WO2022255843A1 - Tm4sf19 억제제 및 이의 용도 - Google Patents
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- WO2022255843A1 WO2022255843A1 PCT/KR2022/007939 KR2022007939W WO2022255843A1 WO 2022255843 A1 WO2022255843 A1 WO 2022255843A1 KR 2022007939 W KR2022007939 W KR 2022007939W WO 2022255843 A1 WO2022255843 A1 WO 2022255843A1
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- tm4sf19
- cancer
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- obesity
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Definitions
- the present invention relates to a composition for preventing or treating bone disease, obesity or obesity-mediated metabolic disease, cancer, cancer metastasis, comprising an inhibitor of the expression or activity of TM4SF19 (transmembrane 4 L six family member 19; transmembrane 4L 6 family member 19), the above It relates to a method for screening a drug for treating a disease.
- TM4SF19 transmembrane 4 L six family member 19; transmembrane 4L 6 family member 19
- Bone is an active tissue that continuously changes throughout life. Bone is visually divided into external cortical bone (compact bone) and internal trabecular bone (cancellous bone, spongy bone). Cortical bone has strong physical strength and plays a role in protecting and supporting the body. plays a role in maintaining constant changes in
- bone remodeling Even after bone growth is stopped, old bones are destroyed and lost (bone resorption), and fixation (bone formation) filling in the missing new bones is repeated throughout life, this phenomenon is called bone remodeling.
- osteoblast The interaction between bone formation by osteoblast and bone resorption by osteoclast is balanced to maintain bone homeostasis and maintain a constant level of calcium in the blood. related diseases occur.
- Bone mass is determined by The process of bone remodeling occurs in a balanced manner by osteoclasts undergoing bone resorption and then bone formation by osteoblasts being coupled. When this efficient balance is disrupted, bone loss occurs.
- Osteoclasts become multinucleated osteoclasts through a differentiation process, and bone resorption occurs by multinucleated osteoclasts. Dysfunction of osteoclasts causes osteopetrosis, and increased bone resorption due to overactivation of osteoclasts causes osteoporosis and inflammatory arthritis due to menopause. So far, bisphosphonates and anti-RANKL antibodies have been developed to target the overactivation of osteoclasts, but the overall inhibition of osteoclasts affects the balance between osteoclasts and osteoblasts and has a profound effect on bone formation. , and side effects due to this overall inhibition are also accompanied. Therefore, the development of a therapeutic agent capable of more selectively inhibiting osteoclasts is required.
- Osteoclasts originate from hematopoietic stem cells, and monocytes/macrophages are activated by M-CSF (monocyte/macrophage colony-stimulating factor) stimulation and RANK (receptor activator of nuclear factor ⁇ B) ligand (RANKL). It is a multinucleated cell that differentiates from the lineage. In the differentiation of osteoclasts, osteoclast precursors differentiate into TRAP-positive mononuclear osteoclasts, which then mature into multinucleated osteoclasts through cell-cell fusion [see Fig. 2c]. . These mature, multinucleated osteoclasts cause bone resorption [Int J Mol Sci. 2020 Aug 8;21(16):5685.].
- Obesity is a biological phenomenon caused by complex interactions of genetic, metabolic, environmental, and behavioral factors, and is defined as abnormal or excessive fat accumulation, which can adversely affect health.
- obesity is known to be an important risk factor that causes various adult diseases such as hypertension, type 2 diabetes, cancer, liver disease, hyperlipidemia, and arteriosclerosis.
- Obesity is a result of chronic imbalance between caloric intake and energy consumption, accompanied by loss of adipose tissue metabolism, endocrine and immune functions, and metabolic abnormalities and reduced responsiveness (resistance) to insulin, a major fat storage signaling hormone. This causes fat accumulation in metabolic organs other than adipose tissue, resulting in various lipotoxicities.
- the most representative pathology of lipotoxicity is an inflammatory response, which can be characterized as a chronic low-grade inflammation.
- adipose tissue macrophages are activated and enter the pre-inflammatory stage. As such, obesity induces a low-intensity chronic inflammatory state in the body, causing various metabolic diseases.
- macrophages in adipose tissue play an important role in the occurrence of chronic inflammation and metabolic complications in the body caused by obesity.
- Fat stored in adipocytes is used as an important energy source in the body. Obesity is induced by excessive differentiation of fat cells and excessive supply of unbalanced energy.
- adipose tissue not only fat cells, but also various cells such as adipose-derived stem cells (ASC), immune cells, and endothelial cells exist in the tissue, which is called the stromal vascular fraction (SVF).
- Adipose tissue stores energy in the form of lipids, but also serves to keep the body warm. It is divided into white adipose tissue (WAT), which stores nutrients, and brown adipose tissue (BAT), which consumes nutrients and generates heat.
- WAT white adipose tissue
- BAT brown adipose tissue
- adipose tissue also produces leptin, resistin, adiponectin, tumor necrosis factor- ⁇ (TNF ⁇ ), and the like.
- Adipocyte differentiation is very complex through the interaction of various hormones and various transcription factors. It is promoted by stimulation of insulin, insulin like growth factor-1, or growth hormone, and CCAAT is involved 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.
- C/EBP C/EBP
- PPAR peroxisome proliferator-activated receptor
- These transcription factors along with adipocyte regulators, promote differentiation of adipocytes and increase the expression of enzymes such as aP2, a fatty acid binding protein, and fatty acid synthase. On the other hand, it has been reported that the accumulation of excessive neutral fat is involved in the progression of fatty liver [J. Clin. Invest., 98, 1575 15
- Fat absorption inhibitors such as Xenical of Roche in Switzerland and anorexia agents such as Meridia of Abbott in the United States are currently used as obesity treatment drugs, but these drugs have problems such as headache, blood pressure increase, and diarrhea. . Therefore, it is necessary to develop a new target anti-obesity drug without side effects.
- Cancer is one of the greatest diseases threatening human health, and is a disease caused by cell proliferation and immortality in an unlimited and uncontrolled way through a series of mutation processes. In the case of cancer detected at an early stage, there are treatments such as surgery, radiation therapy, and chemotherapy, but the side effects are emerging as a big problem.
- TM4SF19 is a transmembrane 4L six family member 19 (TM4SF19), and Korean Patent No. 10-1781200 [Korean Patent No. 10-1781200] describes the use of TM4SF19 as a marker for diagnosis of obesity. have been initiated However, there has been no report on the treatment of bone-related diseases, the treatment of obesity or obesity-related metabolic diseases, the treatment of cancer, or the inhibitory effect of cancer metastasis with respect to TM4SF19.
- the present inventors aimed to find genes related to bone diseases, obesity and various metabolic diseases mediated by obesity, cancer treatment or fundamental treatment of cancer metastasis inhibition, targeting various genes for bone diseases, obesity or obesity-mediated metabolic diseases, cancer A study was conducted on the relationship with, and as a result, the present invention was completed by confirming various functions of TM4SF19.
- An object of the present invention is to provide a pharmaceutical composition for preventing or treating bone disease comprising a TM4SF19 expression or activity inhibitor as an active ingredient.
- Another object of the present invention is to treat a bone disease-suspected specimen with a candidate substance for treating bone disease; and comparing the mRNA or protein expression level of the TM4SF19 gene with that of the control group.
- Another object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity or obesity-mediated metabolic diseases comprising a TM4SF19 expression or activity inhibitor as an active ingredient.
- Another object of the present invention is to treat obesity or obesity-mediated metabolic disease suspected sample with a candidate for treatment of obesity or obesity-mediated metabolic disease, and compare the expression level of mRNA or protein of the TM4SF19 gene with a control group. , To provide a screening method for drugs for treating obesity or obesity-mediated metabolic diseases.
- Another object of the present invention is to provide a pharmaceutical composition for preventing, treating or inhibiting cancer metastasis comprising a TM4SF19 expression or activity inhibitor as an active ingredient.
- Another object of the present invention is to treat cancer prevention, treatment, or cancer metastasis-suspected specimens with candidates for cancer prevention, treatment, or inhibition of cancer metastasis, and to compare the mRNA or protein expression levels of the TM4SF19 gene with a control group. It is to provide a method for screening a drug for cancer treatment or cancer metastasis treatment, including.
- Another object of the present invention is to provide a fusion protein for inhibiting TM4SF19 expression or activity.
- Another object of the present invention is to provide a method for preventing or treating bone disease comprising administering to a subject a composition for preventing or treating bone disease comprising a therapeutically effective amount of a TM4SF19 expression or activity inhibitor.
- Another object of the present invention is to prevent or prevent obesity or obesity-mediated metabolic diseases, comprising administering to a subject a composition for preventing or treating obesity or obesity-mediated metabolic diseases comprising a therapeutically effective amount of a TM4SF19 expression or activity inhibitor. It is to provide a way to treat.
- Another object of the present invention is to prevent or treat cancer, including administering to a subject a composition for preventing, treating, or inhibiting cancer metastasis comprising a therapeutically effective amount of a TM4SF19 expression or activity inhibitor. It is to provide a way to treat.
- TM4SF19 expression or activity inhibitors according to the present invention can be used for preventing or treating bone diseases, obesity, obesity-mediated metabolic diseases, cancer, and cancer metastasis.
- candidates for treating bone diseases, obesity, obesity-mediated metabolic diseases, cancer, and cancer metastasis can be efficiently selected.
- TM4SF19 gene expression by qPCR after cells were extracted from mouse bone marrow, differentiated over time by treatment with M-CSF and RANKL (Receptor activator of nuclear factor kappa- ⁇ ligand).
- FIG. 2a shows the results of TRAP staining after differentiation of wild-type (WT) mice and mouse bone marrow-derived cells of TM4SF19KO, a CRISPR knockout mouse, by treatment with M-CSF (25 ng/ml) + RANKL (100 ng/ml).
- Figure 2b shows that cells were extracted from the bone marrow of TM4SF19KO, a TM4SF19 knockout mouse using CRISPR, and a wild-type (WT) mouse, and the concentration of RANKL was fixed at 100 ng/ml and the concentration of M-CSF was varied at 25, 40, and 60 ng/ml.
- TRAP staining tissuerate-resistant acid phosphatase staining
- an osteoclast-related marker are shown.
- Figure 2c is a schematic diagram showing the osteoclast differentiation process.
- FIG. 3 shows that cells were extracted from the bone marrow of wild-type (WT) mice and TM4SF19KO mice and treated with M-CSF or M-CSF + RANKL (M-CSF 25 ng / ml, RANKL 100 ng / ml). This is the result of confirming the expression of Ctsk, Acp5, c-Fos, and Nfatc1, which are target genes related to osteoclast (OC) differentiation, after 4 days of differentiation by qPCR.
- Figure 4a confirms the formation of the actin belt formed during multinuclear osteoclast differentiation after differentiating wild-type and TM4SF19KO mouse bone marrow by F-actin staining.
- 4b shows pit formation confirmed by 1% toluidine blue staining after BMM cells were plated and differentiated on dentin discs.
- FIG. 5a shows the results of micro CT analysis after fixing the femurs of 8-week-old wild-type (WT) and TM4SF19KO female mice on day 31 by shaming or ovariectomy (OVX) without removing the ovary.
- Figure 5b shows the bone volume (BV) obtained by analyzing the micro-CT images with a 3-dimensional image analysis program, the ratio of bone volume to tissue volume (% BV/TV), the average number of bone trabeculae (Tb.N), and bone trabeculae It shows 3-dimensional micro CT indicators (bone microstructure indicators) including gap (Tb.Sp) and bone trabecular thickness (Tb.Th).
- BV bone volume
- % BV/TV the ratio of bone volume to tissue volume
- Tb.N average number of bone trabeculae
- Tb.Th bone trabecular thickness
- FIG. 6 shows the results of TRAP staining after fixing and decalcification of femurs of 8-week-old wild-type (WT) mice and TM4SF19KO mice.
- Figure 7a shows cells extracted from the bone marrow of wild-type (WT) mice and TM4SF19EC2 ⁇ mice knocked out TM4SF19 extracellular domain 2 (116-165) by CRISPR, and treated with 25 ng/ml M-CSF or 100 ng/ml M-CSF + RANKL. This is the result of TRAP staining after differentiation.
