamer, a fusion protein, and an antibody that specifically bind to TM4SF19 protein, but the present invention is not limited thereto.

The term "compound" as used herein includes all compounds that specifically bind to the TM4SF19 protein to inhibit its activity.

The term "peptide" as used herein has the advantage of high binding strength to a target substance and does not undergo denaturation even during heat/chemical treatment. Furthermore, due to its small molecular size, it can be used as a fusion protein by being linked to another protein. In particular, since it can be used by being linked to a polymer protein chain, it can be used as a diagnostic kit and a drug delivery substance.

The term "peptidomimetic" as used herein inhibits the binding domain of the TM4SF19 protein, thereby inhibiting the activity of the TM4SF19 protein. The peptidomimetic can be peptide or non-peptide and consists of amino acids bound by non-peptide bonds (e.g., psi bonds). Furthermore, the peptidomimetic can be a "conformationally constrained" peptide, a cyclic mimetic, or a cyclic mimetic comprising at least one exocyclic domain, a binding moiety (binding amino acid), and an active moiety. A peptidomimetic is a novel small molecule that has a structure similar to the secondary structural features of the TM4SF19 protein, mimics the inhibitory properties of macromolecules (e.g., antibodies or soluble receptors), and has an effect equivalent to that of a natural antagonist.

The term "aptamer" as used herein refers to a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) that itself has a stable tertiary structure and is capable of binding a target molecule with high affinity and specificity.

The term "fusion protein" as used herein is a novel protein prepared in the form of a binding of two or more different proteins or proteins of the same type, also known as chimeric proteins. Two or more heterologous proteins are bound in a part-to-part, part-to-whole or whole-to-whole manner. In many cases, the sequences of one gene and another gene are codon aligned and ligated to form a hybrid gene that is expressed to produce a protein, if desired.

In one embodiment, a fusion protein according to the invention may comprise a fragment of TM4SF19 that specifically binds to TM4SF19 protein.

In addition to this fragment, an immunoglobulin Fc domain may be included.

The fragment may be all or a portion of an extracellular loop 2 (EC 2) -derived fragment of TM4SF 19. The region of the amino acid sequence corresponding to the entire EC2 may be the region of amino acids 120-169 of the human TM4SF19 protein or the region of amino acids 116-165 of the mouse TM4SF19 protein. The region of the amino acid sequence corresponding to a portion of EC2 may include the region of amino acids 145-169 or 131-169 of the human TM4SF19 protein.

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

As one embodiment, the invention may include fusion proteins wherein the immunoglobulin Fc domain binds to a TM4SF19 fragment, e.g., fusion proteins wherein the immunoglobulin Fc domain binds to all or part of the extracellular loop region of TM4SF 19; or a fusion protein in which the immunoglobulin Fc domain binds to all or part of the extracellular loop region and all or part of the membrane protein of TM4SF 19. Herein, fusion proteins in which an immunoglobulin Fc domain binds to a TM4SF19 fragment include those in which an immunoglobulin Fc domain indirectly binds to a TM4SF19 fragment, and those in which an immunoglobulin Fc domain directly binds to a TM4SF19 fragment. When the immunoglobulin Fc domain indirectly binds to the TM4SF19 fragment, there may be an additional 1 to 10 amino acids, 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, in particular 1 amino acid, corresponding to a linker, spacer or enzyme site, between the TM4SF19 fragment and the immunoglobulin Fc domain.

Fragments of TM4SF19 useful in the present invention include, but are not limited to, the various examples (hTm 4SF 19-196, hTm4SF19 131-160, hTm4SF19 145-169, hTm4SF 19-169, hTm4SF19 120-160, hTm4SF19 120-169, hTm4SF19 120-180, hTm4SF19 120-186, hTm4SF19 120-196, hTm SF19 120-209, hTm4SF19 131-196, and hTm SF19 145-196). Meanwhile, for the stability of TM4SF19 protein, an immunoglobulin Fc domain is used as an example, but a fusion partner capable of binding TM4SF19 protein is not limited to an immunoglobulin Fc domain.

Although not limited thereto, the present invention relates to fusion proteins for inhibiting TM4SF19 expression or activity comprising fragments of extracellular loop 2 and immunoglobulin Fc domain from TM4SF19 protein.

An extracellular loop 2 (EC 2) -derived fragment of the TM4SF19 protein may be a region of amino acid sequence corresponding to all or part of EC 2.

The region of the amino acid sequence corresponding to the entire EC2 may be the region of amino acids 120-169 of the human TM4SF19 protein or the region of amino acids 116-165 of the mouse TM4SF19 protein.

The region of the amino acid sequence corresponding to a portion of EC2 may include the region of amino acids 145-169 of the human TM4SF19 protein.

The region of the amino acid sequence corresponding to a portion of EC2 may include the region of amino acids 131-169 of the human TM4SF19 protein.

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

An enzyme site may be included between the extracellular loop 2 (EC 2) fragment of the TM4SF19 protein and the immunoglobulin Fc domain.

For the production of fusion proteins, the extracellular loop 2 site of human TM4SF19 used as a TM4SF19 fragment is merely exemplary, and the present invention is not limited thereto.

The term "Fc domain" as used herein refers to a protein comprising heavy chain constant domain 2 (CH 2) and heavy chain constant domain 3 (CH 3) of an immunoglobulin, and excludes heavy and light chain variable domains of an immunoglobulin and light chain constant domain 1 (CL 1). The Fc domain may further comprise a hinge to the heavy chain constant domain. Although not limited, the "Fc domain" may be an IgG-, igA-, igD-, igE-or IgM-derived Fc domain. In the following embodiments, human IgG1-Fc used to produce fusion proteins is merely exemplary, and the present invention is not limited thereto.

Furthermore, an "Fc domain" may include a "Fc domain" or "Fc domain variant" of a modified immunoglobulin, which is prepared by replacing some amino acids of the Fc region or combining different types of Fc domains. Preferably, it refers to an FC domain in which antibody-dependent cytolysis (ADCC) or complement-dependent cytolysis (CDC) is attenuated by modifying binding to FC receptors and/or binding to complement (complement) as compared to the wild-type FC domain. Here, the Fc domain of the modified immunoglobulin may be selected from the group consisting of IgG1, igG2, igG3, igD, igG4, and combinations thereof.

The term "antibody" as used herein refers to a specific immunoglobulin directed against an antigenic site. "antibody" includes monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, and human antibodies, and includes antibodies known in the art or commercially available in addition to novel antibodies. Antibodies include not only full length versions containing two heavy and two light chains, but also functional fragments of antibody molecules, as long as they specifically bind TM4SF 19. Functional fragments of antibody molecules refer to fragments having at least an antigen binding function and may include Fab, F (ab ') and F (ab') 2, fv, although the invention is not limited thereto.

The present invention relates to an anti-TM 4SF19 antibody specific for all or part of the extracellular loop 2 (EL 2) region of human TM4SF 19. In one exemplary embodiment, the present invention relates to an anti-TM 4SF19 antibody having specificity for the entire region or a portion of the extracellular loop 2 of human TM4SF19 represented by SEQ ID NOs of human TM4SF19 represented by GenBank accession No. NP-612470.2, 115 through 175. In another exemplary embodiment, the present invention relates to an anti-TM 4SF19 antibody specific for the entire region or part of extracellular loop 2 represented by SEQ ID NOs 120 to 169 of human TM4SF19 of GenBank accession No. 138461.4. The target site of extracellular loop 2 of human TM4SF19 for antibody production is merely exemplary, and the present invention is not limited thereto.

According to one embodiment of the invention, the TM4SF19 protein was found to participate in cell-cell interactions by self-binding.

Specifically, the present inventors produced fusion proteins by targeting the extracellular loop region of TM4SF19 protein by fusing self-binding antibodies against TM4SF19 protein capable of inhibiting TM4SF19 protein, TM4SF19 fragments (e.g., extracellular loop region, and membrane protein), and Fc, and evaluated the efficacy of inhibitors on TM4SF19 protein activity.

The term "bone disease" as used herein may be one or more selected from the group consisting of metabolic bone disease, orthopedic bone disease, aplastic bone disease, degenerative arthritis, rheumatoid arthritis, psoriatic spondylitis, age-related bone loss, osteoporosis, osteogenesis imperfecta, osteomalacia, sarcopenia, bone fracture, bone defect and hip joint disease, rickets, paget's disease, periodontal disease, and bone damage caused by cancer cell bone metastasis.

The term "obesity" as used herein refers to a condition with abnormal or excessive fat accumulation.

The term "obesity-mediated metabolic disease" as used herein includes diabetes, hypertension, hyperlipidemia, nonalcoholic steatohepatitis and specific cancers, more broadly, hypertension, diabetes, insulin resistance syndrome, metabolic syndrome, obesity-related gastroesophageal reflux disease, arteriosclerosis, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, lipodystrophy, nonalcoholic steatohepatitis, cardiovascular disease and polycystic ovary syndrome. When the composition of the present invention is used, the treatment of the above-mentioned diseases can also be performed simultaneously. Furthermore, therapeutic goals for these obesity-related diseases include those desiring to lose weight.

The term "cancer" as used herein includes cell-mediated diseases that have the aggressive characteristics of dividing and growing cells by ignoring normal growth restrictions, invasive characteristics of invading surrounding tissues, and metastatic characteristics of spreading to other parts of the body.

Although there is no limitation on the type of cancer used in the present invention, the cancer may include one or more 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, lymphoid cancer, bladder cancer, gall bladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, sarcoma of soft tissue, cancer of the urinary tract, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvis cancer, central Nervous System (CNS) tumors, spinal cord tumors, brain stem glioma, and pituitary adenoma.

The invention also includes the use of TM4SF19 in connection with inhibiting "cancer" and "cancer metastasis".

Meanwhile, the term "prevention" as used herein means preventing a desired symptom or disease or delaying all of its occurrence or expression by administering the composition of the present invention.

The term "treatment" as used herein refers to all actions that are taken to reduce or eliminate the symptom or disease of interest by administering the compositions of the present invention.

Meanwhile, the composition of the present invention may further include a pharmaceutically acceptable carrier, and may be formulated together with the carrier.

The term "pharmaceutically acceptable carrier" as used herein refers to a carrier or diluent that does not stimulate an organism and does not impair the biological activity and properties of the compound being administered. As a pharmaceutical carrier useful for formulating the liquid composition, which is suitable for sterilization and living body, saline, sterilized water, ringer's solution, buffered saline, albumin injection solution, dextrose solution, glycerol, ethanol, or a mixture of one or more thereof may be used. Other conventional additives such as antioxidants, buffers and bacteriostats may be added as desired. In addition, the pharmaceutically acceptable carrier may be formulated into injectable preparations such as aqueous solutions, suspensions or emulsions, pills, capsules, granules or tablets by further adding diluents, dispersants, surfactants, binders and lubricants.

The composition comprising the TM4SF19 expression or activity inhibitor according to the invention and a pharmaceutically acceptable carrier may be formulated into any dosage form comprising it as active ingredient, may be prepared into oral or parenteral formulations, and may be formulated into unit dosage forms for easy administration and uniform dosage. Pharmaceutical formulations of the invention include suitable forms for oral, rectal, intranasal, topical (including buccal and sublingual administration), subcutaneous, vaginal or parenteral (including intramuscular, subcutaneous and intravenous administration), or for administration by inhalation or insufflation. Oral formulations comprising the compositions of the present invention as an active ingredient may be prepared, for example, as tablets, troches, lozenges, aqueous or oily suspensions, powders or granules, emulsions, hard or soft capsules, syrups or elixirs.

