Variant of bone morphogenetic protein-9, 10 in which therapeutic effect is enhanced by alleviation of heterotopic ossification side effect, and pharmaceutical composition containing the same

Abstract

To provide a bone morphogenetic protein-9 (BMP-9) variant in which heterotopic ossification side effect is alleviated, and a fusion thereof.SOLUTION: Provided are a bone morphogenetic protein-9 (BMP-9) variant expressed by a specific amino acid sequence, BMP-9 variant-Fc fusion protein in which an Fc fragment of immunoglobulin is connected to the BMP-9 variant, and a pharmaceutical composition for treating cardiopulmonary disease which contains the BMP-9 variant or the BMP-9 variant-Fc fusion protein as an active ingredient. The cardiopulmonary disease is one or more kinds selected from the group consisting of cardiac infarction, hypertension, pulmonary hypertension, myocardial fibrosis, and pulmonary fibrosis.SELECTED DRAWING: Figure 4b

Description

[Technical field]

[0001] The present invention relates to BMP-9 variants and derivatives thereof, and more particularly to BMP-9 variants and derivatives thereof that have enhanced therapeutic effects by reducing ectopic bone formation side effects in the treatment of various diseases, disorders and disorders including tumors, cardiovascular diseases, fibrotic diseases and metabolic diseases.

[0002] [Background technology]

[0003] BMP-9 is distinguished from other BMPs because it has the property of binding to a unique receptor, not to the existing BMP receptors, and plays various roles in many intracellular processes. For example, BMP-9 is produced in the liver and can suppress lipid metabolism and control glucose production in the liver, and in the cardiovascular system, it regulates the growth and migration of endothelial cells and suppresses fibrosis of cardiac myoblasts. In particular, in the cardiovascular system, it binds to the ALK-1 receptor, BMPR-II, and endoglin of endothelial cells together with BMP-10, and is also involved in vascular homeostasis and blood pressure regulation. Many literature reports that when BMP-9 and BMP-10 are applied to pulmonary hypertension and myocardial fibrosis caused by the deficiency of the corresponding receptors, the corresponding symptoms are alleviated, so that BMP-9 can be highly useful as a therapeutic substance. In addition, BMP-9 is one of the powerful BMPs that can induce the death of prostate cancer cells depending on the type of tumor and regulate osteogenic differentiation in bone tissue. It also has the effect of improving insulin sensitivity, and is known to have high potential as a target for new anti-diabetes or anti-obesity therapeutic agents.

[0004] Accordingly, in order to develop new therapeutic drugs targeting growth factors including BMP-9, attempts have been made to enhance the biological activity of growth factors by creating mutations by substituting, introducing, and deleting some amino acids based on wild-type growth factors (WO2010 / 065439). However, a problem associated with the direct use of growth factors including BMP-9 as biotherapeutics is that ectopic bone formation and transformation growth factor action are simultaneously expressed. This is due to the complexity of the receptors of growth factors including BMP-9, and there are still few cases in which side effects related to ectopic bone formation have been alleviated. The receptors of BMP-9 and BMP-10 are mainly present in vascular endothelial cells, and are known to be involved in vascular function regulation by acting as ligands for Alk-1 and BMPR-II. That is, BMPR-II forms a complex with ALK-1 and selectively reacts with BMP-9 and 10, and in this case, diseases that frequently occur when the receptor is missing or BMP-9 and 10 are deficient are pulmonary hypertension and myocardial fibrosis. In particular, BMP-9 acts directly on endothelial cells to promote the integrity of the vascular inner wall and inhibit vascular cell death and neovascularization by promoting vascular stability. In particular, unlike other BMPs such as BMP-2, 4, and 6, BMP-9 is known to promote endothelial cell activity even at low concentrations that do not induce bone formation. However, when applied at high concentrations, BMP-9 can also induce ectopic bone formation, and when actually applied in clinical practice, it is necessary to apply a mutant with a controlled ectopic bone formation function.

