Polypeptides and pharmaceutical compositions containing mutant activin type IIB receptors

Abstract

Polypeptides and pharmaceutical compositions comprising mutant activin type IIB receptors are provided. [Solution] The polypeptides of the present invention include extracellular activin receptor type IIB (ActRIIB) mutants comprising a specific amino acid sequence, the C-terminus of which is fused to a human IgG1 Fc domain monomer. The present invention also features pharmaceutical compositions and methods using the polypeptides to treat diseases and conditions, including muscle weakness and atrophy, bone damage, low red blood cell levels (e.g., anemia or blood loss), fibrosis, and / or pulmonary hypertension.

Description

[Technical Field]

[0001] The present invention relates to activin type IIB receptor mutants and methods of their use. [Background technology]

[0002] Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), and amyotrophic lateral sclerosis (ALS) are examples of muscle diseases that cause muscle weakness and atrophy and / or involve the motor neurons that control voluntary muscle movement. DMD is caused by mutations in the X-linked dystrophin gene and is characterized by progressive muscle degeneration and weakness in all skeletal muscles. FSHD particularly affects the skeletal muscles of the face, shoulders, upper arms, and lower limbs. IBM is an inflammatory muscle disease that primarily affects the muscles of the thighs and arms that control flexion of the fingers and wrists. ALS is a motor neuron disease characterized by muscle stiffness, muscle spasms, and muscle atrophy throughout the body due to the degeneration of motor neurons. Efforts to improve the treatment and survival of subjects with these devastating muscle diseases have not been successful.

[0003] Healthy bone undergoes continuous remodeling, including both bone breakdown and bone growth. Bone growth is mediated by osteoblasts, while osteoclasts resorb bone. Pathologies occur when these systems become unbalanced, resulting in net bone loss, due to downregulation of the anabolic program, upregulation of the catabolic system, or a combination of both. Thus, controlling the balance of bone remodeling may be useful for promoting bone injury healing and treating disorders such as osteoporosis, which are associated with bone loss and bone mineralization.

[0004] Bone damage can result from a range of underlying causes, including bone loss related to aging and cancer, genetic conditions, and adverse side effects of drug treatments. The World Health Organization estimates that osteoporosis alone affects 75 million people in the United States, Europe, and Japan and is a significant risk factor for fractures. Generally, bone loss itself is a pathological condition with few effective treatments. Instead, treatment focuses on immobility, exercise, and dietary modification rather than drugs that directly promote bone growth and increase bone density. Regarding osteoporosis, estrogen, calcitonin, osteocalcin in combination with vitamin K, or high-dose dietary calcium have all been used as therapeutic interventions. Other therapeutic approaches for osteoporosis include bisphosphonates, parathyroid hormone, parathyroid hormone-related protein (PTHrP), calcium receptor agonists, statins, anabolic steroids, lanthanum and strontium salts, and sodium fluoride. However, these treatments are often associated with unwanted side effects.

[0005] Fibrosis is the formation of excess connective tissue in organs and tissues. Connective tissue, which can form in response to damage (e.g., injury) or as part of an immune response (e.g., inflammatory response), can disrupt the structure and function of the organ or tissue in which it is formed and increase tissue stiffness. Fibrosis can occur in many organs and tissues in the body, including the lungs (e.g., pulmonary fibrosis, cystic fibrosis), liver (e.g., cirrhosis), heart (e.g., endomyocardial fibrosis or post-myocardial infarction fibrosis), brain (e.g., glial scar formation), skin (e.g., keloid formation), kidneys (e.g., renal fibrosis), and eyes (e.g., corneal fibrosis), among others, and is known to be associated with certain medical treatments (e.g., chemotherapy, radiation therapy, and surgery). Treatment options for patients with fibrosis are limited, with most treatments focusing on improving quality of life or temporarily delaying disease progression.

[0006] Anemia is a global health problem with health consequences that affect both morbidity and mortality. In the United States alone, the prevalence of anemia nearly doubled from 2003 to 2012. Symptoms of anemia include fatigue, weakness, shortness of breath, palpitations, and decreased cognitive ability. Children, pregnant women, women of reproductive age, and the elderly are at highest risk of developing anemia. The most common form of anemia is iron deficiency anemia, but anemia can also be caused by chronic disease, blood loss, and red blood cell destruction. While iron deficiency anemia can be treated with iron supplements, many other forms of anemia, such as aplastic anemia, anemia of chronic disease, and hemolytic anemia, may require blood transfusions.

[0007] Pulmonary hypertension (PH) is a serious condition characterized by higher-than-normal pressure in the blood vessels between the lungs and heart. PH can be classified into five major types, also known as WHO Groups I–V: arterial (PAH), venous (PH secondary to left-sided heart disease), hypoxic (PH caused by lung disease), thromboembolic (PH caused by chronic arterial obstruction, such as a blood clot), or other (PH with unknown mechanisms or multifactorial causes). PAH is characterized by elevated pulmonary vascular pressure due to blockage or narrowing of small blood vessels in the lungs caused by scarring. This increases resistance to blood flow through the lungs and forces the right side of the heart to work harder, potentially leading to heart failure, reduced blood oxygenation, and shortened life expectancy. PAH can be idiopathic (e.g., without an identifiable cause), hereditary (e.g., familial, often due to a genetic mutation), or associated with drug use (e.g., methamphetamine or cocaine use), infection (e.g., HIV infection or schistosomiasis), cirrhosis, congenital heart abnormalities, and connective tissue / autoimmune diseases (such as scleroderma and lupus). Treatments for PH include vasodilators, anticoagulants, and supplemental oxygen; however, these treatments manage the symptoms of the disease rather than target the underlying biological mechanisms. Summary of the Invention [Problem to be solved by the invention]

[0008] There is a need for new and effective treatments for muscle diseases, bone diseases, anemia, fibrosis, and PH. [Means for solving the problem]

[0009] The present invention relates to polypeptides comprising an extracellular activin receptor type IIB (ActRIIB) mutant. In some embodiments, the polypeptides of the present invention comprise an extracellular ActRIIB mutant fused to the N- or C-terminus of an Fc domain monomer or another moiety. Such moieties may be attached by amino acids or other covalent bonds and increase the stability of the polypeptide. Polypeptides comprising an extracellular ActRIIB mutant fused to an Fc domain monomer may also form dimers (e.g., homodimers or heterodimers) through interactions between the two Fc domain monomers. The polypeptides of the present invention can be used to increase muscle mass and strength in subjects with, or at risk of developing, diseases or conditions involving muscle weakness and atrophy, such as Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia. The polypeptides of the present invention can be used to increase bone mass or bone mineral density in subjects with, or at risk of developing, diseases or conditions involving bone damage, such as primary osteoporosis, secondary osteoporosis, osteopenia, osteopetrosis, fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low-gravity-related bone loss, or immobility.Additionally, the polypeptides of the invention may be used to increase red blood cell levels (e.g., increase hemoglobin levels, increase hematocrit, and / or increase red blood cell count) in a subject in need thereof, e.g., a subject having or at risk of developing anemia or blood loss, to prevent or reduce fibrosis in a subject having or at risk of developing fibrosis, or to treat, prevent, or delay the onset or progression of pulmonary hypertension (e.g., arterial, venous, hypoxic, thromboembolic, or other pulmonary hypertension) in a subject having or at risk of developing pulmonary hypertension. Additionally, the polypeptides of the present invention can also be used to affect myostatin, activin, and / or bone morphogenetic protein 9 (BMP9) signaling in subjects at risk of developing or having a disease or condition associated with muscle weakness and muscle atrophy, bone damage or demineralization, low blood cell levels (e.g., low hemoglobin levels, low hematocrit, and / or low red blood cell count), fibrosis, or pulmonary hypertension (e.g., arterial, venous, hypoxic, thromboembolic, or other pulmonary hypertension).

[0010] In a first aspect, the present invention features a polypeptide comprising an extracellular ActRIIB mutant having one or more amino acid substitutions compared to the sequence GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 17), wherein the mutant comprises one or more amino acid substitutions that confer reduced BMP9 binding relative to wild-type extracellular ActRIIB, and one or more additional amino acid substitutions, wherein the substitutions that reduce BMP9 binding are one or more of: (a) amino acid substitution E75K; (b) amino acid substitutions Q69T and E70D; or (c) amino acid substitutions Q69D and E70T.

[0011] In some embodiments, the one or more additional amino acid substitutions are selected from the group consisting of I11L, Y12F, L19K, E20D, S25T, L27V, R29P, E31Y, E33D, Q34K, L38R, Y41F, R45K, S47I, S48T, T50S, I51L, L53I, K56Q, F63I, T74K, E76D, N77S, Q79E, and F89M.

[0012] In some embodiments, the variant comprises the amino acid substitution E75K and the additional amino acid substitutions E20D and F63I. In some embodiments, the variant comprises the amino acid substitution E75K and additional amino acid substitutions that reduce BMP9 binding. In some embodiments of any of the above aspects, the additional amino acid substitutions that reduce BMP9 binding comprise T74K, E76D, N77S, and Q79E.

[0013] In some embodiments, the variant comprises one or more additional amino acid substitutions. In some embodiments, the variant comprises the additional amino acid substitutions Y41F, R45K, and K56Q, hi some embodiments, the variant comprises the additional amino acid substitutions Y12F, L19K, E20D, R29P, E31Y, E33D, L38R, and F63I.

[0014] In some embodiments, the variant comprises the additional amino acid substitutions S25T and S47I. In some embodiments, the variant comprises the additional amino acid substitution S48T.

[0015] In some embodiments, the variant comprises the additional amino acid substitution R29P. In some embodiments, the variant comprises the additional amino acid substitutions E31Y, E33D and Q34K.

[0016] In some embodiments, the variant comprises the additional amino acid substitutions Y12F, L19K, and E20D. In some embodiments, the variant comprises the additional amino acid substitutions E31Y, E33D and L38R.

[0017] In some embodiments, the variant comprises amino acid substitutions Q69T and E70D, and additional amino acid substitutions I11L, L27V, Q34K, T50S, I51L, L53I, and F89M.

[0018] In some embodiments, the variant comprises amino acid substitutions Q69D and E70T, and additional amino acid substitutions I11L, L27V, Q34K, T50S, I51L, L53I, and F89M.

[0019] In some embodiments, the variant further comprises the amino acid substitution E75K. The following embodiments E1 to E59 illustrate other features of the present invention. E1. A polypeptide comprising an ActRIIB mutant, wherein the mutant is GRGEAETRECX1X2YNANWEX3X4RTNQX5GX6EX7CX8GX9X 10 DKRX 11 HCX 12 ASWX 13 NX 14 X 15 GX 16 X 17 EX 18 VKX 19 GCWLDDX 20 NCYDRX 21 X 22 CVAX 23 X 24 X 25 X 26 PX 27 VYFCCEGNX 28 X1 is I or L; X2 is F or Y; X3 is L or K; X4 is D or E; X5 is T or S; X6 is L or V; X7 is P or R; X8 is Y or E; X9 is D or E; 10 is K or Q;X 11is R or L; X 12 is Y or F;X 13 is R or K; X 14 is S or I; X 15 is S or T;X 16 is S or T;X 17 is I or L; X 18 is I or L; X 19 is K or Q;X 20 is F or I;X 21 is Q, T, or D; X 22 is E, D, or T; X 23 is K or T; X 24 is K or E;X 25 is D or E;X 26 is S or N; X 27 is E or Q;X 28 is F or M, and X 24 is E and / or X 21 is T and X 22 is D or X 21 is D and X 22 is T, polypeptide.

[0020] A mutant of E1 in which E2.X1 is I. A mutant of E1 in which E3.X1 is L. E4. A mutant of any one of E1 to E3, wherein X2 is F.

[0021] E5. A mutant of any one of E1 to E3, wherein X2 is Y. E6. A mutant of any one of E1 to E5, wherein X3 is L. E7. A mutant of any one of E1 to E5, wherein X3 is K.

[0022] A mutant of any one of E1 to E7, wherein E8.X4 is D. A mutant of any one of E1 to E7, wherein E9.X4 is E. A mutant of any one of E1 to E9, in which E10.X5 is T.

[0023] A mutant of any one of E1 to E9, in which E11.X5 is S. A mutant of any one of E1 to E11, in which E12.X6 is L. A mutant of any one of E1 to E11, in which E13.X6 is V.

[0024] A mutant of any one of E1 to E13, in which E14.X7 is P. A mutant of any one of E1 to E13, in which E15.X7 is R. E16. A mutant of any one of E1 to E15, in which X8 is Y.

[0025] A mutant of any one of E1 to E15, where E17.X8 is E. A mutant of any one of E1 to E17, in which E18.X9 is D. A mutant of any one of E1 to E17, wherein E19.X9 is E.

[0026] E20.X 10 A mutant of any one of E1 to E19, wherein E21.X 10 A mutant of any one of E1 to E19, wherein E22.X 11 any one of the mutants E1 to E21, wherein

[0027] E23.X 11 any one of the mutants E1 to E21, wherein is L. E24.X 12 A mutant of any one of E1 to E23, wherein E25.X 12 A mutant of any one of E1 to E23, wherein:

[0028] E26.X 13 any one of E1 to E25 mutants, wherein E27.X 13 A mutant of any one of E1 to E25, wherein is K. E28.X 14A mutant of any one of E1 to E27, wherein is S.

[0029] E29.X 15 A mutant of any one of E1 to E27, wherein is S. E30.X 15 A mutant of any one of E1 to E29, wherein: E31.X 15 A mutant of any one of E1 to E29, wherein

[0030] E32.X 16 A mutant of any one of E1 to E31, wherein: E33.X 16 A mutant of any one of E1 to E31, wherein E34.X 17 A mutant of any one of E1 to E33, wherein:

[0031] E35.X 17 A mutant of any one of E1 to E33, wherein is L. E36.X 18 A mutant of any one of E1 to E35, wherein: E37.X 18 A mutant of any one of E1 to E35, wherein is L.

[0032] E38.X 19 A mutant of any one of E1 to E37, wherein is K. E39.X 19 A mutant of any one of E1 to E37, wherein is Q. E40.X 20 A mutant of any one of E1 to E39, wherein:

[0033] E41.X 20 A mutant of any one of E1 to E39, wherein: E42.X 21 A variant of any one of E1 to E41, wherein is Q. E43.X 21 A mutant of any one of E1 to E41, wherein

[0034] E44.X 21 A mutant of any one of E1 to E41, wherein E45.X 22 A mutant of any one of E1 to E42, wherein E46.X 22 A mutant of any one of E1 to E41 and E43, wherein

[0035] E47.X 22 A mutant of any one of E1 to E41 and E44, wherein E48.X 23 A mutant of any one of E1 to E47, wherein is K. E49.X 23 A mutant of any one of E1 to E47, wherein

[0036] E50.X 24 A mutant of any one of E1 to E49, wherein E51.X 24 A mutant of any one of E1 to E41, E43, E44, E46, and E47 to E49, wherein E is E.

[0037] E52.X 25 A mutant of any one of E1 to E51, wherein E53.X 25 A mutant of any one of E1 to E51, wherein E54.X 26 A mutant of any one of E1 to E53, wherein:

[0038] E55.X 26 A mutant of any one of E1 to E53, wherein E56.X 27 A mutant of any one of E1 to E55, wherein E57.X 27 A variant of any one of E1 to E55, wherein is Q.

[0039] E58.X 28 any one of the mutants E1 to E57, wherein the is F. E59.X 28 A mutant of any one of E1 to E57, wherein is M. In some embodiments, the variant has the sequence of any one of SEQ ID NOs: 2-15.

[0040] In some embodiments, the polypeptide further comprises an Fc domain monomer fused to the C-terminus of the polypeptide (e.g., the C-terminus of the variant) via a linker. In some embodiments, the Fc domain monomer has the sequence of SEQ ID NO: 19. In some embodiments, the polypeptide forms a dimer.

[0041] In some embodiments, the polypeptide further comprises a wild-type Fc domain fused to the C-terminus of the polypeptide (e.g., the C-terminus of the variant) via a linker. In some embodiments, the wild-type Fc domain has the sequence of SEQ ID NO: 71.

[0042] In some embodiments, the polypeptide further comprises an Fc domain having an amino acid substitution fused to the C-terminus of the polypeptide (e.g., the C-terminus of the variant) via a linker. In some embodiments, the Fc domain does not form a dimer.

[0043] In some embodiments, the polypeptide further comprises an albumin-binding peptide fused to the C-terminus of the polypeptide (e.g., the C-terminus of the variant) via a linker. In some embodiments, the albumin-binding peptide has the sequence of SEQ ID NO: 72.