- Figure 7b shows cells extracted from the bone marrow of wild-type (WT) mice and TM4SF19EC2 ⁇ mice, the concentration of RANKL was fixed at 100 ng/ml, and the concentration of M-CSF was treated at 25 ng/ml or 100 ng/ml to differentiate, and TRAP It is the result of dyeing.
- FIG. 1 shows cells extracted from the bone marrow of wild-type (WT) mice and TM4SF19EC2 ⁇ mice knocked out TM4SF19 extracellular domain 2 (116-165) by CRISPR, and treated with 25 ng/ml M-C
- FIG. 7c shows whether the actin belt formed during differentiation of multinucleated osteoclasts after differentiating wild-type and TM4SF19EC2 ⁇ mouse bone marrow was confirmed by F-actin staining.
- FIG. 7d shows wild-type and TM4SF19EC2 ⁇ BMM cells plated on dentin discs and differentiated, and pit formation was confirmed by 1% toluidine blue staining.
- FIG. 9a shows micro CT analysis results of 8-week-old WT and TM4SF19EC2 ⁇ female mice by fixing the femurs 31 days after sham or ovariectomy (OVX) without removing the ovary.
- BV bone volume
- % BV/TV ratio of bone volume to tissue volume
- Tb.N average number of bone trabeculae
- Tb.Th bone trabecular thickness
- FIG. 10 shows micro CT analysis results of 8-week-old wild-type (WT), TM4SF19KO, and TM4SF19EC2 ⁇ female mice after shaming or ovariectomy (OVX) without removing the ovaries and fixing the femur 31 days later, bone microstructure show indicators.
- FIG. 11 shows micro CT scan images of 8-week-old wild-type (WT), TM4SF19KO, and TM4SF19EC2 ⁇ female mice after sham or ovariectomy (OVX) without removing the ovary and fixing the femur 31 days later.
- WT wild-type
- TM4SF19KO wild-type
- OVX ovariectomy
- FIG. 12a is a diagram schematically illustrating structures of hTm4sf19 variants (hTM4SF19 115-175 ⁇ , hTm4sf19 105-186 ⁇ , hTm4sf19 105-196 ⁇ , hTm4sf19 94-186 ⁇ , and hTm4sf19 94-196 ⁇ ) in which a part of TM4SF19 sequence was deleted.
- Figure 12b is a result showing that TM4SF19 binds itself and is involved in cell-to-cell interactions. This is a result showing that the 94-196 deletion mutant among mutants in which some sequences of TM4SF19 are deleted does not bind to TM4SF19 itself.
- Figure 12c is of hTm4sf19 The remaining sequences were deleted leaving only the 94-196 sequence. The results (bottom) show that hTm4sf19 94-196 (top) binds itself similarly to the wild type (WT) and is involved in cell-to-cell interactions.
- FIG. 12c is of hTm4sf19 The remaining sequences were deleted leaving only the 94-196 sequence. The results (bottom) show that hTm4sf19 94-196 (top) binds itself similarly to the wild type (WT) and is involved in cell-to-cell interactions.
- 12D and 12E are HTM4SF19 131 ⁇ 160, HTM4SF19 145 ⁇ 169, HTM4SF19 131 ⁇ 169, HTM4SF19 120 ⁇ 160, HTM4SF19 120 ⁇ 169, HTM4SF19 120 ⁇ 180, HTM4SF19 120 ⁇ 166, HTM4SF19 120 ⁇ 166 , The structures of hTm4sf19 120-209, hTm4sf19 131-196, and hTm4sf19 145-196 and the interaction between TM4SF19 tagged with 3Flag at the N-terminus and the hIgG1-Fc fusion protein of the TM4SF19 fragment were confirmed by immunoprecipitation.
- 12f shows the result of immunoprecipitation of the interaction between integrin ⁇ v tagged with 3HA at the C-terminus and the hIgG1-Fc fusion protein of the TM4SF19 fragment.
- 12g shows the result of immunoprecipitation of the interaction between integrin ⁇ 3 tagged with 3HA at the C-terminus and the hIgG1-Fc fusion protein of the TM4SF19 fragment.
- 12h shows integrin ⁇ v or integrin ⁇ 3, a protein that regulates TM4SF19 and osteoclast function and is involved in cytoskeleton rearrangement during multinuclear osteoclast formation, in 293T cells or osteoclast precursor Raw 264.7 cells, and then confirms the interaction. .
- Figure 13 shows the results of confirming cell proliferation, colony formation, and cell migration by overexpressing TM4SF19 in mouse-derived breast cancer cells E0771 [LPCX: retroviral control vector name].
- FIG. 15 shows bone marrow from wild-type (WT) mice and TM4SF19KO mice, which are differentiated into bone marrow-derived macrophages, co-cultured with breast cancer cells, and cell migration.
- Target genes involved in cell migration CDH2, SNAI1, SNAI2 expression (Fig. 15a) was confirmed by qPCR (Fig. 15a) and metastasis-related proteins (vimentin, slug, bnail, E-cadherin, beta-actin) expression This is the result confirmed by Western blotting (FIG. 15B) [BMDM: bone marrow derived macrophages].
- Figure 16 shows that after differentiating human adipose-derived mesenchymal stem cells (hADMSC) into adipocytes, gene (C/EBP ⁇ , PPAR ⁇ ) expression was confirmed by qPCR (upper part) and protein (TM4SF19, PPAR ⁇ , FABP4) expression was confirmed by Western blotting. This is the result of checking (bottom).
- Figure 17 shows the results of confirming the body weight gain while feeding a high-fat diet (60% fat) for 16 weeks to 6-week-old wild-type (WT) mice and TM4SF19KO mice fed a normal diet.
- Figure 18 is a 6-week-old wild-type (WT) mouse and TM4SF19KO mice fed a normal diet were fed a high-fat diet for 12 weeks, and then insulin resistance was confirmed by HOMA-IR (A), liver phenotype was confirmed, and liver tissue After fixation, H&E staining (B), and the result of measuring the triglyceride level in the liver (C) are shown.
- WT wild-type
- TM4SF19KO mice fed a normal diet were fed a high-fat diet for 12 weeks, and then insulin resistance was confirmed by HOMA-IR (A), liver phenotype was confirmed, and liver tissue After fixation, H&E staining (B), and the result of measuring the triglyceride level in the liver (C) are shown.
- FIG 19 shows the weight gain (a) and the weight of each tissue (b) in 6-week-old wild-type (WT) mice and TM4SF19KO mice fed a normal diet and fed a high-fat diet for 18 weeks, subcutaneous fat and visceral fat weight ( c), and H&E staining of epididymal white adipose tissue (eWAT) confirmed the phenotype of fat and macrophages of adipose tissue around fat (d), and anti-obesity cytokine in serum The secretion of adiponectin was also confirmed [rtWAT: retroperitoenal white adipose tissue, sWAT: subcutaneous white adipose tissue, ingWAT: inguinal white adipose tissue, iWAT: interscapular white adipose tissue, iBAT: interscapular brown adipose tissue].
- eWAT epididymal white adipose tissue
- sWAT subcutaneous white adipose tissue
- Figure 21 shows macrophage (A) % and dendritic cell (B) % confirmed by FACS analysis of epididymal white adipose tissue after 6-week-old wild-type (WT) mice and TM4SF19KO mice fed a normal diet were fed a high-fat diet for 12 weeks. to be.
- FIG. 22 shows the expression of macrophage marker genes MCP1 and F4/80 in the stromal vascular fraction (SVF) in epididymal white adipose tissue after feeding wild-type (WT) and TM4SF19KO mice a high-fat diet for 12 weeks, M1- This is a result of confirming the expression of IL6 and TNF ⁇ , which are macrophage-like markers, and the expression of IL10, which is an M2-like macrophage marker.
- SVF stromal vascular fraction
- Figure 23 is a result showing the role of TM4SF19 on inflammation, insulin resistance and fatty liver in eWAT.
- 24a is a result confirming the conversion from epididymal white fat to beige or brown fat phenotype in TM4SF19KO mice in a high-fat diet-induced obese mouse model fed for 12 weeks by starting to give a high-fat diet to 6-week-old mice fed a normal diet.
- Figure 24b is a result confirming that Ucp1 expression increased in white fat of TM4SF19KO mice in a high-fat diet-induced obese mouse model.
- 24c shows a manufacturing process of a brown fat differentiation in vitro model.
- 25a shows white fat digested with collagen type I, centrifuged, and divided into adipose tissue, infranatant, and SVF fractions. It is a schematic diagram of cell types in each fraction.
- Figure 25b is the result of confirming the expression of TM4SF19 by dividing adipocytes and SVF in epididymal white fat and subcutaneous white fat of mice fed a high-fat diet of 60% fat for 24 weeks.
- FIG. 27 shows the results of confirming marker genes such as macrophage, inflammation, and adiponectin (left) after contact co-culture with macrophage (right) after differentiating adipocytes as an in vitro model [Control: macrophage and adipocytes, respectively After growing, RNA was extracted and combined and used].
- Figure 29 is the result of adipogenesis by extracting SVF from wild-type (WT) mice and TM4SF19KO mice, and differentiation into macrophages by extracting bone marrow from each mouse, followed by contact co-culture to induce inflammatory changes.
- Figure 30 is the result of inducing inflammatory changes by extracting SVF from wild-type (WT) mice and TM4SF19KO mice to induce adipogenesis, extracting bone marrow from each mouse and differentiating them into macrophages, and then performing individual or contact co-culture [control group (ctrl) : After growing macrophages and adipocytes, RNA is extracted and combined and used].
- control group (ctrl) After growing macrophages and adipocytes, RNA is extracted and combined and used].
- FIG. 31 shows the in vitro insulin resistance model production process (top) and differentiation of adipocytes for 12 days and treatment with TNF ⁇ for 24 hours, confirming the fat differentiation marker C/EBP ⁇ and the insulin resistance in vitro model marker C/EBP ⁇ ( Center), and the increase in TM4SF19 was also confirmed (bottom).
- 32 is an in vitro insulin resistance model showing a decrease in insulin resistance in TM4SF19KO compared to wild type.
- FIG. 34 shows macrophage differentiation by extracting bone marrow from wild-type (WT) mice and TM4SF19KO mice, proceeding with M1 polarization, and confirming markers involved in M1 polarization after extracting bone marrow from wild-type mice and TM4SF19KO mice and differentiating macrophages ( bottom) is.
- TM4SF19 shows the polyclonal mouse TM4SF19 antibody prepared above after transiently overexpressing human TM4SF19 (hTM4SF19), human EC2 ⁇ (hTM4SF19 115-175 ⁇ ), mouse TM4SF19 (mTM4SF19), and mouse EC2 ⁇ (mTM4SF19 116-165 ⁇ ) in 293T cells. It shows the result of performing Western blotting to confirm the expression of .
- E1 Sample eluted with Elution buffer (20 mM glycine).
- E1-1 Samples subjected to Ultrafiltration/Diafiltration (UF/DF) with Buffer A (50 mM phosphate, 50 mM NaCl, pH 7.0) (Fc may be more stable)].
- 38b shows the inhibition of multinucleated osteoclast formation by TRAP staining by treating hTM4SF19 120-169 -Fc during osteoclast differentiation.
- 38c shows that hIgG1-Fc, hTM4SF19 120-169 -Fc, and mTM4SF19 116-165 -Fc were treated during osteoclast differentiation, and hTM4SF19 120-169 -Fc and mTM4SF19 116-165 -Fc inhibited the formation of multinucleated osteoclasts. This is the result confirmed by TRAP staining.
- FIG. 38D shows the formation of an actin belt formed during multinuclear osteoclast differentiation by F-actin staining after treatment with 10 ⁇ g/ml of IgG-Fc and mTM4SF19-Fc while differentiating wild-type mouse bone marrow.
- Figure 38e confirms that bone resorption is blocked by mTM4SF19-Fc by Toluidine blue staining.
- 39a is a micro CT image showing the bone loss inhibitory effect of mouse TM4SF19-Fc.
- FIG. 39b shows the bone volume (BV) obtained by analyzing the micro CT image of FIG. 39a with a 3D image analysis program, the ratio of bone volume to tissue volume (% BV/TV), and the average number of bone trabeculae (Tb.N ) and 3D micro CT indicators including trabecular gap (Tb.Sp).