Parenteral formulations comprising the compositions of the present invention as an active ingredient may be prepared in injectable form for subcutaneous, intravenous or intramuscular injection, in suppository injectable form or as a spray, such as an inhalable aerosol using a respirator. For the formulation of injectable formulations, the compositions of the invention may be prepared as solutions or suspensions by mixing with stabilizers or buffers in water and may be formulated for unit administration in ampoules or vials.

The compositions of the present invention are 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 a disease, and the effective dosage level may be determined by the type, severity, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of excretion, duration of treatment, concomitant medication, and other factors well known in the medical arts. The compositions of the invention may be administered as a sole therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, or may be administered in single or multiple doses. That is, the total effective amount of the compositions of the present invention may be administered to a patient in a single dose or multiple doses via a long-term fractionated treatment regimen. With all of the above factors in mind, it is important to administer an amount that can achieve the maximum effect in a minimum amount without side effects, as can be readily determined by one of ordinary skill in the art.

The dosage of the pharmaceutical composition of the present invention may vary according to the weight, age and sex of the patient, health condition, diet, administration time, administration method, excretion rate and severity of the disease. In parenteral administration, the daily dose is preferably from 0.01 μg to 100mg, more preferably from 1 μg to 50mg, per kg body weight. However, the above dose does not limit the scope of the present invention in any way, since the dose may be increased or decreased according to the administration route, severity of obesity, sex, weight or age.

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

In another aspect, the invention also relates to a method of preventing or treating bone disease, the method comprising: a therapeutically effective amount of a composition for preventing or treating a bone disease comprising a substance for inhibiting TM4SF19 expression or activity is administered to a subject.

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

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

The term "administration" as used herein means that a predetermined substance is introduced into an individual by a suitable method, and the administration route of the prophylactic or therapeutic composition according to the invention may be oral administration or parenteral administration by a general route capable of reaching the tissue of interest. Furthermore, the composition for preventing or treating cartilage related diseases according to the present invention may be applied by any device capable of moving the active ingredient to the target cells.

The term "subject" as used herein includes mammals, such as mice, rats, rabbits, cattle, horses, pigs, goats, camels, antelopes, or dogs, suffering from a related disease, or humans, whose symptoms are alleviated by administration of the pharmaceutical compositions of the present invention.

The route of administration of the compositions of the present invention may be by conventional means via various oral or parenteral routes capable of reaching the tissue of interest, in particular oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, intranasal, inhalation, intraocular or intradermal routes.

The prophylactic or therapeutic methods of the invention comprise administering the compositions of the invention in a therapeutically effective amount. A therapeutically effective amount refers to an amount that increases the effect of reducing the weight or size of an adipocyte. It will be apparent to those of ordinary skill in the art that a physician can determine the appropriate total daily amount within the scope of the correct medical judgment. Preferably, the specific therapeutically effective amount for a particular patient depends on the type and extent of the response to be achieved, whether another agent is used when needed, the age, weight, general health, sex and diet of the patient, the time of administration, the route of administration and the rate of secretion of the composition, the duration of the treatment, various factors and other factors well known in the medical arts. Accordingly, an effective amount of a pharmaceutical composition suitable for the purposes of the present invention is preferably determined in view of the foregoing. Furthermore, in some cases, the therapeutic effect may be enhanced by administering the compositions of the present invention in combination with therapeutic agents for known related diseases.

In another aspect, the invention relates to a method of screening for a drug for treating a bone disorder, the method comprising: treating a sample suspected of bone disease with a candidate for treating bone disease; and

mRNA or protein expression levels of the TM4SF19 gene were compared to controls.

In another aspect, the invention relates to a method of screening for a drug for treating obesity or an obesity-mediated metabolic disease, the method comprising: treating a sample suspected of being obese or obesity-mediated metabolic disease with a candidate for treating obesity or obesity-mediated metabolic disease; and

mRNA or protein expression levels of the TM4SF19 gene were compared to controls.

In another aspect, the invention relates to a method of screening for a drug for treating cancer or cancer metastasis, the method comprising:

treating a sample suspected of cancer or cancer metastasis with a candidate for preventing or treating cancer or inhibiting cancer metastasis; and

mRNA or protein expression levels of the TM4SF19 gene were compared to controls.

The term "sample" as used herein refers to an individual or sample used to screen for candidates for treatment of a related disorder, including but not limited to mammals, including dogs, cattle, pigs, rabbits, chickens, mice, and humans, and includes samples isolated from individuals, such as whole blood, serum, blood, plasma, saliva, urine, sputum, lymph, cells, or tissue.

The term "control" as used herein refers to a sample that has not been treated with a candidate. Furthermore, the term "candidate" as used herein refers to a drug for testing changes in expression or activity of TM4SF19, as well as a target for measuring the ability to prevent or treat a related disease by directly or indirectly altering the expression level or activity of TM4SF19, and includes any molecule, such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides, and a wide range of compounds. These candidates also include natural and synthetic substances.

The term "therapeutic agent" as used herein refers to a substance used for the prevention or treatment of a related disease.

The screening method of the present invention can be carried out by determining a substance that reduces the expression of mRNA or protein of the TM4SF19 gene as compared to a control as a therapeutic agent for a related disease, treating a subject suspected of the related disease with a therapeutic candidate, and comparing the expression level of mRNA or protein of the TM4SF19 gene with a control not treated with the candidate. Analytical methods for measuring mRNA levels include reverse transcription polymerase reaction, competitive reverse transcription polymerase reaction, real-time reverse transcription polymerase reaction, RNase protection assay, northern blot, DNA chip, but the present invention is not limited thereto.

Analytical methods for measuring protein levels include Western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation assay, complement fixation assay, FACS, and protein chip assay, but the present invention is not limited thereto.

Furthermore, according to one embodiment of the present invention, it was confirmed that TM4SF19 expression was increased during osteoclast differentiation. In the case of the TM4SF19 deficiency model (TM 4SF19 knockout mice, TM4SF19KO and TM4SF19 extracellular domain 2 (116-165) knockout mice, TM4SF19EC 2. DELTA.) it was confirmed that the formation of mononuclear osteoclasts was inhibited in osteoclast differentiation. Osteoclasts that do not differentiate into polynuclear osteoclasts have an osteoclast phenotype and maintain a low level of bone resorption activity. That is, the TM4SF19 inhibitor of the present invention can selectively inhibit the formation of polynuclear osteoclasts, can more selectively control bone resorption, without completely eliminating bone resorption function, and significantly reduce side effects, as compared to a therapeutic agent that inhibits the formation of mononuclear osteoclasts (e.g., an anti-RANKL antibody). In addition, it was demonstrated that the TM4SF19 deficient model prevented bone loss caused by ovariectomy. Furthermore, as a result of treating an anti-mouse TM4SF19 polyclonal antibody and a mouse TM4SF19EC2 (116-165) -Fc fusion protein of GenBank accession No. np_001153874.1, human TM4SF19EC2 (120-169 isoform 1 of GenBank accession No. np_ 612470.2)) Fc fusion protein in osteoclast differentiation, it was confirmed that these inhibitors inhibit multinucleated osteoclast formation during osteoclast differentiation. Therefore, a substance for inhibiting expression of TM4SF19 or a substance for inhibiting activity of TM4SF19 is effective in preventing or treating bone diseases.

Furthermore, according to one embodiment of the present invention, TM4SF19 expression is increased during adipocyte differentiation of human adipose-derived mesenchymal stem cells (hadmscs). It was confirmed that weight gain was inhibited in a high fat diet-induced model of TM4SF19 knockout mice, and that TM4SF19 acts on adipocyte differentiation and inflammatory responses including macrophages were confirmed due to a decrease in the amount of adipose tissue-related macrophages caused by obesity. As a result, it was confirmed that TM4SF19KO mice reduced insulin resistance and inhibited fatty liver in a high-fat diet-induced obesity mouse model. Thus, it can be seen that substances which inhibit the expression or activity of TM4SF19 are effective in preventing or treating obesity and obesity-mediated metabolic diseases.

Furthermore, according to one embodiment of the present invention, it was confirmed that TM4SF19 overexpression increases proliferation, migration, and colony formation of mouse-derived breast cancer and human osteosarcoma cells, and that TM4SF19 defects are effective in inhibiting lung metastasis of breast cancer cells, inhibiting cell proliferation, migration, and colony formation of osteosarcoma cells, and inhibiting bone metastasis of breast cancer cells, and that substances that inhibit TM4SF19 expression or activity are effective in preventing and treating cancer or cancer metastasis.

Furthermore, according to one embodiment of the present invention, it was confirmed that treatment with human TM4SF19 EC2 (120-169 (isoform 1) of GenBank accession No. NP-612470.2) Fc fusion protein can inhibit the growth and colonization ability of pancreatic cancer cells, and inhibit cell proliferation, migration, and colony formation of osteosarcoma cells. Thus, it can be seen that substances for inhibiting TM4SF19 expression or TM4SF19 activity are effective in the prevention and treatment of cancer or cancer metastasis.

Advantages and features of the invention and methods of accomplishing the same may be understood clearly hereinafter by reference to the detailed description of exemplary embodiments and the accompanying drawings. However, the invention is not limited to the exemplary embodiments disclosed below and may be embodied in many different forms. These exemplary embodiments are provided only to complete the disclosure of the present invention and to fully convey the scope of the invention to those skilled in the art.

Examples (example)

Example 1: roles of TM4SF19 in osteoclast differentiation

FIG. 1 shows the expression of the TM4SF19 gene over time confirmed by qPCR after treatment of cells isolated from mouse bone marrow with M-CSF and nuclear factor kappa-beta receptor activator ligand (RANKL) to differentiate. As shown in FIG. 1, it was confirmed that the expression of TM4SF19 increased with the progress of osteoclast differentiation. The expression of the osteoclast differentiation-related genes ACP5 and CTSK increases with the progress of osteoclast differentiation.

Example 2: confirmation of the action of TM4SF19 Using TM4SF19 knockout mice

(1) Confirmation of TM4SF19 knockout and inhibition of Multi-core osteoclast formation

Cells were isolated from bone marrow of CRISPR-induced TM4SF19 knockout mice TM4SF19KO and wild-type (WT) mice, treated with M-CSF (25 ng/ml) +rankl (100 ng/ml) to induce differentiation, followed by staining with tartrate-resistant acid phosphatase (TRAP), an osteoclast-associated marker.

As shown in FIG. 2A, when TM4SF19KO mouse bone marrow cells are induced to differentiate into osteoclasts, they differentiate into mononuclear osteoclasts, not polynuclear osteoclasts.

In the same manner as described above, cells were isolated from bone marrow of CRISPR-induced TM4SF19 knockout mice TM4SF19KO and wild-type (WT) mice, RANKL concentrations were fixed at 100ng/ml, and cell differentiation was induced at various M-CSF concentrations of 25, 40 and 60ng/ml, followed by staining with TRAP.

As shown in FIG. 2B, it was confirmed that even with the treatment of M-CSF at a high concentration, the TM4SF19KO mouse bone marrow cells were unable to form multinucleated osteoclasts.

Fig. 2C is a schematic diagram illustrating an osteoclast differentiation process. As shown in fig. 2C, the osteoclast precursor first differentiated into TRAP positive single-core osteoclasts, and then matured into giant multinucleated cells by intercellular fusion and incomplete cytokinesis. Polynuclear osteoclasts have high bone resorption activity, whereas osteoclasts that do not differentiate into polynuclear osteoclasts have their own phenotype and express osteoclast-related markers such as TRAP and cathepsin K, but maintain low levels of bone resorption activity.