[0044] In some embodiments, the polypeptide further comprises a fibronectin domain fused to the C-terminus of the polypeptide (e.g., the C-terminus of the variant) via a linker. In some embodiments, the fibronectin domain has the sequence of SEQ ID NO: 73.

[0045] In some embodiments, the polypeptide further comprises human serum albumin fused to the C-terminus of the polypeptide (e.g., the C-terminus of the variant) via a linker. In some embodiments, the human serum albumin has the sequence of SEQ ID NO: 74.

[0046] In some embodiments, the linker is an amino acid spacer. In some embodiments of any of the above aspects, the amino acid spacer is GGG, GGGA (SEQ ID NO:20), GGGG (SEQ ID NO:22), GGGAG (SEQ ID NO:52), GGGAGG (SEQ ID NO:53), or GGGAGGG (SEQ ID NO:54).

[0047] In some embodiments, the amino acid spacer is GA, GS, GG, GGA, GGS, GGG, GGGS (SEQ ID NO:21), GGGGA (SEQ ID NO:23), GGGGS (SEQ ID NO:24), GGGGG (SEQ ID NO:25), GGAG (SEQ ID NO:26), GGSG (SEQ ID NO:27), AGGG (SEQ ID NO:28), SGGG (SEQ ID NO:29), GAGA (SEQ ID NO:30), GSGS (SEQ ID NO:31), GAGAGA (SEQ ID NO:32), GSGSGS (SEQ ID NO:33), GAGAGAGA (SEQ ID NO:34), GS GSGSGS (SEQ ID NO: 35), GAGAGAGAGA (SEQ ID NO: 36), GSGSGSGSGS (SEQ ID NO: 37), GAGAGAGAGAGA (SEQ ID NO: 38), GSGSGSGSGSGS (SEQ ID NO: 39), GGAGGA (SEQ ID NO: 40), GGSGGS (SEQ ID NO: 41), GGAGGAGGA (SEQ ID NO: 42), GGSGGSGGS (SEQ ID NO: 43), GGAGGAGGAGGA (SEQ ID NO: 44), and GGSGGSGGSGGS (SEQ ID NO: 45), GGAGGGAG (SEQ ID NO: 46), GGSGGG SG (SEQ ID NO: 47), GGAGGGAGGGAG (SEQ ID NO: 48), andGGSGGGSGGGSG (SEQ ID NO: 49), GGGGAGGGGAGGGGA (SEQ ID NO: 50), GGGGSGGGGSGGGGS (SEQ ID NO: 51), AAAL (SEQ ID NO: 55), AAAK (SEQ ID NO: 56), AAAR (SEQ ID NO: 57), EGKSSGSGSESKST (SEQ ID NO: 58), GSAGSAAGSGEF (SEQ ID NO: 59), AEAAAKEAAAKA (SEQ ID NO: 60), KESGSVSSEQLAQFRSLD (SEQ ID NO: 61), No.: 61), GENLYFQSGG (SEQ ID NO: 62), SACYCELS (SEQ ID NO: 63), RSIAT (SEQ ID NO: 64), RPACKIPNDLKQKVMNH (SEQ ID NO: 65), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 66), AAANSSIDLISVPVDSR (SEQ ID NO: 67), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 68), EAAAK (SEQ ID NO: 69), or PAPAP (SEQ ID NO: 70).

[0048] In some embodiments, the polypeptide has a serum half-life of at least 7 days. In some embodiments, the polypeptide has increased or decreased binding to one or more ActRIIB ligands (eg, activin, myostatin, GDF-11, or BMP9) compared to wild-type ActRIIB.

[0049] In some embodiments, the polypeptide has a K of 200 pM or greater. D It binds to human bone morphogenetic protein 9 (BMP9). In some embodiments, the polypeptide binds to activin and / or myostatin and has low or weak binding to human BMP9.

[0050] In some embodiments, the polypeptide does not substantially bind to human BMP9. In some embodiments, the polypeptide has a K of 800 pM or less. D It binds to human activin A at

[0051] In some embodiments, the polypeptide has a K of 800 pM or less. D It binds to human activin B at In some embodiments, the polypeptide has a K of 5 pM or greater. D It binds to human GDF-11.

[0052] In another aspect, the present invention relates to a nucleic acid molecule encoding a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIB mutant having any one of the sequences of SEQ ID NOs: 1 to 15 (e.g., SEQ ID NOs: 2 to 15)).

[0053] In another aspect, the present invention also relates to a vector comprising the nucleic acid molecule described herein. In another aspect, the invention relates to a host cell expressing a polypeptide as described herein, which host cell comprises a nucleic acid molecule or vector as described in the previous two aspects, which nucleic acid molecule or vector is expressed in the host cell.

[0054] In another aspect, the invention relates to a method of making a polypeptide as described herein, the method comprising: a) providing a host cell comprising a nucleic acid molecule or vector as described herein; and b) expressing the nucleic acid molecule or vector in the host cell under conditions that allow the formation of the polypeptide.

[0055] In another aspect, the invention relates to a pharmaceutical composition comprising a polypeptide, nucleic acid molecule, or vector described herein and one or more pharmaceutically acceptable carriers or excipients, in some embodiments of the pharmaceutical composition, the polypeptide, nucleic acid molecule, or vector is in a therapeutically effective amount.

[0056] In another aspect, the present invention relates to a construct (e.g., a homodimer) comprising two identical polypeptides, each comprising an extracellular ActRIIB variant described herein (e.g., an ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) fused to the N-terminus or C-terminus of an Fc domain monomer (e.g., the sequence of SEQ ID NO: 19). The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0057] In another aspect, the present invention relates to a construct (e.g., a heterodimer) comprising two different polypeptides, each comprising an extracellular ActRIIB variant described herein (e.g., an ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) fused to the N-terminus or C-terminus of an Fc domain monomer (e.g., the sequence of SEQ ID NO: 19). The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0058] In another aspect, the present invention relates to a method for increasing lean mass in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0059] In another aspect, the present invention relates to a method for increasing muscle mass in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0060] In some embodiments of the methods of increasing lean mass or muscle mass in a subject, the subject has or is at risk of developing Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia.

[0061] In another aspect, the present invention relates to a method for treating a subject having or at risk of developing a muscle disease, comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein. In some embodiments, the muscle disease is DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia.

[0062] In another aspect, the invention relates to a method of affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their endogenous receptors) in a subject having or at risk of developing a disease or condition involving muscle weakness and muscle wasting, comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein. In some embodiments of this aspect, the disease or condition is DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia.

[0063] In another aspect, the present invention relates to a method of treating a subject having or at risk of developing DMD by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0064] In another aspect, the present invention relates to a method of treating a subject having or at risk of developing FSHD by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0065] In another aspect, the invention relates to a method of treating a subject having or at risk of developing IBM by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0066] In another aspect, the present invention relates to a method of treating a subject having or at risk of developing ALS by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0067] In another aspect, the present invention relates to a method of treating a subject having or at risk of developing sarcopenia by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0068] In another aspect, the present invention relates to a method of treating a subject having or at risk of developing cancer cachexia by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0069] In another aspect, the present invention relates to a method for increasing bone mineral density in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0070] In another aspect, the present invention relates to a method of reducing bone resorption in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0071] In another aspect, the present invention relates to a method for increasing bone formation in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0072] In another aspect, the present invention relates to a method for increasing bone strength in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0073] In another aspect, the present invention relates to a method for reducing the risk of fracture in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0074] In some embodiments of any of the above aspects, the subject has or is at risk of developing primary osteoporosis, secondary osteoporosis, osteopenia, osteopetrosis, fracture, bone cancer or cancer metastasis-associated bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with treatment of obesity, low gravity-associated bone loss, or immobility-associated bone loss. In some embodiments, the subject has or is at risk of developing osteoporosis.

[0075] In another aspect, the present invention relates to a method for treating a subject having or at risk of developing a bone disease. The method comprises administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein. In some embodiments, the bone disease is primary osteoporosis, secondary osteoporosis, osteopenia, osteopetrosis, bone fracture, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with the treatment of obesity, low gravity-related bone loss, or immobility-related bone loss. In some embodiments, the bone disease is osteoporosis.

[0076] In another aspect, the invention relates to a method of affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their endogenous receptors) in a subject having or at risk of developing a disease or condition involving bone damage, comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein. In some embodiments of this aspect, the disease or condition is primary osteoporosis, secondary osteoporosis, osteopenia, osteopetrosis, bone fracture, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with the treatment of obesity, low gravity-related bone loss, or immobility-related bone loss. In some embodiments, the disease or condition is osteoporosis.

[0077] In another aspect, the present invention relates to a method of treating a subject having or at risk of developing primary osteoporosis by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0078] In another aspect, the invention relates to a method of treating a subject having or at risk of developing secondary osteoporosis by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0079] In another aspect, the present invention relates to a method of treating a subject having or at risk of developing osteopenia by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0080] In another aspect, the present invention relates to a method of treating a subject having or at risk of developing a fracture by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0081] In another aspect, the present invention relates to a method of treating a subject having or at risk of developing bone cancer or cancer metastasis-associated bone loss by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0082] In another aspect, the invention relates to a method of treating a subject having or at risk of developing Paget's disease by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0083] In another aspect, the invention relates to a method of treating a subject having or at risk of developing renal osteodystrophy by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0084] In another aspect, the invention relates to a method of treating a subject having or at risk of developing treatment-related bone loss by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0085] In another aspect, the invention relates to a method of treating a subject having or at risk of developing diet-related bone loss by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0086] In another aspect, the present invention relates to a method of treating a subject having or at risk of developing low gravity-associated bone loss by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0087] In another aspect, the invention relates to a method of treating a subject having or at risk of developing immobility-related bone loss by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0088] In some embodiments of any of the above aspects, the primary osteoporosis is age-related osteoporosis or hormone-related osteoporosis. In some embodiments of any of the above aspects, the secondary osteoporosis is immobility-induced osteoporosis or glucocorticoid-induced osteoporosis.

[0089] In some embodiments of any of the above aspects, the cancer is multiple myeloma. In some embodiments of any of the above aspects, the treatment is FGF-21 treatment, GLP-1 treatment, cancer treatment, or treatment for obesity or treatment for type 2 diabetes.

[0090] In some embodiments of any of the above aspects, the diet-related bone loss is rickets. In some embodiments of any of the above aspects, the subject is at risk of fracture.

[0091] In some embodiments of any of the above aspects, the method increases bone formation in the subject. In some embodiments of any of the above aspects, the method reduces bone resorption in the subject. In some embodiments of any of the above aspects, the method reduces bone loss in the subject. In some embodiments of any of the above aspects, the method increases osteoblast activity or osteoblast formation. In some embodiments of any of the above aspects, the method reduces osteoclast activity or osteoclast formation. In some embodiments of any of the above aspects, the method reduces the risk of fracture. In some embodiments of any of the above aspects, the method increases bone strength.

[0092] In some embodiments of any of the above aspects, the bone is cortical bone. In some embodiments of any of the above aspects, the bone is trabecular bone. In another aspect, the present invention relates to a method of reducing or preventing fibrosis in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0093] In another aspect, the present invention relates to a method of slowing or inhibiting the progression of fibrosis in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0094] In another aspect, the present invention relates to a method of reducing the risk of developing fibrosis in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0095] In another aspect, the invention relates to a method of treating a subject having or at risk of developing fibrosis by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0096] In some embodiments of any of the above aspects, the fibrosis is chemotherapy-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis, bone marrow fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, osteoarthrofibrosis, tissue fibrosis, tumor stroma, desmoplastic tumor, surgical adhesion, hypertrophic scar, or keloid.

[0097] In some embodiments of any of the above aspects, the fibrosis is fibrosis associated with a wound, a burn, hepatitis B or C infection, fatty liver disease, schistosome infection, kidney disease (e.g., chronic kidney disease), heart disease, macular degeneration, Crohn's disease, retinal or vitreoretinopathy, systemic or localized scleroderma, atherosclerosis, or restenosis. In some embodiments of any of the above aspects, the fibrosis is caused by chronic kidney disease.

[0098] In another aspect, the invention relates to a method of affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their endogenous receptors) in a subject having or at risk of developing fibrosis, or a disease or condition associated with fibrosis, wherein the method comprises administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein. In some embodiments of any of the above aspects, the fibrosis is chemotherapy-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis, bone marrow fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, osteoarthrofibrosis, tissue fibrosis, tumor stroma, desmoplastic tumor, surgical adhesion, hypertrophic scar, or keloid. In some embodiments of this aspect, the disease or condition is fibrosis associated with a wound, a burn, hepatitis B or C infection, fatty liver disease, schistosome infection, kidney disease (e.g., chronic kidney disease), heart disease, macular degeneration, Crohn's disease, retinal or vitreoretinopathy, systemic or localized scleroderma, atherosclerosis, or restenosis. In some embodiments of this aspect, the fibrosis is caused by chronic kidney disease.

[0099] In some embodiments of any of the above aspects, the tissue fibrosis is fibrosis affecting a tissue selected from the group consisting of muscle tissue, skin epidermis, skin dermis, tendon, cartilage, pancreatic tissue, uterine tissue, nervous tissue, testes, ovaries, adrenal glands, arteries, veins, colon, small intestine, large intestine, biliary tract, and intestine.

[0100] In some embodiments of any of the above aspects, the method improves the function of a fibrotic tissue or organ. In some embodiments of any of the above aspects, the method slows or inhibits the progression of fibrosis. In some embodiments of any of the above aspects, the method reduces one or more symptoms of fibrosis (e.g., reduces their frequency or severity).

[0101] In another aspect, the present invention relates to a method of increasing red blood cell levels (e.g., increasing hemoglobin levels, red blood cell count, or hematocrit) in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0102] In another aspect, the present invention relates to a method of promoting or increasing red blood cell formation in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0103] In some embodiments of any of the above aspects, the subject suffers from or is at risk of developing anemia or blood loss. In another aspect, the present invention relates to a method for affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of myostatin, activin, and / or BMP9 to their endogenous receptors) in a subject having or at risk of developing a disease or condition associated with low red blood cell levels (e.g., low hemoglobin levels, low red blood cell count, or low hematocrit), wherein the method comprises administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0104] In some embodiments of the above aspects, the disease or condition is anemia or blood loss. In some embodiments of any of the above aspects, the anemia or blood loss is due to cancer, cancer treatment, kidney disease or renal failure (e.g., chronic kidney disease or acute kidney disease or failure), myelodysplastic syndrome, thalassemia, nutritional deficiency, adverse reaction to a drug, inflammatory or autoimmune disease, splenomegaly, porphyria, vasculitis, hemolysis, bone marrow deficiency, bone marrow transplant, diabetes, liver disease (e.g., acute liver disease or chronic liver disease), bleeding (e.g., acute or chronic bleeding), infection, hemoglobinopathies, drug use, alcohol abuse, elderly, Churg-Strauss syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplant, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schönlein purpura Schoenlein-Henoch, Shwachman syndrome (e.g., Shwachman-Diamond syndrome), contraindications to blood transfusion, associated with surgery, trauma, wounds, ulcers, urinary tract bleeding, gastrointestinal bleeding, frequent blood donations, or heavy menstrual bleeding.

[0105] In another aspect, the present invention relates to a method of treating a subject having or at risk of developing anemia by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein. In some embodiments, the anemia or blood loss is associated with cancer, cancer treatment, kidney disease or renal failure (e.g., chronic kidney disease or acute kidney disease or failure), myelodysplastic syndrome, thalassemia, nutritional deficiency, adverse reaction to a drug, inflammatory or autoimmune disease, splenomegaly, porphyria, vasculitis, hemolysis, bone marrow deficiency, bone marrow transplant, diabetes, liver disease (e.g., acute liver disease or chronic liver disease), bleeding (e.g., acute or chronic bleeding), infection, hemoglobinopathies, drug use, alcohol abuse, advanced age, Churg-Strauss syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplant, myelofibrosis, pancytopenia, pure red blood cell aplasia, Henoch-Schönlein purpura, Shwachman syndrome (e.g., Shwachman-Diamond syndrome), contraindications to blood transfusion, surgery, trauma, wound, ulcer, urinary tract bleeding, gastrointestinal bleeding, frequent blood donations, or heavy menstrual bleeding.

[0106] In some embodiments, the anemia is caused by chronic kidney disease. In some embodiments of any of the above aspects, the anemia is aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, anemia of chronic disease, anemia associated with bone marrow disease, hemolytic anemia, sickle cell anemia, microcytic anemia, hypochromic anemia, sideroblastic anemia, Diamond Blackfan anemia, Fanconi anemia, or refractory anemia with excess blasts.