- 39c is a histopathological analysis of mouse femur tissue through H&E staining.
- 39d shows that bone loss was significantly suppressed after tail vein injection of hIgG1-Fc, mouse TM4SF19-Fc (116-165), and human TM4SF19-Fc (131-169) to 8-week-old female mice after ovariectomy.
- Figure 40a is a graph representing the mRNA expression of Ctsk and Acp5 in cells differentiated under conditions of treatment with 10 ⁇ g/ml of mouse TM4SF19-Fc while treatment with M-CSF or M-CSF and RANKL.
- Figure 40b shows that mTM4SF19-Fc was isolated from the bone marrow of wild-type (WT) mice and differentiated under conditions of treatment with M-CSF or M-CSF and RANKL (M-CSF 25 ng/ml, RANKL 100 ng/ml). The result of confirming the expression of the osteoclast differentiation marker protein after ⁇ g/ml treatment is shown.
- Figure 41 confirms surface binding to mTM4SF19-Fc before and after differentiation of Raw264.7 cells by FACS analysis.
- 42a schematically illustrates an experimental schedule for confirming the prevention and treatment of rheumatoid arthritis using TM4SF19-Fc.
- Figure 42b 42 days after the induction of collagen induced arthritis (CIA), the result of checking the joint condition of the mouse is shown.
- Figure 42c shows the results of analyzing the CIA score and the number of swollen joints investigated during the experimental period.
- 42d is a micro CT image showing that inflammation and bone damage in the foot of a mouse model with collagen-induced arthritis were inhibited by treatment with mTM4SF19-Fc in a dose-dependent manner.
- 42e is a result showing that inflammation and bone damage occurring in the foot of a collagen-induced arthritis mouse model were dose-dependently inhibited by treatment with mTM4SF19-Fc.
- FIG. 42f shows bone volume (BV), ratio of bone volume to tissue volume (% BV/TV), average number of bone trabeculae ( Tb.N) and trabecular gap (Tb.Sp), 3-dimensional micro CT indicators (bone microstructure indicators) are shown.
- Figure 42g is the result of confirming the cartilage damage of the collagen-induced arthritis mouse model with toluidine blue. It shows that cartilage damage in the mTM4SF19-Fc treated group was suppressed compared to the hIgG1-Fc treated group.
- 42h shows the semi-therapeutic effect of mTM4SF19-Fc and hTM4SF19-Fc (120-169) in an arthritis-induced mouse model injected with LPS 3 days after collagen antibody treatment was confirmed by arthritis score.
- TM4SF19-Fc treatment resulted in a lower arthritis disease score and a decrease in paw thickness. 42i confirms the therapeutic effect of mTM4SF19-Fc after 8 days when the arthritis disease score is maximum in an arthritis-induced mouse model injected with LPS after 3 days of collagen antibody treatment. Compared to the untreated group, TM4SF19-Fc treatment lowered arthritis disease score, decreased paw thickness, and suppressed cartilage damage with toludine blue, confirming the treatment effect.
- 43a schematically illustrates an experimental schedule for confirming the effect of suppressing breast cancer bone metastasis using mTM4SF19-Fc.
- Figure 43b shows the results of confirming the cancer progression state through bioluminescence image analysis.
- 43c shows the result of a micro CT scan of the joint of a mouse in which bone metastasis of breast cancer occurred.
- 43D shows the results of histological analysis through H&E staining of the joints of mice in which bone metastasis of breast cancer occurred.
- 44a schematically illustrates an experimental schedule for confirming the inhibitory effect of breast cancer bone metastasis using hTM4SF19-Fc (145-169).
- Figure 44b shows the results of confirming the cancer progression state through bioluminescence image analysis.
- 44c shows the results of luminescence analysis on the joints of mice with bone metastasis of breast cancer.
- 44D shows the result of a micro CT scan of the joint of a mouse in which bone metastasis of breast cancer occurred.
- lung metastasis was induced by injecting mouse-derived E0771 breast cancer cells into the tail vein of wild-type mice, and lung metastasis was significantly inhibited by mTM4SF19-Fc (116-165) and hTM4SF19-Fc (145-169) that showed the result.
- Figure 46a shows that obesity caused by a high-fat diet is suppressed by mTM4SF19-Fc treatment.
- Figure 46b confirms the effect of reducing the weight of white fat in organs and suppressing fatty liver by high-fat diet by mTM4SF19-Fc treatment by organ weight compared to organ weight and body weight.
- Figure 47 shows the effect of reducing fatty liver in liver tissue and reducing triglyceride in plasma according to mTM4SF19-Fc treatment confirmed by H&E staining and Masson & trichrome staining.
- Figure 50a confirms the inhibition of cancer cell migration by osteoclast differentiation in TM4SF19KO by staining with Crystal violet solution 0.05%.
- Figure 50b confirms the inhibition of cancer cell migration by osteoclast differentiation in TM4SF19EC2 ⁇ by staining with Crystal violet solution 0.05%.
- 53 shows the results of confirming the growth of cells of different clones (#4 and #6) by MTT analysis after knocking out TM4SF19 in 143b osteosarcoma cell line by CRISPR, and confirming the colony formation by colony formation.
- FIG. 54 shows that 143b osteosarcoma cells were treated with hTM4SF19-Fc (120-169 aa), cell growth was confirmed by MTT analysis (FIG. 54a) and cell counting (FIG. 54c), and colony formation was confirmed by colony formation (FIG. 54c). 54b), and the result of confirming cell migration (FIG. 54d).
- HOS osteosarcoma cells confirms that the cell migration ability of HOS osteosarcoma cells is inhibited by treatment with 10 ⁇ g/ml hTM4SF19-Fc (120-169 aa) and hTM4SF19-Fc (145-169 aa).
- Figure 57a confirms the inhibition of pancreatic cancer cell colony formation by human TM4SF19-Fc (120-169 aa).
- Figure 57b confirms the inhibition of pancreatic cancer cell growth by human TM4SF19-Fc (120-169 aa) by cell counting.
- Figures 58a-d show the sequence information of the TM4SF19 fragment and TM4SF19-Fc fusion protein used in the examples herein [The corresponding target sequence is indicated in bold in each figure, SEQ ID NOs: 8, 12, 15, 18 marked; The enzyme site is the site between the target sequence and the immunoglobulin Fc region; Immunoglobulin Fc region is highlighted and indicated as SEQ ID NO: 9].
- the present invention relates to a pharmaceutical composition for preventing or treating bone disease comprising a TM4SF19 expression or activity inhibitor as an active ingredient.
- the present invention relates to a pharmaceutical composition for preventing or treating obesity or obesity-mediated metabolic diseases comprising a TM4SF19 expression or activity inhibitor as an active ingredient.
- the present invention relates to a pharmaceutical composition for preventing, treating or inhibiting cancer metastasis comprising a TM4SF19 expression or activity inhibitor as an active ingredient.
- TM4SF19 refers to transmembrane 4L six family member 19 (TM4SF19, OCTM4) and belongs to the transmembrane 4L 6 superfamily. These superfamily members are known to be involved in various cellular processes including cell proliferation, motility and adhesion through interaction with integrins, and are involved in diseases such as liver fibrosis and cancer. However, TM4SF19 has not been properly studied in relation to its function.
- TM4SF19 protein can be expressed not only in humans (Homo sapiens) or mice (Mus musculus), but also in other mammals such as monkeys, cows, horses, dogs, and cats.
- Human-derived TM4SF19 is GenBank Accession No. mRNAs containing NM_138461.4, NM_001204897.2, and NM_001204898.2 can be translated into peptides or proteins containing amino acid sequences represented by NP_612470.2, NP_001191826.1, and NP_001191827.1, respectively. That is, human-derived TM4SF19 has three transcript variants; GenBank Accession No. There are NM_138461.4 (transcript variant 1), NM_001204897.2 (transcript variant 2), and NM_001204898.2 (transcript variant 3), and there are three isoforms; GenBank Accession No.
- TM4SF19 used in the examples of the present invention is GenBank Accession No. NP_612470.2 (isoform 1) obtained from mRNA containing NM_138461.4 (transcript variant 1). isoform 1 is highly homologous to mouse TM4SF19.
- Mouse TM4SF19 was GenBank Accession No. NP_001153874.1, indicated by SEQ ID NO: 7.
- the TM4SF19 expression or activity inhibitor of the present invention refers to a substance that reduces the expression of TM4SF19 gene or the activity of TM4SF19 protein.
- the inhibitor for inhibiting the expression of the TM4SF19 gene of the present invention directly acts on the TM4SF19 gene or indirectly acts on an upstream regulator of the TM4SF19 gene to reduce the expression of the TM4SF19 gene at the transcriptional level, or the expressed TM4SF19 gene It refers to a substance that reduces the expression level or activity of the TM4SF19 gene by increasing the degradation or interfering with its activity.
- antisense nucleotides complementary to the mRNA of the TM4SF19 gene may be one or more selected from the group consisting of, but is not limited thereto.
- antisense nucleotide refers to a DNA or RNA sequence that is complementary to a specific gene and can bind to TM4SF19 mRNA. Since antisense nucleotides are long chains of monomeric units, they can be easily synthesized against the target gene sequence.
- siRNA small interfering RNA
- siRNA refers to a short double-stranded RNA capable of inducing RNA interference (RNAi) through cleavage of a specific mRNA.
- siRNA includes a sense RNA strand having a sequence homologous to the mRNA of a target gene and an antisense RNA strand having a sequence complementary thereto. Since siRNA can suppress the expression of a target gene, it is used in a gene knockdown method or a gene therapy method.
- siRNA short hairpin RNA
- microRNA refers to 21 to 23 non-coding RNAs that regulate gene expression after transcription by promoting degradation of target RNAs or inhibiting their translation.
- ribozyme refers to an RNA molecule having a function such as an enzyme that recognizes a specific nucleotide sequence and cleave it itself.
- a ribozyme is composed of a region that binds with specificity to a complementary nucleotide sequence of a target messenger RNA strand and a region that cleaves the target RNA.
- the inhibitor for inhibiting the activity of the TM4SF19 protein of the present invention may be at least one selected from the group consisting of compounds, peptides, peptidomimetics, aptamers, fusion proteins and antibodies that specifically bind to the TM4SF19 protein, It is not limited thereto.
- the term "compound” includes any compound capable of specifically binding to and inhibiting the activity of TM4SF19 protein.
- the term "peptide” has the advantage of high binding force to a target substance, and does not undergo denaturation even during heat/chemical treatment.
- it can be attached to other proteins and used as a fusion protein. Specifically, since it can be attached to a polymer protein chain and used, it can be used as a diagnostic kit and a drug delivery material.
- peptide mimetics refers to inhibiting the activity of TM4SF19 protein by inhibiting the binding domain of TM4SF19 protein.
- Peptide mimetics may be peptides or non-peptides, such as psi binding, It may consist of amino acids linked by peptide bonds, and may also include “conformationally constrained” peptides, cyclic mimetics, at least one exocyclic domain, binding moiety It can be a cyclic mimetic comprising (binding amino acids) and an active site.
- Peptide mimics are structured similarly to the secondary structure properties of TM4SF19 protein and can mimic the properties of inhibiting large molecules such as antibodies or water soluble receptors, and can mimic natural It may be a novel small molecule that can act with an effect equivalent to that of an antagonist.
- aptamer refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) that has a stable tertiary structure and is capable of binding to a target molecule with high affinity and specificity. do.
- fusion protein is a new protein made in the form of combining two or more different proteins or proteins of the same kind, and is often referred to as a chimera protein.
- Two or more heterologous proteins combine part and part, part and whole, or whole and whole.
- the sequence of one gene and the sequence of another gene are spliced by matching codons to create a hybrid gene and express it as a protein to produce it.
- the fusion protein according to the present invention may include a fragment of TM4SF19 that specifically binds to the TM4SF19 protein.
- an immunoglobulin Fc region may be further included.
- the fragment may be all or part of a fragment derived from extracellular loop 2 (EC2) of TM4SF19.
- the amino acid sequence region corresponding to the entirety of EC2 may be the 120-169th amino acid region of the human TM4SF19 protein or the 116-165th amino acid region of the mouse TM4SF19 protein.
- the amino acid sequence region corresponding to part of EC2 may include amino acid regions 145-169 or 131-169 of human TM4SF19 protein.