Thus, the fact that TM4SF19 knockout prevents formation of polynuclear osteoclasts suggests that prevention or treatment of diseases associated with bone loss may be attempted by inhibiting TM4SF 19. In particular, TM4SF19 inhibitors can selectively inhibit multi-core osteoclast formation and more selectively modulate bone resorption rather than completely abrogate the bone resorption function of osteoclasts, thereby significantly reducing side effects compared to therapeutic agents that inhibit single-core osteoclast formation (e.g., anti-RANKL antibodies).

(2) Confirmation of inhibition of expression of Gene involved in osteoclast differentiation in TM4SF19KO mice

Cells were isolated from CRISPR-mediated TM4SF19 knockout mice TM4SF19KO and bone marrow of wild-type (WT) mice and expression of Ctsk, acp5, c-Fos and nfatt 1 involved in osteoclast differentiation was confirmed by qPCR 4 days after differentiation in the presence or absence of M-CSF and RANKL treatment.

FIG. 3 shows a set of graphs illustrating mRNA expression of Ctsk, acp5, c-Fos, and Nfatc1 in osteoclast differentiation in the presence or absence of M-CSF and RANKL treatment.

As shown in fig. 3, during the osteoclast formation, the expression of the osteoclast differentiation-related gene was significantly inhibited in TM4SF19KO compared to the control (WT).

(3) Confirmation of actin band formation and bone resorption inhibition

After bone marrow differentiation of Wild Type (WT) and TM4SF19KO mice, actin band formation was confirmed by F-actin staining. Although Wild Type (WT) formed normal actin bands, TM4SF19KO did not form actin bands (fig. 4A).

After the BMM cells were spread on the dentin disk and differentiated, pit formation was confirmed. Although bone resorption was confirmed in the Wild Type (WT), it was confirmed that bone resorption was inhibited in TM4SF19KO (fig. 4B).

(4) Confirmation of OVX inhibition of bone loss

Wild Type (WT) and TM4SF19KO female mice at 8 weeks of age were dissected without ovariectomy (sham) or Ovariectomy (OVX), and then the femur was fixed on day 31 for micro CT analysis.

In addition, 3D micro-CT parameters (bone micro-structure parameters) including Bone Volume (BV), ratio of bone volume to tissue volume (% BV/TV), average trabecular number (tb.n), and trabecular separation (tb.sp) were obtained by analyzing the micro-CT images using a 3D imaging procedure.

It was confirmed that bone loss caused by ovariectomy was inhibited in TM4SF19KO mice (fig. 5).

Regardless of ovariectomy, TM4SF19KO mice have higher bone densities than Wild Type (WT) mice.

(5) Confirmation of osteoclast formation by femoral staining with TRAP

Femur of 8 week old Wild Type (WT) and TM4SF19KO mice were fixed and decalcified, and then stained with TRAP.

As a result, as shown in fig. 6, the lack of multinucleated osteoclasts was confirmed by TRAP staining of the femur of TM4SF19KO mice.

Example 3-1: confirmation of TM4SF19 extracellular loop 2 (EC 2) deficient mice were deficient in multinucleated osteoclast formation

(1) Confirming defect of multi-nucleation of osteoclast

Cells were isolated from bone marrow of wild-type (WT) mice and TM4SF19EC2 Del (also denoted TM4SF19EC2 delta) mice in which CRISPR knockdown of M4SF19 extracellular domain 2 (116-165) was used, and TRAP staining was performed after differentiation with or without treatment with M-CSF (25 ng/ml) and RANKL (100 ng/ml).

As a result, as shown in fig. 7A, it was confirmed that when the BMM of the TM4SF19EC2 delta mouse was able to differentiate into single-core osteoclasts, but not multi-core osteoclasts.

FIG. 7B is TRAP staining results obtained from BMM of Wild Type (WT) mice and TM4SF19EC 2.DELTA.mice in which the CRISPR knockout M4SF19 extracellular domain 2 (116-165) was used, RANKL concentrations were fixed at 100ng/ml and cells were treated with either 25ng/ml or 100ng/ml of M-CSF to induce differentiation. Even with high concentrations of M-CSF treatment, TM4SF19EC2 delta osteoclasts were unable to rescue the defect of multinucleated osteoclast formation.

Example 3-2: cytoskeletal rearrangement by TM4SF19EC2 delta

(1) Confirmation of actin band formation and bone resorption inhibition

When polynuclear osteoclasts (mature osteoclasts) are formed during osteoclast differentiation, actin bands consisting of podocytes form and adhere to bone, resulting in bone resorption.

Cells were isolated from bone marrow of wild-type and TM4SF19EC2 delta mice, treated with MCSF (60 ng/ml) and RANKL (100 ng/ml) to induce differentiation, and then differentiated cells were fixed and stained with phalloidin FITC, and examined for F-actin bands using confocal microscopy (400 fold).

It was confirmed that TM4SF19EC2 delta inhibits polynuclear osteoclast formation, affecting cytoskeletal rearrangement associated with integrin signaling. As shown in fig. 7C, actin bands were formed by the multinuclear formation of osteoclasts in wild-type mice, but not in TM4SF19EC2 delta.

After differentiation by plating the BMM cells on dentin discs, pit formation was confirmed by 1% toluidine blue staining. As a result, although bone resorption was confirmed in wild-type (WT) mice, bone resorption was blocked in TM4SF19EC2 delta (fig. 7D).

(2) Confirmation of inhibition of osteoclast-associated Gene expression

To confirm the expression of osteoclast differentiation-related target genes (Ctsk, acp5, c-Fos, and nfatt 1) in wild-type (WT) mice and TM4SF19EC2 delta mice using CRISPR knock-out TM4SF19 extracellular domain 2 (116-165), qPCR was performed.

As shown in fig. 8, the expression of osteoclast differentiation-related genes (Ctsk, acp5, c-Fos, and nfatt 1) was significantly inhibited during osteoclast differentiation in TM4SF19EC2 delta mice compared to the control.

(3) Confirmation of inhibition of bone loss

Wild Type (WT) and TM4SF19EC2 delta female mice at 8 weeks of age were dissected without ovariectomy (sham) or Ovariectomy (OVX), and then the femur was fixed on day 31 for micro CT analysis.

In addition, 3D micro-CT parameters (bone micro-structure parameters) including Bone Volume (BV), ratio of bone volume to tissue volume (% BV/TV), average trabecular number (tb.n), and trabecular separation (tb.sp) were obtained by analyzing the micro-CT images using a 3D imaging procedure.

Bone loss due to ovariectomy was confirmed to be inhibited in TM4SF19EC2 delta mice (fig. 9).

Wild Type (WT) TM4SF19KO and TM4SF19EC2 delta female mice at 8 weeks of age were dissected without ovariectomy (sham) or Ovariectomy (OVX), and then the femur was fixed on day 31 for micro CT (μct) analysis.

Fig. 10 shows 3D micro-CT parameters (bone micro-structural parameters), including Bone Volume (BV), bone volume to tissue volume ratio (% BV/TV), average trabecular number (tb.n), trabecular separation (tb.sp) and trabecular thickness (tb.th), obtained by analyzing micro-CT images using a 3D imaging procedure.

Fig. 11 shows a micro CT image.

As a result, as shown in fig. 10 and 11, it was confirmed that bone loss caused by ovariectomy was suppressed in TM4SF19KO and TM4SF19EC 2a mice, and that the bone density of TM4SF19KO and TM4SF19EC 2a mice was higher than that of Wild Type (WT) mice in the sham surgery group without Ovariectomy (OVX).

Example 4: confirmation of self-binding and interaction with integrin of TM4SF19

(1) Confirmation of self-interaction of TM4SF19 with TM4SF19

To confirm whether TM4SF19 is involved in the interaction between cells, HA-or flag-tagged TM4SF19 was produced. TM4SF19 (wherein 3HA is tagged at the N-terminus), wild type (wherein 3Flag is tagged at the N-terminus) and TM4SF19 deletion mutants were transiently overexpressed in 293T cells to confirm binding by immunoprecipitation. Preparation of TM4SF19 deletion mutant is shown in FIG. 12A. After 3Flag was tagged to each hTm SF19 variant (hTM 4SF19115-175 a, hTm4SF19 105-186 a, hTm4SF19 105-196 a, hTm4SF19 94-186 a, and hTm4SF19 94-196 a) shown in fig. 12A in which a portion of the TM4SF19 sequence was deleted, the intercellular interactions were analyzed by immunoprecipitation.

As a result, it was confirmed that TM4SF19 participates in the intercellular interaction and binds to itself (fig. 12B). However, it was confirmed that mutants (hTm 4SF19 94-196. Delta.) in which TM4SF19 transmembrane 3 (TM 3), extracellular region 2 (EC 2) and transmembrane 4 (TM 4) were deleted did not undergo self-binding. From the above results, it was confirmed that the transmembrane 3-extracellular domain 2-transmembrane 4 region of TM4SF19 is important for the self-interaction of TM4SF19, and aa 94 to 196 of TM4SF19 are particularly important regions for the self-interaction between TM4SF19 and TM4SF19.

(2) Confirmation of region involved in self-interaction of TM4SF19 with TM4SF19

To confirm that aa 94-196 of TM4SF19 is an important region of self-interaction between TM4SF19 and TM4SF19, in this case, hTm SF19 94-196 was generated as a deletion mutant of TM4SF19, in which only the sequence of aa 94-196 of hTm4SF19 was retained, and the other sequences were deleted, unlike 3Flag-hTm SF19 94-196 delta (FIG. 12C, upper panel). Binding was confirmed by immunoprecipitation by transiently overexpressing TM4SF19 (where 3HA is labeled at the N-terminus), wild-type (where 3Flag is labeled at the N-terminus) and TM4SF19 deletion mutants in 293T cells in the same manner as described above. As a result, it was confirmed that transmembrane 3, extracellular region 2 (EC 2) and mutants of transmembrane 4 (hTm sf 19-196) expressing Tm4sf19 self-bind similarly to wild-type (WT) (FIG. 12C, lower panel). From these results, it was confirmed again that the aa 94-196 region of TM4SF19 is a very important part for the self-interaction between TM4SF19 and TM4SF 19.

(3) Interaction between TM4SF19 and TM4SF19-Fc

Interactions between hIgG1-Fc fusion proteins of 3 Flag-labeled TM4SF19 and TM4SF19 fragments at the N-terminus in 293T cells were confirmed by immunoprecipitation in the same manner as described above. As TM4SF19 fragment, hTm4SF19 region 131-160, hTm4SF19 region 145-169, hTm4SF19131-169, hTm4SF19 region 120-160, hTm4SF19 region 120-169, hTm SF19 region 120-180, hTm4SF19 region 120-186, hTm4SF19 region 120-196, hTm SF19 region 120-209, hTm4SF19131-196, or hTm4SF19 region 145-196 are used (FIG. 12D, upper panel). As a result, as shown in the lower graphs of FIGS. 12D and 12E, it was confirmed that the aa 160-169 region of hTm SF19 plays an important role in binding, and it was also confirmed that the aa 145-196 region of hTM4SF19-Fc plays an important role in increasing binding to TM4SF 19.