[0107] In some embodiments of any of the above aspects, the subject does not respond well to treatment with erythropoietin (EPO) or is sensitive to the side effects of EPO. In some embodiments of any of the above aspects, the method increases erythropoiesis, red blood cell count, hemoglobin level, or hematocrit.

[0108] In some embodiments of any of the above aspects, the method reduces the subject's need for blood transfusions. In another aspect, the present invention relates to a method of reducing pulmonary hypertension (PH) in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0109] In another aspect, the present invention relates to a method of reducing the risk of developing PH in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0110] In another aspect, the present invention relates to a method of slowing or inhibiting the progression of PH in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0111] In another aspect, the present invention relates to a method of treating a subject having or at risk of developing PH by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0112] In another aspect, the invention relates to a method of affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their receptors) in a subject having or at risk of developing PH by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0113] In another aspect, the present invention relates to a method of reducing vascular remodeling in a subject having or at risk of developing PH by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0114] In another aspect, the invention relates to a method of reducing right ventricular hypertrophy in a subject having or at risk of developing PH by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0115] In another aspect, the present invention relates to a method of decreasing pulmonary vascular resistance in a subject having or at risk of developing PH by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0116] In some embodiments of any of the above aspects, the PH is pulmonary arterial hypertension (PAH). In some embodiments, the PAH is idiopathic PAH. In some embodiments, the PAH is hereditary PAH. In some embodiments, the PAH is associated with HIV infection, schistosomiasis, liver cirrhosis, congenital heart abnormalities, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, connective tissue disorders, autoimmune disorders (e.g., scleroderma or lupus), or drug use or abuse (e.g., cocaine or methamphetamine use).

[0117] In some embodiments of any of the above aspects, the PH is venous PH. In some embodiments, the PH is associated with left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital or acquired pulmonary vein stenosis.

[0118] In some embodiments of any of the above aspects, the PH is hypoxic PH. In some embodiments, the hypoxic PH is associated with chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), pulmonary disease (e.g., pulmonary fibrosis), alveolar hypoventilation, chronic exposure to high altitude, or developmental abnormalities.

[0119] In some embodiments of any of the above aspects, the PH is thromboembolic PH. In some embodiments, the thromboembolic PH is associated with chronic thromboembolic pulmonary hypertension, pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary stenosis, or parasitic infection.

[0120] In some embodiments of any of the above aspects, the PH is other PH. In some embodiments, the other PH is associated with a blood disorder (e.g., chronic hemolytic anemia, sickle cell disease), a systemic disease (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), a metabolic disorder (e.g., glycogen storage disease, Gaucher disease, or thyroid disease), pulmonary neoplastic thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension.

[0121] In some embodiments of any of the above aspects, the method reduces the frequency or severity of one or more symptoms of PH (e.g., reduces the severity or frequency of shortness of breath (dyspnea), fatigue, swelling of the legs, feet, abdomen (ascites), or neck (e.g., edema), chest pain or pressure, rapid pulse or heart palpitations, bluish color of the lips or skin (cyanosis), dizziness, or fainting).

[0122] In some embodiments of any of the above aspects, the method reduces pulmonary vascular remodeling. In some embodiments of any of the above aspects, the method reduces vascular remodeling in the heart.

[0123] In some embodiments of any of the above aspects, the method reduces right ventricular hypertrophy. In some embodiments of any of the above aspects, the method reduces pulmonary vascular resistance (eg, reduces pulmonary vascular resistance compared to a measurement taken before treatment).

[0124] In some embodiments of any of the above aspects, the method improves performance in a 6-minute walk test (eg, improves performance compared to a measurement taken before treatment). In some embodiments of any of the above aspects, the method reduces or inhibits binding of activin and / or myostatin to their receptors.

[0125] In some embodiments of any of the above aspects, the polypeptide, nucleic acid, vector, or pharmaceutical composition increases muscle mass and / or strength, increases bone mineral density, decreases bone resorption, decreases bone loss, decreases the rate of bone resorption, increases bone formation, increases the rate of bone formation, decreases osteoclast activity, increases osteoblast activity, reduces risk of fracture, increases bone strength, reduces fibrosis, prevents the onset of fibrosis, delays the onset of fibrosis, delays or inhibits the progression of fibrosis, reduces the risk of developing fibrosis, reduces one or more symptoms of fibrosis, improves the function of a fibrotic tissue or organ, increases red blood cell levels, increases hemoglobin levels, increases hematocrit, reduces the need for blood transfusions, increases erythropoiesis, increases red blood cell count, treats anemia, and prevents PH. The compound is administered in an amount sufficient to prevent, reduce the risk of developing PH, reduce the severity or frequency of one or more symptoms of PH, delay the onset of PH, delay or inhibit the progression of PH, treat PH, reduce pulmonary vascular remodeling, reduce vascular remodeling in the heart, reduce right ventricular hypertrophy, reduce pulmonary vascular resistance, improve performance in a 6-minute walk test, affect myostatin, activin, and / or BMP-9 signaling in a subject, or reduce or inhibit the binding of activin and / or myostatin to their receptors. In some embodiments, the PH is PAH. In some embodiments, the PH is venous PH. In some embodiments, the PH is hypoxic PH. In some embodiments, the PH is thromboembolic PH. In some embodiments, the PH is other PH.

[0126] In some embodiments of any of the above aspects, the method does not cause a vascular complication in the subject. In some embodiments, the method does not increase vascular permeability or leakage.

[0127] definition As used herein, the term "extracellular activin type IIB receptor (ActRIIB) mutant" refers to a peptide comprising a soluble extracellular portion of a single-pass transmembrane receptor, ActRIIB, having at least one amino acid substitution relative to wild-type extracellular ActRIIB (e.g., the bolded portion of the sequence of SEQ ID NO: 18 shown below). The sequence of wild-type human ActRIIB is shown below (SEQ ID NO: 18), in which the signal peptide is italicized and the extracellular portion is bolded.

[0128] Wild-type human ActRIIB (SEQ ID NO: 18):

[0129] [ka]

[0130] The extracellular ActRIIB mutant has the sequence of any one of SEQ ID NOs: 1 to 15. In certain embodiments, the extracellular ActRIIB mutant has the sequence of any one of SEQ ID NOs: 2 to 15 (Table 2). In some embodiments, the extracellular ActRIIB mutant may have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or more) amino acid sequence identity to the sequence of wild-type extracellular ActRIIB (SEQ ID NO: 17).

[0131] As used herein, the term "linker" refers to a bond between two elements, e.g., peptide or protein domains. The polypeptides described herein may contain an extracellular ActRIIB variant fused to a moiety (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)). This moiety can increase the stability or improve the pharmacokinetic properties of the polypeptide. The moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin) may be fused to the polypeptide via a linker. The linker can be a covalent bond or a spacer. The term "bond" refers to any type of bond created by a chemical bond, e.g., an amide bond or a disulfide bond, or a chemical reaction, e.g., chemical conjugation. The term "spacer" refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or amino acid sequence (e.g., a sequence of 1 to 200 amino acids) that exists between two elements, e.g., peptide or protein domains, to provide space and / or flexibility between the two elements. An amino acid spacer is part of the primary sequence of a polypeptide (e.g., fused to a peptide spaced apart by the polypeptide backbone). For example, the formation of a disulfide bond between two hinge regions that form an Fc domain is not considered a linker.

[0132] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers. An Fc domain comprises at least C H 2 domain and C H The Fc domain monomer has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, or 100% sequence identity) with a human Fc domain comprising three domains. The Fc domain monomer comprises a second and a third antibody constant domain (C H 2 and C HIn some embodiments, the Fc domain monomer also includes a hinge domain. The Fc domain does not include any portion of an immunoglobulin that can serve as an antigen recognition region, such as a variable domain or a complementarity determining region (CDR). In a wild-type Fc domain, two Fc domain monomers are joined by two C H Dimers are formed through interactions between the three antibody constant domains and one or more disulfide bonds formed between the hinge domains of two dimerizing Fc domain monomers. In some embodiments, the Fc domain may be mutated to lack effector function, exemplified by a "dead Fc domain." In certain embodiments, each Fc domain monomer of the Fc domain is mutated to reduce interaction or binding between the Fc domain and Fcγ receptors. H The Fc domain comprises an amino acid substitution within the antibody constant domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce or inhibit dimerization of the Fc domain. The Fc domain may be of any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain may be of an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain may also be a non-natural Fc domain, for example, a recombinant Fc domain.

[0133] As used herein, the term "albumin-binding peptide" refers to an amino acid sequence of 12 to 16 amino acids that has affinity for and functions to bind to serum albumin. Albumin-binding peptides may be of different origins, e.g., human, mouse, or rat. In some embodiments, the albumin-binding peptide has the sequence DICLPRWGCLW (SEQ ID NO: 72).

[0134] As used herein, the term "endogenous" refers to a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, e.g., a human cell, e.g., a human hair cell).

[0135] As used herein, the term "fibronectin domain" refers to a high molecular weight glycoprotein of the extracellular matrix, or a fragment thereof, that binds to transmembrane receptor proteins, such as integrins, and extracellular matrix components, such as collagen and fibrin. In some embodiments, the fibronectin domain is a fibronectin type III domain having amino acids 610-702 of the sequence of UniProt ID NO:P02751 (SEQ ID NO:73). In other embodiments, the fibronectin domain is an Adnectin protein.

[0136] As used herein, the term "human serum albumin" refers to the albumin protein present in human plasma. Human serum albumin is the most abundant protein in blood. Human serum albumin accounts for approximately half of serum proteins. In some embodiments, human serum albumin has the sequence of UniProt ID NO: P02768 (SEQ ID NO: 74).

[0137] As used herein, the term "fused" is used to refer to the combination or joining of two or more elements, components, or protein domains, e.g., peptides or polypeptides, by means including chemical conjugation, recombinant means, and chemical bonds, e.g., amide bonds. For example, two single peptides can be fused in tandem via chemical conjugation, chemical bonds, peptide linkers, or any other covalent bonding means to form one continuous protein structure, e.g., a polypeptide. In some embodiments of the polypeptides described herein, an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 1 to 15 (e.g., SEQ ID NOs: 2 to 15)) may be fused in tandem via a linker to the N-terminus or C-terminus of a moiety (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 19), a wild-type Fc domain (e.g., the sequence of SEQ ID NO: 71), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide (e.g., the sequence of SEQ ID NO: 72), a fibronectin domain (e.g., the sequence of SEQ ID NO: 73), or human serum albumin (e.g., the sequence of SEQ ID NO: 74)). For example, an extracellular ActRIIB mutant is fused to a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin) via a peptide linker, in which the N-terminus of the peptide linker is fused to the C-terminus of the extracellular ActRIIB mutant via a chemical bond, e.g., a peptide bond, and the C-terminus of the peptide linker is fused to the N-terminus of the moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin) via a chemical bond, e.g., a peptide bond.

[0138] As used herein, the terms "bone mineral density (BMD)," "bone mineral density," and "bone mass" refer to a measure of the amount of bone mineral (e.g., calcium) in bone tissue. BMD can be measured by well-established clinical techniques known to those skilled in the art (e.g., by single- or dual-energy photon or X-ray absorptiometry). The concept of BMD relates to the amount of mineral mineral per volume of bone, but clinically, it is measured, as a proxy, according to the optical density per square centimeter of bone surface during imaging. BMD measurements are used in clinical medicine as an indirect indicator of osteoporosis and fracture risk. In some embodiments, BMD test results are presented as a T-score, which represents the subject's BMD compared to the ideal or peak bone mineral density of a healthy 30-year-old adult. A score of 0 indicates that the BMD is equivalent to the normal reference value for healthy young adults. The difference between the measured BMD of a subject and the reference value for healthy young adults is evaluated in standard deviation units (SD). Thus, a T-score between +1SD and -1SD may indicate normal BMD, a T-score between -1SD and -2.5SD may indicate low bone mass (e.g., osteopenia), and a T-score below -2.5SD may indicate osteoporosis or severe osteoporosis. In some embodiments, a polypeptide of the present invention comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOS: 1 to 15 (e.g., SEQ ID NOS: 2 to 15)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule is administered to a subject in need thereof, wherein the patient has low bone mass (e.g., a T-score between -1SD and -2.5SD). In some embodiments, a polypeptide of the present invention comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOS: 1 to 15 (e.g., SEQ ID NOS: 2 to 15)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule is administered to a subject in need thereof, wherein the patient has osteoporosis (e.g., a T-score less than -2.5SD).In some embodiments, administration of a polypeptide of the present invention comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule treats a subject by increasing their BMD. In some embodiments, administration of a polypeptide of the present invention comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule increases the subject's BMD, resulting in an increase in the subject's T-score (e.g., an increase in the subject's T-score of 0.1 or more, 0.2 or more, 0.3 or more, 0.4 or more, 0.5 or more, 1.0 or more, or 2.0 or more).

[0139] As used herein, the term "bone strength" refers to a bone measurement determined by bone mineral density as well as bone quality. Bone quality is influenced by bone shape, microstructure, and tissue characteristics. Bone strength can be used to assess bone fracture risk.

[0140] As used herein, the term "bone disease" refers to a condition characterized by bone damage (e.g., decreased bone mineral density, decreased bone strength, and / or bone loss). Such diseases or pathologies may be caused by an imbalance in osteoblast and / or osteoclast activity (e.g., increased bone resorption or decreased bone formation). Bone diseases include primary osteoporosis, secondary osteoporosis, osteopenia, osteopetrosis, bone fracture, bone loss associated with bone cancer or cancer metastasis (e.g., bone loss associated with multiple myeloma), Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, hypogravity-related bone loss, or immobility-related bone loss.

[0141] As used herein, the terms "bone remodeling" or "bone metabolism" refer to the process of maintaining bone strength and ionic homeostasis by replacing discontinuous portions of old bone with packets of newly synthesized proteinaceous matrix. Bone is resorbed by osteoclasts and deposited by osteoblasts in a process called ossification. Osteocyte activity plays an important role in this process. Conditions leading to bone loss can occur through either increased resorption or decreased ossification. In healthy individuals, bone formation exceeds resorption during childhood. With aging, resorption exceeds formation. Additionally, the rate of bone resorption is generally much higher in older, postmenopausal women due to the estrogen deficiency associated with menopause.

[0142] As used herein, the term "bone resorption" or "bone catabolic activity" refers to the process by which osteoclasts break down bone tissue, releasing minerals and resulting in the transfer of minerals (e.g., calcium) from bone tissue to the blood. Increased bone resorption rates are associated with aging, including aging in postmenopausal women. High bone resorption rates, or bone resorption rates that exceed the ossification rate, are associated with bone disorders such as decreased bone mineral density, including osteopenia and osteoporosis, and consequent bone loss. In some embodiments, a polypeptide of the present invention comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule is administered to a subject in need thereof to reduce bone resorption (e.g., the amount or rate of bone resorption in the subject) (e.g., reduce bone mass).

[0143] As used herein, the terms "bone formation," "ossification," "osteogenesis," or "bone anabolic activity" refer to the process of forming new bone tissue by osteoblasts. In some embodiments, a polypeptide of the present invention comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule is administered to a subject in need thereof to increase bone formation (e.g., to increase the amount or rate of bone formation or osteogenesis in the subject). A decrease in the rate of bone formation, or a rate of bone formation that exceeds the rate of bone resorption, can result in bone loss.

[0144] As used herein, the terms "increase" and "reduce" refer to modulating, resulting in a greater or lesser amount of function, expression, or activity of a metric, respectively, compared to a reference. For example, following administration of a polypeptide of the present invention comprising an extracellular ActRIIB variant in the manner described herein, the amount of a marker for a metric described herein (e.g., lean mass) may increase or decrease in a subject compared to the amount of the marker before administration. Generally, the metric is measured at least one week, one month, three months, or six months after administration, when administration has shown the recited effect, e.g., after the treatment regimen has begun.

[0145] As used herein, the term "fibrosis" refers to a pathological process in which excessive fibrous connective tissue is formed. Fibrosis is characterized by the accumulation of fibroblasts and the deposition of collagen in excess of normal levels in a particular tissue. In response to inflammation or tissue injury, nearby fibroblasts migrate into the wound, proliferate, and produce large amounts of collagenous extracellular matrix. When fibrosis occurs in response to injury, the term "scarring" can be used synonymously. Fibrosis can occur in many tissues of the body, including, for example, the lung, skin, liver, kidney, heart, eye, tendon, cartilage, pancreatic tissue, uterine tissue, nervous tissue, testes, ovaries, adrenal glands, arteries, veins, colon, small and large intestines, biliary tract, and intestines.

