Bone morphogenetic protein-9 and -10 variants with improved therapeutic effects due to reduced side effects of ectopic bone formation and pharmaceutical compositions 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 improved therapeutic effects by reducing the side effect of ectopic bone formation 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 distinct from other BMPs because it binds to a unique receptor rather than to other known BMP receptors, and plays diverse roles in numerous intracellular processes. For example, BMP-9 is produced in the liver and can inhibit lipid metabolism and regulate hepatic glucose production. In the cardiovascular system, BMP-9 regulates endothelial cell growth and migration and inhibits cardiomyoblast fibrosis. In particular, in the cardiovascular system, it binds to the ALK-1 receptor, BMPR-II, and endoglin on endothelial cells, along with BMP-10, and is involved in vascular homeostasis and blood pressure regulation. Numerous studies have reported that the administration of BMP-9 and BMP-10 can alleviate symptoms of pulmonary hypertension and myocardial fibrosis caused by deficiency of the corresponding receptors, demonstrating the potential for BMP-9 as a therapeutic agent. 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-diabetic or anti-obesity therapeutic agents.

[0004] To develop new therapeutic agents targeting growth factors, including BMP-9, attempts have been made to enhance the biological activity of growth factors by creating mutations based on wild-type growth factors, such as substituting, introducing, or deleting certain amino acids (WO 2010 / 065439). However, a problem associated with the direct use of growth factors, including BMP-9, as biotherapeutics is the simultaneous occurrence of ectopic bone formation and transforming growth factor activity. This is due to the complexity of the receptors for growth factors, including BMP-9, and there have been few cases in which side effects associated with ectopic bone formation have been alleviated. Receptors for BMP-9 and BMP-10 are primarily found on vascular endothelial cells and are known to regulate vascular function by acting as ligands for Alk-1 and BMPR-II. BMPR-II forms a complex with ALK-1 and selectively reacts with BMP-9 and BMP-10. Receptor deficiency or BMP-9 and BMP-10 deficiency frequently result in pulmonary hypertension and myocardial fibrosis. In particular, BMP-9 acts directly on endothelial cells to promote the integrity of the vascular lining and inhibit vascular cell death and neovascularization by promoting vascular stability. 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, at high concentrations, BMP-9 can also induce ectopic bone formation. Therefore, for clinical application, it is necessary to develop mutants with controlled ectopic bone formation.

[0005] Furthermore, the extremely short half-life of wild-type growth factors has been cited as a barrier to their commercialization as therapeutics (Kharitonenkov, A. et al., Journal of Clinical Investigation, 115:1627-1635, 2005). The in vivo half-life of BMP-9 is short—10 minutes to 1 hour in mice and 1.5 to 2 hours in monkeys—so developing it as a therapeutic agent would require daily administration. To date, various techniques have been reported to increase the in vivo half-life of recombinant proteins. In one example, the polymer polyethylene glycol (PEG) was linked to the protein to increase its molecular weight, thereby inhibiting renal excretion and increasing its residence time in the body (WO 2012 / 066075). Another example was the fusing of a fatty acid that binds to human albumin to a growth factor molecule, thereby improving its half-life (WO 2012 / 010553). Furthermore, there have been cases where agonist antibodies that specifically bind to human growth factor receptors alone or in complex with beta-Klotho have been created, demonstrating pharmacological activity identical to the mechanism of action of growth factors while increasing their half-life (WO2012 / 170438).In addition, there have been cases where long-acting fusion proteins have been created by linking the Fc of immunoglobulin IgG to growth factor molecules, thereby improving their half-life (WO2013 / 188181).

[0006] Therefore, the inventors have been researching BMP9-based therapeutic mutant proteins to suppress ectopic bone formation and extend 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 solely to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to those skilled in the art to which the present 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 product 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 yet another object of the present invention to provide a pharmaceutical composition for preventing or treating tumors, inflammatory diseases, metabolic diseases or autoimmune diseases, which comprises the BMP-9 mutant or a fusion product thereof.

[0017] It is yet 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 use of the BMP-9 variants and fusions thereof in the manufacture of medicaments 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) variant 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 the BMP-9 mutant is linked to an Fc fragment of immunoglobulin.

[0022] The present invention also provides a pharmaceutical composition for tumor treatment, which comprises 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 inflammatory diseases, 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 metabolic diseases, 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 autoimmune diseases, 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, which comprises 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 use of the BMP-9 mutant or the BMP-9 mutant-Fc fusion protein in the manufacture of a medicament for the treatment of tumors, inflammatory diseases, metabolic diseases, or autoimmune diseases. [Brief explanation of the drawings]

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

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

[0031] [Figure 3] Figure 3a shows the SDS-PAGE and Western blot results of the expressed and purified BMP-9 mutant, and Figure 3b shows the SDS-PAGE and Western blot results of the 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 its variants on vascular endothelial cells, and FIG. 4b shows the osteogenic signal transduction activity.