(4) Confirmation of interaction between integrins αv-TM4SF19

The interaction between TM4SF19 and integrin αv was confirmed to play an important role in osteoclast differentiation. To confirm the important binding region for TM4SF19-Fc to integrin αv-TM4SF19, interactions between 3 HA-labeled integrin αv and the igg1-Fc fusion protein of TM4SF19 fragment in 293T cells were confirmed by immunoprecipitation (fig. 12F).

(5) Confirmation of interaction between integrin beta 3-TM4SF19

It was confirmed that the interaction between TM4SF19 and integrin β3 plays an important role in osteoclast differentiation. To confirm the important binding region for TM4SF19-Fc to integrin β3-TM4SF19, interactions between igg1-Fc fusion proteins of integrin β3 and TM4SF19 fragments labeled with 3HA in 293T cells were confirmed by immunoprecipitation (fig. 12G).

Fig. 12H is a result of confirming interactions after overexpression of TM4SF19 and integrin αv or integrin β3 (which regulate osteoclast function and participate in cytoskeletal rearrangement during formation of polynuclear osteoclasts) in 293T cells or osteoclast precursor Raw 264.7 cells. This is a result of confirming that TM4SF19 binds to integrin αv and also to integrin β3.

TM4SF19 is a member of the four transmembrane protein (tetraspin) family, whose extracellular domain 2 (the large extracellular loop) is known to play an important role in binding to another binding partner. To confirm whether the extracellular domain of TM4SF19 is necessary for interaction with the partner, the interaction between TM4SF19 wt or the protein lacking extracellular domain 2 (aa 115-175) and integrin αv or integrin β3 was confirmed by immunoprecipitation. The results confirm that TM4SF19 wt shows interaction with it, but no binding to hTM4SF19115-175 delta is formed.

FIG. 12I shows confirmation of interactions between TM4SF19 and DC-Stamp and siglec-15 by immunoprecipitation, which are membrane proteins involved in the regulation of osteoclast function and cytoskeletal rearrangement during the formation of polynuclear osteoclasts. This is an immunoprecipitation result confirming the interaction of TM4SF19 labeled with 3HA at the N-terminus, dc-Stamp labeled with 3Flag at the N-terminus, or Siglec-15 labeled with 3Flag at the C-terminus in 293T cells.

Example 5: confirmation of increased proliferation, colony formation and migration of cancer cells by TM4SF19 overexpression

After overexpression of TM4SF19 in murine breast cancer cells E0771 (hTM 4SF 19), cell proliferation and colony formation assays and cell migration assays were performed over time.

Fig. 13A is a set of graphs showing a significant increase in cell proliferation of breast cancer cells E0771 after 24, 48 or 72 hours by TM4SF19 overexpression. FIG. 13B shows that after inoculation of 1000 breast cancer cells E0771 overexpressing TM4SF19 and control cells, the colony forming ability was confirmed and that colony formation was increased due to TM4SF19 overexpression. FIG. 13C shows the change in cell migration capacity over time, assessed after plating TM4SF19 overexpressing breast cancer cells E0771 and control cells in the migration chamber, indicating increased cell migration due to TM4SF19 overexpression.

Example 6: confirmation of TM4SF19 deficiency inhibiting metastasis of cancer cells

(1) Confirmation of TM4SF19 deficiency inhibiting metastasis of breast cancer cells to the lung

After 12 days after injection of breast cancer cells E0771 into the tail vein Wild Type (WT), TM4SF19KO and TM4SF19EC2 delta mice were sacrificed, the lungs were stained with the ink to confirm lung metastasis.

The lung metastasis phenotype of breast cancer cells of wild-type (WT), TM4SF19KO, and TM4SF19EC2 delta mice was confirmed, and the number of nodules produced in the lung was counted.

As a result, it was confirmed that TM4SF19KO and TM4SF19EC2 delta mice significantly inhibited lung metastasis of breast cancer cells compared to wild-type (WT) mice (fig. 14).

(2) Confirmation of TM4SF19 deficiency inhibiting expression of genes and proteins involved in metastasis of breast cancer cells

The environment surrounding cancer cells is an important factor in cancer metastasis, among which the extent of metastasis may vary due to antitumor factors released by macrophages. For co-culture, bone marrow was obtained from TM4SF19KO mice (TMKO) and Wild Type (WT) to differentiate into bone marrow-derived macrophages (BMDM) and placed in the lower part of the migration chamber and breast cancer cells in the upper part of the chamber. Expression of target genes involved in cell migration (CDH 2, SNAI1 and SNAI 2) in migrating cells was confirmed by qPCR, and western blotting was performed to confirm expression of transfer-related proteins (vimentin, slug, bnail, E-cadherin and β -actin).

As a result, it was confirmed that expression of target genes (CDH 2, SNAI1, and SNAI 2) associated with cell migration was inhibited (fig. 15A) and expression of transfer-related proteins (vimentin, slug, bnail, E-cadherin, and β -actin) was inhibited (fig. 15B) by co-culture with TM4SF19KO mouse macrophages. This indicates that the expression of tumor cell metastasis associated genes and proteins is inhibited by a defect in TM4SF 19.

Example 7: confirmation of involvement of TM4SF19 in adipogenic differentiation of human ADMSCs

Human adipose-derived mesenchymal stem cells (hADMSCs) were differentiated into adipocytes on days 0, 14 and 21 after growth in DMEM/F12 supplemented with 10. Mu.g/ml insulin, 1nM3,3', 5-triiodo-L-thyroxine, 1. Mu.M dexamethasone and 1. Mu.M rosiglitazone, expression of genes (C/EBP. Alpha. And PPARgamma.) was confirmed by qPCR and expression of proteins (PPARgamma and FABP 4) was confirmed by Western blotting.

As a result, it was confirmed that the expression level of TM4SF19 was increased by adipogenic differentiation of human ADMSCs (fig. 16).

Example 8: confirmation of the role of TM4SF19 in obesity and metabolic diseases related diseases

(1) Confirmation of weight gain of high fat raised TM4SF19KO

Wild Type (WT) and TM4SF19KO mice (which had been fed a normal diet) with 6 weeks of age were fed a high fat diet (60% fat) for 16 weeks and weight gain was confirmed.

As a result, the TM4SF19KO mice were resistant to high fat diet-induced obesity (FIG. 17).

(2) Confirmation of insulin resistance and fatty liver

After Wild Type (WT) and TM4SF19KO mice were fed a high fat diet, insulin resistance was confirmed by HOMA-IR (a), liver phenotype was confirmed, and liver tissue was fixed and stained with H & E (B), and triglyceride levels in the liver were measured (C).

As a result, in the case of TM4SF19KO mice, insulin resistance was reduced and fatty liver formation was inhibited in a high fat diet-induced obese mouse model. Thus, it was confirmed that TM4SF19KO mice (prepared by feeding 6-week old mice that had been fed a normal diet, a high fat diet for 12 weeks) were resistant to high fat diet-induced insulin resistance and fatty liver (fig. 18).

(3) Confirmation of resistance to high fat diet-induced obesity

When 6 week old Wild Type (WT) and TM4SF19KO mice that had been fed normal diet were fed high fat diet for 18 weeks, their body weight increased (fig. 19A), after 18 weeks, the mice were sacrificed to confirm the weight of each tissue (fig. 19B), confirm the weight of subcutaneous fat and visceral fat (fig. 19C), confirm the fat phenotype and adipose tissue macrophages around fat by H & E staining of epididymal white adipose tissue (eaft) (fig. 19D), and also confirm the secretion of anti-obesity cytokine adiponectin in serum (fig. 19E) [ rtWAT: retroperitoneal white adipose tissue, sWAT: subcutaneous white adipose tissue, ingWAT: inguinal white adipose tissue, iWAT: inter-scapular white adipose tissue, iBAT: inter-scapular brown adipose tissue ].

As a result, it was confirmed that in the case of TM4SF19KO mice, obesity was inhibited in a high fat diet-induced obese mouse model, and the adiponectin secretion amount in serum was also increased. Thus, it was confirmed that TM4SF19KO mice were resistant to high fat diet-induced obesity (18 weeks) (fig. 19).

(4) Confirmation of adipogenic differentiation marker and macrophage/M1-like macrophage marker expression

After 6 weeks of large Wild Type (WT) and TM4SF19KO mice (which had been fed normal diet) were fed high fat diet for 12 weeks, expression of adipogenic differentiation markers (upper panel) and macrophage/M1-like macrophage markers (lower panel) in epididymal white adipose tissue (eWAT) and subcutaneous white adipose tissue (sWAT) was confirmed by qPCR.

As a result, expression of adipogenic differentiation markers and macrophage/M1-like macrophage markers was reduced in epididymis and subcutaneous white fat of TM4SF19KO mice despite the high fat diet (fig. 20).

(5) Confirmation of adipose tissue-related macrophage population

After 6 weeks of Wild Type (WT) and TM4SF19KO mice (which had been fed normal food) were fed high fat food for 12 weeks,% macrophages (a) and dendritic cells (B) were confirmed by FACS analysis of epididymal white adipose tissue (eWAT).

As a result, although on a high fat diet, the TM4SF19KO mice had reduced% macrophages and dendritic cells in epididymal white fat, and the amount of adipose tissue-related macrophages induced by obesity was reduced in TM4SF19KO (fig. 21).

(6) Expression of marker genes in stromal vascular fraction of white adipose tissue

After 6 weeks of large Wild Type (WT) and TM4SF19KO mice (which had been fed a normal diet) were fed a high fat diet for 12 weeks, qPCR was performed on epididymal white adipose tissue (heft) in order to confirm the expression of macrophage marker genes in the Stromal Vascular Fraction (SVF).

As a result, stromal Vascular Fraction (SVF) was isolated from epididymal white fat of TM4SF19KO mice, and it was confirmed that the expression of macrophage markers MCP1 and F4/80 (fig. 22A) and M1-like macrophage markers IL6 and tnfα (fig. 22B) was decreased, and the expression of M2-like macrophage marker IL10 (fig. 22C) was increased.

(7) Confirmation of the role of TM4SF19 in eWAT inflammation, insulin resistance and fatty liver

To confirm the role of TM4SF19 in hewat inflammation, insulin resistance and fatty liver, experiments were performed with high fat diet-induced obese mouse models.

As a result, TM4SF19 regulates adipocyte differentiation and inflammatory response including macrophages, and thus, it was confirmed that TM4SF19 deficiency reduced insulin resistance and inhibited fatty liver (fig. 23).

(8) Confirmation of white to beige adipocyte transformation in TM4SF19KO mice

In a high fat diet induced obese mouse model (prepared by feeding high fat diet to 6 week old mice that had been fed normal diet for 12 weeks), the conversion of epididymal white fat to a beige or brown fat phenotype was confirmed in TM4SF19KO mice. Further, it was confirmed that Ucp1 expression in white fat of TM4SF19KO mice was increased in a 12-week high fat diet-induced obese mouse model, and Ucp1 in TM4SF19KO was increased in an in vitro brown fat differentiation model, confirming that TM4SF19 was involved in changing white adipocytes into beige adipocytes (fig. 24).

FIG. 24A shows the beige or brown fat phenotype and fatty liver formation in epididymal white fat of high fat diet induced obese WT or TM4SF19KO mice confirmed by H & E staining.

Fig. 24B shows the results of confirming the expression of brown adipocyte marker Ucp1 in epididymal white adipose tissue and subcutaneous white adipose tissue of high fat diet-induced obese WT or TM4SF19KO mice. It was confirmed that the expression of Ucp1 was increased in white adipose tissue of TM4SF19 KO.