[0146] As used herein, the term "pulmonary hypertension" or "PH" refers to a disorder characterized by elevated blood pressure between the heart and lungs, and may include elevated blood pressure in the pulmonary arteries (pulmonary arterial hypertension), pulmonary veins, or pulmonary capillaries. Pulmonary hypertension is associated with many symptoms, including shortness of breath (dyspnea), fatigue, swelling in the legs, feet, abdomen (e.g., ascites), or neck (e.g., edema), chest pain or tightness, rapid pulse or palpitations, bluish discoloration of the lips or skin (cyanosis), dizziness, or fainting. PH also reduces exercise tolerance and can lead to heart failure.

[0147] As used herein, the term "pulmonary arterial hypertension" or "PAH" refers to a form of pulmonary hypertension characterized by narrowing or obstruction of small pulmonary arteries, often caused by scarring, and elevated pulmonary artery blood pressure. PAH is also known as WHO Group I pulmonary hypertension. PAH can be diagnosed based on a resting mean pulmonary artery pressure greater than 25 mmHg with a normal pulmonary artery capillary wedge pressure. PAH can cause shortness of breath, dizziness, syncope, and other symptoms, all of which worsen with exertion. PAH can be a severe disease with markedly reduced exercise tolerance and heart failure. The two main types of PAH are idiopathic PAH (e.g., PAH without an identified predisposing factor) and hereditary PAH (e.g., PAH associated with mutations in BMPR2, ALK1, SMAD9, caveolin-1, KCNK3, or EIF2AK4). In 70% of familial PAH cases, mutations are located in the BMPR2 gene. Risk factors for developing PAH include a family history of PAH, drug use (e.g., methamphetamine or cocaine use), infection (e.g., HIV infection or schistosomiasis), liver cirrhosis, congenital heart abnormalities, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, or connective tissue / autoimmune disorders (e.g., scleroderma or lupus).

[0148] As used herein, the terms "venous pulmonary hypertension" and "venous PH" refer to forms of pulmonary hypertension secondary to left heart disease. Venous PH is also known as WHO Group II PH. Venous PH can be associated with or caused by left ventricular systolic dysfunction (e.g., left ventricular failure), left ventricular diastolic dysfunction, valvular heart disease (e.g., mitral valve disease or aortic valve disease), congenital cardiomyopathy, or congenital or acquired pulmonary vein stenosis.

[0149] As used herein, the terms "hypoxic pulmonary hypertension" and "hypoxic PH" refer to a form of pulmonary hypertension resulting from lung disease or chronic hypoxia. This form of PH is also known as WHO Group III PH. Hypoxic PH can be associated with or caused by chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), lung disease (e.g., pulmonary fibrosis), alveolar hypoventilation disorders, chronic exposure to high altitude, or developmental abnormalities.

[0150] As used herein, the terms "thromboembolic pulmonary hypertension" and "thromboembolic PH" refer to a form of pulmonary hypertension associated with chronic arterial obstruction (e.g., a blood clot). Thromboembolic PH is also known as WHO Group IV PH. Thromboembolic PH can be associated with or caused by chronic thromboembolic pulmonary hypertension or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary stenosis, or parasitic infection).

[0151] As used herein, the terms "miscellaneous pulmonary hypertension" and "other PH" refer to forms of pulmonary hypertension with an unknown or multifactorial mechanism. This form of PH is classified as WHO Group V PH. Other PH is associated with hematological disorders (e.g., chronic hemolytic anemia, sickle cell disease), systemic diseases (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), metabolic disorders (e.g., glycogen storage disease, Gaucher disease, or thyroid disease), pulmonary neoplastic thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension.

[0152] As used herein, the terms "increasing red blood cell levels" and "promoting red blood cell formation" refer to clinically observable metrics such as hematocrit, red blood cell count, and hemoglobin measurements, and are intended to be neutral with respect to the mechanism by which such changes occur. As used herein, the term "low red blood cell levels" refers to red blood cell count, hematocrit, and hemoglobin measurements that are below the range of values ​​considered normal for a subject's age and sex.

[0153] As used herein, the terms "red blood cell formation" and "red blood cell production" refer to the generation of red blood cells, such as the process of erythropoiesis in which red blood cells are produced in the bone marrow.

[0154] As used herein, the term "anemia" refers to an abnormality in hemoglobin or red blood cells that results in a decrease in the oxygen level in the blood. Anemia may be related to abnormal production, processing, or performance of red blood cells and / or hemoglobin. The term anemia refers to any decrease in the number of red blood cells and / or hemoglobin levels in the blood compared to normal blood levels.

[0155] As used herein, the term "percent (%) identity" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence that are identical to those in a reference sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment to determine percent identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for assessing alignment, including the algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. In some embodiments, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence (alternatively, this can be expressed as a given candidate sequence having or containing a particular percent amino acid (or nucleic acid) sequence identity to a given reference sequence) is calculated as follows: 100 x (fraction A / B) where A is the number of amino acid (or nucleic acid) residues assigned an identity score in the alignment of the candidate sequence with the reference sequence, and B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments, if the length of the candidate sequence is not equal to the length of the reference sequence, the percent amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence will not be equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.

[0156] In certain embodiments, a reference sequence aligned for comparison to a candidate sequence may show 50% to 100% identity over the entire length of the candidate sequence or over a selected portion of consecutive amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. If a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0157] As used herein, the term "serum half-life," in the context of administering a therapeutic protein to a subject, refers to the time required for the plasma concentration of that protein to decrease by half in the subject. Proteins may be redistributed or eliminated from the bloodstream, or may be degraded, for example, by proteolysis. As described herein, a polypeptide comprising an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15) exhibits a serum half-life of 7 days in humans.

[0158] As used herein, the term "lean mass" refers to components of body composition, including, for example, lean mass, body fat, and body fluid. Lean mass is usually calculated by subtracting the weight of body fat and body fluid from total body weight. Typically, a subject's lean mass is between 60% and 90% of their total body weight. In the present invention, administering to a subject a polypeptide comprising an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOS: 1 to 15 (e.g., SEQ ID NOS: 2 to 15), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOS: 1 to 15 (e.g., SEQ ID NOS: 2 to 15)), or a vector containing such a nucleic acid molecule increases the subject's lean mass.

[0159] As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. Generally, binding affinity refers to the strength of the total non-covalent interactions between a molecule and its binding partner, for example, an extracellular ActRIIB mutant and BMP9 or activin A. Unless otherwise indicated, binding affinity refers to the intrinsic binding affinity, which represents a 1:1 interaction between members of a binding pair. The binding affinity between two molecules is generally determined by the dissociation constant (K D ) or affinity constant (K A ) Two molecules that have low binding affinity for each other generally tend to bind slowly and dissociate easily, with a large K D Two molecules that have a high affinity for each other generally bind more easily and tend to remain bound longer, exhibiting a small K D The K of two interacting molecules is shown. D can be determined using methods and techniques well known in the art, such as, for example, surface plasmon resonance. D is k off / k on It is calculated as the ratio of

[0160] As used herein, the term "muscle mass" refers to a component of body composition. Typically, muscle mass is calculated by subtracting the weight of body fat and fluid from total body weight. The percentage of muscle mass can vary greatly between individuals depending on the subject's genetic makeup, age, race, and health status. Typically, a subject's muscle mass can be 20% to 50% of their total body weight.

[0161] As used herein, the phrase "affecting myostatin, activin, and / or BMP9 signaling" means altering the binding of myostatin, activin, and / or BMP9 to their receptors, such as ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIB). In some embodiments, a polypeptide comprising an extracellular ActRIIB mutant described herein reduces or inhibits the binding of myostatin, activin, and / or BMP9 to their receptors (e.g., ActRIIA, ActRIIB, and BMPRII (e.g., endogenous ActRIIB)). As described herein, a polypeptide of the present invention comprising an extracellular ActRIIB mutant having the sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15) may have a weak binding affinity for BMP9 (e.g., a K of 200 pM or greater). D ).

[0162] As used herein, the term "vascular complications" refers to vascular disorders or any damage to blood vessels, for example, damage to the vascular wall. Damage to the vascular wall can lead to increased vascular permeability or leakage. The term "vascular permeability or leakage" refers to the ability of the vascular wall to allow small molecules, proteins, and cells to enter and exit the blood vessel. Increased vascular permeability or leakage can occur due to an increase in the gaps between endothelial cells lining the vascular wall (e.g., an increase in the size and / or number of gaps) and / or thinning of the vascular wall.

[0163] As used herein, the term "polypeptide" refers to a single polymer in which the monomers are amino acid residues covalently linked to one another through amide bonds. Polypeptide is intended to encompass any amino acid sequence that is naturally occurring, recombinant, or synthetically produced.

[0164] As used herein, the term "homodimer" refers to a molecular construct formed by two identical macromolecules, such as proteins or nucleic acids. These two identical monomers can form a homodimer through covalent or non-covalent bonds. For example, an Fc domain can be a homodimer of two Fc domain monomers if the two Fc domain monomers contain the same sequence. In another example, a polypeptide described herein comprising an extracellular ActRIIB variant fused to an Fc domain monomer can form a homodimer through the interaction of the two Fc domain monomers, which form the Fc domain in the homodimer.

[0165] As used herein, the term "heterodimer" refers to a molecular construct formed by two different macromolecules, such as proteins or nucleic acids. These two monomers can form a heterodimer through covalent or non-covalent bonds. For example, a polypeptide described herein comprising an extracellular ActRIIB variant fused to an Fc domain monomer can form a heterodimer through the interaction of two Fc domain monomers, each fused to a different ActRIIB variant, to form an Fc domain in the heterodimer.

[0166] As used herein, the term "host cell" refers to a vehicle containing necessary cellular components, e.g., organelles required for expressing a protein from a corresponding nucleic acid. The nucleic acid is generally contained within a nucleic acid vector, which can be introduced into the host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). The host cell can be a prokaryotic cell, e.g., a bacterial cell, or a eukaryotic cell, e.g., a mammalian cell (e.g., a CHO cell or HEK293 cell).

[0167] As used herein, the term "therapeutically effective amount" refers to an amount of the polypeptide, nucleic acid, or vector of the present invention, or a pharmaceutical composition containing the polypeptide, nucleic acid, or vector of the present invention, which is effective to treat a muscle disease or condition accompanied by muscle weakness and muscle atrophy (e.g., Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia or cancer cachexia), bone damage (e.g., osteoporosis or a condition involving bone damage, e.g., primary osteoporosis, secondary osteoporosis, osteopenia, "A therapeutically effective amount" refers to an amount effective to achieve a desired therapeutic effect in treating a patient having or at risk of developing a disease, such as a disease or condition involving osteopetrosis, bone fracture, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with the treatment of obesity, hypogravity-related bone loss, or immobility-related bone loss, a disease or condition involving low red blood cell levels (e.g., anemia or blood loss), a disease or condition involving fibrosis, or a disease or condition involving PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). In particular, a therapeutically effective amount of a polypeptide, nucleic acid, or vector avoids adverse side effects.

[0168] As used herein, the term "pharmaceutical composition" refers to a medicament or pharmaceutical formulation containing an active ingredient and excipients and diluents that can make the active ingredient suitable for the administration method. The pharmaceutical composition of the present invention contains pharmaceutically acceptable ingredients that are compatible with the polypeptide, nucleic acid, or vector. The pharmaceutical composition may be in the form of a tablet or capsule for oral administration, or in the form of an aqueous dosage form for intravenous or subcutaneous administration.

[0169] As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to an excipient or diluent in a pharmaceutical composition. A pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not harmful to the recipient. In the present invention, a pharmaceutically acceptable carrier or excipient must provide sufficient pharmaceutical stability to a polypeptide containing an extracellular ActRIIB mutant, a nucleic acid molecule encoding the polypeptide, or a vector containing such a nucleic acid molecule. The nature of the carrier or excipient will vary depending on the mode of administration. For example, aqueous carriers are commonly used for intravenous administration, while solid carriers are preferred for oral administration.

[0170] As used herein, the term "treating and / or preventing" refers to the treatment and / or prevention of a disease, e.g., a muscle disease (e.g., DMD, FSHD, IBM, and ALS), a bone disease (e.g., a disease or condition associated with bone damage, e.g., osteoporosis, osteopenia, osteopetrosis, fracture, bone cancer or cancer metastasis-associated bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with the treatment of obesity, hypogravity-associated bone loss, or immobility-associated bone loss), a disease associated with low red blood cell levels (e.g., anemia or blood loss), fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), using the methods and compositions of the invention. Generally, treatment of a muscle, bone, hypoxic, or fibrotic disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) occurs after a subject has developed and / or been diagnosed with the muscle, bone, hypoxic, or fibrotic disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). Prevention of a muscle, bone, hypoxic, or fibrotic disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) refers to methods or procedures taken when a subject is at risk of developing the muscle, bone, hypoxic, or fibrotic disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH).The subject exhibits signs or mild symptoms that a physician determines to be an indication or risk factor for developing a muscle, bone, hypocytic, or fibrotic disease, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH); has other diseases or conditions associated with the development of a muscle, bone, hypocytic, or fibrotic disease, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH); has undergone treatment (e.g., surgery, chemotherapy, or radiation) that may cause anemia, fibrosis, or loss of bone density; or has a family history or genetic predisposition to developing a muscle, bone, hypocytic, or fibrotic disease, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), but has not yet developed the disease.

[0171] As used herein, the term "subject" refers to a mammal, such as, preferably, a human. Mammals include, but are not limited to, humans and domestic and farm animals, such as monkeys (e.g., cynomolgus monkeys), mice, dogs, cats, horses, and cows. [Effects of the Invention]

[0172] According to the present invention, a polypeptide and a pharmaceutical composition comprising an activin type IIB receptor mutant can be provided. [Brief explanation of the drawings]

[0173] [Figure 1] Figure 1 shows a sequence alignment of the wild-type sequences of extracellular ActRIIA and ActRIIB and the amino acid substitutions of ActRIIB mutants. The percent weight change resulting from treatment of mice with the corresponding mutants is also shown. [Figure 2]Figure 2 is a bar graph showing the effect of extracellular ActRIIB mutants on body weight at the end of day 28. Mice received a single hydrodynamic injection of a plasmid construct encoding the indicated ActRIIB mutant or a control plasmid. The legend lists the bars in the bar graph from left to right ("Vehicle" is the bar closest to the y-axis, and "pLEV-113-ActRIIb-2.10" is the bar furthest from the y-axis). [Figure 3A] 3A and 3B are bar graphs showing the effect of extracellular ActRIIB variants on muscle mass in individuals by tissue analysis. [Figure 3B] 3A and 3B are bar graphs showing the effect of extracellular ActRIIB variants on muscle mass in individuals by tissue analysis. [Figure 4] FIG. 4 is a graph showing the effect of extracellular ActRIIB variants ActRIIB 2.11-Fc and ActRIIB 2.12-Fc on percent body weight over 28 days. [Figure 5A] 5A-5C are a series of graphs showing the effect of ActRIIB variants ActRIIB 2.11-Fc and ActRIIB 2.12-Fc on red blood cell counts, hemoglobin levels, and hematocrit. [Figure 5B] 5A-5C are a series of graphs showing the effect of ActRIIB variants ActRIIB 2.11-Fc and ActRIIB 2.12-Fc on red blood cell counts, hemoglobin levels, and hematocrit. [Figure 5C] 5A-5C are a series of graphs showing the effect of ActRIIB variants ActRIIB 2.11-Fc and ActRIIB 2.12-Fc on red blood cell counts, hemoglobin levels, and hematocrit. [Figure 6A] 6A-6D are a series of graphs showing the effect of the ActRIIB variant ActRIIB 2.12-Fc on trabecular bone in a mouse model of osteoporosis. [Figure 6B]6A-6D are a series of graphs showing the effect of the ActRIIB variant ActRIIB 2.12-Fc on trabecular bone in a mouse model of osteoporosis. [Figure 6C] 6A-6D are a series of graphs showing the effect of the ActRIIB variant ActRIIB 2.12-Fc on trabecular bone in a mouse model of osteoporosis. [Figure 6D] 6A-6D are a series of graphs showing the effect of the ActRIIB variant ActRIIB 2.12-Fc on trabecular bone in a mouse model of osteoporosis. [Figure 7A] 7A-7C are a series of graphs showing the effect of the ActRIIB variant ActRIIB 2.12-Fc on red blood cell mass parameters in wild-type rats. [Figure 7B] 7A-7C are a series of graphs showing the effect of the ActRIIB variant ActRIIB 2.12-Fc on red blood cell mass parameters in wild-type rats. [Figure 7C] 7A-7C are a series of graphs showing the effect of the ActRIIB variant ActRIIB 2.12-Fc on red blood cell mass parameters in wild-type rats. [Figure 8A] Figures 8A-8E are a series of graphs showing the effect of ActRIIB variant ActRIIB 2.12-Fc on trabecular bone in wild-type rats. [Figure 8B] Figures 8A-8E are a series of graphs showing the effect of ActRIIB variant ActRIIB 2.12-Fc on trabecular bone in wild-type rats. [Figure 8C] Figures 8A-8E are a series of graphs showing the effect of ActRIIB variant ActRIIB 2.12-Fc on trabecular bone in wild-type rats. [Figure 8D] Figures 8A-8E are a series of graphs showing the effect of ActRIIB variant ActRIIB 2.12-Fc on trabecular bone in wild-type rats. [Figure 8E]Figures 8A-8E are a series of graphs showing the effect of ActRIIB variant ActRIIB 2.12-Fc on trabecular bone in wild-type rats. DETAILED DESCRIPTION OF THE INVENTION

[0174] The present invention relates to polypeptides comprising extracellular activin type IIB receptor (ActRIIB) mutants. In some embodiments, the polypeptides of the present invention comprise an extracellular ActRIIB mutant fused to a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin). Polypeptides comprising an extracellular ActRIIB mutant fused to an Fc domain monomer can also form dimers (e.g., homodimers or heterodimers) through interaction between two Fc domain monomers. The ActRIIB mutants described herein exhibit reduced binding to bone morphogenetic protein 9 (BMP9) compared to wild-type extracellular ActRIIB, or exhibit weaker or no binding affinity to BMP9 compared to activin and myostatin. The present invention also includes methods for treating diseases and conditions associated with muscle weakness or muscle atrophy by increasing muscle mass and strength; treating or preventing bone damage by increasing bone mineral density, increasing bone formation, or decreasing bone resorption; treating or preventing fibrosis; treating or preventing low blood cell levels (such as anemia or blood loss) by increasing red blood cell levels (e.g., red blood cell count, hemoglobin level, or hematocrit) or red blood cell production; treating or preventing pulmonary hypertension (PH) (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH); or affecting myostatin, activin, and / or BMP9 signaling in a subject by administering to the subject a polypeptide comprising an extracellular ActRIIB variant described herein.