- a preferred fusion protein according to the present invention may include an amino acid sequence represented by SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16 or SEQ ID NO: 19.
- the present invention provides a fusion protein in which an immunoglobulin Fc region is coupled to a fragment of TM4SF19, for example, a fusion protein in which an immunoglobulin Fc region is coupled to all or part of an extracellular loop of TM4SF19; Alternatively, it may include a fusion protein in which an immunoglobulin Fc region is coupled to all or part of the extracellular loop of TM4SF19 and all or part of the membrane protein.
- the fusion protein in which the immunoglobulin Fc region is coupled to the TM4SF19 fragment includes a protein in which the immunoglobulin Fc region is indirectly coupled to the TM4SF19 fragment in addition to direct binding of the immunoglobulin Fc region to the TM4SF19 fragment.
- the immunoglobulin Fc region is indirectly bound to a fragment of TM4SF19, 1 to 10 corresponding to a linker, spacer or enzyme site, for example, 1 to 9, 1 to 8, 1 to 7, 1 to 7 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, eg, 1 amino acid sequences may additionally be present between the fragment of TM4SF19 and the immunoglobulin Fc region.
- Fragments of TM4SF19 usable in the present invention are not limited thereto, but various examples (hTm4sf19 94 ⁇ 196, HTM4SF19 131 ⁇ 160, HTM4SF19 145 ⁇ 169, HTM4SF19 131 ⁇ 169, HTM4SF19 120 ⁇ 160, HTM4SF19 120 ⁇ 169, HTM4SF19 120 ⁇ 180, HTM4SF19 120 ⁇ 186, HTM4SF19 120 ⁇ 196 131-196 and hTm4sf19 145-196).
- the immunoglobulin Fc region is used as an example for the stability of the TM4SF19 protein, fusion partners capable of binding with the TM4SF19 protein are not limited to the immunoglobulin Fc region.
- the present invention also relates to a fusion protein for inhibiting TM4SF19 expression or activity comprising a fragment derived from extracellular loop 2 of the TM4SF19 protein and an immunoglobulin Fc region.
- the extracellular loop 2 (EC2)-derived fragment of the TM4SF19 protein may be an amino acid sequence corresponding to all or part of EC2.
- the amino acid sequence region corresponding to the entirety of EC2 may be the 120-169th amino acid region of the human TM4SF19 protein or the 116-165th amino acid region of the mouse TM4SF19 protein.
- the amino acid sequence region corresponding to part of EC2 may include amino acid regions 145-169 of human TM4SF19 protein.
- the amino acid sequence region corresponding to part of EC2 may include amino acid regions 131 to 169 of human TM4SF19 protein.
- a preferred fusion protein according to the present invention may include an amino acid sequence represented by SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16 or SEQ ID NO: 19.
- An enzyme site may be included between the fragment derived from the extracellular loop 2 (EC2) of the TM4SF19 protein and the immunoglobulin Fc region.
- the site of extracellular loop 2 of human TM4SF19 used as a fragment of TM4SF19 for the preparation of the fusion protein is exemplary only, and is not limited thereto.
- the term "Fc region” includes immunoglobulin heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3), and immunoglobulin heavy and light chain variable regions and light chain constant region 1 (CL1) refers to proteins that do not contain It may further comprise the hinge region of the heavy chain constant region.
- the “Fc region” may be an Fc region derived from IgG, IgA, IgD, IgE or IgM.
- the human IgG1-Fc used for constructing the fusion protein is exemplary only, but is not limited thereto.
- the "Fc region” may include a "modified immunoglobulin Fc region” or "Fc region variant", which is prepared by substituting some amino acids in the Fc region or combining different types of Fc regions.
- it refers to an Fc region in which antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) is attenuated compared to the wild-type Fc region by modifying binding affinity with an Fc receptor and/or binding force with complement.
- the Fc region of the modified immunoglobulin may be selected from the group consisting of IgG1, IgG2, IgG3, IgD, IgG4, and combinations of these sequences.
- the term “antibody” refers to a specific immunoglobulin directed against an antigenic site.
- “Antibody” includes monoclonal antibodies, polyclonal antibodies, bispecific antibodies, multispecific antibodies, chimeric antibodies, humanized antibodies, and human antibodies, and includes antibodies already known or commercially available in the art in addition to novel antibodies. do.
- the antibody includes a full-length form comprising two heavy chains and two light chains, as well as functional fragments of the antibody molecule, as long as it specifically binds to TM4SF19.
- the functional fragment of the antibody molecule refers to a fragment having at least an antigen-binding function, which may include Fab, F(ab'), F(ab')2, Fv, etc., but is not limited thereto.
- an anti-TM4SF19 antibody specific for all or part of the extracellular loop 2 of human TM4SF19 As a specific form, the present invention GenBank Accession No. An anti-TM4SF19 antibody specific to all or part of the extracellular loop 2 region of SEQ ID NOs: 115 to 175 of human TM4SF19 of NP_612470.2. In another embodiment, the present invention provides GenBank Accession No. An anti-TM4SF19 antibody specific for all or part of the extracellular loop 2 region of SEQ ID NOs: 120-169 of human TM4SF19 of 138461.4.
- the target site of extracellular loop 2 of human TM4SF19 used for antibody construction is illustrative, but not limited thereto.
- the TM4SF19 protein was found to be involved in cell-to-cell interactions through self-binding.
- the present inventors target the extracellular loop region of the TM4SF19 protein, antibodies to the TM4SF19 protein capable of inhibiting the self-binding of the TM4SF19 protein, and fragments of TM4SF19 (e.g., extracellular loop region, extracellular loop Region and membrane proteins) and Fc were fused to prepare a fusion protein, and the efficacy of the TM4SF19 protein activity inhibitor was evaluated.
- TM4SF19 e.g., extracellular loop region, extracellular loop Region and membrane proteins
- bone disease refers to metabolic bone disease, orthopedic bone disease, aplastic bone disease, degenerative bone disease, degenerative arthritis, rheumatoid arthritis, psoriatic arthritis, psoriatic spondylitis, age-related osteopenia bone loss), osteoporosis, osteogenesis imperfecta, osteomalacia, bone mass loss, fracture, bone defect and hip joint loss, rickets, Paget's bone disease, periodontal disease, and one or more days selected from the group consisting of bone damage caused by bone metastasis of cancer cells.
- the term "obesity” refers to a state of abnormal or excessive fat accumulation.
- the term "obesity-mediated metabolic disease” includes diabetes, hypertension, hyperlipidemia, non-alcoholic steatohepatitis and certain cancers, and more broadly includes hypertension, diabetes, insulin resistance syndrome, metabolic syndrome, obesity-related gastroesophageal reflux disease, These include arteriosclerosis, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, lipodystrophy, non-alcoholic steatohepatitis, cardiovascular disease, polycystic ovary syndrome, and the like.
- the treatment of the disease may be performed simultaneously.
- treatment targets for these obesity-related diseases include those who have a desire to lose weight.
- cancer refers to aggressive characteristics in which cells divide and grow beyond normal growth limits, invasive characteristics infiltrating surrounding tissues, and metastatic characteristics spreading to other parts of the body. It is a general term for diseases caused by cells with metastatic characteristics.
- cancer used in the present invention, but colorectal cancer, stomach cancer, colon cancer, breast cancer, lung cancer, non-small cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, melanoma, uterine cancer, ovarian cancer, small intestine cancer, Rectal cancer, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, esophageal cancer, lymphatic cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penis cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, spinal cord tumor, brainstem glioma, and pituitary adenoma. It may be one or more selected from the group consisting of
- the present invention also includes the use of TM4SF19 with respect to inhibition of "cancer” as well as “cancer metastasis”.
- prevention refers to all actions to prevent the occurrence of desired symptoms or diseases by administering the composition of the present invention, or to delay the occurrence or expression thereof.
- treatment refers to any action that improves or disappears a desired symptom or disease by administering the composition of the present invention.
- composition of the present invention may further include a pharmaceutically acceptable carrier, and may be formulated with the carrier.
- the term "pharmaceutically acceptable carrier” refers to a carrier or diluent that does not stimulate organisms and does not inhibit the biological activity and properties of the administered compound.
- Acceptable pharmaceutical carriers for compositions formulated as liquid solutions are sterile and biocompatible, and include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and these One or more of the components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary.
- diluents such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.
- composition comprising the TM4SF19 expression or activity inhibitor of the present invention and a pharmaceutically acceptable carrier can be applied in any dosage form containing the same as an active ingredient, and can be prepared as an oral or parenteral dosage form, with ease of administration and It can be formulated in unit dosage form for uniformity of dosage.
- the pharmaceutical formulations of the present invention may be taken oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal or parenteral (intramuscular, subcutaneous). and intravenous) or forms suitable for administration by inhalation or insufflation.
- Formulations for oral administration containing the composition of the present invention as an active ingredient include, for example, tablets, troches, lozenges, aqueous or oily suspensions, prepared powders or granules, emulsions, hard or soft capsules, syrups or elixirs. can do.
- Formulations for parenteral administration containing the composition of the present invention as an active ingredient include injection forms such as subcutaneous, intravenous, or intramuscular injections, suppository injections, or sprays such as aerosols that enable inhalation through the respiratory tract. can be formulated into
- the composition of the present invention may be mixed in water with a stabilizer or buffer to prepare a solution or suspension, which may be formulated for unit administration in an ampoule or vial.
- composition of the present invention is administered in a pharmaceutically effective amount, that is, a therapeutically effective amount.
- therapeutically effective amount means an amount sufficient to treat a disease
- the effective dose level is the type of patient's disease, severity, drug activity, drug sensitivity, administration time, administration route and factors including excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical arts.
- the composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple times.
- the total effective amount of the composition of the present invention can be administered to the patient in a single dose, or it can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. . Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.
- the dosage of the pharmaceutical composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.
- a daily dose it is preferably 0.01 ⁇ g to 100 mg, more preferably 1 ⁇ g to 50 mg per 1 kg of body weight per day for parenteral administration.
- the dosage is not limited to the scope of the present invention in any way.
- composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.
- the present invention provides a method for preventing or treating bone disease comprising administering to a subject a composition for preventing or treating bone disease comprising a therapeutically effective amount of a substance that inhibits the expression or activity of TM4SF19 It is about.
- the present invention provides a composition for preventing or treating obesity or obesity-mediated metabolic diseases comprising a therapeutically effective amount of an inhibitor of TM4SF19 expression or activity as an active ingredient, comprising administering to a subject a composition for obesity or obesity-mediated It relates to methods for preventing or treating metabolic diseases.
- the present invention provides a composition for preventing, treating, or inhibiting cancer metastasis comprising a therapeutically effective amount of a TM4SF19 expression or activity inhibitor as an active ingredient for preventing, treating, or preventing cancer, including administering to a subject a composition for inhibiting cancer metastasis. It relates to a method for preventing or treating cancer metastasis.
- the term "administration” means introducing a predetermined substance into a subject by any appropriate method, and the administration route of the preventive or therapeutic composition according to the present invention can be any general route as long as it can reach the target tissue. It can be administered orally or parenterally through.
- the composition for preventing or treating cartilage-related diseases according to the present invention may be administered by any device capable of moving active ingredients to target cells.
- subject of the present invention refers to mice, rats, rabbits, cows, horses, sheep, pigs, goats, camels, antelopes, dogs with related diseases whose symptoms can be improved by administration of the pharmaceutical composition according to the present invention. including mammals or humans.
- the administration route of the composition of the present invention may be administered through various oral or parenteral routes as long as it can reach the target tissue, specifically, oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, It can be administered in a conventional manner via transdermal, intranasal, inhalational, intraocular or intradermal routes.
- the prophylactic or therapeutic method of the present invention comprises administering a therapeutically effective amount of a composition of the present invention.
- the therapeutically effective amount refers to an amount that enhances the effect of reducing body weight or reducing the size of adipocytes. It is obvious to those skilled in the art that an appropriate total daily amount can be determined by a treating physician within the scope of sound medical judgment.