[0005] In addition, the wild type growth factor has a very short half-life in the body, which has been pointed out as a factor that makes it difficult to commercialize it as a therapeutic agent (Kharitonenkov, A. et al., Journal of Clinical Investigation, 115: 1627-1635, 2005). The half-life of BMP-9 in the body is short, 10 minutes to 1 hour in mice and 1.5 to 2 hours in monkeys, so if it is developed as a therapeutic agent, it has the disadvantage of having to be administered every day. To date, various techniques have been reported to increase the half-life of recombinant proteins in the body. In one example, the molecular weight was increased by linking the polymeric substance polyethylene glycol (PEG) to the protein, and the residence time in the body was increased by suppressing renal excretion (WO2012 / 066075). In another example, the half-life was improved by fusing a fatty acid that binds to human albumin to the growth factor molecule (WO2012 / 010553). Furthermore, there are examples where agonist antibodies that specifically bind to human growth factor receptors alone or in complex with beta-Klotho have been created, and have shown the same pharmacological activity as the mechanism of action of growth factors while increasing their half-life (WO2012 / 170438). There are also examples where a sustained fusion protein has been created by linking the Fc of immunoglobulin IgG to a growth factor molecule, improving its half-life (WO2013 / 188181).

[0006] Therefore, the inventors have devoted themselves to researching BMP9-based therapeutic mutant proteins for suppressing ectopic bone formation and extending their duration of action, and as a result, have confirmed that in the case of some mutants, ectopic bone formation is inhibited and the in vivo half-life is increased, thereby completing the present invention.

[0007] The information provided in this Background section is intended to enhance understanding of the background of the invention only and may not contain information that constitutes prior art already known to a person of ordinary skill in the art to which the invention pertains. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2010 / 065439

[0009] [Patent Document 2] WO2012 / 066075

[0010] [Patent Document 3] WO2012 / 010553

[0011] [Patent Document 4] WO2012 / 170438

[0012] [Patent Document 5] WO2013 / 188181 [Non-patent literature]

[0013] [Non-Patent Document 1] Kharitonenkov, A. et al., Journal of Clinical Investigation, 115:1627-1635, 2005 Summary of the Invention [Problem to be solved by the invention]

[0014] An object of the present invention is to provide a bone morphogenetic protein-9 (BMP-9) variant and a fusion thereof that have reduced side effects of ectopic bone formation.

[0015] Another object of the present invention is to provide various therapeutic uses of the bone morphogenetic protein-9 (BMP-9) variants and fusions thereof.

[0016] It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating tumors, inflammatory diseases, metabolic diseases or autoimmune diseases, comprising the BMP-9 mutant or a fusion product thereof.

[0017] It is still another object of the present invention to provide a method for preventing or treating tumors, inflammatory diseases, metabolic diseases or autoimmune diseases, which comprises the step of administering the BMP-9 mutant or a fusion thereof.

[0018] It is still another object of the present invention to provide uses of the BMP-9 variants and fusions thereof for the prevention or treatment of tumors, inflammatory diseases, metabolic diseases or autoimmune diseases.

[0019] It is yet another object of the present invention to provide the use of said BMP-9 variants and fusions thereof in the manufacture of a medicament for the treatment of tumors, inflammatory diseases, metabolic diseases or autoimmune diseases. [Means for solving the problem]

[0020] To achieve the above object, the present invention provides a bone morphogenetic protein-9 (BMP-9) mutant represented by any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2 to 29.

[0021] The present invention also provides a BMP-9 mutant-Fc fusion protein in which an Fc fragment of immunoglobulin is linked to the BMP-9 mutant.

[0022] The present invention also provides a pharmaceutical composition for tumor treatment comprising the BMP-9 mutant or the BMP-9 mutant-Fc fusion protein as an active ingredient.

[0023] The present invention also provides a pharmaceutical composition for treating an inflammatory disease, which comprises the BMP-9 mutant or the BMP-9 mutant-Fc fusion protein as an active ingredient.

[0024] The present invention also provides a pharmaceutical composition for treating a metabolic disease, which comprises the BMP-9 mutant or the BMP-9 mutant-Fc fusion protein as an active ingredient.

[0025] The present invention also provides a pharmaceutical composition for treating an autoimmune disease, which comprises the BMP-9 mutant or the BMP-9 mutant-Fc fusion protein as an active ingredient.