[0175] I. Extracellular activin type IIB receptor mutants The activin type II receptor is a single-transmembrane domain receptor that mediates signaling for ligands in the transforming growth factor β (TGF-β) superfamily. TGF-β superfamily ligands are involved in many physiological processes in the host, such as muscle growth, vascular growth, cell differentiation, homeostasis, and bone formation. Examples of TGF-β superfamily ligands include activins (e.g., activin A and activin B), inhibins, growth differentiation factors (GDFs) (e.g., GDF8, also known as myostatin), and bone morphogenetic proteins (BMPs) (e.g., BMP9). Myostatin and activins are known to play a role in regulating skeletal muscle growth. For example, myostatin-null mice exhibit a significant increase in skeletal muscle mass. Myostatin is also involved in promoting fibrosis. For example, myostatin-deficient mice exhibit reduced muscle fibrosis, and injection of myostatin-coated beads induces muscle fibrosis in mice. Mice overexpressing activin subunits that induce the production of diffusible activin A also exhibit fibrosis. Furthermore, activin is abundantly expressed in bone tissue and regulates bone formation by controlling both osteoblast and osteoclast function. Activin is reported to be upregulated in bone diseases and inhibit osteoblast activity. Myostatin has also been associated with bone homeostasis through increased bone formation and inhibition of osteoblast activity. Elevated activin A has also been observed in clinical and experimental pulmonary hypertension. Therefore, methods to reduce or inhibit activin or myostatin signaling can be used to treat diseases and conditions including muscle atrophy or weakness, fibrosis, bone damage, low red blood cell levels (e.g., anemia), or pulmonary hypertension (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH).

[0176] There are two types of activin type II receptors: ActRIIA and ActRIIB. Studies have shown that BMP9 binds to ActRIIB with approximately 300-fold higher binding affinity than ActRIIA (see, for example, Townson et al., J. Biol. Chem. 287:27313, 2012). ActRIIA is known to have a longer half-life than ActRIIB. The present invention describes extracellular ActRIIB variants constructed by introducing amino acid residues of ActRIIA into ActRIIB or by introducing novel amino acid substitutions, with the aim of reducing BMP9 binding to prevent or reduce disruption of endogenous BMP9 signaling. Amino acid substitutions may also confer beneficial physiological and pharmacokinetic properties of ActRIIA, such as a longer half-life or the ability to increase red blood cell levels. An optimal peptide will result in a significant increase in lean mass, muscle mass, bone mineral density and / or red blood cell levels (e.g., increased red blood cell production), a reduction in fibrosis, or treatment of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), while, for example, decreasing binding affinity to BMP9. Preferred ActRIIB variants also exhibit similar or improved binding to activin and / or myostatin compared to wild-type ActRIIB, allowing them to compete with endogenous activin receptors for ligand binding and reduce or inhibit endogenous activin receptor signaling.These variants can be used to treat disorders in which activin receptor signaling is elevated, such as bone disease (e.g., a disease or condition involving bone damage), muscle disease, fibrosis, PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), and / or anemia, resulting in decreased bone resorption or osteoclast activity, increased bone formation or bone mineral density, increased muscle mass or strength, reduced fibrosis (e.g., reduced fibrosis or slowed or stopped the progression of fibrosis), increased red blood cell levels (e.g., increased hemoglobin levels, hematocrit, or red blood cell count, e.g., increased red blood cell production), or reduced symptoms or progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). In some embodiments, amino acid substitutions can be introduced into extracellular ActRIIB variants to reduce or eliminate the variant's binding affinity for BMP9. The wild-type amino acid sequences of the extracellular portions of human ActRIIA and ActRIIB are shown below.

[0177] Human ActRIIA extracellular portion (SEQ ID NO: 16):

[0178] [ka]

[0179] Human ActRIIB extracellular portion (SEQ ID NO: 17):

[0180] [ka]

[0181] The polypeptides described herein include extracellular ActRIIB mutants having at least one amino acid substitution relative to wild-type extracellular ActRIIB having the sequence of SEQ ID NO: 17. Potential amino acid substitutions can be introduced into the extracellular ActRIIB mutants at 28 different positions (Table 1). The extracellular ActRIIB mutants can have one or more (e.g., 1-28, 1-25, 1-23, 1-21, 1-19, 1-17, 1-15, 1-13, 1-11, 1-9, 1-7, 1-5, 1-3, or 1-2; e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27) amino acid substitutions relative to the sequence of wild-type extracellular ActRIIB (SEQ ID NO: 17). In some embodiments, an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of SEQ ID NO: 1) may contain amino acid substitutions at all 28 positions as listed in Table 1. In some embodiments, an extracellular ActRIIB variant may contain amino acid substitutions at multiple positions, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 18, 20, 22, 24, 26, or 27, of the 28 positions as listed in Table 1. In some embodiments, the substitution is a substitution of an amino acid from ActRIIA for the same position in ActRIIB. In some embodiments, the substitution is a novel change (e.g., a substitution of an amino acid not present in the corresponding position in ActRIIA, e.g., S48T, I51L, Q69D, or E70T).

[0182] The amino acid substitution may reduce or improve the activity and / or binding affinity of the ActRIIB mutant of the present invention (e.g., an extracellular ActRIIB mutant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)). In some embodiments, the amino acid substitution worsens the binding affinity of the ActRIIB mutant to BMP9 (e.g., the mutant has reduced binding to BMP9 compared to wild-type extracellular ActRIIB, or lower binding to BMP9 compared to other ActRIIB ligands (e.g., activin A or B, myostatin, or GDF-11)). In some embodiments, the ActRIIB mutant has reduced, weak, or substantially no binding to BMP9. In some embodiments, the amino acid substitution improves the binding affinity of ActRIIB to myostatin, activin A or B, and / or GDF-11 (e.g., the mutant has improved binding affinity compared to wild-type extracellular ActRIIB, or binds more strongly to myostatin, activin A or B, or GDF-11 than to BMP9). In some embodiments, the amino acid substitution reduces the binding affinity of ActRIIB to myostatin, activin A or B, and / or GDF-11 (e.g., the mutant has reduced binding affinity compared to wild-type extracellular ActRIIB, or binds less strongly to myostatin, activin A or B, or GDF-11 than to BMP9). In some embodiments, the amino acid substitution does not substantially alter extracellular ActRIIB function (e.g., the ActRIIB variant increases lean mass, muscle mass, or bone mineral density by the same amount as wild-type extracellular ActRIIB, or reduces or prevents fibrosis; e.g., the ActRIIB variant is functionally equivalent to wild-type extracellular ActRIIB). In some embodiments, the amino acid substitution confers a property or activity of ActRIIA to the ActRIIB variant (e.g., the ActRIIB variant can increase red blood cell levels or has a longer half-life than WT extracellular ActRIIB).Preferably, the ActRIIB variant has one or more, two or more, or three or more of the above properties (e.g., reduced BMP9 binding and improved binding to activin A or B, myostatin and / or GDF-11, or reduced BMP9 binding and reduced functional equivalence to wild-type ActRIIB, resulting in increased lean mass, muscle mass, or bone mineral density, or reduced or prevented fibrosis).

[0183] The ActRIIB mutants of the present invention (e.g., extracellular ActRIIB mutants having any one of the sequences of SEQ ID NOS: 1 to 15 (e.g., SEQ ID NOS: 2 to 15)) preferably have one or more amino acid substitutions that reduce BMP9 binding. In some embodiments, the amino acid substitution that reduces BMP9 binding is E75K (e.g., X in SEQ ID NOS: 1). 24 In some embodiments, the amino acid substitutions that decrease BMP9 binding are Q69T and E70D (e.g., X in SEQ ID NO: 1). 21 is T and X 22 In some embodiments, the amino acid substitutions that decrease BMP9 binding are Q69D and E70T (e.g., X in SEQ ID NO: 1). 21 is D and X 22 In some embodiments, amino acid substitutions that reduce BMP9 binding are T74K, E75K, E76D, N77S, and Q79E (e.g., X 23 , X 24 , X 25 , X 26 , and X 28are K, K, D, S, and E, respectively, in SEQ ID NO: 1. In some embodiments, the ActRIIB variant has two or more of the foregoing amino acid substitutions that reduce BMP9 binding (e.g., substitution E75K and substitutions Q69D and E70T, or substitution E75K and substitutions Q69T and E70D). In some embodiments, the ActRIIB variant of the present invention has one or more amino acid substitutions that reduce BMP9 binding and one or more additional amino acid substitutions. The additional amino acid substitutions may confer other beneficial properties, such as altered binding to activin or myostatin or improved activity. For example, amino acid substitutions T74K, E75K, E76D, N77S, and Q79E result in reduced ActRIIB mutant activity (e.g., the mutant has less effect on lean mass and muscle mass compared to wild-type extracellular ActRIIB), while including additional substitutions S25T and S47I; E31Y, E33D and Q34K; or Y41F, R45K, and K56Q improves the effect of the ActRIIB mutant on lean mass and / or muscle mass. Further amino acid substitutions may include one or more of the substitutions I11L, Y12F, L19K, E20D, S25T, L27V, R29P, E31Y, E33D, Q34K, L38R, Y41F, R45K, S47I, S48T, T50S, I51L, L53I, K56Q and F63I, T74K, E76D, N77S, Q79E or F89M.

[0184] In some embodiments, the polypeptides described herein comprise an extracellular ActRIIB mutant having the sequence of SEQ ID NO:1.

[0185] [Table 1]

[0186] In some embodiments, the polypeptides described herein comprise an extracellular ActRIIB mutant having the sequence of any one of SEQ ID NOs: 2-15 (Table 2).

[0187] [Table 2-1]

[0188] [Table 2-2]

[0189] In some embodiments, a polypeptide of the present invention comprising an extracellular ActRIIB variant may further comprise a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin), which can be fused to the N-terminus or C-terminus (e.g., the C-terminus) of the extracellular ActRIIB variant via a linker or other covalent bond. A polypeptide comprising an extracellular ActRIIB variant fused to an Fc domain monomer can form a dimer (e.g., a homodimer or heterodimer) through interaction between two Fc domain monomers, which combine to form the Fc domain in the dimer.

[0190] Furthermore, in some embodiments, the polypeptides described herein have a serum half-life in humans of at least 7 days. D The polypeptide can bind to bone morphogenetic protein 9 (BMP9) at a K of 10 pM or greater. D In some embodiments, the polypeptide can bind to activin A at a specific binding site. In some embodiments, the polypeptide does not bind to BMP9 or activin A. In some embodiments, the polypeptide binds to activin and / or myostatin and exhibits low (e.g., weak) binding to BMP9.

[0191] Additionally, in some embodiments, the polypeptide has a K of about 200 pM or greater. D (e.g., K of about 200, 300, 400, 500, 600, 700, 800, or 900 pM or greater)D , e.g., a K of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nM or greater D , e.g., a K between about 200 pM and about 50 nM D In some embodiments, the polypeptide does not substantially bind to human BMP9. In some embodiments, the polypeptide has a K of about 800 pM or less. D (e.g., a K of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less) D , e.g., K between about 800 pM and about 200 pM D In some embodiments, the polypeptide may bind to human activin A with a K of 800 pM or less. D (e.g., a K of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less) D , for example, a K of about 800 pM to about 200 pM D The polypeptide can bind to human activin B with a K of approximately 5 pM or greater. D (e.g., a K of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or more) D ) and can also bind to growth and differentiation factor 11 (GDF-11).

[0192] II. Fc Domain In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIB variant fused to an Fc domain monomer or a fragment of an Fc domain of an immunoglobulin to extend the serum half-life of the polypeptide. A polypeptide comprising an extracellular ActRIIB variant fused to an Fc domain monomer can form a dimer (e.g., a homodimer or a heterodimer) through interaction between two Fc domain monomers, which form an Fc domain in the dimer. As conventionally known in the art, an Fc domain is a protein structure found at the C-terminus of an immunoglobulin. An Fc domain is a C HThe Fc domain comprises two Fc domain monomers that form a dimer through interaction between three antibody constant domains. A wild-type Fc domain forms the minimal structure required for binding to an Fc receptor, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, or FcγRIV. In some embodiments, the Fc domain can be mutated to lack effector function, typically resulting in a "dead" Fc domain. For example, the Fc domain may contain specific amino acid substitutions known to minimize interaction between the Fc domain and an Fcγ receptor. In some embodiments, the Fc domain is derived from an IgG1 antibody and contains amino acid substitutions L234A, L235A, and G237A. In some embodiments, the Fc domain is derived from an IgG1 antibody and contains amino acid substitutions D265A, K322A, and N434A. The above amino acid positions are defined according to Kabat (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Kabat numbering of amino acid residues can be determined for a given antibody by aligning homologous regions of the antibody sequence with the "standard" Kabat numbering sequence. Furthermore, in some embodiments, the Fc domain does not induce any immune system-related response. For example, the Fc domain in a polypeptide dimer comprising an extracellular ActRIIB variant fused to an Fc domain monomer may be modified to reduce the interaction or binding between the Fc domain and the Fcγ receptor. The sequence of an Fc domain monomer that can be fused to an extracellular ActRIIB variant is shown below (SEQ ID NO: 19):

[0193] [ka]

[0194] In some embodiments, the Fc domain is derived from an IgG1 antibody and contains amino acid substitutions L12A, L13A, and G15A relative to the sequence of SEQ ID NO: 19. In some embodiments, the Fc domain is derived from an IgG1 antibody and contains amino acid substitutions D43A, K100A, and N212A relative to the sequence of SEQ ID NO: 19. In some embodiments, an extracellular ActRIIB variant described herein (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) can be fused to the N- or C-terminus of an Fc domain monomer (e.g., SEQ ID NO: 19) by conventional genetic or chemical means, for example, chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIB variant and the Fc domain monomer. The Fc domain monomer can be fused to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRIIB variant.

[0195] In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIB variant fused to an Fc domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce or inhibit dimerization of the Fc domain. In some embodiments, the Fc domain comprises a hinge domain. The Fc domain may be of the immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain may be of an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain may also be a non-natural Fc domain, for example, a recombinant Fc domain.

[0196] Methods for creating Fc domains with reduced dimerization are known in the art. In some embodiments, C is substituted with C to impair dimerization through steric clashes. H 3-C H One or more amino acids with large side chains (e.g., tyrosine or tryptophan) may be introduced into the C3 dimer interface to remove favorable interactions.H 3-C H 3 One or more amino acids with small side chains (e.g., alanine, valine, or threonine) may be introduced into the dimer interface. H Methods for introducing amino acids with large or small side chains into the three domains are described, for example, in Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Application Publication No. 2006 / 0074225, U.S. Patent No. 8,216,805 and U.S. Patent No. 5,731,168, Ridgway et al. (Protein Eng. 9:617-612, 1996), Atwell et al. (J Mol Biol. 270:26-35, 1997), and Merchant et al. (Nat Biotechnol. 16:677-681, 1998), all of which are incorporated herein by reference in their entirety.