- a specific therapeutically effective amount for a particular patient depends on the type and extent of the response to be achieved, the specific composition including whether other agents are used as the case may be, the patient's age, weight, general health condition, sex and diet, time of administration, It is preferable to apply differently according to the route of administration and secretion rate of the composition, treatment period, and various factors and similar factors well known in the medical field. Therefore, the effective amount of the pharmaceutical composition suitable for the purpose of the present invention is preferably determined in consideration of the above. In some cases, the therapeutic effect may be enhanced by co-administering a known therapeutic agent for a related disease together with the composition of
- the present invention includes the steps of treating a bone disease-suspected specimen with a candidate substance for treating bone disease;
- It relates to a method for screening a drug for treating bone disease, comprising comparing the expression level of mRNA or protein of a control and TM4SF19 gene.
- the present invention comprises the steps of treating a candidate for obesity or obesity-mediated metabolic disease treatment in a suspected sample of obesity or obesity-mediated metabolic disease;
- It relates to a screening method for a drug for treating obesity or obesity-mediated metabolic diseases, comprising the step of comparing the expression level of mRNA or protein of a control and TM4SF19 gene.
- the present invention includes the steps of treating cancer prevention, treatment, or cancer metastasis-suspected specimens with candidates for cancer prevention, treatment, or cancer metastasis inhibition;
- It relates to a method for screening a drug for treating cancer or cancer metastasis, comprising the step of comparing the mRNA or protein expression levels of a control group and the TM4SF19 gene.
- the term "specimen” refers to an individual or sample used to screen a candidate for treatment of a related disease, which includes, without limitation, mammals including dogs, cows, pigs, rabbits, chickens, mice, and humans. and samples such as whole blood, serum, blood, plasma, saliva, urine, sputum, lymph, cells, and tissues isolated from the subject.
- control group means a sample not treated with a candidate substance.
- candidate means a drug to be tested for changes in the expression or activity of TM4SF19, and the ability to prevent or treat related diseases by directly or indirectly changing the expression level or activity of TM4SF19.
- objects to be measured include arbitrary molecules such as proteins, oligopeptides, small organic molecules, polysaccharides, polynucleotides and a wide range of compounds.
- candidate materials also include both natural as well as synthetic materials.
- therapeutic drug refers to a substance that prevents or treats a related disease.
- the screening method of the present invention compares the mRNA or protein expression level of the TM4SF19 gene with a control group that is not treated with the candidate substance after treating a subject suspected of a related disease with a candidate substance for treatment, It can be performed by judging a substance that reduces expression as a therapeutic agent for a related disease.
- Assay methods for measuring mRNA levels include, but are not limited to, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, northern blotting, DNA chip, and the like.
- Assay methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunoassay, Oukteroni immunodiffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chips, etc., but are not limited thereto.
- TM4SF19 increased during osteoclast differentiation.
- TM4SF19 deficiency model TM4SF19 knockout mouse, TM4SF19KO and TM4SF19 extracellular domain 2 (116-165) knockout mouse, TM4SF19EC2 ⁇
- mononuclear osteoclasts were formed during osteoclast differentiation, while multinucleated osteoclasts Cell (multinucleated osteoclast) formation was confirmed to be inhibited. Osteoclasts that have not differentiated into multinucleated osteoclasts retain their phenotype as osteoclasts and maintain a low level of bone resorption activity.
- the TM4SF19 inhibitor of the present invention which can selectively inhibit the formation of multinuclear osteoclasts, can more selectively regulate bone resorption rather than completely abolish the bone resorption function, resulting in the formation of mononuclear osteoclasts like anti-RANKL antibodies.
- the TM4SF19 deficiency model prevents bone loss due to ovariectomy.
- anti-mouse TM4SF19 polyclonal antibody and mouse TM4SF19 EC2 (116-165 of GenBank Accession No. NP_001153874.1)-Fc fusion protein, human TM4SF19 EC2 (120 of GenBank Accession No.
- NP_612470.2 (isoform 1)
- TM4SF19 expression was increased during adipocyte differentiation of hADMSC (human adipose-derived mesenchymal stem cells).
- hADMSC human adipose-derived mesenchymal stem cells.
- TM4SF19 knockout mice it was confirmed that weight gain was suppressed in a high-fat diet-induced obesity model, and the amount of adipose tissue-related macrophages caused by obesity was reduced, confirming that TM4SF19 acts on adipocyte differentiation and inflammatory responses including macrophages. did As a result, it was confirmed that TM4SF19KO mice lowered insulin resistance and suppressed fatty liver in a high-fat diet-induced obese mouse model. Therefore, it can be seen that substances that inhibit TM4SF19 expression or activity are effective in preventing or treating obesity and obesity-mediated metabolic diseases.
- TM4SF19 overexpression increases mouse-derived breast cancer and human osteosarcoma cell proliferation, migration, and colony formation
- TM4SF19 deficiency suppresses lung metastasis of breast cancer cells, and osteosarcoma cell proliferation, migration, and colony Since the formation and bone metastasis inhibitory effect of breast cancer cells was confirmed, it can be seen that substances that inhibit TM4SF19 expression or activity are effective in preventing and treating cancer and cancer metastasis.
- pancreatic cancer cells are inhibited by treatment with human TM4SF19 EC2 (120-169 of GenBank Accession No. NP_612470.2 (isoform 1))-Fc fusion protein, and osteosarcoma Effects of inhibiting cell proliferation, migration, and colony formation were confirmed. Accordingly, it can be seen that a substance that inhibits the expression of TM4SF19 or a substance that inhibits the activity of TM4SF19 is effective in preventing and treating cancer and cancer metastasis.
- human TM4SF19 EC2 120-169 of GenBank Accession No. NP_612470.2 (isoform 1)
- osteosarcoma Effects of inhibiting cell proliferation, migration, and colony formation were confirmed. Accordingly, it can be seen that a substance that inhibits the expression of TM4SF19 or a substance that inhibits the activity of TM4SF19 is effective in preventing and treating cancer and cancer metastasis.
- Figure 1 shows the mRNA of TM4SF19 according to the differentiation induction time after inducing differentiation into osteoclasts (OC) by extracting cells from mouse bone marrow and treating them with M-CSF and RANKL (Receptor activator of nuclear factor kappa- ⁇ ligand). This is the result of confirming the expression by qPCR. As can be seen in Figure 1, it was confirmed that the expression of TM4SF19 increased with the progress of osteoclast differentiation. The expression of ACP5 CTSK, a gene related to osteoclast differentiation, also increased with the progress of osteoclast differentiation.
- TM4SF19KO TM4SF19 knockout mouse using CRISPR
- WT wild-type mice
- M-CSF 25 ng/ml + RANKL 100 ng/ml M-CSF 25 ng/ml + RANKL 100 ng/ml.
- TRAP staining heartrate-resistant acid phosphatase staining
- an osteoclast-related marker was performed.
- TM4SF19KO TM4SF19 knockout mouse using CRISPR
- WT wild type mice
- concentration of RANKL was fixed at 100 ng/ml and the concentration of M-CSF was varied at 25, 40, and 60 ng/ml.
- TRAP staining titanium-resistant acid phosphatase staining
- an osteoclast-related marker was performed.
- FIG. 2c is a schematic diagram showing the differentiation process of osteoclasts.
- osteoclast precursors first differentiate into TRAP-positive mononuclear osteoclasts and then mature into giant multinucleated cells through cell-cell fusion and incomplete cytokinesis. Mature, multinucleated osteoclasts have high bone resorption activity, whereas osteoclasts that have not differentiated into multinucleated osteoclasts retain the osteoclast phenotype and express osteoclast-associated markers such as TRAP and cathepsin K. However, it has been reported to maintain a low level of bone resorption activity.
- TM4SF19 knockout prevents the differentiation and maturation of multinucleated osteoclasts during the differentiation process of osteoclasts suggests that prevention or treatment of bone loss-related diseases can be attempted through inhibition of TM4SF19.
- TM4SF19 inhibitors can selectively inhibit the formation of multinuclear osteoclasts, which can more selectively regulate bone resorption rather than completely abolish the bone resorption function of osteoclasts. Side effects can be significantly lowered compared to therapeutic agents that suppress from the formation stage.
- TM4SF19KO TM4SF19 knockout mouse using CRISPR
- WT wild-type mice
- Ctsk a gene involved in osteoclast differentiation
- Figure 3 is a graph representing the mRNA expression of Ctsk, Acp5, c-Fos, and Nfatc1 in cells differentiated under conditions with or without M-CSF and RANKL treatment.
- BMM cells were plated on dentin discs, differentiated, and pit formation was confirmed. Bone resorption was confirmed in the wild type (WT), but bone resorption was blocked in TM4SF19KO (FIG. 4b).
- micro CT images were analyzed with a 3D image analysis program to determine bone volume (BV), ratio of bone volume to tissue volume (% BV/TV), average number of trabeculae (Tb.N), trabecular gap 3-dimensional micro CT indicators (bone microstructure indicators) including (Tb.Sp) were obtained.
- BV bone volume
- % BV/TV ratio of bone volume to tissue volume
- Tb.N average number of trabeculae
- Tb.Sp trabecular gap 3-dimensional micro CT indicators
- TM4SF19KO mice Regardless of ovariectomy, the bone mineral density of TM4SF19KO mice is higher than that of wild-type (WT) mice.
- the femurs of 8-week-old wild-type (WT) mice and TM4SF19KO mice were fixed and decalcified, followed by TRAP staining.
- Example 3-1 TM4SF19 extracellular loop 2 (EC2) deficient mouse multinucleated osteoclast formation damage confirmed
- TM4SF19EC2 Del also denoted TM4SF19EC2 ⁇ knocked out TM4SF19 extracellular domain 2 (116-165) by CRISPR and treated or treated with 25 ng/ml M-CSF and 100 ng/ml RANKL. After differentiation without staining, TRAP staining was performed.
- Figure 7b shows cells from the bone marrow of wild-type (WT) mice and TM4SF19EC2 ⁇ mice in which TM4SF19 extracellular domain 2 (116-165) was knocked out by CRISPR, and the concentration of RANKL was fixed at 100 ng/ml and the concentration of M-CSF was 25 ng. This is the result of TRAP staining after treatment with /ml or 100 ng/ml for differentiation. When the bone marrow of TM4SF19EC2 ⁇ mice was differentiated into osteoclasts, multinucleated osteoclast formation damage was not recovered even by high-concentration M-CSF treatment.
- osteoclast differentiation process when multinucleated osteoclasts (mature osteoclasts) are formed, an actin belt made up of podosomes is formed, and the podosomes adhere to bone for bone resorption.
- Cells were extracted from the bone marrow of wild-type mice and TM4SF19EC2 ⁇ mice, differentiated with MCSF 60 ng/ml and RANKL 100 ng/ml, and then the cells were fixed and phalloidin FITC-stained to confirm the formation of F-actin belts under a confocal microscope (400x). .
- TM4SF19EC2 ⁇ inhibits the formation of multinucleated osteoclasts and affects cytoskeleton rearrangement related to integrin signaling. As shown in FIG. 7c , it was confirmed that the actin belt was formed by differentiation of multinucleated osteoclasts in the case of wild type, but not in TM4SF19EC2 ⁇ .
- qPCR was performed to confirm the expression of target genes (Ctsk, Acp5, c-Fos, Nfatc1) related to osteoclast differentiation in wild-type (WT) mice and TM4SF19EC2 ⁇ mice in which TM4SF19 extracellular domain 2 (116-165) was knocked out by CRISPR. performed.
- target genes Ctsk, Acp5, c-Fos, Nfatc1 related to osteoclast differentiation in wild-type (WT) mice and TM4SF19EC2 ⁇ mice in which TM4SF19 extracellular domain 2 (116-165) was knocked out by CRISPR. performed.
- micro CT images were analyzed with a 3D image analysis program to determine bone volume (BV), ratio of bone volume to tissue volume (% BV/TV), average number of trabeculae (Tb.N), trabecular gap 3-dimensional micro CT indicators (bone microstructure indicators) including (Tb.Sp) were obtained.
- BV bone volume
- % BV/TV ratio of bone volume to tissue volume
- Tb.N average number of trabeculae
- Tb.Sp trabecular gap 3-dimensional micro CT indicators
- BV bone volume
- % BV/TV ratio of bone volume to tissue volume
- Tb.N average number of bone trabeculae
- Tb.Th bone trabecular thickness
- 11 shows a photographed image of micro CT.