[0026] The present invention also provides a method for preventing or treating tumors, inflammatory diseases, metabolic diseases, or autoimmune diseases, comprising the step of administering the BMP-9 mutant or the BMP-9 mutant-Fc fusion protein.

[0027] The present invention also provides use of the BMP-9 mutant or the BMP-9 mutant-Fc fusion protein for the prevention or treatment of tumors, inflammatory diseases, metabolic diseases or autoimmune diseases.

[0028] The present invention also provides the use of said BMP-9 variant or said BMP-9 variant-Fc fusion protein in the manufacture of a medicament for the treatment of a tumor, an inflammatory disease, a metabolic disease or an autoimmune disease. [Brief description of the drawings]

[0029] [Figure 1] FIG. 1 is a schematic diagram showing an overview of the present invention.

[0030] [Diagram 2] FIG. 1 shows expression vectors into which BMP-9, BMP-9 mutants, and BMP-9 fused with Fc are introduced.

[0031] [Diagram 3] Figure 3a shows the SDS-PAGE and western blot results of expressed and purified BMP-9 mutants, and Figure 3b shows the SDS-PAGE and western blot results of expressed and purified BMP-9 fused to Fc.

[0032] [Figure 4] FIG. 4a shows the signal transduction activity of expressed and purified BMP-9 and mutants on vascular endothelial cells, and FIG. 4b shows the osteogenic signal transduction activity.

[0033] [Diagram 5] FIG. 1 shows the inhibitory effect of BMP-9 mutants on fibrosis-related markers in cardiac fibrocytes.

[0034] [Figure 6] 1 is a graph showing the measurement of blood concentrations of blood-purified BMP-9 and Fc-fused BMP-9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs. In general, the nomenclature used herein and the experimental methods described below are those well known and commonly used in the art.

[0036] In the present invention, the BMP-9 gene was cloned, various mutants were prepared, and then the mutants were expressed and purified in mammalian cells (CHO cells and human fetal kidney cell lines). BMP-9 mutants that are effective against tumors, inflammatory diseases, cardiovascular diseases, metabolic diseases, and autoimmune diseases were selected, and the blood half-life was increased by fusing Fc to the mutants. Specifically, in the present invention, one or more amino acids were replaced in the amino acid sequence corresponding to the wrist epitope and knuckle epitope of the BMP-9 protein, and the receptor binding strength was adjusted to induce optimal BMP-9 therapeutic effects. That is, the present invention aimed to select mutants that can suppress ectopic bone formation to less than one-tenth while maintaining the signal transmission specific to endothelial cells. In addition, it was confirmed through alkaline phosphatase activity and Alizarin Red staining that the mutant according to the present invention has a significantly reduced ability to form ectopic bone in mouse cardiac fibrocytes (C2C12) compared to the wild type.

[0037] Therefore, in one aspect, the present invention relates to bone morphogenetic protein-9 (BMP-9) or a variant thereof represented by any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 29, and in another aspect, the present invention relates to a BMP-9 variant-Fc fusion protein in which an Fc fragment of an immunoglobulin is linked to the bone morphogenetic protein-9 (BMP-9) or a variant thereof.

[0038] The amino acid sequence of SEQ ID NO: 1 represents the wild type of BMP-9, and includes the signal sequence shown in bold and the Pro-BMP-9 sequence shown in underlined, as shown below: The FFPLADDVTPTKHAIVQTLVHLKF sequence is a region that binds to ALK-1 and belongs to the list epitope when viewed from the overall structure of BMP-9, and the KVGKACCVPTKLSPISVLYK sequence belongs to the knuckle epitope that binds to the BMP-2 receptor.

[0039] JPEG2025081327000002.jpg47164

[0040] The mutant sequence derived from the wild-type BMP-9 sequence may be represented by any one of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 29. In addition, these mutants are obtained by replacing a portion of the amino acid sequence of the wild-type BMP-9 sequence. However, it will be obvious to those skilled in the art that the mutant according to the present invention is not limited to the specific amino acid sequence represented by the SEQ ID NO:, and any amino acid sequence that can be considered equivalent to the corresponding amino acid sequence falls within the scope of the present invention. For example, the 335th amino acid in SEQ ID NO: 2 is replaced by Ala instead of Asp compared to the wild-type. If a portion of the remaining sequence excluding the core structure of such a mutant is mutated so as not to affect the function and structure of the BMP-9 protein, it will be obvious to those skilled in the art that the corresponding mutant also falls within the scope of the present invention. Therefore, it will be understood that the present invention also includes mutants that have at least 95% or more, at least 90% or more, at least 80% or more, or at least 70% or more homology with the remaining sequence excluding the replacement of the core amino acid of the mutant in any one of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 29.