Fig. 24C is a schematic diagram illustrating a method of beige/brown adipocyte differentiation of adipocytes extracted from white adipose tissue. Adipocyte progenitor cells isolated from white adipose tissue of WT and TM4SF19KO mice were grown in DMEM/F-12 medium supplemented with T3 and insulin. Two days after fusion, cells were treated with DMI and indomethacin to initiate differentiation, starting from day 2 and growing to day 7 or 8, while being treated with rosiglitazone to induce beige/brown adipocyte differentiation.

FIGS. 24D and 24E show the results of confirming Ucp gene and protein expression after differentiation of beige/brown adipocytes.

(9) Confirmation of expression of TM4SF19 in adipocytes and Stromal Vascular Fraction (SVF) of white adipose tissue

Expression of TM4SF19 was confirmed by adipocytes and SVFs isolated in epididymal white fat and subcutaneous white fat of mice fed high fat diet for 24 weeks.

In the high fat diet induced (24 hours of administration of 6 week old mice (fed normal diet)) mice of eWAT and sWAT, the expression of TM4SF19 in adipose tissue and SVF including macrophages was confirmed, indicating that TM4SF19 functions not only in adipocytes but also in SVF including macrophages and monocytes (FIG. 25).

FIG. 25A is a schematic representation of the cell types in each fraction after digestion of white fat with type I collagen and separation by centrifugation into adipose tissue, lower fat and SVF fractions.

Fig. 25B is a result of confirming expression of TM4SF19 after epididymis and subcutaneous white adipose tissue of mice fed a high fat diet (60% fat) for 24 weeks are separated into adipocytes and SVF.

(10) Brown fat differentiation is induced by treatment of Stromal Vascular Fraction (SVF) isolated from TM4SF19KO and sWAT of wild-type mice with isoprenaline

SVF was isolated from subcutaneous white fat of wild-type (WT) and TM4SF19KO mice, induced to differentiate into brown fat with isoproterenol, and qPCR was performed to confirm thermogenic gene expression and beige fat marker gene expression.

Increased thermogenic gene expression and beige fat marker gene expression were observed in SVF in TM4SF19KO mice, suggesting that TM4SF19KO mice may be involved in the conversion of white fat to brown or beige fat (fig. 26).

(11) Expression of TM4SF19 in Co-culture of macrophages and 3T3-L1 adipocytes

In high-fat diet-induced obesity, the interaction between 3T3-L1 adipose tissue and macrophages plays an important role.

As shown on the right side of fig. 27, differentiated adipocytes were co-cultured with macrophages in a contact system. qPCR confirms the expression of macrophage, inflammatory and anti-obesity marker genes [ macrophage markers; inflammatory marker MCP-1; tnfα, IL6, MMP3, anti-obesity markers; adiponectin ]. As a control, macrophages and adipocytes were cultured separately, and RNA was extracted and pooled.

By co-culturing 3T3-L1 adipocytes and macrophages, the expression of the inflammation-associated genes MMP3 and TM4SF19 is increased.

(12) Induction of inflammatory changes by co-culturing adipocytes and macrophages in contact systems

After SVF was extracted from wild-type (WT) mice and adipogenesis, wild-type (WT) bone marrow differentiated into macrophages, followed by induction of inflammatory changes in either culture alone or in contact co-culture. As a control, macrophages and adipocytes were cultured separately, and RNA was extracted and pooled.

After adipocyte differentiation and macrophage differentiation of SVF, expression of TM4SF19 was increased by co-culture (FIG. 28).

SVF was extracted from wild-type (WT) and TM4SF19KO mice and adipogenesis was performed, and inflammatory changes were induced by contact co-culture after bone marrow was extracted from each mouse and differentiated into macrophages.

As a result, as shown in fig. 29, the expression of MCP1 and IL6 was reduced in the differentiation of TM4SF19KO differentiated adipocytes+macrophages in the co-culture system, compared to the differentiation of WT differentiated adipocytes+macrophages in the co-culture system.

SVF was extracted from wild-type (WT) and TM4SF19KO mice and adipogenesis was performed, and after bone marrow was extracted from each mouse and differentiated into macrophages, inflammatory changes were induced by control (ctrl) culture or co-culture. As a control (control culture), macrophages and adipocytes were cultured separately, and RNA was extracted and pooled.

In the co-culture model after adipocyte differentiation and bone marrow macrophage differentiation of SVF, TM4SF19KO BMDM reduced expression of inflammatory response markers as compared to WT adipocyte-WT macrophages, and inflammatory response was reduced in the TM4SF19KO adipocyte-TM 4SF19KO macrophage co-culture (FIG. 30).

In the case of adipocytes and BMDM co-culture, the inflammatory signal increases.

(13) Confirmation of expression of TM4SF19 in an in vitro insulin resistance model

An in vitro insulin resistance model was made.

3T3-L1 adipocytes differentiated for 12 days, treated with TNF alpha for 24 hours, and adipogenic differentiation markers C/EBP alpha and insulin resistance in vitro model markers C/EBP beta and TM4SF19 were confirmed by qPCR.

Tnfα induces inflammation, a model that mimics the increase in inflammatory signals in the surrounding environment when obesity is induced in vivo.

SVF was extracted from wild-type (WT) and TM4SF19KO mice, adipogenic differentiation was performed, and treatment with TNF alpha was performed for 24 hours, and adipogenic differentiation markers C/EBP alpha and insulin resistance in vitro model markers C/EBP beta and TM4SF19 were confirmed by qPCR. As a result, it was confirmed that TM4SF19 was increased in the in vitro insulin resistance model (FIGS. 31 and 32). It was confirmed that TM4SF19KO reduced insulin resistance (C/EBP. Beta. Is an insulin resistance marker).

(14) Roles of TM4SF19 in macrophage differentiation and polarization

Markers involved in M1 polarization were confirmed after bone marrow was extracted from wild-type (WT) and TM4SF19KO mice and differentiated into macrophages. M1 macrophages are involved in pre-inflammatory signaling.

It was confirmed that inflammatory markers and TM4SF19 increased by differentiation of M1 macrophages derived from bone marrow of wild-type (WT) mice (fig. 33), and that bone marrow-derived M1 macrophages differentiation markers were decreased in TM4SF19KO (fig. 34).

Preparation example 1: preparation and verification of TM4SF19 antibody

(1) Preparation of polyclonal mouse TM4SF19 antibody

After determining available targets by predicting antibody immunogenicity, polyclonal mouse TM4SF19 antibodies targeting aa 141-159 region in mouse TM4SF19 extracellular loop 2 were prepared (mouse TM4SF19 protein is represented by SEQ ID NO: 7).

(2) Validation of TM4SF19 antibody

After transient overexpression of human TM4SF19 (hTM 4SF 19), human EC 2. Delta (hTM 4SF19 115-175. Delta.), mouse TM4SF19 (mTM SF 19) and mouse EC 2. Delta (mTM 4SF19 116-165. Delta.) in 293T cells, western blotting was performed in order to confirm expression using the previously produced polyclonal mouse TM4SF19 antibody.

Polyclonal mouse TM4SF19 antibodies recognize human TM4SF19 and mouse TM4SF19. However, no extracellular loop 2 deletion mutant was identified (fig. 35).

Example 9: verification of the Effect of TM4SF19 Using TM4SF19 antibody

(1) Confirmation of inhibition of osteoclast differentiation by TM4SF19 antibody

Bone marrow was extracted from Wild Type (WT) mice, differentiated into osteoclasts, then treated with TM4SF19 antibody, followed by TRAP staining.

As a result, TM4SF19 antibody dose-dependently inhibited osteoclast multinucleation (fig. 36).

Preparation example 2: manufacture of TM4SF19-Fc

The TM4SF19-Fc fusion proteins of human TM4SF19 and mouse TM4SF19 extracellular loop 2 were made using the TM4SF family of sequences and phylogenetic tree information.

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

Since mouse TM4SF19-Fc (116-165) was secreted well, it was purified for in vitro experiments.

Since human TM4SF19-Fc (115-175) was not secreted well, the Fc of the new region (120-169) was produced.

Mouse TM4SF19-Fc was secreted well and thus purified.

Mouse TM4SF19EC2-Fc (116-165) was manufactured and used in subsequent experiments.

Human TM4SF19-EC2-Fc (115-175) was not well expressed, so this expression was confirmed by remanufacturing with mouse TM4SF19-EC2-Fc and a conserved sequence. Human TM4SF19-Fc (120-169) was secreted more into the medium than extracellular loop 2 (115-175) and therefore was also used in subsequent experiments (FIG. 37).

The fusion protein used in the embodiment according to the present invention is shown in fig. 58.

Hereinafter, "hTM4SF19-Fc" refers to the hTM4SF19-Fc (aa 120-169) fusion protein. "mTM SF19-Fc" refers to the mouse TM4SF19-Fc (aa 116-165) fusion protein.

Example 10: verification of the Effect of TM4SF19 Using TM4SF19-Fc

hIgG1-Fc, hTM4SF19 120-169-Fc, and mTM SF19116-165-Fc were treated in osteoclast differentiation. For osteoclast differentiation, bone marrow-derived cells were treated with MCSF (60 ng/ml) and RANKL (100 ng/ml) and TRAP stained.

The results are shown in fig. 38A to 38C. Inhibition of multinucleated osteoclast formation was confirmed by treatment of purified mTM SF19 EC2-Fc during osteoclast differentiation [ E1; samples eluted with elution buffer (20 mM glycine). E1-1; samples (Fc may be more stable) were ultrafiltered/diafiltered (UF/DF) with buffer a (50 mM phosphate, 50mM nacl, ph 7.0). In addition, inhibition of the formation of multinucleated osteoclasts was confirmed by treatment of purified hTM4SF19 120-169-Fc.

After bone marrow differentiation in wild-type mice and treatment with 10. Mu.g/ml of IgG-Fc and mTM SF19-Fc, actin band formation, which occurred during multinucleated osteoclast differentiation, was confirmed by F-actin staining. It was confirmed that the formation of actin bands was inhibited by mTM SF19-Fc, and that TM4SF19 was localized in actin bands using the mouse TM4SF19 antibody (FIG. 38D).

BMM was plated on dentin discs, differentiated and then treated with 10. Mu.g/ml IgG-Fc or mTM SF 19-Fc. Pit formation was confirmed by 1% toluidine blue staining. As shown in fig. 38E, mTM SF19-Fc was confirmed to block bone resorption.

Example 11: confirmation of rescue of bone loss Using TM4SF19-Fc

One week after ovariectomy of 8 week old female mice, mice TM4SF19-Fc were injected into the tail vein once a week for 3 weeks, and microct was measured after one week after the last injection, sacrificed and the femur fixed. The concentrations of mouse TM4SF19-Fc used herein were 2.5mg/kg and 5mg/kg.

FIG. 39A is a set of microscopic CT images showing rescue of bone loss in mouse TM4SF 19-Fc.

The 3D micro CT parameters (bone micro structure parameters) including Bone Volume (BV), bone volume to tissue volume ratio (% BV/TV), average trabecular number (tb.n) and trabecular separation (tb.sp) were obtained by analyzing the micro CT images using the 3D imaging procedure of fig. 39A (fig. 39B).

As shown in fig. 39B, it was confirmed that total BMD (bone mineral density),% BV/TV (bone volume per tissue volume) and tb.n (trabecular number) decreased due to ovariectomy and increased in dose dependence due to mTM SF 19-Fc. On the other hand, mTM SF19-Fc was confirmed to decrease Tb.Sp (trabecular separation). From the above results, it was confirmed that mouse TM4SF19-Fc saved ovariectomy-induced bone loss.