[0197] In yet another embodiment, a C between two Fc domains H 3-C H 3C that constitutes the interface H One or more amino acid residues in the three domains are substituted with positively charged amino acid residues (e.g., lysine, arginine, or histidine) or negatively charged amino acid residues (e.g., aspartic acid or glutamic acid) so that the interaction is electrostatically unfavorable due to the introduced amino acid of the particular charge. H Methods for introducing charged amino acids into the three domains are described, for example, in Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Application Publication No. 2006 / 0074225, U.S. Patent Application Publication No. 2012 / 0244578, and U.S. Patent Application Publication No. 2014 / 0024111, all of which are incorporated herein by reference in their entirety.

[0198] In some embodiments of the invention, the Fc domain contains the following amino acid substitutions relative to the sequence of human IgG1: T366W, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L352K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E , L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T, and K409I. In one specific embodiment, the Fc domain comprises the amino acid substitution T366W relative to the sequence of human IgG1. The sequence of the wild-type Fc domain is shown in SEQ ID NO: 71:

[0199] [ka]

[0200] III. Albumin-binding peptides In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIB variant fused to a serum protein-binding peptide. Binding to the serum protein peptide can improve the pharmacokinetics of protein drugs.

[0201] By way of example, albumin-binding peptides that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the albumin-binding peptide comprises the sequence DICLPRWGCLW (SEQ ID NO: 72).

[0202] In the present invention, to extend the serum half-life of an extracellular ActRIIB mutant, an albumin-binding peptide can be linked to the N-terminus or C-terminus (e.g., the C-terminus) of an extracellular ActRIIB mutant described herein (e.g., an extracellular ActRIIB mutant having any one of the sequences of SEQ ID NOs: 1 to 15 (e.g., SEQ ID NOs: 2 to 15)). In some embodiments, the albumin-binding peptide is linked to the N-terminus or C-terminus of the extracellular ActRIIB mutant directly or via a linker.

[0203] In some embodiments, an extracellular ActRIIB variant described herein (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) can be fused to the N- or C-terminus of an albumin-binding peptide (e.g., SEQ ID NOS: 72) by conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIB variant and the albumin-binding peptide. Without being bound by theory, it is believed that the inclusion of an albumin-binding peptide in the extracellular ActRIIB variant described herein may result in extended retention of the therapeutic protein via its binding to serum albumin.

[0204] IV. Fibronectin Domains In some embodiments, the polypeptides described herein can comprise an extracellular ActRIIB variant fused to a fibronectin domain, which can improve the pharmacokinetics of protein drugs.

[0205] A fibronectin domain is, for example, a high-molecular-weight glycoprotein of the extracellular matrix, or a fragment thereof, that binds to transmembrane receptor proteins such as integrins and extracellular matrix components such as collagen and fibrin. In some embodiments of the present invention, to extend the serum half-life of an extracellular ActRIIB mutant, a fibronectin domain is linked to the N-terminus or C-terminus (e.g., the C-terminus) of an extracellular ActRIIB mutant described herein (e.g., an extracellular ActRIIB mutant having any one of the sequences of SEQ ID NOS: 1 to 15 (e.g., SEQ ID NOS: 2 to 15)). The fibronectin domain can be linked to the N-terminus or C-terminus of the extracellular ActRIIB mutant directly or via a linker.

[0206] By way of example, fibronectin domains that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the fibronectin domain is a fibronectin type III domain having amino acids 610 to 702 of the sequence of UniProt ID NO:P02751 (SEQ ID NO:73):

[0207] [ka]

[0208] In another embodiment, the fibronectin domain is an adnectin protein. In some embodiments, an extracellular ActRIIB variant described herein (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) can be fused to the N- or C-terminus of a fibronectin domain (e.g., SEQ ID NOS: 73) by conventional genetic or chemical means, e.g., chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIB variant and the fibronectin domain. Without being bound by theory, it is believed that the inclusion of a fibronectin domain in the extracellular ActRIIB variant described herein may result in extended retention of the therapeutic protein through its binding to integrins and extracellular matrix components such as collagen and fibrin.

[0209] V. Serum Albumin In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIB variant fused to serum albumin. Binding to serum albumin can improve the pharmacokinetics of protein drugs.

[0210] Serum albumin is a globular protein that is the most abundant blood protein in mammals. It is produced in the liver and accounts for approximately half of serum proteins. Serum albumin is monomeric and soluble in blood. Some of the most important functions of serum albumin include transporting hormones, fatty acids, and other proteins in the body, buffering pH, and maintaining the osmotic pressure required for proper distribution of body fluids between blood vessels and body tissues. In a preferred embodiment, the serum albumin is human serum albumin. In some embodiments of the present invention, to extend the serum half-life of an extracellular ActRIIB mutant described herein (e.g., an extracellular ActRIIB mutant having any one of the sequences of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)), human serum albumin is linked to the N-terminus or C-terminus (e.g., C-terminus) of the extracellular ActRIIB mutant. Human serum albumin can be linked to the N-terminus or C-terminus of the extracellular ActRIIB mutant directly or via a linker.

[0211] By way of example, serum albumin that can be used in the methods and compositions described herein is generally known in the art. In one embodiment, the serum albumin comprises the sequence of UniProt ID NO:P02768 (SEQ ID NO:74):

[0212] [ka]

[0213] In some embodiments, an extracellular ActRIIB variant described herein (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) can be fused to the N-terminus or C-terminus of human serum albumin (e.g., SEQ ID NOS: 74) by conventional genetic or chemical means, for example, chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIB variant and human serum albumin. Without being bound by theory, it is believed that the inclusion of human serum albumin in the extracellular ActRIIB variant described herein may result in extended retention of the therapeutic protein.

[0214] VI. Linker The polypeptides described herein may include an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) fused to a moiety via a linker. In some embodiments, the moiety increases the stability of the polypeptide. Exemplary moieties include an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin. In the present invention, the linker between a certain portion (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 19), a wild-type Fc domain (e.g., SEQ ID NO: 71), an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide (e.g., SEQ ID NO: 72), a fibronectin domain (e.g., SEQ ID NO: 73), or human serum albumin (e.g., SEQ ID NO: 74)) and an extracellular ActRIIB mutant (e.g., an extracellular ActRIIB mutant having a sequence of any one of SEQ ID NOs: 1 to 15 (e.g., SEQ ID NOs: 2 to 15)) can be an amino acid spacer containing 1 to 200 amino acids. Suitable peptide spacers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine. In some embodiments, the spacer may include a GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO:20), GGGS (SEQ ID NO:21), GGGG (SEQ ID NO:22), GGGGA (SEQ ID NO:23), GGGGS (SEQ ID NO:24), GGGGG (SEQ ID NO:25), GGAG (SEQ ID NO:26), GGSG (SEQ ID NO:27), AGGG (SEQ ID NO:28), or SGGG (SEQ ID NO:29) motif, e.g., multiple or repeated motifs.In some embodiments, the spacer can include 2 to 12 amino acids containing a GA or GS motif, e.g., GA, GS, GAGA (SEQ ID NO: 30), GSGS (SEQ ID NO: 31), GAGAGA (SEQ ID NO: 32), GSGSGS (SEQ ID NO: 33), GAGAGAGA (SEQ ID NO: 34), GSGSGSGS (SEQ ID NO: 35), GAGAGAGAGA (SEQ ID NO: 36), GSGSGSGSGS (SEQ ID NO: 37), GAGAGAGAGAGA (SEQ ID NO: 38), and GSGSGSGSGSGS (SEQ ID NO: 39). In some embodiments, the spacer can include 3 to 12 amino acids containing a GGA or GGS motif, e.g., GGA, GGS, GGAGGA (SEQ ID NO: 40), GGSGGS (SEQ ID NO: 41), GGAGGAGGA (SEQ ID NO: 42), GGSGGSGGS (SEQ ID NO: 43), GGAGGAGGAGGA (SEQ ID NO: 44), and GGSGGSGGSGGS (SEQ ID NO: 45). Further, in some embodiments, the spacer can comprise 4 to 12 amino acids including the motifs GGAG (SEQ ID NO: 26), GGSG (SEQ ID NO: 27), e.g., GGAG (SEQ ID NO: 26), GGSG (SEQ ID NO: 27), GGAGGGAG (SEQ ID NO: 46), GGSGGGSG (SEQ ID NO: 47), GGAGGGAGGGAG (SEQ ID NO: 48), and GGSGGGSGGGSG (SEQ ID NO: 49). In some embodiments, the spacer can comprise the motifs GGGGA (SEQ ID NO: 23) or GGGGS (SEQ ID NO: 24), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 50) and GGGGSGGGGSGGGGGS (SEQ ID NO: 51). In some embodiments of the present invention, the amino acid spacer between a certain portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or serum albumin) and an extracellular ActRIIB mutant (e.g., an extracellular ActRIIB mutant having any one of SEQ ID NOs: 1 to 15 (e.g., SEQ ID NOs: 2 to 15)) may be GGG, GGGA (SEQ ID NO: 20), GGGG (SEQ ID NO: 22), GGGAG (SEQ ID NO: 52), GGGAGG (SEQ ID NO: 53), or GGGAGGG (SEQ ID NO: 54).

[0215] In some embodiments, the spacer may also include amino acids other than glycine, alanine, and serine, such as AAAL (SEQ ID NO:55), AAAK (SEQ ID NO:56), AAAR (SEQ ID NO:57), EGKSSGSGSESKST (SEQ ID NO:58), GSAGSAAGSGEF (SEQ ID NO:59), AEAAAKEAAAKA (SEQ ID NO:60), KESGSVSSEQLAQFRSLD (SEQ ID NO:61), GENLYFQSGG (SEQ ID NO:62), SACYCELS (SEQ ID NO:63), RSIAT (SEQ ID NO:64), RPACKIPNDLKQKVMNH (SEQ ID NO:65), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO:66), AAANSSIDLISVPVDSR (SEQ ID NO:67), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO:68). In some embodiments, the spacer may include a motif, e.g., a multiple or repeated motif, of EAAAK (SEQ ID NO:69). In some embodiments, the spacer is (XP) n (wherein X can be any amino acid (e.g., A, K, or E) and n is 1 to 5), and can include motifs, e.g., multiple or repeated motifs, of proline-rich sequences such as PAPAP (SEQ ID NO: 70).

[0216] The length of the peptide spacer and amino acids used can be adjusted depending on the two proteins involved and the degree of flexibility desired in the final protein fusion polypeptide. The length of the spacer can be adjusted to ensure proper protein folding and avoid the formation of aggregates.

[0217] VII. Vectors, Host Cells, and Protein Production The polypeptides of the present invention can be produced from host cells. A host cell refers to a vehicle containing necessary cellular components, e.g., organelles required for expressing the polypeptides and fusion polypeptides described herein from the corresponding nucleic acids. These nucleic acids may be contained within a nucleic acid vector, which can be introduced into host cells by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, or infection). The choice of nucleic acid vector will depend in part on the host cell used. Generally, preferred host cells are of eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origin.

[0218] Nucleic acid vector construction and host cells Nucleic acid sequences encoding the amino acid sequences of the polypeptides of the present invention can be prepared by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. Nucleic acid molecules encoding the polypeptides of the present invention can be obtained using standard techniques, such as gene synthesis. Alternatively, nucleic acid molecules encoding wild-type extracellular ActRIIB can be mutated to contain specific amino acid substitutions using standard techniques in the art, such as QuikChange™ mutagenesis. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR technology.

[0219] The nucleic acid sequence encoding the polypeptide of the present invention can be inserted into a vector capable of replicating and expressing the nucleic acid molecule in a prokaryotic or eukaryotic host cell. Many vectors are available in the art and can be used for the purposes of the present invention. Each vector may contain various components, which can be adjusted and optimized to suit a specific host cell. For example, vector components include, but are not limited to, a replication origin, a selectable marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence encoding a target protein, and a transcription termination sequence.

[0220] In some embodiments, mammalian cells can be used as host cells of the present invention. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK293F) cells, Chinese hamster ovary (CHO) cells, HeLa cells, COS cells, PC3 cells, Vero cells, MC3T3 cells, NS0 cells, Sp2 / 0 cells, VERY cells, BHK cells, MDCK cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20 cells, T47D cells, NS0 cells (a mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O cells, and HsS78Bst cells. In some embodiments, Escherichia coli (E. coli) cells can also be used as host cells of the present invention. E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ1776 (ATCC® 31,537), E. coli BL21(DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC® 31,608). Different host cells have characteristic and specific mechanisms for post-translational processing and modification (e.g., glycosylation) of protein products. An appropriate cell line or host system can be selected to ensure proper modification and processing of the expressed polypeptide. The above-described expression vectors can be introduced into appropriate host cells using conventional techniques in the art, such as transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vector has been introduced into the host cells for protein production, the host cells are cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify the gene encoding the desired sequence.Methods for expression of therapeutic proteins are known in the art, see, e.g., Paulina Balbas, Argelia Lorence (eds.), Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd Edition (2004), and Vladimir Voynov and Justin A. Caravella (eds.), Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology), Humana Press; 2nd Edition (2012).

[0221] Protein Production, Recovery and Purification Host cells used to produce the polypeptides of the present invention can be grown in media known in the art and suitable for culturing the selected host cells. Examples of suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Expi293™ Expression Medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria broth (LB) with any necessary supplementation of a selection agent, e.g., ampicillin. Host cells are cultured at a suitable temperature, e.g., about 20°C to about 39°C, e.g., 25°C to about 37°C, preferably 37°C, and at a CO2 level, e.g., 5-10%. The pH of the medium is generally about 6.8 to 7.4, e.g., 7.0, depending primarily on the host organism. When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activation of the promoter.

[0222] In some embodiments, depending on the expression vector and host cell used, the expressed protein can be secreted from the host cell (e.g., mammalian host cell) into the cell culture medium. Protein recovery can include filtering the cell culture medium to remove cellular debris. These proteins may be further purified. The polypeptides of the present invention can be purified by any method known in the art of protein purification, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Proteins can be isolated and purified, for example, by appropriately selecting an affinity column, such as a Protein A column (e.g., POROS Protein A chromatography), in combination with a chromatography column (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, salting out, and dialysis.

[0223] In other embodiments, host cells can be disrupted, for example, by osmotic shock, sonication, or lysis, to recover the expressed protein. Once the cells are disrupted, the cellular debris can be removed by centrifugation or filtration. In some cases, the polypeptide can be linked to a marker sequence, such as a peptide, to facilitate purification. An example of a marker amino acid sequence is a hexahistidine peptide (His tag), which binds with micromolar affinity to nickel-functionalized agarose affinity columns. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767, 1984).

[0224] Alternatively, the polypeptides of the present invention can be produced by cells of a subject (e.g., a human) by administering a vector (e.g., a viral vector (e.g., a retroviral vector, an adenoviral vector, a poxvirus vector (e.g., a vaccinia virus vector, e.g., Modified Vaccinia Ankara (MVA)), an adeno-associated virus vector, and an alphavirus vector)) containing a nucleic acid molecule encoding a polypeptide of the present invention, e.g., in gene therapy. Once in the subject's cells (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), the vector promotes expression of the polypeptide, which is then secreted from the cells. If treatment of the disease or disorder is the desired outcome, no further action may be required. If collection of protein is desired, blood can be drawn from the subject and the protein can be purified from the blood by methods known in the art.

[0225] VIII. Pharmaceutical Compositions and Formulations The present invention relates to pharmaceutical compositions comprising the polypeptides described herein (e.g., polypeptides comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15))). In some embodiments, the pharmaceutical compositions of the present invention comprise, as a therapeutic protein, a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) fused to a moiety (e.g., an Fc domain monomer or dimer thereof, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or serum albumin). In some embodiments, the pharmaceutical compositions of the present invention comprising the polypeptides of the present invention can be used in combination with other agents (e.g., therapeutic biologics and / or small molecules) or compositions in therapy. In addition to a therapeutically effective amount of a polypeptide, the pharmaceutical composition may contain one or more pharmaceutically acceptable carriers or excipients and can be formulated by methods known to those skilled in the art. In some embodiments, a pharmaceutical composition of the present invention comprises a nucleic acid molecule (DNA or RNA, eg, mRNA) encoding a polypeptide of the present invention, or a vector containing such a nucleic acid molecule.