[0041] On the other hand, the variants of the present invention may also be applicable to other subtypes of bone morphogenetic proteins, such as BMP-7 and -10.

[0042] In the present invention, in order to increase the half-life in blood of wild-type or mutant BMP-9, an Fc fragment of immunoglobulin may be fused to the wild-type or mutant BMP-9. Here, the Fc fragment of immunoglobulin may be characterized by being represented by the amino acid sequence of SEQ ID NO: 30, but is not limited thereto. Meanwhile, in addition to the Fc fragment of immunoglobulin, various peptides or proteins that can be fused to a protein to increase the half-life in blood, such as albumin, may be fused to the wild-type or mutant BMP-9.

[0043] In the present invention, the Fc fragment of the immunoglobulin may be linked to the N-terminus or C-terminus of the bone morphogenetic protein-9 (BMP-9) or variant thereof.

[0044] In the present invention, the bone morphogenetic protein-9 (BMP-9) or a variant thereof; and the Fc fragment of immunoglobulin may be characterized as being linked by a linker, but is not limited thereto.

[0045] In the present invention, the linker may be characterized by being represented by the amino acid sequence of SEQ ID NO: 31, but is not limited thereto.

[0046] In the present invention, the BMP-9 mutant-Fc fusion protein may be characterized by being represented by the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33, but is not limited thereto.

[0047] The BMP-9 or variants, or fusion proteins thereof, of the present invention may be used as therapeutic, diagnostic, or research reagents, but are most preferably used therapeutically.

[0048] Specifically, the present invention provides a pharmaceutical composition for tumor treatment comprising the bone morphogenetic protein-9 (BMP-9) or a mutant thereof; or the BMP-9 mutant-Fc fusion protein as an active ingredient.

[0049] In the present invention, the tumor may be characterized as being one or more types selected from the group consisting of breast cancer, lung cancer, colon cancer, colorectal cancer, liver cancer, pancreatic cancer, brain tumor, prostate cancer, skin cancer, osteosarcoma, and blood cancer, but is not limited thereto.

[0050] The present invention also provides a pharmaceutical composition for treating an inflammatory disease, comprising the bone morphogenetic protein-9 (BMP-9) or a mutant thereof, or the BMP-9 mutant-Fc fusion protein as an active ingredient.

[0051] In the present invention, the inflammatory disease may be characterized as one or more selected from the group consisting of steatohepatitis, hepatitis, and enteritis, but is not limited thereto.

[0052] The present invention also provides a pharmaceutical composition for treating a metabolic disease, comprising the bone morphogenetic protein-9 (BMP-9) or a mutant thereof, or the BMP-9 mutant-Fc fusion protein as an active ingredient.

[0053] In the present invention, the metabolic disease may be characterized as being one or more selected from the group consisting of obesity, weight loss, diabetes, atherosclerosis, arteriosclerosis, cardiopulmonary disease, neurological disease, Alzheimer's disease, cognitive impairment, oxidative stress, skin disease, skin aging, damage caused by UV radiation, hypertension, hypercholesterolemia (LDL, HDL, VLDL), hyperlipidemia (triglyceride), immune deficiency, cancer and metabolic syndrome, but is not limited thereto.

[0054] In the present invention, the cardiopulmonary disease may be characterized as one or more selected from the group consisting of myocardial infarction, hypertension, pulmonary hypertension, myocardial fibrosis and pulmonary fibrosis, but is not limited thereto.

[0055] The present invention also provides a pharmaceutical composition for autoimmune therapy, comprising the bone morphogenetic protein-9 (BMP-9) or a mutant thereof, or the BMP-9 mutant-Fc fusion protein as an active ingredient.