- TM4SF19KO mice and TM4SF19EC2 ⁇ mice suppress bone loss induced by ovariectomy, and even in the sham group without ovariectomy (OVX), TM4SF19KO mice and TM4SF19EC2 ⁇ mice It was confirmed that the bone density of the TM4SF19EC2 ⁇ mouse was higher than that of the wild type (WT).
- TM4SF19 tagged with HA and flag, respectively was manufactured.
- 3HA-tagged TM4SF19 at the N-terminus, wild type and TM4SF19 deletion mutants tagged with 3Flag at the N-terminus were transiently overexpressed, and binding was confirmed by immunoprecipitation.
- TM4SF19 deletion mutant was constructed as shown in FIG. 12a. As shown in FIG.
- the hTm4sf19 mutants in which some sequences of TM4SF19 were deleted (hTM4SF19 115-175 ⁇ , hTm4sf19 105-186 ⁇ , hTm4sf19 105-196 ⁇ , hTm4sf19 94-186 ⁇ and hTm4sf19 94-196 ⁇ ) were each tagged with 3 Flags, and then cell-to-cell binding was analyzed by immunoprecipitation.
- TM4SF19 is involved in cell-to-cell interaction and binds to itself (FIG. 12b).
- mutant (hTm4sf19 94-196 ⁇ ) in which transmembrane 3 (TM3), extracellular region 2 (EC2), and transmembrane 4 (TM4) of TM4SF19 were deleted did not self-bind. From these results, it was confirmed that the transmembrane 3-extracellular region 2-trasmembrane 4 region of TM4SF19 is important for TM4SF19 self interaction, and positions 94 to 196 of TM4SF19 are particularly important for self interaction between TM4SF19 and TM4SF19.
- TM4SF19 TM4SF19 deletion mutant (top of Fig. 12c).
- TM4SF19 tagged with 3HA at the N-terminus, wild-type and TM4SF19 deletion mutants tagged with 3Flag at the N-terminus were transiently overexpressed in 293T cells, and binding was confirmed by immunoprecipitation.
- transmembrane 3 and EC2 extracellular region 2 of TM4SF19 and a mutant expressing transmembrane 4 (hTm4sf19 94-196 ) self-binding similarly to the wild type (WT) (bottom of FIG. 12c). From these results, it was reconfirmed that the 94 ⁇ 196 aa region of TM4SF19 is an important site for TM4SF19-TM4SF19 self interaction.
- TM4SF19 tagged with 3Flag at the N-terminus of 293T cells was confirmed by immunoprecipitation using the same method as above.
- TM4SF19 is a member of the Tetraspanins family, and it is known that the extracellular region 2 (Large extracellular loop) of this family plays an important role in binding to other binding partners.
- the interaction between TM4SF19 wt or integrin ⁇ v or integrin ⁇ 3 was confirmed by immunoprecipitation.
- TM4SF19 wt is the result of confirming that the interaction with them is confirmed, whereas the binding with hTM4SF19 115 ⁇ 175 ⁇ is not made.
- Figure 12i shows the result of confirming the interaction between TM4SF19 and DC-Stamp and siglec-15, which are membrane proteins involved in osteoclast function regulation and cytoskeletal rearrangement during multinucleated osteoclast formation, by immunoprecipitation.
- TM4SF19 is N-terminal 3HA tagged
- DC-Stamp is N-terminus tagged with 3Flag
- Siglec-15 is C-terminus tagged with 3Flag, expressed in 293T, and the interaction was confirmed by immunoprecipitation.
- Example 5 Confirmation of cancer cell proliferation, colony formation, and migration increase according to TM4SF19 overexpression
- TM4SF19 overexpressing TM4SF19 (hTM4SF19) in mouse-derived breast cancer cells E0771, cell proliferation and colony formation assays over time and cell migration assays were performed.
- FIG. 13a is a graph showing the significant increase in cell proliferation of breast cancer cells E0771 after 24, 48 and 72 hours by overexpression of TM4SF19.
- FIG. 13B shows that colony formation ability is confirmed after plating breast cancer cells E0771 cells overexpressing TM4SF19 and 1000 control cells, indicating that colony formation is increased by overexpression of TM4SF19.
- Figure 13c shows that cells overexpressing TM4SF19 in breast cancer cells E0771 and control cells were plated in a migration chamber and then the migration ability of the cells was evaluated over time, showing that TM4SF19 overexpression increased cell migration. indicate
- the lung metastasis phenotype of breast cancer cells of wild-type (WT) mice, TM4SF19KO mice, and TM4SF19EC2 ⁇ mice was confirmed, and the number of nodules formed in the lungs was measured.
- Bone marrow was obtained from TM4SF19KO mice (TMKO) and wild-type (WT) mice, differentiated into bone marrow derived macrophages (BMDM), placed in the lower part of the migration chamber, and breast cancer cells were placed in the upper chamber.
- TMKO TM4SF19KO mice
- WT wild-type mice
- BMDM bone marrow derived macrophages
- target genes CDH2, SNAI1, SNAI2
- metastasis-related proteins vimentin, slug, bnail, E-cadherin, Beta-actin
- Example 7 Confirmation of involvement of TM4SF19 in adipocyte differentiation in human ADMSC
- hADMSC Human adipose-derived mesenchymal stem cells
- Example 8 Identification of the role of TM4SF19 in obesity and metabolic diseases
- TM4SF19KO was fed a high-fat diet and the weight gain was confirmed.
- TM4SF19KO mice lowered insulin resistance and suppressed fatty liver formation in a high-fat diet-induced obese mouse model. Accordingly, it was confirmed that TM4SF19KO mice were resistant to insulin resistance and fatty liver induced by a high-fat diet (6-week-old mice fed a normal diet and fed a high-fat diet for 12 weeks) (FIG. 18).
- rtWAT retroperitoneal white adipose tissue
- sWAT subcutaneous white adipose tissue
- ingWAT inguinal white adipose tissue
- iWAT interscapular white adipose tissue
- iBAT interscapular brown adipose tissue
- TM4SF19KO mice are resistant to high fat-induced obesity (18 weeks) (FIG. 19).
- WT epidermal growth factor
- sWAT subcutaneous white adipose tissue
- qPCR to confirm the expression of macrophage marker genes in the stromal vascular fraction (SVF) in epididymal white adipose tissue after 6-week-old wild-type (WT) mice fed a normal diet and TM4SF19KO mice fed a high-fat diet for 12 weeks was performed.
- SVF stromal vascular fraction
- TM4SF19 acts on the inflammatory response including adipocyte differentiation and macrophages, and thus, TM4SF19 deficiency lowers insulin resistance and suppresses fatty liver (FIG. 23).
- 24a shows the phenotype of beige or brown fat and fatty liver formation in epididymal white fat of high-fat diet-induced obese WT or TM4SF19KO mice by H&E staining.
- 24c is a schematic diagram showing a method for differentiating beige/brown adipocytes in white fat.
- Adipocyte progenitor cells extracted from white adipose tissue of WT and TM4SF19KO mice were grown in DMEM/F-12 media with T3 and insulin. Two days after they became confluent, they were treated with DMI and indometacine to initiate differentiation, and from day 2, rosiglitazone was treated. while culturing from day 7 to day 8 to induce beige/brown adipocyte differentiation.
- 24d and 24e show the results of confirming gene expression and protein expression of Ucp1 after beige/brown adipocyte differentiation.
- TM4SF19 was confirmed by dividing adipocytes and SVF in epididymal white fat and subcutaneous white fat of mice fed a high-fat diet for 24 weeks.
- TM4SF19 functions not only in adipocytes but also in SVF containing macrophages and monocytes. means (Fig. 25).
- 25a shows white fat digested with collagen type I, centrifuged, and divided into adipose tissue, infranatant, and SVF fractions. It is a schematic diagram of cell types in each fraction.
- Figure 25b is the result of confirming the expression of TM4SF19 by dividing adipocytes and SVF in epididymal white fat and subcutaneous white fat of mice fed a high-fat diet of 60% fat for 24 weeks.
- SVF was extracted from subcutaneous white fat of wild-type (WT) and TM4SF19KO mice, and differentiation into brown fat was induced with isoproterenol, and qPCR was performed to confirm thermogenic gene expression and beige fat marker gene expression.
- thermogenic genes and beige fat markers were increased in SVF of TM4SF19KO mice, suggesting that TM4SF19KO mice may be involved in brown or beige localization of white fat (FIG. 26).
- 3T3-L1 adipose tissue and macrophages plays an important role in high-fat diet-induced obesity.
- adipocytes were differentiated as an in vitro model, contact co-culture was performed with macrophages, and marker genes such as macrophages, inflammation, and anti-obesity [Macrophage maker; MCP-1, inflammation marekr; TNF ⁇ , IL6, MMP3, anti-obesity marker; adiponectin] was confirmed by qPCR.
- macrophages and adipocytes were grown separately, and then RNA was extracted and combined.
- Co-culture of 3T3-L1 adipocytes and macrophages increased the expression of MMP3, a gene related to inflammation, and also increased TM4SF19.
- SVF was extracted from wild-type (WT) mice, adipogenesis was performed, wild-type (WT) bone marrow was differentiated into macrophages, and inflammatory changes were induced by individual or contact co-culture.
- WT wild-type
- WT bone marrow
- inflammatory changes were induced by individual or contact co-culture.
- macrophages and adipocytes were grown separately, and then RNA was extracted and combined.
- TM4SF19 was increased by co-culture after differentiation of adipocytes of SVF and macrophages of bone marrow (FIG. 28).
- Adipogenesis was performed by extracting SVF from wild-type (WT) mice and TM4SF19KO mice, and bone marrow from each mouse was differentiated into macrophages, followed by contact co-culture to induce inflammatory changes.
- MCP1 and IL6 were decreased in differentiated adipocytes + macrophages of TM4SF19KO compared to the co-culture of differentiated WT adipocytes + macrophages.
- Adipogenesis was performed by extracting SVF from wild-type (WT) mice and TM4SF19KO mice, and bone marrow from each mouse was differentiated into macrophages, followed by ctrl culture or co-culture to induce inflammatory changes.
- a control ctrl culture
- macrophages and adipocytes were grown separately, and then RNA was extracted and combined.
- TM4SF19KO BMDM reduced the expression of inflammatory response markers, and the inflammatory response was reduced in TM4SF19KO adipocyte-TM4SF19KO macrophage co-culture compared to wt adipocyte-wt macrophage cells. was (FIG. 30).
- Inflammatory signals increased when differentiated adipocytes and BMDM were co-cultured.
- TNF ⁇ induces inflammation and is a mimic model for the increase of peripheral inflammation signals when obesity is induced in vivo.
- SVF was extracted from wild-type (WT) mice and TM4SF19KO mice, subjected to adipose differentiation, treated with TNF ⁇ for 24 hours, and then the adipose differentiation marker C/EBP ⁇ , insulin resistant in vitro model marker C/EBP ⁇ , and TM4SF19 were confirmed by qPCR.
- TM4SF19 increased in the in vitro insulin resistance model (Fig. 31. Fig. 32).
- TM4SF19KO decreased insulin resistance (C/EBP ⁇ is an insulin resistance marker).
- Bone marrow was extracted from wild-type (WT) mice and TM4SF19KO mice, differentiated into macrophages, and markers involved in M1 polarization were identified. M1 macrophages are involved in pro-inflammatory signaling.
- inflammatory markers and TM4SF19 were increased by differentiation of wild-type (WT) mouse bone marrow-derived M1 macrophages (FIG. 33), and bone marrow-derived M1 macrophage differentiation markers were decreased in TM4SF19KO (FIG. 34).
- TM4SF19 antibody targeting the 141-159 aa region in mouse TM4SF19 extracellular loop 2 was prepared. (See SEQ ID NO: 7 for mouse TM4SF19 protein.)
- Human TM4SF19 (hTM4SF19), human EC2 ⁇ (hTM4SF19 115-175 ⁇ ), mouse TM4SF19 (mTM4SF19), and mouse EC2 ⁇ (mTM4SF19 116-165 ⁇ ) were transiently overexpressed in 293T cells, and expression was then expressed with the previously prepared polyclonal mouse TM4SF19 antibody. Western blotting was performed to confirm.