In addition, histopathological analysis of mouse femoral tissue was performed by H & E staining. As a result, as shown in fig. 39C, it was confirmed that tb.n was reduced by ovariectomy (small Liang Shuliang), and that tb.n reduction was saved by TM4SF19-Fc treatment in a dose-dependent manner.

In addition, after ovariectomy was performed on 8-week-old female mice, mouse TM4SF19-Fc (116-165) and human TM4SF19-Fc (131-169) were intravenously injected. Human and mouse TM4SF19-Fc was confirmed to significantly inhibit bone loss compared to untreated groups (fig. 39D). Mouse TM4SF19-Fc (116-165) was treated at 10mg/kg, and human TM4SF19-Fc (131-169) was treated at 25 mg/kg.

The aa 131-169 region of human TM4SF19 can rescue ovariectomy-induced bone loss.

The above results indicate the possibility of developing TM4SF19-Fc as an osteoporosis therapeutic agent.

Example 12: verification of the Effect of TM4SF19 Using TM4SF19-Fc

(1) Osteoclast generation

Cells were isolated from bone marrow of wild-type (WT) mice and differentiated under conditions of treatment with M-CSF or M-CSF and RANKL, and then after 10. Mu.g/ml of mTM SF19-Fc treatment (control: treatment with buffer), expression of Ctsk and Acp5, which are genes involved in osteoclast differentiation, was confirmed by qPCR.

FIG. 40 is a set of graphs showing mRNA expression of Acp5 and Ctsk in osteoclast differentiation under conditions of treatment of M-CSF or M-CSF and RANKL (WT 1,2, 3, 4 and 5 represent results obtained from osteoclast differentiation of bone marrow cells obtained from each of five different wild-type mice; the group treated with M-CSF alone refers to an undifferentiated group).

As shown in FIG. 40A, the expression of genes involved in osteoclast differentiation was inhibited by mTM SF19-Fc treatment compared to control (WT).

Also, as shown in FIG. 40B, after cells isolated from bone marrow of wild-type (WT) mice were treated with 5. Mu.g/ml of mTM SF19-Fc while differentiation was performed under the conditions of treatment of M-CSF or M-CSF and RANKL, as a result of confirming the expression of osteoclast differentiation marker protein, it was confirmed that the expression of osteoclast differentiation marker protein was more significantly inhibited by mTM SF19-Fc than that of control (W).

(2) Surface binding of mTM SF19-Fc

The reason why TM4SF19-Fc blocks the multinuclear formation of osteoclasts was determined to be due to its increased surface binding capacity during differentiation, and thus the surface binding of mTM SF19-Fc before and after the differentiation of raw264.7 cells was analyzed by FACS. As shown in fig. 41, although there was little difference in surface binding between control IgG1 (dark gray plot) and mouse TM4SF19-Fc (light gray plot) before differentiation, mTM SF19-Fc (light gray plot) migrated rightward after differentiation compared to control IgG1 (dark gray plot), confirming that in fact surface binding was increased at mTM SF19-Fc after differentiation.

As shown in fig. 41, an increase in binding to mTM SF19-Fc after differentiation was confirmed.

Example 13: prevention and treatment of rheumatoid arthritis with TM4SF19-Fc

According to the experimental protocol of fig. 42A, the semi-therapeutic effect of mTM SF19-Fc was confirmed in a collagen-induced arthritis mouse model.

On day 18 after 14 days of acclimation of the DBA/1 mice, chicken collagen II was mixed with Complete Freund's Adjuvant (CFA) and injected, and chicken collagen II was mixed with Incomplete Freund's Adjuvant (IFA) for booster injection. mTM4SF19-Fc was administered starting 15 days after the first injection, intravenously at 10mg/kg or 25mg/kg twice weekly. CIA scores were checked twice weekly until day 63 mice were sacrificed.

CIA score was evaluated by the following method: score 0 (normal paw), score 1 (one or two toes inflamed and swollen), score 2 (3+ toe inflamed but no paw swollen, or whole paw mild swollen), score 3 (whole paw inflamed and swollen), score 4 (severely swollen paw and all toes, or stiff paw and toe)

As shown in fig. 42B, as a result of examining the joint status of mice 42 days after collagen-induced arthritis (CIA), both toes and feet were swollen in the IgG 1-treated control group, but it was confirmed that toe and foot edema were somewhat suppressed in the group treated with mTM SF19-Fc of 10mg/kg, and edema phenomenon was significantly suppressed in the group treated with mTM SF19-Fc of 25mg/kg, compared to the sham-operated group treated without any substance.

Meanwhile, the graph of fig. 42C shows the analysis results of CIA score and number of swollen joints investigated during the experiment. It was confirmed that CIA score and edema dose-dependently decreased in mTM SF19-Fc treated group compared to IgG1 treated control group.

In addition, the feet of the mouse model of collagen-induced arthritis were subjected to micro-CT photographing and the images were analyzed.

Fig. 42D is a set of microscopic CT images showing that inflammation and bone damage occurring in the paw of a collagen-induced arthritis mouse model was dose-dependently inhibited by mTM SF19-Fc treatment. The toes are bent due to collagen-induced arthritis, and the toe joints and ankle are bone-damaged due to inflammation. However, joint inflammation and bone injury in mTM SF 4 19-Fc treated mice were dose-dependently inhibited compared to control hIgG1-Fc treated mice.

Referring to fig. 42E, inflammation and bone damage occurring in the paw of a collagen-induced arthritis mouse model was dose-dependently inhibited by mTM SF19-Fc treatment, and it was confirmed that bone damage was inhibited in mice with the same RA fraction as in control hig 1-Fc treated mice.

Furthermore, 3D micro CT parameters (bone micro structural parameters) including Bone Volume (BV), bone volume to tissue volume ratio (% BV/TV), average trabecular number (tb.n) and trabecular separation (tb.sp) were obtained by analyzing micro CT images of the femur of a collagen induced arthritis mouse model using a 3D imaging procedure.

Fig. 42F shows that BMD and tb.n were reduced by collagen-induced arthritis, but bone loss was inhibited by TM4SF19-Fc compared to sham.

Fig. 42G is the result of confirming cartilage damage in collagen-induced arthritic mice using toluidine blue. Cartilage damage was shown to be inhibited in the mTM4SF19-Fc treated group compared to the hIgG1-Fc treated group.

In addition, the semi-therapeutic effects of mTM SF19-Fc and hTM4SF19-Fc (120-169) were confirmed by arthritis scores in a collagen antibody-induced arthritis mouse model. Three days after administration of collagen antibodies to Balbc mice, arthritis was induced by LPS injection. Arthritis inhibitory effects were shown at 25mg/kg of mTM SF19-Fc and 25mg/kg of hTM4SF19-Fc (120-169) compared to the control group (FIG. 42H). It was confirmed that by TM4SF19-Fc treatment, the arthritic disease score was reduced and the foot thickness was reduced compared to hIgG 1-Fc.

In addition, the therapeutic effect of mTM SF19-Fc was confirmed by arthritis scoring after induction of arthritis by LPS injection three days after administration of collagen antibodies to collagen-induced arthritis mice models.

To confirm the therapeutic effect of mTM SF19-Fc after induction of arthritis by LPS injection 3 days after collagen antibody administration, 50mg/kg of mTM SF19-Fc was treated starting from day 8 where the arthritis disease score was highest, and the arthritis disease score was checked every 2 days from day 9 to day 19.

Compared to the untreated group, it was confirmed that by mTM SF19-Fc treatment, the arthritis score was reduced, the foot thickness was reduced, and cartilage damage confirmed by toluidine blue was suppressed (fig. 42I). That is, the therapeutic effect of mTM SF19-Fc on arthritis was shown.

Example 14: inhibition of breast cancer bone metastasis using TM4SF19-Fc

According to the protocol of FIG. 43A, MDA-MB231-luc breast cancer cells (a cell line expressing luciferase in MDA-MB231 for imaging) were injected into the tail of 6 week old female NOD-SCID mice via the tail artery (CA) route to induce bone metastasis. After induction of bone metastasis, hIgG1 or TM4SF19-Fc was injected intravenously. The administration of TM4SF19-Fc at 10mpk or 25mpk was started intravenously two days after cancer cell injection, twice weekly. The status of progression of cancer was confirmed by bioluminescence imaging analysis.

Bone metastasis was detected by bioluminescence imaging analysis 45 days after cancer cell injection. As shown in fig. 43B (left panel), it was confirmed that bone metastasis was inhibited in the hTM4SF19-Fc administration group, compared to the hig 1 administration group as a control. As shown in fig. 43B (right panel), the results of luminescence analysis of the excised legs of mice sacrificed after injection of fluorescein on day 45 confirm that TM4SF19-Fc treatment inhibited bone metastasis.

Fig. 43C shows the results of a micro CT scan of a mouse joint with bone metastasis from breast cancer. From the results of the micro-CT analysis, it was confirmed that bone damage occurred due to bone metastasis of breast cancer, and that bone damage was saved in a dose-dependent manner by TM4SF19-Fc treatment.

In addition, histological analysis of joints of mice with bone metastases of breast cancer was performed by H & E staining. As a result, as shown in fig. 43D, it was confirmed that bone metastasis and tumor growth of breast cancer were dose-dependently inhibited by TM4SF19-Fc treatment.

According to the protocol of 44A, MDA-MB231-luc breast cancer cells (a cell line expressing luciferase in MDA-MB231 by imaging) were injected into the tail artery (CA) of 6-week-old female NOD-SCID mice to induce bone metastasis. From 30 days after induction, intravenous administration of 50mg/kg of control hIgG1 or TM4SF19-F was started.

Bioluminescence imaging was analyzed every 7 days after injection, up to 21 days, and inhibition of bone metastasis by hTM4SF19-Fc (120-169) was confirmed (fig. 44B). After injection of fluorescein, luminescence analysis was performed on the excised legs of mice sacrificed after injection of fluorescein, confirming that TM4SF19-Fc treatment inhibited bone metastasis (fig. 44C). The micro-CT images showed that bone destruction occurred due to bone metastasis of breast cancer and was saved by TM4SF19-Fc treatment (fig. 44D).

Example 15: inhibition of breast cancer lung metastasis using TM4SF19-Fc

Murine E0771 breast cancer cells were injected into the tail vein of wild type mice to induce lung metastasis. Inhibition of breast cancer cell lung metastasis was confirmed by staining cells with printing ink and counting the number of nodules in the lung after intravenous injection of hIgG1-Fc, mouse TM4SF19-Fc (116-165), and human TM4SF19-Fc (145-169) at 10mg/kg each. As shown in FIG. 45, it was confirmed that mouse TM4SF19-Fc (116-165) and human TM4SF19-Fc (145-169) significantly inhibited lung metastasis.

Example 16: inhibition of high fat diet induced obesity and fatty liver by TM4SF19-Fc

After purchasing 6-week old mice fed a high fat diet for 7 weeks and adapting them by feeding a high fat diet for 1 week, mice TM4SF19-Fc were administered to them twice weekly by subcutaneous or intravenous injection while feeding a high fat diet for 9 weeks. As a result, as can be seen from fig. 46A, it was confirmed that obesity caused by high-fat diet was inhibited by administration of 10mg/kg of mTM SF 19-Fc.