[0056] In the present invention, the autoimmune disease may be one or more selected from the group consisting of insulin-dependent diabetes mellitus, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis, osteoarthritis, myasthenia gravis, thyroiditis, uveitis, Hashimoto's thyroiditis, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, autoimmune hemolytic anemia, idiopathic leukopenia, primary sclerosing cholangitis, alcoholic / non-alcoholic steatohepatitis, inflammatory bowel disease, Crohn's disease, ulcerative bowel disease, psoriasis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid LE, and systemic lupus erythematosus, but is not limited thereto.

[0057] For the above-mentioned therapeutic purposes, the active BMP-9 mutants and derivatives of the present invention may be administered alone, but are preferably administered in the form of a pharmaceutical composition (dosage form), preferably in the form of a sterile dosage form.

[0058] In the present invention, the pharmaceutical composition may be characterized by being formulated into any one of the dosage forms selected from the group consisting of injections, oral preparations, liquids (e.g., for injection) such as aqueous solutions, suspensions, and emulsions, capsules, granules, tablets, and preparations for mucosal administration, but is not limited thereto. These preparations can be prepared by conventional methods used for formulation in the art or by methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA, and can be formulated into various preparations depending on each disease or component.

[0059] Meanwhile, the pharmaceutical composition of the present invention may further comprise one or more pharma- ceutical acceptable carriers in addition to the therapeutic BMP-9 variants and derivatives, which may include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, or a mixture of one or more of these components.

[0060] The pharmaceutical composition of the present invention may further contain a pharma- ceutically acceptable auxiliary agent, if necessary, which may be one or more selected from the group consisting of excipients, diluents, dispersants, buffers, antibacterial preservatives, bacteriostatic agents, surfactants, binders, lubricants, antioxidants, thickeners, and viscosity modifiers, but is not limited thereto.

[0061] The pharmaceutical composition according to the present invention may be administered orally or parenterally (e.g., intravenously, subcutaneously, intramuscularly, intraperitoneally or topically) depending on the intended method, and the dosage may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease, etc., within the range determined by a specialist.

[0062] In one embodiment of the present invention, the protein is administered at a single dose of 1 μg / kg to 100 mg / kg, preferably 5 μg / kg to 50 mg / kg, once a day or once to three times a week, but the dose and administration interval are not limited thereto.

[0063] In yet another aspect, the present invention relates to a method for preventing or treating tumors, inflammatory diseases, metabolic diseases or autoimmune diseases, comprising the step of administering the BMP-9 mutant or the BMP-9 mutant-Fc fusion protein.

[0064] In yet another aspect, the present invention relates to use of the BMP-9 mutant or the BMP-9 mutant-Fc fusion protein for the prevention or treatment of tumors, inflammatory diseases, metabolic diseases, or autoimmune diseases.

[0065] In yet another aspect, the present invention relates to the use of the BMP-9 mutant or the BMP-9 mutant-Fc fusion protein in the manufacture of a medicament for the treatment of a tumor, an inflammatory disease, a metabolic disease or an autoimmune disease. EXAMPLES

[0066] Working Example The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0067] Example 1. Production of recombinant human proBMP-9 and proBMP10

[0068] The entire cDNA including the open reading frame of human pre-proBMP9 was inserted and cloned into the pcDNA3.4 vector (Figure 2), which was confirmed by DNA sequencing. Pro-BMP9 mutants were obtained using the QuickChange site-directed mutagenesis kit, which was also confirmed by DNA sequencing.

[0069] The BMP-9 wild-type and mutant sequences used in the present invention are as follows:

[0070] SEQ ID NO: 1. Wild type latent BMP-9 (NIBEC-J)

[0071] JPEG2025081327000003.jpg46170

[0072] In the wild-type sequence, bold indicates the signal peptide sequence, underline indicates the Pro-BMP sequence, and no indication is given in the BMP-9 sequence.

[0073] The plasmid containing PreproBMP9 was transfected into CHO-S cells with polyethyleneimine, and the cells were cultured for 8 days after adding enhancer. The expressed ProBMP9 and ProBMP10 were measured by Western blot using antiBMP9 and antiBMP10 antibodies.