[0226] Acceptable carriers and excipients for pharmaceutical compositions are non-toxic to recipients at the dosage and concentration used.Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES, and TAE; antioxidants such as ascorbic acid and methionine; preservatives such as hexamethonium chloride, octadecyldimethylbenzylammonium chloride, resorcinol, and benzalkonium chloride; proteins such as human serum albumin, gelatin, dextran, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, histidine, and lysine; and carbohydrates such as glucose, mannose, sucrose, and sorbitol.The pharmaceutical composition of the present invention can be administered parenterally in the form of an injection preparation.Injectable pharmaceutical compositions can be prepared using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, saline, and cell culture medium (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-Modified Eagle Medium (α-MEM), F-12 medium). Formulation methods are known in the art, see, for example, Banga (ed.), Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (3rd Edition), Taylor & Francis Group, CRC Press (2015).

[0227] The pharmaceutical compositions of the present invention may be formulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules. The pharmaceutical compositions of the present invention may also be formulated in other drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules. Such techniques are described in Remington: The Science and Practice of Pharmacy, 22nd Edition (2012). Pharmaceutical compositions used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0228] The pharmaceutical compositions of the present invention may also be prepared as sustained-release formulations. Suitable sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptides of the present invention. Examples of sustained-release matrices include polyesters, hydrogels, polyactides, copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., LUPRON DEPOT™), and poly-D-(-)-3-hydroxybutyric acid. Some sustained-release formulations enable release of molecules over several months, e.g., 1 to 6 months, while other formulations release the pharmaceutical compositions of the present invention over shorter periods, e.g., days to weeks.

[0229] The pharmaceutical composition can be prepared in a unit dosage form, if necessary. The amount of the active ingredient, e.g., the polypeptide of the present invention, contained in the pharmaceutical preparation is such that an appropriate dose is provided within the specified range (e.g., a dose within the range of 0.01 to 100 mg / kg body weight).

[0230] Pharmaceutical compositions for gene therapy may be in an acceptable diluent or may comprise a slow-release matrix in which the gene delivery vehicle is embedded. When hydrodynamic injection is used as the delivery method, pharmaceutical compositions containing nucleic acid molecules encoding the polypeptides described herein or vectors (e.g., viral vectors) containing the nucleic acid molecules are easily delivered intravenously in large liquid volumes. Vectors that can be used as in vivo gene delivery vehicles include, but are not limited to, retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors, e.g., mutant vaccinia Ankara), adeno-associated virus vectors, and alphavirus vectors.

[0231] IX. Route, Dosage, and Administration The pharmaceutical composition comprising the polypeptide of the present invention as therapeutic protein can be prepared for, for example, intravenous administration, parenteral administration, subcutaneous administration, intramuscular administration, intraarterial administration, intrathecal administration or intraperitoneal administration.The pharmaceutical composition can also be prepared for or administered via oral administration, nasal administration, spray administration, aerosol administration, rectal or vaginal administration.For injection preparations, various effective pharmaceutical carriers are known in the art.For example, see ASHP Handbook on Injectable Drugs, Toissel, 18th Edition (2014).

[0232] In some embodiments, pharmaceutical compositions comprising nucleic acid molecules encoding the polypeptides of the present invention or vectors containing such nucleic acid molecules can be delivered by gene delivery. Methods of gene delivery are well known to those skilled in the art. Vectors that can be used for in vivo gene delivery and expression include, but are not limited to, retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors, e.g., mutated vaccinia Ankara (MVA)), adeno-associated virus vectors, and alphavirus vectors. In some embodiments, mRNA molecules encoding the polypeptides of the present invention can be directly administered to a subject.

[0233] In some embodiments of the present invention, nucleic acid molecules encoding the polypeptides described herein or vectors containing such nucleic acid molecules can be administered using a hydrodynamic injection platform. In hydrodynamic injection, nucleic acid molecules encoding the polypeptides described herein are placed under the control of a strong promoter within an engineered plasmid (e.g., a viral plasmid). Plasmids are often easily delivered intravenously in large fluid volumes. Hydrodynamic injection uses controlled hydrodynamic pressure within a vein to increase cell permeability, so that the high pressure resulting from the rapid injection of a large fluid volume results in extravasation of the fluid and plasmid from the vein. Expression of nucleic acid molecules is primarily driven by the liver. In mice, hydrodynamic injection is often performed by injecting the plasmid into the tail vein. In certain embodiments, mRNA molecules encoding the polypeptides described herein can be administered using hydrodynamic injection.

[0234] The dosage of the pharmaceutical composition of the present invention depends on factors including the route of administration, the disease being treated, and the subject's physical characteristics, such as age, weight, and health condition. The pharmaceutical composition of the present invention may contain a polypeptide of the present invention at a dose ranging from 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg), or in more specific embodiments, about 0.1 to about 30 mg / kg, and in more specific embodiments, about 0.3 to about 30 mg / kg. The dosage can be adjusted by a physician according to conventional factors, such as the extent of the subject's disease and various parameters.

[0235] Pharmaceutical compositions are administered in a manner appropriate for the dosage form and in a therapeutically effective amount to improve or remedy symptoms. Pharmaceutical compositions are administered in a variety of dosage forms, including intravenous, subcutaneous, and oral dosage forms (e.g., ingestible solutions, drug-release capsules). Generally, therapeutic proteins are administered at 0.1-100 mg / kg, e.g., 1-50 mg / kg. Pharmaceutical compositions comprising the polypeptides of the present invention can be administered to a subject in need thereof, for example, daily, weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, yearly, or one or more times (e.g., 1-10 times or more) as medically necessary. In some embodiments, pharmaceutical compositions comprising the polypeptides of the present invention can be administered to a subject in need thereof weekly, biweekly, monthly, bimonthly, or quarterly. Doses can be provided in single or multiple dosing regimens. The timing between doses can be decreased as the medical condition improves or increased as the patient's health declines.

[0236] X. Treatment method The present invention is based on the discovery that amino acid substitution from the extracellular portion of ActRIIA to the extracellular portion of ActRIIB results in ActRIIB variants with improved properties. ActRIIB variants created by introducing residues from ActRIIA into ActRIIB retain the beneficial properties of ActRIIB, such as the ability to increase muscle mass and high binding affinity for activin A and B, and may acquire some of the beneficial properties of ActRIIA, such as reduced binding affinity for BMP9 or the ability to increase red blood cell levels. Because ActRIIB variants contain the extracellular portion of the receptor, they are soluble and can compete with endogenous activin receptors by binding to and sequestering ligands (e.g., activin A and B, myostatin, GDF11) without activating intracellular signaling pathways. Therefore, the extracellular ActRIIB variants described herein can be used to treat diseases or conditions in which elevated activin signaling is involved in the pathogenesis (e.g., diseases or conditions in which increased expression of activin receptors or activin receptor ligands has been observed). For example, loss of myostatin has been shown to increase skeletal muscle mass, suggesting that myostatin inhibits skeletal muscle growth. Loss of myostatin reduces fibrosis, while increases in myostatin or activin have been shown to induce fibrosis, suggesting that myostatin also promotes fibrosis. Additionally, activin has been found to be upregulated in bone disease and is known to inhibit osteoblast activity, suggesting that increased activin levels are involved in bone disease. Activin A has also been found to be elevated in clinical and experimental pulmonary hypertension. In another example, the activin receptor ligand GDF11 has been overexpressed in a mouse model of hemolytic anemia and is associated with defects in red blood cell production.Without wishing to be bound by theory, therapeutic agents that bind to activin receptor ligands (e.g., GDF11, myostatin, and / or activin) and reduce binding to or interaction with endogenous activin receptors may have therapeutic utility for treating or preventing various diseases or conditions, such as muscle diseases, bone diseases, fibrosis, anemia, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH).

[0237] The compositions and methods described herein can be used to treat and / or prevent (e.g., prevent the onset of or treat a subject diagnosed with) a medical condition, such as a muscle disorder, a bone disorder, low red blood cell levels (e.g., low hemoglobin levels or low red blood cell count, e.g., anemia), fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15))) can be administered to a subject in need thereof to increase muscle mass and strength. In some embodiments, a polypeptide described herein can be administered to increase lean mass. The polypeptides described herein can increase muscle mass or lean mass compared to measurements obtained before treatment. In some embodiments, the subject has or is at risk of developing a disease that causes muscle weakness or muscle atrophy (e.g., skeletal muscle weakness or atrophy). In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors) in a subject with a disease or condition associated with muscle weakness and atrophy.

[0238] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)), e.g., an effective amount of an ActRIIB variant) can be administered to a subject in need thereof to increase bone mineral density, increase bone formation, enhance bone strength, reduce the risk of fracture, or reduce bone resorption. The polypeptide described herein can increase bone mineral density, increase bone formation, or reduce bone resorption compared to measurements obtained before treatment. In some embodiments, the subject has or is at risk for developing a disease that causes bone damage (e.g., osteoporosis or osteopenia). In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors) in a subject with a disease or condition associated with bone damage.

[0239] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)), e.g., an effective amount of an ActRIIB variant) can be administered to increase red blood cell levels (e.g., increase hemoglobin levels, increase red blood cell count, increase hematocrit, or increase red blood cell formation or production) in a subject in need thereof. The polypeptide described herein can increase red blood cell levels (e.g., increase hemoglobin levels, red blood cell count, hematocrit, or red blood cell formation) compared to measurements obtained before treatment. In some embodiments, the subject can have a disease or condition associated with low red blood cell levels (e.g., anemia or blood loss). In some embodiments, the subject has or may be at risk of developing anemia or blood loss (e.g., the subject may be at risk of developing anemia due to other diseases or conditions, such as chronic kidney disease, rheumatoid arthritis, cancer, or inflammatory diseases (e.g., Crohn's disease, SLE, ulcerative colitis), or due to medical treatments, such as chemotherapy, radiation therapy, or surgery). In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors) in a subject with a disease or condition associated with low red blood cell levels (e.g., anemia or blood loss).

[0240] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)), e.g., an effective amount of an ActRIIB variant) can be administered to prevent or reduce fibrosis in a subject in need thereof. In some embodiments, a polypeptide described herein can be administered to slow or halt the progression of fibrosis, reduce the risk of developing fibrosis, or reduce one or more symptoms of fibrosis (e.g., reduce the frequency or severity thereof). The polypeptides described herein can reduce fibrosis or slow the progression of fibrosis, at least as compared to the progression of fibrosis before treatment or as compared to the progression of fibrosis in an untreated subject. In some embodiments, the subject may have or be at risk for developing fibrosis (e.g., the subject may have a disease or condition associated with fibrosis, such as a wound, hepatitis B or C, fatty liver disease, kidney disease (e.g., chronic kidney disease), heart disease, or atherosclerosis, or may be undergoing a treatment associated with the development of fibrosis, such as chemotherapy, radiation therapy, or surgery). In some embodiments, the polypeptides described herein prevent or delay the onset of fibrosis in a subject at risk for developing fibrosis (e.g., the subject may be being treated with chemotherapy, radiation therapy, or surgery, or the subject may have a disease or condition associated with fibrosis, such as a wound, hepatitis B or C, fatty liver disease, kidney disease (e.g., chronic kidney disease), heart disease, or atherosclerosis). In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors) in a subject with fibrosis or a disease or condition associated with fibrosis.

[0241] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 1 to 15 (e.g., SEQ ID NOs: 2 to 15)), e.g., an effective amount of an ActRIIB variant) can be administered to a subject in need thereof to treat PH, reduce PH (e.g., reduce the severity or frequency of one or more symptoms of PH, such as shortness of breath (dyspnea), fatigue, swelling in the legs, feet, or abdomen (e.g., ascites), or neck (e.g., edema), chest pain or tightness, rapid pulse or palpitations, bluish color of the lips or skin (cyanosis), dizziness or fainting), prevent PH (e.g., prevent the onset of PH), reduce the risk of developing PH, or slow or stop the progression of PH. The polypeptides described herein can reduce the symptoms of PH (e.g., reduce the severity or frequency of one or more symptoms of PH, such as shortness of breath (dyspnea), fatigue, swelling in the legs, feet, abdomen (e.g., ascites), neck (e.g., edema), chest pain or pressure, rapid pulse or heart palpitations, bluish color of the lips or skin (cyanosis), dizziness or fainting), or slow the progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), compared to the symptoms or progression observed before treatment or compared to the symptoms or progression of PH in an untreated subject.In some embodiments, the subject may have PH or be at risk for developing PH (e.g., the subject may have idiopathic PAH); the subject may have a disease or condition associated with PAH (e.g., a disease or condition that induces an increased risk of developing PAH), such as HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, cirrhosis, congenital heart abnormalities, connective tissue / autoimmune diseases (e.g., scleroderma and lupus), or drug use or abuse (e.g., methamphetamine or cocaine use). The subject may have a condition (e.g., a family history of PH (e.g., hereditary PAH); the subject may have a disease or condition associated with venous PH (e.g., a disease or condition that induces an increased risk of developing venous PH), such as left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital / acquired pulmonary vein stenosis; the subject may have chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), pulmonary disease (e.g., pulmonary fibrosis), alveolar hypoventilation disorder, or exposure to high altitude. The subject may have a disease or condition associated with hypoxic PH (e.g., a disease or condition that increases the risk of developing hypoxic PH), such as chronic exposure to pulmonary thromboembolic agents, or a developmental abnormality; the subject may have a disease or condition associated with thromboembolic PH (e.g., a disease or condition that increases the risk of developing thromboembolic PH), such as chronic thromboembolic pulmonary hypertension, or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or parasitic infection); or the subject may have a blood disorder (e.g., Patients may have other diseases or conditions associated with PH (e.g., diseases or conditions that increase the risk of developing other PH), such as: chronic hemolytic anemia, sickle cell disease), systemic diseases (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), metabolic diseases (e.g., glycogen storage disease, Gaucher disease, thyroid disease), pulmonary neoplastic thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension (pulmonary hypertension limited to one or more lobes of the lung).In some embodiments, the polypeptides described herein prevent or delay the onset of PH in a subject at risk of developing PH (e.g., the subject has a family history of PH (e.g., hereditary PAH), or the subject has a pre-existing condition known as PAH (e.g., HIV infection, schistosomiasis, cirrhosis of the liver, congenital heart defects, connective tissue / autoimmune diseases such as scleroderma and lupus), or drug use or abuse (e.g., methamphetamine or cocaine use), venous PH (e.g., left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital / acquired pulmonary vein stenosis), hypoxic PH (e.g., chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), pulmonary disease (e.g., pulmonary fibrosis), impaired alveolar hypoventilation, chronic exposure to high altitude, or developmental abnormalities), thromboembolic PH (e.g., chronic thromboembolic pulmonary hypertension), or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or or parasitic infection), or other PH (e.g., having a disease or condition that causes an increased risk of developing a blood disorder (e.g., chronic hemolytic anemia, sickle cell disease), a systemic disease (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), a metabolic disease (e.g., glycogen storage disease, Gaucher disease, thyroid disease), pulmonary neoplastic thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, segmental pulmonary hypertension). In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors) in a subject with PH or a disease or condition associated with PH. In some embodiments, the PH is PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH.

[0242] In some embodiments, polypeptides comprising the extracellular ActRIIB mutants described herein reduce or inhibit the binding of myostatin, activin, and / or BMP9 to their respective receptors (e.g., ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIB)). The polypeptides described herein may reduce the binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors compared to the binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors in the absence of the polypeptides of the present invention. In some embodiments, affecting myostatin, activin, and / or BMP9 signaling (e.g., decreasing or inhibiting the binding of myostatin, activin, and / or BMP9 to their respective endogenous receptors, e.g., ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIB)) can result in an increase in muscle mass in a subject, an increase in bone mineral density or bone formation in a subject, a decrease in bone resorption in a subject, an increase in red blood cell levels (e.g., hemoglobin level, hematocrit, or red blood cell count, e.g., promoting the formation or production of red blood cells), or an increase in muscle mass in a subject, an increase in bone mineral density or bone formation in a subject, a decrease in bone resorption in a subject, an increase in red blood cell levels (e.g., hemoglobin level, hematocrit, or red blood cell count, e.g., promoting the formation or production of red blood cells). the progression of PH (e.g., slowing or inhibiting progression), reducing fibrosis or the risk of developing fibrosis in a subject, delaying the onset of fibrosis, reducing the progression of fibrosis (e.g., slowing or inhibiting progression), reducing symptoms of PH (e.g., reducing shortness of breath (dyspnea), fatigue, swelling of the legs, feet, abdomen (ascites), or neck (e.g., edema), chest pain or tightness, rapid pulse or heart palpitations, bluish discoloration to the lips or skin (cyanosis), dizziness, or fainting), reducing the risk of developing PH, delaying the onset of PH, and / or reducing the progression of PH (e.g., slowing or inhibiting progression). PH can be PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH.