- the polyclonal mouse TM4SF19 antibody recognized human TM4SF19 and mouse TM4SF19. However, the extracellular loop 2 deletion mutant was not recognized (FIG. 35).
- Bone marrow was extracted from wild-type (WT) mice, treated with TM4SF19 antibody while differentiating into osteoclasts, and confirmed by TRAP staining.
- the TM4SF19 antibody inhibited osteoclast multinucleation in a dose-dependent manner (FIG. 36).
- TM4SF19-Fc fusion proteins for human TM4SF19 and mouse TM4SF19 extracellular loop2 were constructed using the sequence definition and phylogenetic information of the TM4SF family.
- TM4SF19-Fc is a form in which human IgG1-Fc is fused to the c-terminus of human TM4SF19 or mouse TM4SF19 extracelluar loop2.
- Mouse TM4SF19-Fc was well secreted and purified.
- Mouse TM4SF19EC2-Fc (116-165) was constructed and purified, and was used in subsequent experiments.
- TM4SF19-EC2-Fc 115-175 was not well expressed, it was reconstructed with mouse TM4SF19-EC2-Fc and a conserved sequence to confirm expression. Human TM4SF19-Fc (120-169) secreted more into the media than extracellular loop 2 (115-175), so it was used in subsequent experiments (FIG. 37).
- the fusion proteins used in Examples according to the present invention are shown in FIG. 58 .
- hTM4SF19-Fc means hTM4SF19-Fc (120-169 aa) fusion protein.
- mTM4SF19-Fc means mouse TM4SF19-Fc (116-165 aa) fusion protein.
- hIgG1-Fc, hTM4SF19 120-169 -Fc and mTM4SF19 116-165 -Fc were treated in the course of osteoclast differentiation.
- bone marrow-derived cells were treated with MCSF 60 ng/ml and RANKL 100 ng/ml, and then TRAP staining was performed.
- the results are as shown in FIGS. 38A to 38C.
- the purified mTM4SF19 EC2-Fc was treated during osteoclast differentiation to confirm inhibition of multinucleated osteoclast formation [E1; Sample eluted with Elution buffer (20 mM glycine). E1-1; Samples subjected to UF/DF (Ultrafiltration/Diafiltration) with Buffer A (50 mM phosphate, 50 mM NaCl, pH 7.0) (Fc may be more stable)].
- UF/DF Ultrafiltration/Diafiltration
- Buffer A 50 mM phosphate, 50 mM NaCl, pH 7.0
- Fc may be more stable
- Wild-type mouse bone marrow was treated with 10 ⁇ g/ml of IgG-Fc and mTM4SF19-Fc while differentiating, and the formation of an actin belt formed during differentiation of multinucleated osteoclasts was confirmed by F-actin staining. Actin belt formation was inhibited by mTM4SF19-Fc, and it was confirmed that TM4SF19 was localized to the actin belt by mouse TM4SF19 antibody (FIG. 38d).
- BMM cells were plated on dentin discs and treated with 10 ⁇ g/ml of IgG-Fc and mTM4SF19-Fc while differentiating, and pit formation was confirmed by 1% toluidine blue staining. As shown in Figure 38e, it was confirmed that bone resorption was blocked by mTM4SF19-Fc.
- mice After oophorectomy in 8-week-old female mice, mice were injected with TM4SF19-Fc through the tail vein once a week for 3 weeks starting one week later, sacrificed one week after the last injection, and micro CT was measured after fixing the femur.
- the mouse TM4SF19-Fc concentrations were 2.5 mg/kg and 5 mg/kg.
- 39A is a micro CT image showing recovery of bone loss by mouse TM4SF19-Fc.
- the micro CT image of FIG. 39a was analyzed with a 3D image analysis program to determine bone volume (BV), ratio of bone volume to tissue volume (% BV/TV), average number of trabeculae (Tb.N), trabecular gap 3D micro CT indicators (bone microstructure indicators) including (Tb.Sp) were obtained (FIG. 39b).
- BV bone volume
- % BV/TV ratio of bone volume to tissue volume
- Tb.N average number of trabeculae
- Tb.Sp trabecular gap 3D micro CT indicators
- total bone mineral density (BMD), % bone volume per tissue volume (BV/TV), and trabecular number (Tb.N) were decreased by ovariectomy and increased by mTM4SF19-Fc in a dose-dependent manner. It was confirmed that On the other hand, Tb. Sp (trabecular separation) was confirmed to decrease by mTM4SF19-Fc. From these results, it can be confirmed that mouse TM4SF19-Fc rescued bone loss induced by ovariectomy.
- mouse femur tissue was histopathologically analyzed by H&E staining.
- H&E staining As a result, as can be seen in FIG. 39c , it can be seen that the number of trabeculars was reduced by the ovariectomy, and the reduction in the number of trabeculars was restored in a dose-dependent manner by the TM4SF19-Fc treatment.
- mouse TM4SF19-Fc (116-165) and human TM4SF19-Fc (131-169) were iv injected. Compared to the untreated group, it was confirmed that bone loss was significantly suppressed by human and mouse TM4SF19-Fc (FIG. 39d).
- Mouse TM4SF19-Fc (116-165) was treated with 10 mg/kg, and human TM4SF19-Fc (131-169) with 25 mg/kg.
- the human TM4SF19 region can rescue bone loss by ovariectomy.
- M-CSF or M-CSF and RANKL treatment are 5 wild-type mice, respectively. Refers to the results obtained by differentiating bone marrow cells obtained from osteoclasts; the group treated with only M-CSF means the non-differentiated group).
- Example 13 Prevention and treatment of rheumatoid arthritis using TM4SF19-Fc
- the CIA score is assessed using the following methods: Score 0 (normal paw), Score 1 (one or two toes inflamed and swollen), Score 2 (3+ toes inflamed w/no paw swelling, or mild swelling of entire paw). , Score 3 (entire paw inflamed and swollen), Score 4 (severely swollen paw and all toes, or ankylosed paw and toes)
- the graph of FIG. 42c shows the results of analyzing the CIA scores and the number of swollen joints examined during the experiment period. It was confirmed that the mTM4SF19-Fc-treated group decreased CIA score and edema in a dose-dependent manner compared to the IgG1-treated control group.
- a micro CT image was taken of the foot of a femur, a mouse model of collagen-induced arthritis, and the images were analyzed.
- 42d is a micro CT image showing that inflammation and bone damage in the foot of a mouse model with collagen-induced arthritis were inhibited by treatment with mTM4SF19-Fc in a dose-dependent manner.
- Collagen-induced arthritis resulted in bent toes and bone damage due to inflammation in the toes and ankles, but joint inflammation and bone damage in mice treated with mTM4SF19-Fc compared to mice treated with hIgG1-Fc (control group). inhibited in a dose-dependent manner.
- micro CT images of the collagen-induced arthritis mouse model femur were analyzed with a 3-dimensional image analysis program to determine bone volume (BV), the ratio of bone volume to tissue volume (% BV/TV), and the average number of bone trabeculae ( Tb.N) and trabecular gap (Tb.Sp) were obtained.
- BV bone volume
- % BV/TV ratio of bone volume to tissue volume
- Tb.N average number of bone trabeculae
- Tb.Sp trabecular gap
- Figure 42f shows that BMD (Bone mineral density) and Tb.N (trabecular number) were reduced by collagen-induced arthritis compared to the sham group, but bone loss was suppressed by TM4SF19-Fc.
- 42g is a result of confirming cartilage damage in a mouse model of collagen-induced arthritis with toluidine blue. It shows that cartilage damage in the mTM4SF19-Fc treated group was suppressed compared to the hIgG1-Fc treated group.
- mTM4SF19-Fc and hTM4SF19-Fc were confirmed by arthritis score in the collagen antibody-induced arthritis mouse model.
- Balbc mice were injected with LPS 3 days after collagen antibody administration to induce arthritis.
- 25 mg/kg of mTM4SF19-Fc and 25 mg/kg of hTM4SF19-Fc showed an anti-arthritis effect (FIG. 42h).
- TM4SF19-Fc treatment lowered the arthritis disease score and decreased paw thickness.
- arthritis was induced by injecting LPS 3 days after collagen antibody administration, and the therapeutic effect of mTM4SF19-Fc was confirmed by arthritis score.
- mTM4SF19-Fc After 3 days of collagen antibody administration, LPS was injected to induce arthritis. In order to confirm the therapeutic effect of mTM4SF19-Fc, mTM4SF19-Fc was treated at 50 mg/kg from the 8th day when the arthritis disease score was the highest, and from the 9th to the 19th Arthritis disease scores were checked every 2 days.
- mTM4SF19-Fc Compared to the untreated group, it was confirmed that mTM4SF19-Fc treatment lowered the arthritis score, decreased the paw thickness, and suppressed cartilage damage confirmed by toluidine blue (FIG. 42i). In other words, mTM4SF19-Fc showed an anti-arthritis effect.
- Example 14 Inhibition of breast cancer bone metastasis using TM4SF19-Fc
- MDA-MB231-luc breast cancer cells (a cell line expressing luciferase in MDA-MB231 for imaging) were injected into the tail of NOD-SCID 6-week-old female mice in the caudal artery (CA) to bone. metastasis was induced. After bone metastasis induction, hIgG1 or TM4SF19-Fc was intravenously injected. TM4SF19-Fc was injected intravenously two days after the cancer cell injection, and 10 mpk or 25 mpk was administered twice a week. Cancer progression was confirmed through bioluminescence imaging analysis.
- 43C shows the result of a micro CT scan of the joint of a mouse in which bone metastasis of breast cancer occurred. According to the micro CT analysis results, bone damage was caused by bone metastasis of breast cancer, and it was confirmed that bone damage was restored in a dose-dependent manner by TM4SF19-Fc treatment.
- MDA-MB231-luc breast cancer cells (a cell line in which luciferase was expressed in MDA-MB231 for imaging) were injected into the tail of NOD-SCID 6-week-old female mice in the caudal artery (CA) to bone. metastasis was induced. 50 mg/kg iv administration of control hIgG1 or TM4SF19-Fc was started 30 days after induction.
- Bioluminescence imaging was performed every 7 days up to 21 days after injection, and it was confirmed that bone metastasis was inhibited by hTM4SF19-Fc (120-169) (FIG. 44B). After luciferin injection, the legs were sacrificed and luminescence was analyzed. As a result, it was confirmed that bone metastasis was inhibited by TM4SF19-Fc treatment (FIG. 44c). It was confirmed by micro CT that bone destruction occurred due to breast cancer bone metastasis and was rescued by TM4SF19-Fc treatment (FIG. 44d).
- Example 15 Inhibition of lung metastasis of breast cancer using TM4SF19-Fc
- Lung metastasis was induced by injecting mouse-derived E0771 breast cancer cells into the tail vein of wild-type mice. After iv injection of hIgG1-Fc, mouse TM4SF19-Fc (116 ⁇ 165), and human TM4SF19-Fc (145 ⁇ 169) at 10 mg/kg each, the inhibition of lung metastasis of breast cancer cells was stained with Indian ink, and the results The number of nodules was checked by leaking. As can be seen in Figure 45, it was confirmed that lung metastasis was significantly inhibited by mouse TM4SF19-Fc (116 to 165) and human TM4SF19-Fc (145 to 169).
- Example 16 Inhibition of high-fat diet-induced obesity and inhibition of fatty liver by TM4SF19-Fc treatment
- a 6-week-old mouse fed a high-fat diet for 7 weeks was purchased, fed a high-fat diet for 1 week and acclimatized, and then fed a high-fat diet for 9 weeks while mice TM4SF19-Fc were injected subcutaneously or intravenously twice a week. administered.
- mice TM4SF19-Fc were injected subcutaneously or intravenously twice a week. administered.
- Figure 46a it was confirmed that obesity caused by a high-fat diet was suppressed by the administration of 10 mg/kg of mTM4SF19-Fc.
- Example 17 Inhibition of adipocyte differentiation by TM4SF19-Fc treatment
- the control group was not treated with differentiation inducing factors, and the experimental group was treated with differentiation inducing factors (dexamethasone 1 ⁇ M, IBMX 500 ⁇ M, insulin 4 ⁇ g/ml (DMI), Rosiglitazone 5 ⁇ M during adipocyte differentiation) and mouse TM4SF19 115-165 -Fc.