[0074] The expressed protein was separated through a Q-Sepharose column that had already reached equilibrium using 1-5 liters of conditioned media as a buffer. The target protein attached to the column was fractionated through a sodium chloride gradient, and the fractionated sample was concentrated and passed through gel chromatography to obtain the target molecular weight. The protein obtained through this process was confirmed to have a purity of 95% or more.

[0075] Example 2. Construction of recombinant human proBMP-9 mutants

[0076] BMP9 and BMP10 selectively bind to the ALK1 receptor of vascular endothelial cells, which makes them promising therapeutic agents for cardiovascular diseases. However, they still have the potential to stimulate mesenchymal cells and myoblasts to promote bone formation, so this property must be neutralized when they are developed as therapeutic agents. To date, it has not been clarified which receptor is related to the bone differentiation ability of BMP9 and 10. The present researcher has determined that the wrist epitope and knuckle epitope in the structure of BMP-9 act as ligands that mediate the main signal transmission, and confirmed that by adjusting these sequences, it is possible to suppress the bone differentiation ability while maintaining the endothelial cell signal transmission (sequence 1-29, Figure 4). It was determined that such a mutant has similar physiological activity in vivo, and it is expected to provide the advantage of minimizing the concerns of side effects while improving the therapeutic effect.

[0077] Example 3. Signal transduction by BMP-9 mutants and derivatives in endothelial cells

[0078] The amount of each ProBMP9 mutant was measured by ELISA before treatment with cells. The mutants were added to HUVEC cells in the indicated concentration range without serum. After 8 hours of treatment, the cells were harvested and mRNA was extracted to measure the expression of ID1 and BMPR-II by quantitative PCR. For the expression of pSmad1 / 5 / 8, the cells were treated with BMP9 mutants in a serum-free state and observed after 1 hour. The expression level of the protein obtained after treating the cells with a lysis buffer was measured by immunoblotting with anti-pSmad1 / 5 / 8 antibody. As a result of the observation (Figure 4), it was confirmed that each mutant significantly increased ID1 and BMPR-II, and the increased amount did not show a significant difference compared to wild-type BMP-9. As confirmed in a previous report, it was confirmed that BMP9 and BMP10 act as ligands for the ALK1 receptor in vascular endothelial cells. According to the literature, BMP9 is known to inhibit endothelial cell migration, proliferation and angiogenesis by endothelial cells in the circulatory system.

[0079] Example 4. Bone differentiation signaling by BMP9 mutants in C2C12 cell cultures

[0080] C2C12, a mouse cardiomyocyte, was selected to observe the osteogenic differentiation ability because it has a differentiation ability similar to that of mesenchymal stem cells. C2C12 cells were cultured in DMEM medium with 0.25% FBS for 16 hours and then treated with each BMP9 variant. After further culturing for 72 hours, the cells were disrupted with 1% Triton X-100 / PBS, and the ALP enzyme activity was measured for the protein obtained. The ALP enzyme activity was confirmed by measuring the absorbance at 405 nm of the water-soluble substance produced by reacting with 4-nitrophenyl phosphate disodium salt, which is the substrate of the enzyme, and BMP-9 was purchased and used as a control. As a result of the observation, it was confirmed that each variant had a significantly reduced osteogenic differentiation ability compared to the standard substance (Figure 4). It was determined that each variant thus formed maintained the vascular endothelial cell signal transmission in Example 3, but had abolished osteogenic ability, providing a therapeutic effect without causing ectopic bone formation side effects.

[0081] Example 5. Effect of extending the half-life of BMP9-Fc derivatives in vivo

[0082] BMP-9 and its Fc-fused derivatives were injected into the tail vein of mice at a dose of 1 mg / kg, and blood was collected at regular intervals to measure the amount of BMP-9 in the blood using a BMP-9 quantification kit. As a result of the measurement (FIG. 5), it was confirmed that the Fc-bound BMP-9 derivatives (SEQ ID NOs: 32-33) showed a significantly increased blood concentration curve.