[0033] [Figure 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 INVENTION

[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. Generally, the nomenclature used herein and the experimental procedures described below are well known and commonly used in the art.

[0036] In the present invention, we cloned the BMP-9 gene and constructed various mutants. We then expressed and purified these in mammalian cells (CHO cells and human embryonic kidney cell lines). We then selected BMP-9 mutants that are effective against tumors, inflammatory diseases, cardiovascular diseases, metabolic diseases, and autoimmune diseases. We then fused these mutants to Fc to increase their serum half-life. Specifically, in the present invention, we substituted one or more amino acids in the amino acid sequences corresponding to the wrist epitope and knuckle epitope of the BMP-9 protein to regulate receptor binding, thereby optimizing BMP-9 therapeutic effects. Specifically, our goal was to select mutants that could suppress ectopic bone formation by more than one-tenth while maintaining endothelial cell-specific signaling. Furthermore, alkaline phosphatase activity and Alizarin Red staining confirmed that the mutants of the present invention had significantly reduced ectopic bone formation ability 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 the bone morphogenetic protein-9 (BMP-9) or a variant thereof is linked to an Fc fragment of an immunoglobulin.

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

[0039] JPEG0007802978000001.jpg47164

[0040] The mutant sequences derived from the wild-type BMP-9 sequence may be represented by any one of the amino acid sequences set forth in SEQ ID NOs: 2 to 29. These mutants are obtained by substituting a portion of the amino acid sequence of the wild-type BMP-9 sequence. However, it will be apparent to those skilled in the art that the mutants of the present invention are not limited to the specific amino acid sequences set forth in SEQ ID NOs. 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 substituted with Ala instead of Asp compared to the wild-type. However, it will be apparent to those skilled in the art that such mutants also fall within the scope of the present invention if a portion of the remaining sequence, excluding the core structure of the mutant, is mutated in a manner that does not affect the function and structure of the BMP-9 protein. Therefore, it will be understood that the scope of 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 any one of the amino acid sequences set forth in SEQ ID NOs: 2 to 29, excluding the core amino acid substitution of the mutant.

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

[0042] In the present invention, to increase the serum half-life 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, but is not limited to, the amino acid sequence of SEQ ID NO: 30. Meanwhile, in addition to the Fc fragment of immunoglobulin, various peptides or proteins, such as albumin, that can be fused to proteins to increase serum half-life may also be fused.

[0043] In the present invention, the Fc fragment of the immunoglobulin can 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 having 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, according to the present invention can be used as therapeutic, diagnostic, or research reagents, but are most preferably used for therapeutic purposes.

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

[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 inflammatory diseases, which comprises 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 metabolic diseases, which comprises the bone morphogenetic protein-9 (BMP-9) or a variant thereof, or the BMP-9 variant-Fc fusion protein as an active ingredient.

[0053] In the present invention, the metabolic disease may be characterized as 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, which comprises the bone morphogenetic protein-9 (BMP-9) or a variant thereof, or the BMP-9 variant-Fc fusion protein as an active ingredient.

[0056] In the present invention, the autoimmune disease may be 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 colitis, psoriasis, Sjogren's syndrome, scleroderma, Wegener's granulomatosis, polymyositis, dermatomyositis, discoid leukemia, 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), and 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 dosage form selected from the group consisting of injections, oral administration preparations, liquid preparations (e.g., for injection) such as aqueous solutions, suspensions, and emulsions, capsules, granules, tablets, and mucosal administration preparations, but is not limited thereto. These preparations can be prepared by conventional methods used in the art for formulation or by the methods disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA, and can be formulated into various preparations depending on the disease or component.

[0059] Meanwhile, the pharmaceutical composition of the present invention may further comprise one or more pharmaceutically acceptable carriers in addition to the therapeutic BMP-9 variants and derivatives, such as 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 pharmaceutically acceptable auxiliary agent, if necessary, which may be one or more selected from the group consisting of excipients, diluents, dispersants, buffers, antimicrobial preservatives, bacteriostats, 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., and may be used within a range that varies according to the opinion of a specialist.

[0062] In one embodiment of the present invention, the single dose of the protein is 1 μg / kg to 100 mg / kg, preferably 5 μg / kg to 50 mg / kg, and the protein may be administered once a day or once to three times a week, although the dose and 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 the use of the BMP-9 variant or the BMP-9 variant-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 tumors, inflammatory diseases, metabolic diseases, or autoimmune diseases. [Example]

[0066] Example The present invention will be described in more detail below through 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 should not be construed as limiting the scope of the present invention.