As a result of killing the mice and analyzing the white fat distribution in the organs, as shown in fig. 46B, it was confirmed that the weight of white fat was reduced by the TM4SF19-Fc treatment, and also a significant fatty liver inhibitory effect was confirmed.

Furthermore, as a result of observing liver tissue by H & E staining and Masson and trichromatic staining, it can be seen from fig. 47 (left panel) that the effect of reducing fatty liver is clearly shown compared to the control of the hig 1-Fc treatment. Further, triglyceride reduction in plasma was confirmed (fig. 47 (right panel)).

Example 17: inhibition of adipocyte differentiation by TM4SF19-Fc treatment

Treatment of C3H10T1/2 cells with purified mouse TM4SF19-Fc (116-165) confirmed that adipocyte differentiation was inhibited dose-dependently (FIG. 48).

For the control, the differentiation-inducing factor was not treated, for the experimental group, mouse TM4SF 19-165-Fc was treated at 0ng/ml, 1.25ng/ml, 2.5ng/ml, 5ng/ml or 10ng/ml, and the differentiation-inducing factor (treated with dexamethasone (1. Mu.M), IBMX (500. Mu.M), insulin (4. Mu.g/ml, DMI), rosiglitazone (5. Mu.M) in adipocyte differentiation) was treated, and adipocyte differentiation was observed.

Further, as a result of confirming the expression of the major markers of adipocyte differentiation, C/EBP alpha and PPARgamma, it was confirmed that the expression of the DMI-treated group was increased as compared to the undifferentiated group (without DMI), and that the treatment with mouse TM4SF19-Fc (116-165) inhibited the expression.

Example 18: inhibition of cancer cell migration by osteoclast differentiation

(1)TM4SF19-Fc

Inhibition of osteoclast differentiation-induced cancer cell migration by mTM SF19-Fc or hTM4SF19-Fc (120-169) was confirmed by staining with 0.05% crystal violet solution. Bone marrow-derived macrophages were plated on 12 wells and treated with MCSF and RANKL to induce differentiation. At this time, cells were treated with 10. Mu.g/ml mTM SF19-Fc or hTM4SF19-Fc, or cells were not treated with RANKL to disallow differentiation, and then cancer cells MDA-MB231 or PC3M were placed in a migration chamber to allow migration. The migrated cancer cells were stained with crystal violet.

As a result, as shown in fig. 49, it was confirmed that cancer cell migration induced by osteoclast differentiation was inhibited by TM4SF 19-Fc.

(2)TM4SF19KO

Inhibition of cancer cell migration by osteoclast differentiation was confirmed in TM4SF19KO, bone marrow-derived macrophages extracted from WT and TM4SF19KO were treated with MCSF and then induced to differentiate with RANKL, or not treated to differentiate, and then cancer cells MDA-MB231 or PC3M were added in a migration chamber to allow migration. The migrated cancer cells were stained with crystal violet.

In FIG. 50A, the above results show the inhibition of cancer cell migration by osteoclast differentiation in TM4SF19 KO.

(3)TM4SF19EC2Δ

To confirm inhibition of cancer cell migration by osteoclast differentiation in TM4SF19EC2 delta, bone marrow-derived macrophages extracted from WT and TM4SF19EC2 delta were treated with MCSF and then induced to differentiate with RANKL, or not treated to differentiate, and then cancer cells MDA-MB231 or PC3M were placed in a migration chamber to allow migration. The migrated cancer cells were stained with crystal violet.

As a result, fig. 50B shows inhibition of cancer cell migration by osteoclast differentiation in TM4SF19EC2 delta.

Example 19: confirmation of osteosarcoma cell proliferation, migration and colony formation

(1) TM4SF19 overexpression

After stable overexpression of 3Flag-hTM4SF19 in MG63 and HOS osteosarcoma cell lines, cell growth was analyzed by MTT and colony formation and migration were examined. As a result, it was confirmed that TM4SF19 overexpression increased proliferation, migration, and colony formation of osteosarcoma cells (fig. 51, 52).

In addition, after knocking out TM4SF19 in the 143b osteosarcoma cell line by CRISPR, cell growth was checked by MTT analysis and colony formation was checked using different clones (# 4 and # 6).

As a result, it was confirmed that proliferation and colony formation of osteosarcoma cells (143 b) were inhibited by inhibition of expression of TM4SF19 (FIG. 53).

TM4SF19 overexpression increases osteosarcoma cell proliferation and migration. Inhibition of TM4SF19 expression inhibits osteosarcoma cell proliferation and colony formation.

(2) TM4SF19-Fc treatment

Cell growth was checked by MTT assay and cell counting by treatment of 143b osteosarcoma cell line with hTM4SF19-Fc (120-169), colonisation ability was checked by colony formation, and cell migration was checked.

Inhibition of 143B osteosarcoma cell growth by TM4SF19-Fc treatment was confirmed by MTT analysis (fig. 54A) and cell counting (fig. 54C), colonisation ability was confirmed by colony formation (fig. 54B), and cell migration was confirmed (fig. 54D).

It was confirmed that U2OS and MG63 osteosarcoma cell colony formation was inhibited by treatment with human TM4SF19-Fc (aa 120-169) (10. Mu.g/ml) (FIG. 55).

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

It was determined that hTM4SF19-Fc comprising at least 145-169aa was active.

Example 20: confirmation of inhibition of pancreatic cancer cell growth and colony formation

After treatment of pancreatic cancer cell lines Panc-1 and MIA-PaCa2 with 100. Mu.g/ml of hTM4SF19-Fc (120-169), the ability of the cells to colonize was examined by a colony formation assay. As a result, it can be seen from FIG. 57A that hTM4SF19-Fc (120-169) was confirmed to inhibit the colonisation ability of the pancreatic cancer cell line. In addition, pancreatic cancer cell lines Panc-1, MIA-PaCa2 and AsPC-1 were treated with either 62.5. Mu.g/ml or 125. Mu.g/ml of hTM4SF19-Fc (120-169), and inhibition of cell growth was detected by cell counting (FIG. 57B). As a result, it was confirmed that the growth and colonisation ability of pancreatic cancer cells were inhibited by TM4SF19-Fc treatment.

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Lys Asp Gly Pro Phe Cys Met Phe Asp Val Ser Ser Phe Asn Gln Thr