[0083] Although the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, it can be said that the substantial scope of the present invention is defined by the appended claims and their equivalents. [Industrial Applicability]

[0084] In the present invention, a mutant of BMP-9 was designed to maximize the therapeutic effect of BMP-9 and significantly reduce side effects, and was expressed and purified efficiently in mammalian cells. In addition, a fusion protein was prepared by fusing BMP-9 with the Fc region of immunoglobulin to extend the half-life of BMP-9 in the blood. These mutants and fusion proteins have been shown to have excellent therapeutic effects against various diseases such as tumors, fibrosis, cardiopulmonary vascular diseases, obesity, and fatty liver through in vitro and animal experiments, and therefore may be used as novel therapeutic agents for the treatment of these diseases.

[0085] [Sequence List Free Text]

[0086] Electronic file attached.

Claims

1. A bone morphogenetic protein-9 (BMP-9) variant represented by any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 2 to 29.

2. A BMP-9 mutant-Fc fusion protein in which an Fc fragment of an immunoglobulin is linked to the BMP-9 mutant according to claim 1.

3. The BMP-9 mutant-Fc fusion protein according to claim 2, wherein the Fc fragment of immunoglobulin is represented by the amino acid sequence of SEQ ID NO:

30.

4. 3. The BMP-9 mutant-Fc fusion protein according to claim 2, wherein the BMP-9 mutant and the Fc fragment of immunoglobulin are linked by a linker.

5. The BMP-9 mutant-Fc fusion protein according to claim 4, wherein the linker is represented by the amino acid sequence of SEQ ID NO:

31.

6. A pharmaceutical composition for tumor treatment comprising the BMP-9 mutant according to claim 1 or the BMP-9 mutant-Fc fusion protein according to any one of claims 2 to 5 as an active ingredient.

7. The pharmaceutical composition for tumor treatment according to claim 6, wherein the tumor is one or more selected from the group consisting of breast cancer, lung cancer, colon cancer, colorectal cancer, liver cancer, pancreatic cancer, brain cancer, prostate cancer, skin cancer, osteosarcoma, and blood cancer.

8. A pharmaceutical composition for treating an inflammatory disease, comprising as an active ingredient the BMP-9 mutant according to claim 1; or the BMP-9 mutant-Fc fusion protein according to any one of claims 2 to 5.

9. The pharmaceutical composition for treating an inflammatory disease according to claim 8, wherein the inflammatory disease is at least one selected from the group consisting of steatohepatitis, hepatitis, and enteritis.

10. A pharmaceutical composition for treating a metabolic disease, comprising as an active ingredient the BMP-9 mutant according to claim 1; or the BMP-9 mutant-Fc fusion protein according to any one of claims 2 to 5.

11. The pharmaceutical composition for treating metabolic diseases according to claim 10, wherein the metabolic disease is at least one selected from the group consisting of obesity, weight loss, diabetes, atherosclerosis, arteriosclerosis, cardiopulmonary disease, neurological disease, Alzheimer's disease, cognitive impairment, oxidative stress, skin disease, skin aging, damage caused by UV radiation, hypertension, hypercholesterolemia (LDL, HDL, VLDL), hyperlipidemia (triglyceride), immune deficiency, cancer, and metabolic syndromes.

12. The pharmaceutical composition for treating metabolic diseases according to claim 11, wherein the cardiopulmonary disease is one or more selected from the group consisting of myocardial infarction, hypertension, pulmonary hypertension, myocardial fibrosis, and pulmonary fibrosis.

13. A pharmaceutical composition for treating an autoimmune disease, comprising as an active ingredient the BMP-9 mutant according to claim 1; or the BMP-9 mutant-Fc fusion protein according to any one of claims 2 to 5.

14. 14. The pharmaceutical composition for treating an autoimmune disease according to claim 13, wherein the autoimmune disease is at least one selected from the group consisting of insulin-dependent diabetes mellitus, multiple sclerosis, autoimmune encephalomyelitis, rheumatoid arthritis, osteoarthritis, myasthenia gravis, thyroiditis, uveitis, Hashimoto's thyroiditis, thyrotoxicosis, pernicious anemia, autoimmune atrophic gastritis, autoimmune hemolytic anemia, idiopathic leukopenia, primary sclerosing cholangitis, alcoholic / non-alcoholic steatohepatitis, inflammatory bowel disease, Crohn's disease, ulcerative bowel disease, psoriasis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid LE, and systemic lupus erythematosus.

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