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

[0068] The entire cDNA containing the open reading frame of human pre-proBMP9 was inserted into the pcDNA3.4 vector and cloned (Figure 2). Pro-BMP9 mutants were obtained using the QuickChange site-directed mutagenesis kit and were 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] JPEG0007802978000002.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 CHO-S cell line was transfected with a plasmid containing preproBMP9 using polyethyleneimine, and then cultured for 8 days with an enhancer. The expressed proBMP9 and proBMP10 were measured by Western blot analysis using anti-BMP9 and anti-BMP10 antibodies.

[0074] The expressed protein was separated through a Q-Sepharose column equilibrated with 1-5 liters of conditioned medium as a buffer. The target protein attached to the column was fractionated using 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 over 95%.

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

[0076] BMP9 and BMP10 selectively bind to the ALK1 receptor on vascular endothelial cells, making them promising therapeutic agents for cardiovascular disease. However, they still have the potential to stimulate mesenchymal cells and myoblasts to promote bone formation, and this property must be neutralized before they can be developed as therapeutic agents. To date, it remains unclear which receptors are involved in the osteogenic differentiation of BMP9 and BMP10. The present researchers identified the wrist and knuckle epitopes in the BMP-9 structure as ligands mediating key signaling pathways. They demonstrated that by manipulating these sequences, they could suppress osteogenic differentiation while maintaining endothelial cell signaling (sequences 1-29, Figure 4). These variants were determined to have similar physiological activity in vivo, potentially offering the advantage of enhancing therapeutic efficacy while minimizing side effects.

[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 application to cells. The mutants were added to HUVEC cells in the serum-free condition at the indicated concentration range. After 8 hours of treatment, cells were harvested, and mRNA was extracted and the expression of ID1 and BMPR-II was measured by quantitative PCR. For pSmad1 / 5 / 8 expression, the cells were treated with BMP9 mutants under serum-free conditions and then monitored for 1 hour. The protein expression levels obtained after treatment with cell lysis buffer were measured by immunoblotting using anti-pSmad1 / 5 / 8 antibodies. The results (Figure 4) showed that each mutant significantly increased ID1 and BMPR-II, but the increase was not significantly different from that of wild-type BMP-9. This confirms that BMP9 and BMP10 act as ligands for the ALK1 receptor in vascular endothelial cells, as previously reported. According to literature, BMP9 is known to inhibit endothelial cell migration, proliferation, and angiogenesis in the circulatory system.

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

[0080] C2C12, a mouse cardiomyocyte, was selected for the study because it has differentiation potential 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 culture for 72 hours, the cells were disrupted with 1% Triton X-100 / PBS, and the resulting protein was assayed for ALP enzyme activity. ALP enzyme activity was determined by measuring the absorbance at 405 nm of the water-soluble product produced by reacting the cells with the enzyme's substrate, 4-nitrophenyl phosphate disodium salt. BMP-9 was used as a control. The results confirmed that each variant had significantly reduced osteogenic differentiation potential compared to the control (Figure 4). These variants maintained endothelial cell signaling as described in Example 3, while attenuating osteogenic potential, providing therapeutic efficacy without the side effect of ectopic bone formation.

[0081] Example 5. In vivo half-life extension effect of BMP9-Fc derivatives

[0082] BMP-9 and its Fc-fused derivatives were injected into mice via the tail vein at a dose of 1 mg / kg, and blood samples were taken at regular intervals. The amount of BMP-9 in the blood was measured using a BMP-9 quantification kit. The results (Figure 5) confirmed that the Fc-conjugated BMP-9 derivatives (SEQ ID NOs: 32-33) exhibited a significantly increased blood concentration curve.

[0083] Although certain parts of the present invention have been described in detail above, it will be apparent 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, the true scope of the present invention can be said to be defined by the appended claims and their equivalents. [Industrial Applicability]

[0084] In the present invention, we designed mutants of BMP-9 to maximize the therapeutic effect and significantly reduce side effects, and we expressed and purified them with high efficiency in mammalian cells. We also created fusion proteins in which BMP-9 is fused to the Fc region of immunoglobulin to extend its half-life in the blood. These mutants and fusion proteins have demonstrated excellent therapeutic effects in in vitro and animal experiments against various diseases such as tumors, fibrosis, cardiopulmonary vascular disease, obesity, and fatty liver, and 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 the amino acid sequence of SEQ ID NO:

4.

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

3. The BMP-9 variant-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 variant-Fc fusion protein according to claim 2, wherein the BMP-9 variant 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 treating cardiopulmonary diseases, 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.

7. The pharmaceutical composition according to claim 6, wherein the cardiopulmonary disease is one or more selected from the group consisting of myocardial infarction, hypertension, pulmonary hypertension, myocardial fibrosis, and pulmonary fibrosis.

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