1 5 10 15

Gln Ala Trp Lys Phe Gly Tyr Pro Phe Lys Asp Leu His Asn Arg Asn

20 25 30

Tyr Leu Tyr Asp Arg Ser Leu Trp Thr Ser Val Cys Leu Glu Pro Ser

35 40 45

Lys Ala

50

<210> 9

<211> 232

<212> PRT

<213> artificial sequence

<220>

<223> hIgG1-Fc

<400> 9

Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

20 25 30

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

35 40 45

Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val

50 55 60

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

65 70 75 80

Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

85 90 95

Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala

100 105 110

Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro

115 120 125

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr

130 135 140

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

145 150 155 160

Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr

165 170 175

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

180 185 190

Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe

195 200 205

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

210 215 220

Ser Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 10

<211> 284

<212> PRT

<213> artificial sequence

<220>

<223> mouse TM4SF19 (116-165) -Fc fusion protein

<400> 10

Lys Asp Gly Pro Phe Cys Met Phe Asp Val Ser Ser Phe Asn Gln Thr

1 5 10 15

Gln Ala Trp Lys Phe Gly Tyr Pro Phe Lys Asp Leu His Asn Arg Asn

20 25 30

Tyr Leu Tyr Asp Arg Ser Leu Trp Thr Ser Val Cys Leu Glu Pro Ser

35 40 45

Lys Ala Thr Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

50 55 60

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

65 70 75 80

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

85 90 95

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

100 105 110

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

115 120 125

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

130 135 140

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

145 150 155 160

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

165 170 175

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

180 185 190

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

195 200 205

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

210 215 220

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

225 230 235 240

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

245 250 255

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

260 265 270

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

275 280

<210> 11

<211> 855

<212> DNA

<213> artificial sequence

<220>

<223> nucleotide encoding mouse TM4SF19 (116-165) -Fc fusion protein

<400> 11

aaagatggtc ccttttgcat gtttgatgtc tcatccttca atcagacaca agcttggaaa 60

ttcggctatc cctttaaaga tctacacaac aggaattatc tgtatgaccg ttcactttgg 120

acctccgttt gcctggagcc ctctaaggct actagtgagc ccaaatcttg tgacaaaact 180

cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc 240

cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 300

gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 360

gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 420

agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 480

tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc 540

cgagaaccac aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc 600

agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc 660

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 720

ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 780

tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg 840

tctccgggta aatga 855

<210> 12

<211> 50

<212> PRT

<213> Chile person

<400> 12

Lys Asp Gly Pro Phe Cys Met Phe Asp Val Ser Ser Phe Asn Gln Thr

1 5 10 15

Gln Ala Trp Lys Tyr Gly Tyr Pro Phe Lys Asp Leu His Ser Arg Asn

20 25 30

Tyr Leu Tyr Asp Arg Ser Leu Trp Asn Ser Val Cys Leu Glu Pro Ser

35 40 45

Ala Ala

50

<210> 13

<211> 284

<212> PRT

<213> artificial sequence

<220>

<223> human TM4SF19 (120-169) -Fc fusion protein

<400> 13

Lys Asp Gly Pro Phe Cys Met Phe Asp Val Ser Ser Phe Asn Gln Thr

1 5 10 15

Gln Ala Trp Lys Tyr Gly Tyr Pro Phe Lys Asp Leu His Ser Arg Asn

20 25 30

Tyr Leu Tyr Asp Arg Ser Leu Trp Asn Ser Val Cys Leu Glu Pro Ser

35 40 45

Ala Ala Thr Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro

50 55 60

Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe

65 70 75 80

Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val

85 90 95

Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe

100 105 110

Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro

115 120 125

Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr

130 135 140

Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val

145 150 155 160

Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala

165 170 175

Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg

180 185 190

Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly

195 200 205

Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro

210 215 220

Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser

225 230 235 240

Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln

245 250 255

Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His

260 265 270

Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

275 280

<210> 14

<211> 855

<212> DNA

<213> artificial sequence

<220>

<223> nucleotide encoding human TM4SF19 (120-169) -Fc fusion protein

<400> 14

aaagatggtc ctttttgcat gtttgatgtt tcatccttca atcagacaca agcttggaaa 60

tatggttacc cattcaaaga cctgcatagt aggaattatc tgtatgaccg ttcgctctgg 120

aactccgtct gcctggagcc ctctgcagct actagtgagc ccaaatcttg tgacaaaact 180

cacacatgcc caccgtgccc agcacctgaa ctcctggggg gaccgtcagt cttcctcttc 240

cccccaaaac ccaaggacac cctcatgatc tcccggaccc ctgaggtcac atgcgtggtg 300

gtggacgtga gccacgaaga ccctgaggtc aagttcaact ggtacgtgga cggcgtggag 360

gtgcataatg ccaagacaaa gccgcgggag gagcagtaca acagcacgta ccgtgtggtc 420

agcgtcctca ccgtcctgca ccaggactgg ctgaatggca aggagtacaa gtgcaaggtc 480

tccaacaaag ccctcccagc ccccatcgag aaaaccatct ccaaagccaa agggcagccc 540

cgagaaccac aggtgtacac cctgccccca tcccgggatg agctgaccaa gaaccaggtc 600

agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga gtgggagagc 660

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 720

ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg gaacgtcttc 780

tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag cctctccctg 840

tctccgggta aatga 855

<210> 15

<211> 25

<212> PRT

<213> Chile person

<400> 15

Lys Asp Leu His Ser Arg Asn Tyr Leu Tyr Asp Arg Ser Leu Trp Asn

1 5 10 15

Ser Val Cys Leu Glu Pro Ser Ala Ala

20 25

<210> 16

<211> 259

<212> PRT

<213> artificial sequence

<220>

<223> human TM4SF19 (145-169) -Fc fusion protein

<400> 16

Lys Asp Leu His Ser Arg Asn Tyr Leu Tyr Asp Arg Ser Leu Trp Asn

1 5 10 15

Ser Val Cys Leu Glu Pro Ser Ala Ala Thr Ser Glu Pro Lys Ser Cys

20 25 30

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

35 40 45

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

50 55 60

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

65 70 75 80

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

85 90 95

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

100 105 110

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

115 120 125

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

130 135 140

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

145 150 155 160

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

165 170 175

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

180 185 190

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

195 200 205

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

210 215 220

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

225 230 235 240

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

245 250 255

Pro Gly Lys

<210> 17

<211> 780

<212> DNA

<213> artificial sequence

<220>

<223> nucleotide encoding human TM4SF19 (145-169) -Fc fusion protein

<400> 17

aaagacctgc atagtaggaa ttatctgtat gaccgttcgc tctggaactc cgtctgcctg 60

gagccctctg cagctactag tgagcccaaa tcttgtgaca aaactcacac atgcccaccg 120

tgcccagcac ctgaactcct ggggggaccg tcagtcttcc tcttcccccc aaaacccaag 180

gacaccctca tgatctcccg gacccctgag gtcacatgcg tggtggtgga cgtgagccac 240

gaagaccctg aggtcaagtt caactggtac gtggacggcg tggaggtgca taatgccaag 300

acaaagccgc gggaggagca gtacaacagc acgtaccgtg tggtcagcgt cctcaccgtc 360

ctgcaccagg actggctgaa tggcaaggag tacaagtgca aggtctccaa caaagccctc 420

ccagccccca tcgagaaaac catctccaaa gccaaagggc agccccgaga accacaggtg 480

tacaccctgc ccccatcccg ggatgagctg accaagaacc aggtcagcct gacctgcctg 540

gtcaaaggct tctatcccag cgacatcgcc gtggagtggg agagcaatgg gcagccggag 600

aacaactaca agaccacgcc tcccgtgctg gactccgacg gctccttctt cctctacagc 660

aagctcaccg tggacaagag caggtggcag caggggaacg tcttctcatg ctccgtgatg 720

catgaggctc tgcacaacca ctacacgcag aagagcctct ccctgtctcc gggtaaatga 780

780

<210> 18

<211> 39

<212> PRT

<213> Chile person

<400> 18

Ser Phe Asn Gln Thr Gln Ala Trp Lys Tyr Gly Tyr Pro Phe Lys Asp

1 5 10 15

Leu His Ser Arg Asn Tyr Leu Tyr Asp Arg Ser Leu Trp Asn Ser Val

20 25 30

Cys Leu Glu Pro Ser Ala Ala

35

<210> 19

<211> 273

<212> PRT

<213> artificial sequence

<220>

<223> human TM4SF19 (131-169) -Fc fusion protein

<400> 19

Ser Phe Asn Gln Thr Gln Ala Trp Lys Tyr Gly Tyr Pro Phe Lys Asp

1 5 10 15

Leu His Ser Arg Asn Tyr Leu Tyr Asp Arg Ser Leu Trp Asn Ser Val

20 25 30

Cys Leu Glu Pro Ser Ala Ala Thr Ser Glu Pro Lys Ser Cys Asp Lys

35 40 45

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

50 55 60

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

65 70 75 80

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

85 90 95

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

100 105 110

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

115 120 125

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

130 135 140

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

145 150 155 160

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

165 170 175

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

180 185 190

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

195 200 205

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

210 215 220

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

225 230 235 240

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

245 250 255

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

260 265 270

Lys

<210> 20

<211> 822

<212> DNA

<213> artificial sequence

<220>

<223> nucleotide encoding human TM4SF19 (131-169) -Fc fusion protein

<400> 20

tccttcaatc agacacaagc ttggaaatat ggttacccat tcaaagacct gcatagtagg 60

aattatctgt atgaccgttc gctctggaac tccgtctgcc tggagccctc tgcagctact 120

agtgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 180

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 240

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 300

ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 360

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 420

aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 480

accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 540

cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 600

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 660

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 720

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 780

cactacacgc agaagagcct ctccctgtct ccgggtaaat ga 822

<210> 21

<211> 61

<212> PRT

<213> Chile person

<400> 21

Ser Gly Val Ala Leu Lys Asp Gly Pro Phe Cys Met Phe Asp Val Ser

1 5 10 15

Ser Phe Asn Gln Thr Gln Ala Trp Lys Tyr Gly Tyr Pro Phe Lys Asp

20 25 30

Leu His Ser Arg Asn Tyr Leu Tyr Asp Arg Ser Leu Trp Asn Ser Val

35 40 45

Cys Leu Glu Pro Ser Ala Ala Val Val Trp His Val Ser

50 55 60

Claims (29)

1. A pharmaceutical composition for preventing or treating bone diseases, comprising:

an inhibitor of the expression or activity of transmembrane 4L six family member 19 (TM 4SF 19) as an active ingredient.

2. The composition of claim 1, wherein the inhibitor of TM4SF19 expression or activity is one or more selected from the group consisting of antisense nucleotides, small hairpin RNAs (shrnas), small interfering RNAs (sirnas), micrornas (mirnas), and ribozymes that complementarily bind to mRNA of TM4SF19 gene.

3. The composition of claim 1, wherein the inhibitor of expression or activity of TM4SF19 is one or more selected from the group consisting of a compound that specifically binds TM4SF19 protein, a peptide, a peptidomimetic, an aptamer, a fusion protein, and an antibody.

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

5. The composition of claim 4, wherein the TM4SF19 fragment comprises all or a portion of the extracellular loop 2 region of TM4SF 19.

6. The composition of claim 1, wherein the bone disease is one or more selected from the group consisting of metabolic bone disease, orthopedic bone disease, aplastic bone disease, degenerative arthritis, rheumatoid arthritis, psoriatic spondylitis, age-related bone loss, osteoporosis, osteogenesis imperfecta, osteomalacia, sarcopenia, fractures, bone defects and hip arthropathy, rickets, paget's disease, periodontal disease, and bone damage caused by cancer cell bone metastasis.

7. A method of screening for a drug for treating a bone disorder comprising:

Treating a sample suspected of bone disease with a candidate for treating bone disease; and

mRNA or protein expression levels of the TM4SF19 gene were compared to controls.

8. A pharmaceutical composition for preventing or treating obesity or an obesity-mediated metabolic disease comprising:

inhibitors of transmembrane 4L six family member 19 (TM 4SF 19) expression or activity as an active ingredient.

9. The composition of claim 8, wherein the inhibitor of TM4SF19 expression or activity is one or more selected from the group consisting of antisense nucleotides, small hairpin RNAs (shrnas), small interfering RNAs (sirnas), micrornas (mirnas), and ribozymes that complementarily bind to mRNA of TM4SF19 gene.

10. The composition of claim 8, wherein the inhibitor of expression or activity of TM4SF19 is one or more selected from the group consisting of a compound that specifically binds TM4SF19 protein, a peptide, a peptidomimetic, an aptamer, a fusion protein, and an antibody.

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

12. The composition of claim 11, wherein the TM4SF19 fragment comprises all or a portion of the extracellular loop 2 region of TM4SF 19.

13. The composition of claim 8, wherein the obesity-mediated metabolic disease is one or more selected from the group consisting of diabetes, hypertension, hyperlipidemia, nonalcoholic steatohepatitis, and specific cancers, more broadly comprising hypertension, diabetes, insulin resistance syndrome, metabolic syndrome, obesity-related gastroesophageal reflux disease, arteriosclerosis, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, lipodystrophy, nonalcoholic steatohepatitis, cardiovascular disease, and polycystic ovary syndrome.

14. A method of screening for a drug for treating obesity or an obesity-mediated metabolic disease comprising:

treating a sample suspected of being obese or obesity-mediated metabolic disease with a candidate substance for treating obesity or obesity-mediated metabolic disease; and

mRNA or protein expression levels of the TM4SF19 gene were compared to controls.

15. A pharmaceutical composition for preventing or treating cancer or inhibiting cancer metastasis, comprising an inhibitor of transmembrane 4L six family member 19 (TM 4SF 19) expression or activity as an active ingredient.

16. The composition of claim 15, wherein the inhibitor of TM4SF19 expression or activity is one or more selected from the group consisting of antisense nucleotides, small hairpin RNAs (shRNA), small interfering RNAs (siRNA), micrornas (miRNA) and ribozymes that complementarily bind to mRNA of TM4SF19 gene.

17. The composition of claim 15, wherein the inhibitor of expression or activity of TM4SF19 is one or more selected from the group consisting of a compound that specifically binds TM4SF19 protein, a peptide, a peptidomimetic, an aptamer, a fusion protein, and an antibody.

18. The composition of claim 15, wherein the fusion protein that specifically binds TM4SF19 protein comprises a TM4SF19 fragment and the immunoglobulin Fc domain.

19. The composition of claim 18, wherein the TM4SF19 fragment comprises all or a portion of the extracellular loop 2 region of TM4SF 19.

20. The composition of claim 15, wherein the cancer is one or more 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, lymphoid cancer, bladder cancer, gall bladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, sarcoma of soft tissue, cancer of the urinary tract, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, renal or ureteral cancer, renal cell carcinoma, renal pelvis cancer, central Nervous System (CNS) tumors, spinal cord tumors, brain stem glioma, and pituitary adenoma.

21. A method of screening for a drug for treating cancer or cancer metastasis comprising:

treating a sample suspected of cancer or cancer metastasis with a candidate for preventing or treating cancer or inhibiting cancer metastasis; and

mRNA or protein expression levels of the TM4SF19 gene were compared to controls.

22. A fusion protein for inhibiting the expression or activity of a transmembrane 4L six family member 19 (TM 4SF 19) comprising an extracellular loop 2-derived fragment of TM4SF19 protein and the immunoglobulin Fc domain.

23. The fusion protein of claim 22, wherein the extracellular loop 2 (EC 2) -derived fragment of the TM4SF19 protein corresponds to all or a portion of EC 2.

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

25. The fusion protein of claim 22, wherein the region of amino acid sequence corresponding to a portion of EC2 is the region of amino acids 145-169 of the human TM4SF19 protein.

26. The fusion protein of claim 22, wherein the region of amino acid sequence corresponding to a portion of EC2 is the region of amino acids 145-169 of the human TM4SF19 protein.

27. A method of preventing or treating a bone disorder comprising:

a therapeutically effective amount of a composition for preventing or treating a bone disease comprising an inhibitor of transmembrane 4L six family member 19 (TM 4SF 19) expression or activity is administered to a subject.

28. A method of preventing or treating obesity or an obesity-mediated metabolic disease comprising:

a therapeutically effective amount of a composition for preventing or treating obesity or an obesity-mediated metabolic disease comprising an inhibitor of transmembrane 4L six family member 19 (TM 4SF 19) expression or activity is administered to a subject.

29. A method of preventing or treating cancer or cancer metastasis comprising:

a therapeutically effective amount of a composition for preventing or treating cancer or inhibiting cancer metastasis comprising an inhibitor of transmembrane 4L six family member 19 (TM 4SF 19) expression or activity is administered to a subject.