- differentiation inducing factors distaloids, IL-12, IL-12, and mouse TM4SF19 115-165 -Fc.
- Adipocyte differentiation was observed by treatment with 0 ng/ml, 1.25 ng/ml, 2.5 ng/ml, 5 ng/ml, and 10 ng/ml.
- Example 18 Cancer cell migration induced by osteoclast differentiation inhibitory effect
- MCSF was added to bone marrow-derived macrophages extracted from wt and TM4SF19KO, and RANKL was added to differentiate or not to differentiate, and cancer cells MDA-MB231 or PC3M were placed on the migration chamber. put in and move on. Migrated cancer cells were stained with crystal violet.
- cancer cell migration was inhibited by osteoclast differentiation in TM4SF19EC2 ⁇ , and MCSF was added to bone marrow-derived macrophages extracted from wt and TM4SF19EC2 ⁇ , and RANKL was added to differentiate or not differentiated, and cancer cells MDA-MB231 or PC3M were placed on the migration chamber. put in and move on. Migrated cancer cells were stained with crystal violet.
- TM4SF19 overexpression increases osteosarcoma cell proliferation and migration. Inhibition of TM4SF19 expression inhibits osteosarcoma cell proliferation and colony formation.
- HOS osteosarcoma cells were treated with 10 ⁇ g/ml hTM4SF19-Fc (120-169 aa) and hTM4SF19-Fc (145-169 aa) (FIG. 56).
- hTM4SF19-Fc containing at least 145 to 169 aa shows activity.
- Pancreas cancer cell lines Panc-1 and MIA-PaCa2 were treated with 100 ⁇ g/ml of hTM4SF19-Fc (120-169), and the ability of the cells to colonize was confirmed by colony formation assay. As a result, as shown in FIG. 57a, it was confirmed that the colonization ability of the pancreatic cancer cell line was suppressed by hTM4SF19-Fc (120-169). Additionally, pancreatic cancer cell lines Panc-1, MIA-PaCa2, and AsPC-1 were treated with 62.5 ⁇ g/ml or 125 ⁇ g/ml of hTM4SF19-Fc(120-169), and cell growth inhibition was confirmed by cell counting (Fig. 57b). As a result, it was confirmed that the growth and colonization ability of pancreatic cancer cells were inhibited by TM4SF19-Fc treatment.
Abstract
Description
Claims (29)
- TM4SF19(transmembrane 4 L six family member 19) 발현 또는 활성 억제제를 유효성분으로 포함하는 골 질환 예방 또는 치료용 약학적 조성물.
- 제 1 항에 있어서,상기 TM4SF19(transmembrane 4 L six family member 19) 발현 또는 활성 억제제는 TM4SF19 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오티드, 짧은 헤어핀 RNA(small hairpin RNA, shRNA), 작은 간섭 RNA(small interfering RNA, siRNA), 마이크로 RNA(microRNA, miRNA) 및 리보자임(ribozyme)으로 구성된 군에서 선택된 하나 이상인 약학적 조성물.
- 제 1 항에 있어서,상기 TM4SF19(transmembrane 4 L six family member 19) 발현 또는 활성 억제제는 TM4SF19 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 모방체, 앱타머, 융합단백질 및 항체로 구성된 군에서 선택된 하나 이상인 약학적 조성물.
- 제 3 항에 있어서,상기 TM4SF19 단백질에 특이적으로 결합하는 융합단백질은 TM4SF19의 단편 및 면역글로불린 Fc 영역이 포함된 것인 약학적 조성물.
- 제 4 항에 있어서,상기 TM4SF19의 단편은 TM4SF19의 세포외 루프 2(extracellular loop 2) 전체 또는 일부 부위를 포함하는 약학적 조성물.
- 제 1 항에 있어서,상기 골 질환은 대사성 골 질환, 정형외과적 골 질환, 무형성 골 질환, 퇴행성 골 질환, 퇴행성 관절염, 류마티스성 관절염, 건선성 관절염, 건선성 척추염, 노화성 골감소증(age-related bone loss), 골다공증, 골 형성 부전증, 골연화증, 골량 감소증, 골절, 골 결손 및 고관절 감소증, 구루병, 파제트 골 질환, 치주질환, 암세포의 골전이에 의해 초래되는 골 손상으로 이루어진 군으로부터 선택된 하나 이상인 약학적 조성물.
- 골 질환 의심 검체에 골 질환 치료용 후보물질을 처리하는 단계; 및대조군과 TM4SF19 유전자의 mRNA 또는 단백질의 발현 수준을 비교하는 단계를 포함하는, 골 질환 치료 약물의 스크리닝 방법.
- TM4SF19 발현 또는 활성 억제제를 유효성분으로 포함하는 비만 또는 비만 매개 대사성 질환 예방 또는 치료용 약학적 조성물.
- 제 8 항에 있어서,상기 TM4SF19(transmembrane 4 L six family member 19) 발현 또는 활성 억제제는 TM4SF19 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오티드, 짧은 헤어핀 RNA(small hairpin RNA, shRNA), 작은 간섭 RNA(small interfering RNA, siRNA), 마이크로 RNA(microRNA, miRNA) 및 리보자임(ribozyme)으로 구성된 군에서 선택된 하나 이상인 약학적 조성물.
- 제 8 항에 있어서,상기 TM4SF19(transmembrane 4 L six family member 19) 발현 또는 활성 억제제는 TM4SF19 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 모방체, 앱타머, 융합단백질 및 항체로 구성된 군에서 선택된 하나 이상인 약학적 조성물.
- 제 10 항에 있어서,상기 TM4SF19 단백질에 특이적으로 결합하는 융합단백질은 TM4SF19의 단편 및 면역글로불린 Fc 영역이 포함된 것인 약학적 조성물.
- 제 11 항에 있어서,상기 TM4SF19 단편은 TM4SF19의 세포외 루프 2(extracellular loop 2) 전체 또는 일부 부위를 포함하는 약학적 조성물.
- 제 8 항에 있어서,상기 비만 매개 대사성 질환은 고혈압, 당뇨, 인슐린 내성 증후군, 대사 증후군, 비만관련 위식도 역류성 질환, 동맥경화증, 고지혈증, 과중성지방혈증, 과콜레스테롤혈증, 지방이영양증, 비알콜성 지방간염, 심혈관 질환 및 다낭성 난소 증후군으로 이루어진 군에서 선택된 하나 이상인 약학적 조성물.
- 비만 또는 비만 매개 대사성 질환 의심 검체에 비만 또는 비만 매개 대사성 질환 치료용 후보물질을 처리하는 단계; 및대조군과 TM4SF19 유전자의 mRNA 또는 단백질의 발현수준을 비교하는 단계를 포함하는, 비만 또는 비만 매개 대사성 질환 치료 약물의 스크리닝 방법.
- TM4SF19 발현 또는 활성 억제제를 유효성분으로 포함하는 암 예방, 치료 또는 암 전이 억제용 약학적 조성물.
- 제 15 항에 있어서,상기 TM4SF19(transmembrane 4 L six family member 19) 발현 또는 활성 억제제는 TM4SF19 유전자의 mRNA에 상보적으로 결합하는 안티센스 뉴클레오티드, 짧은 헤어핀 RNA(small hairpin RNA, shRNA), 작은 간섭 RNA(small interfering RNA, siRNA), 마이크로 RNA(microRNA, miRNA) 및 리보자임(ribozyme)으로 구성된 군에서 선택된 하나 이상인 약학적 조성물.
- 제 15 항에 있어서,상기 TM4SF19(transmembrane 4 L six family member 19) 발현 또는 활성 억제제는 TM4SF19 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 모방체, 앱타머, 융합단백질 및 항체로 구성된 군에서 선택된 하나 이상인 약학적 조성물.
- 제 17 항에 있어서,상기 TM4SF19 단백질에 특이적으로 결합하는 융합단백질은 TM4SF19의 단편 및 면역글로불린 Fc 영역이 포함된 것인 약학적 조성물.
- 제 18 항에 있어서,상기 TM4SF19의 단편은 TM4SF19의 세포외 루프 2(extracellular loop 2) 전체 또는 일부 부위를 포함하는 약학적 조성물.
- 제 15 항에 있어서,상기 암은 대장암, 위암, 결장암, 유방암, 폐암, 비소세포성폐암, 골암, 췌장암, 피부암, 두부 또는 경부암, 흑색종, 자궁암, 난소암, 소장암, 직장암, 항문부근암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 식도암, 임파선암, 방광암, 담낭암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 전립선암, 만성 또는 급성 백혈병, 림프구림프종, 방광암, 신장 또는 수뇨관 암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS; central nervous system) 종양, 척수 종양, 뇌간 신경교종, 및 뇌하수체 선종으로 이루어진 군에서 선택된 하나 이상인 약학적 조성물.
- 암 예방, 치료 또는 암 전이 의심 검체에 암 예방, 치료 또는 암 전이 억제용 후보물질을 처리하는 단계; 및대조군과 TM4SF19 유전자의 mRNA 또는 단백질의 발현 수준을 비교하는 단계를 포함하는, 암 또는 암 전이 치료 약물의 스크리닝 방법.
- TM4SF19 단백질의 세포외 루프 2(extracellular loop 2) 유래 단편 및 면역글로불린 Fc 영역을 포함하는 TM4SF19 발현 또는 활성 억제용 융합 단백질.
- 제 22 항에 있어서,상기 TM4SF19 단백질의 세포외 루프 2(extracellular loop 2, EC2) 유래 단편은 EC2 전체 또는 일부에 해당하는 아미노산 서열 부위인 융합 단백질.
- 제 22 항에 있어서,상기 EC2 전체에 해당하는 아미노산 서열 부위는 인간 TM4SF19 단백질의 120-169번째 아미노산 부위 또는 마우스 TM4SF19 단백질의 116-165 번째 아미노산 부위인 융합 단백질.
- 제 22 항에 있어서,상기 EC2 일부에 해당하는 아미노산 서열 부위는 인간 TM4SF19 단백질의 145-169번째 아미노산 부위를 포함하는 융합 단백질.
- 제 22 항에 있어서,SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16 또는 SEQ ID NO: 19로 표시되는 아미노산 서열을 포함하는 융합 단백질.
- 치료학적으로 유효한 양의 TM4SF19 발현 또는 활성 억제제를 포함하는 골 질환 예방 또는 치료용 조성물을 대상체에 투여하는 단계를 포함하는 골 질환을 예방 또는 치료하는 방법.
- 치료학적으로 유효한 양의 TM4SF19 발현 또는 활성 억제제를 포함하는 비만 또는 비만 매개 대사성 질환 예방 또는 치료용 조성물을 대상체에 투여하는 단계를 포함하는 비만 또는 비만 매개 대사성 질환을 예방 또는 치료하는 방법.
- 치료학적으로 유효한 양의 TM4SF19 발현 또는 활성 억제제를 포함하는 암 예방, 치료 또는 암 전이 억제용 조성물을 대상체에 투여하는 단계를 포함하는 암 예방, 치료 또는 암 전이를 예방 또는 치료하는 방법.
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IL309059A IL309059A (en) | 2021-06-03 | 2022-06-03 | Inhibitor of TM4SF19 and its uses |
AU2022284686A AU2022284686A1 (en) | 2021-06-03 | 2022-06-03 | Tm4sf19 inhibitor and uses thereof |
EP22816502.3A EP4349358A1 (en) | 2021-06-03 | 2022-06-03 | Tm4sf19 inhibitor and uses thereof |
CA3220026A CA3220026A1 (en) | 2021-06-03 | 2022-06-03 | Tm4sf19 inhibitor and uses thereof |
CN202280038803.9A CN117460522A (zh) | 2021-06-03 | 2022-06-03 | Tm4sf19抑制剂及其用途 |
BR112023025305A BR112023025305A2 (pt) | 2021-06-03 | 2022-06-03 | Composições farmacêuticas, proteína de fusão, e, uso do inibidor do membro 19 da família 4 l seis da transmembrana |
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- 2022-06-03 AU AU2022284686A patent/AU2022284686A1/en active Pending
- 2022-06-03 WO PCT/KR2022/007939 patent/WO2022255843A1/ko active Application Filing
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CA3220026A1 (en) | 2022-12-08 |
BR112023025305A2 (pt) | 2024-02-27 |
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