[0243] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)), e.g., an effective amount of an ActRIIB variant) is administered to a subject for the purposes of increasing muscle mass or strength, increasing bone mineral density, increasing bone formation, increasing bone strength, reducing the risk of fractures, reducing bone resorption, increasing red blood cell levels (e.g., increasing hemoglobin levels, increasing hematocrit, increasing red blood cell count, etc.), or for the purposes of reducing bone mineral density (e.g., increasing bone mineral density, increasing bone formation, increasing bone strength, reducing the risk of fractures, reducing bone resorption, increasing red blood cell levels (e.g., increasing hemoglobin levels, increasing hematocrit, increasing red blood cell count, etc.)). The therapeutic agent may be administered to the subject to increase blood cell counts or induce or increase erythropoiesis), prevent or reduce fibrosis (e.g., reduce fibrosis, prevent or delay the onset of fibrosis, or slow or stop the progression of fibrosis), prevent or treat PH (e.g., to alleviate the symptoms of PH, prevent or delay the onset of PH, or slow or stop the progression of PH such as PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH), or affect myostatin, activin, and / or BMP9 signaling. An extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15), e.g., an effective amount of an ActRIIB variant) can increase muscle mass or strength, increase bone mineral density, increase bone formation, increase bone strength, reduce the risk of fracture, reduce bone resorption, increase red blood cell levels, prevent or reduce fibrosis, or prevent or treat PH, compared to measurements obtained before treatment or compared to measurements obtained from an untreated subject with the same disease or condition. In some embodiments, the methods described herein do not cause vascular complications in the subject, such as increased vascular permeability or leakage.In some embodiments of the methods described herein, the subject has or is at risk of developing a disease or condition associated with muscle weakness and muscle atrophy (e.g., Duchenne muscular dystrophy (DMD), faciocraniohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia). In some embodiments of the methods described herein, the subject has or is at risk of developing a disease or condition associated with bone damage (e.g., primary osteoporosis, secondary osteoporosis, osteopenia, osteopetrosis, fractures, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, low-gravity-related bone loss, or immobility-related bone loss). In some embodiments of the methods described herein, the subject has or is at risk of developing a disease or condition involving low red blood cell levels (e.g., anemia or blood loss, such as anemia associated with cancer (e.g., multiple myeloma, leukemia, breast cancer, lung cancer, colon cancer), cancer treatment (e.g., chemotherapy or radiation therapy), myelodysplastic syndrome, chronic or acute kidney disease or failure (e.g., chronic kidney disease), inflammatory or autoimmune disease (e.g., rheumatoid arthritis, inflammatory bowel disease, e.g., Crohn's disease or ulcerative colitis, SLE), or surgery). In some embodiments of the methods described herein, the subject has or is at risk of developing a disease or condition comprising fibrosis (e.g., chemotherapy-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, liver fibrosis (e.g., cirrhosis), renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis, osteoarthrofibrosis, tissue fibrosis, tumor stroma, desmoplastic tumors, surgical adhesions, hypertrophic scars, keloids, or fibrosis associated with wounds, burns, hepatitis B or C infection, fatty liver disease, schistosome infection, kidney disease, heart disease, macular degeneration, retinal or vitreoretinopathy, systemic or localized scleroderma, atherosclerosis, or restenosis).In some embodiments of the methods described herein, the subject is diagnosed with PH (e.g., PAH, venous PH, hypoxemic PH, thromboembolic PH, or other PH, e.g., idiopathic PAH; hereditary PAH; PAH associated with or resulting from HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, cirrhosis, congenital heart abnormalities, connective tissue / autoimmune diseases (e.g., scleroderma and lupus), drug use or abuse (e.g., methamphetamine or cocaine use); venous PH associated with or resulting from left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital / acquired pulmonary vein stenosis; chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), pulmonary disease (e.g., pulmonary fibrosis), Hypoxic PH associated with or resulting from impaired alveolar ventilation, chronic exposure to high altitude, or developmental abnormalities; thromboembolic PH associated with or resulting from chronic thromboembolic pulmonary hypertension or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or parasitic infection); other PH associated with or resulting from blood disorders (e.g., chronic hemolytic anemia, sickle cell disease), systemic diseases (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), metabolic disorders (e.g., glycogen storage disease, Gaucher disease, thyroid disease), pulmonary neoplastic thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension).

[0244] The present invention also relates to the treatment of Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, cancer cachexia, primary osteoporosis, secondary osteoporosis, osteopenia, osteopetrosis, fractures, bone loss associated with bone cancer or cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with the treatment of obesity, low-gravity-related bone loss, and immobility-related bone loss. The present invention also includes a method for treating a subject having or at risk of developing anemia, blood loss, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) by administering to the subject an effective amount of a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 1 to 15 (e.g., SEQ ID NOs: 2 to 15))).

[0245] In some of the methods described herein, the subject having or at risk of developing a bone disease (e.g., bone injury) has or is at risk of developing a disease or condition, including primary osteoporosis, secondary osteoporosis, osteopenia, osteopetrosis, bone fracture, bone loss associated with bone cancer or cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with obesity treatment, hypogravity-related bone loss, or immobility-related bone loss. In some embodiments, the primary osteoporosis is age-related osteoporosis (e.g., associated with decreased estrogen) or hormone-related osteoporosis. In some embodiments, the secondary osteoporosis is immobility-induced osteoporosis or glucocorticoid-induced osteoporosis. In some embodiments, the bone cancer is multiple myeloma, or the cancer metastasis-related bone loss is caused by multiple myeloma. In some embodiments, the treatment-related bone loss occurs due to treatment with FGF-21 or GLP-1, treatment with an FGF-21 or GLP-1-containing therapeutic, or treatment of type 2 diabetes and / or obesity, or due to cancer treatment (e.g., chemotherapy or radiation). In some embodiments, the diet-related bone loss is rickets (e.g., vitamin D deficiency). In some embodiments, the low-gravity-related bone loss is load-related bone loss. In some embodiments, the methods described herein increase bone mineral density (e.g., increase bone mass) compared to measurements obtained before treatment or compared to bone mineral density typically observed in untreated subjects. In some embodiments, the methods described herein reduce bone resorption (e.g., reduce bone catabolic activity) compared to measurements obtained before treatment or compared to bone resorption typically observed in untreated subjects. In some embodiments, the methods described herein increase bone formation (e.g., increase bone anabolic activity or increase ostepgenesis) compared to measurements obtained before treatment or compared to bone formation typically observed in untreated subjects.In some embodiments, the methods described herein increase osteoblast activity or osteoblast formation (e.g., increase osteoblast activity or osteoblast formation) compared to measurements taken before treatment or compared to osteoblast activity or osteoblast formation typically observed in untreated subjects. In some embodiments, the methods described herein decrease osteoclast activity or osteoclast formation (e.g., decrease osteoclast activity or osteoclast formation) compared to measurements taken before treatment or compared to osteoclast activity or osteoclast formation typically observed in untreated subjects. In some embodiments, the bone is cortical bone or trabecular bone.

[0246] The present invention also includes methods for treating a subject having or at risk of developing anemia or blood loss by administering to the subject an effective amount of a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15))). In any of the methods described herein, the subject has or is at risk of developing low red blood cell levels (e.g., low hemoglobin level, low hematocrit, or low red blood cell count) and has or is at risk of developing anemia or blood loss. In some embodiments, the anemia is due to a nutritional deficiency (e.g., vitamin deficiency), bone marrow deficiency (e.g., paroxysmal nocturnal hemoglobinuria), an adverse reaction to a drug (e.g., antiretroviral HIV drugs), myelodysplastic syndrome, bone marrow transplant, cancer (e.g., solid tumors such as breast cancer, lung cancer, colon cancer, etc.; lymphoid tumors such as chronic lymphocytic leukemia, non-Hodgkin's lymphoma, Hodgkin's lymphoma, etc.; or tumors of the hematopoietic system such as leukemia or multiple myeloma), cancer treatment (e.g., radiation or chemotherapy, e.g., chemotherapy with platinum-containing agents), inflammatory or autoimmune disease (e.g., rheumatoid arthritis, other inflammatory arthritis, systemic lupus erythematosus (SLE), acute or chronic skin disease (e.g., psoriasis), or inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), cystitis, gastritis), idiopathic or congenital conditions associated with acute or chronic renal disease or failure (e.g., chronic kidney disease), acute or chronic liver disease, acute or chronic bleeding, infection (e.g., malaria, osteomyelitis), splenomegaly, porphyria, vasculitis, hemolysis, urinary tract infection, hemoglobinopathies (e.g., sickle cell disease), thalassemia, Churg-Strauss syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplantation, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schönlein purpura, Schwachman syndrome (e.g., Schwachman-Diamond syndrome), drug use or abuse (e.g., alcohol abuse), or transfusion contraindications (e.g., elderly patients, patients with alloantibodies or autoantibodies, pediatric patients, patients with cardiopulmonary disease, and patients who object to transfusions for religious reasons (e.g., some Jehovah's Witnesses)).In some embodiments, the anemia is aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, anemia of chronic disease, anemia associated with bone marrow disease, hemolytic anemia, sickle cell anemia, microcytic anemia, hypochromic anemia, sideroblastic anemia, Diamond-Blackfan anemia, Fanconi anemia, or refractory anemia with excess blasts. The compositions and methods described herein can also be used to treat subjects who do not adequately respond to erythropoietin (EPO) or who are susceptible to EPO's adverse effects (e.g., hypertension, headache, vascular thrombosis, flu-like syndrome, shunt occlusion, myocardial infarction). In some embodiments, the blood loss is due to surgery, trauma, wounds, ulcers, urinary tract bleeding, gastrointestinal bleeding, frequent blood donations, or heavy menstrual bleeding (e.g., menorrhagia). In some embodiments, the methods described herein increase red blood cell levels (e.g., hemoglobin level, hematocrit, or red blood cell count) compared to measurements taken before treatment. In some embodiments, the methods described herein increase or induce red blood cell formation compared to measurements taken before treatment. In some embodiments, the compositions and methods described herein reduce a subject's need for blood transfusions (e.g., the subject no longer requires blood transfusions, or the subject requires less frequent transfusions than before treatment with the compositions and methods described herein). Subjects with normal red blood cell levels can be treated using the methods and compositions described herein to increase their red blood cell levels, thereby allowing blood to be collected and stored for later use in transfusions.

[0247] The present invention also includes methods for treating a subject having or at risk of developing fibrosis by administering to the subject an effective amount of a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIB mutant (e.g., an extracellular ActRIIB mutant having the sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15))). In any of the methods described herein, the subject has fibrosis or is at risk of developing fibrosis. In some embodiments, the fibrosis is chemotherapy-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis (e.g., cystic fibrosis, idiopathic fibrosis, or fibrosis associated with tuberculosis, pneumonia, or coal dust), hepatic fibrosis (such as cirrhosis, biliary atresia, etc.), renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis (e.g., fibrosis associated with endomyocardial fibrosis or myocardial infarction), myelofibrosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, osteoarthrofibrosis, tissue fibrosis (e.g., fibrosis affecting muscle tissue, skin epidermis, skin dermis, tendon, cartilage, pancreatic tissue, uterine tissue, nervous tissue, testes, ovaries, adrenal glands, arteries, veins, colon, small intestine, large intestine, biliary tract, or intestine), tumor stroma, desmoplastic tumor, surgical adhesion, hypertrophic scar, or keloid. In some embodiments, the fibrosis is fibrosis associated with a wound, a burn, hepatitis B or C infection, fatty liver disease, schistosome infection, kidney disease (e.g., chronic kidney disease), heart disease, macular degeneration, retinal or vitreoretinopathy, Crohn's disease, systemic or localized scleroderma, atherosclerosis, or restenosis. In some embodiments, the subject is at risk for developing fibrosis associated with cancer treatment (chemotherapy or radiation therapy), disease or infection (e.g., tuberculosis, pneumonia, myocardial infarction, hepatitis B or C infection, fatty liver disease, schistosome infection, kidney disease (e.g., chronic kidney disease), heart disease, macular degeneration, retinal or vitreoretinopathy, Crohn's disease, systemic or localized scleroderma, atherosclerosis, restenosis), surgery, a wound, or a burn. In some embodiments, the methods described herein reduce fibrosis compared to measurements obtained before treatment or compared to fibrosis in an untreated subject.In some embodiments, the methods described herein prevent the onset of fibrosis or reduce the risk of developing fibrosis (e.g., reduce the risk of developing fibrosis compared to the onset of fibrosis in an untreated subject). In some embodiments, the methods described herein slow or halt the progression of fibrosis (e.g., slow the progression of fibrosis compared to the progression before treatment compared to the progression in the absence of treatment or compared to the progression in an untreated subject). In some embodiments, the methods described herein reduce the frequency or severity of one or more symptoms of fibrosis. In some embodiments, the methods described herein improve organ or tissue function (e.g., the function of an organ or tissue with fibrosis) compared to the organ or tissue function before treatment. Tissue and organ function can be assessed using standard clinical tests commonly used to assess tissue and organ function.

[0248] The present invention also includes methods for treating a subject having or at risk of developing PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) by administering to the subject an effective amount of a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15))). In any of the methods described herein, the subject has or is at risk of developing PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH). In some embodiments, the PH is PAH. In some embodiments, the PAH is idiopathic PAH. In some embodiments, the PAH is hereditary PAH. In some embodiments, the PAH is PAH associated with HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, liver cirrhosis, congenital heart abnormalities, connective tissue / autoimmune diseases (such as scleroderma and lupus), or drug use or abuse (e.g., methamphetamine or cocaine use). In some embodiments, the PH is venous PH. In some embodiments, the venous PH is related to (e.g., caused by or associated with) left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathies, or congenital / acquired pulmonary vein stenosis. In some embodiments, the PH is hypoxic PH. In some embodiments, the hypoxic PH is hypoxic PH associated with (e.g., caused by or associated with) chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), pulmonary disease (e.g., pulmonary fibrosis), alveolar hypoventilation, chronic exposure to high altitude, or developmental abnormality. In some embodiments, the PH is thromboembolic PH. In some embodiments, the thromboembolic PH is associated with (e.g., caused by or associated with) chronic thromboembolic pulmonary hypertension or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary stenosis, or parasitic infection).In some embodiments, the PH is other PH related to (e.g., caused by or associated with) a blood disorder (e.g., chronic hemolytic anemia, sickle cell disease), a systemic disease (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), a metabolic disorder (e.g., glycogen storage disease, Gaucher disease, or thyroid disease), pulmonary neoplastic thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension. In some embodiments, the methods described herein reduce symptoms of PH compared to the frequency or severity of the symptoms before treatment (e.g., reduce the frequency or severity of symptoms such as shortness of breath (dyspnea), fatigue, swelling of the legs, feet, abdomen (ascites), or neck (e.g., edema), chest pain or tightness, rapid pulse or heart palpitations, bluish color of the lips or skin (cyanosis), dizziness, or fainting). In some embodiments, the methods described herein prevent the onset of PH or reduce the risk of developing PH (e.g., reduce the risk of developing PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) compared to the onset of PH in an untreated subject). In some embodiments, the methods described herein slow or halt the progression of PH (e.g., slow the progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or other PH) compared to the progression before treatment or compared to the progression in an untreated or untreated subject). In some embodiments, the methods described herein reduce pulmonary vascular remodeling or cardiac vascular remodeling (e.g., the onset or progression of cardiac or pulmonary vascular remodeling) in a subject compared to pre-treatment vascular remodeling or compared to vascular remodeling in an untreated subject. In some embodiments, the methods described herein reduce right ventricular hypertrophy (e.g., reduce right ventricular hypertrophy or reduce the progression of right ventricular hypertrophy) compared to pre-treatment right ventricular hypertrophy or compared to right ventricular hypertrophy in an untreated subject.Symptoms of PH can be assessed before and after treatment using standard clinical tests. Commonly used tests to assess PH include electrocardiogram, pulmonary function test, echocardiogram, right heart catheterization, computed tomography scan, measurement of pulmonary vascular resistance, and 6-minute walk test. In some embodiments, the methods described herein reduce pulmonary vascular resistance (e.g., result in a reduction in pulmonary vascular resistance compared to pre-treatment pulmonary vascular resistance). In some embodiments, the methods described herein improve performance in a 6-minute walk test compared to performance in a 6-minute walk test before treatment.

[0249] In any of the methods described herein, a dimer (e.g., a homodimer or heterodimer) formed by the interaction of two Fc domain monomers fused to a polypeptide containing an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) can be used as a therapeutic protein. In any of the methods described herein, a polypeptide containing an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) fused to a moiety (e.g., a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or serum albumin) can be used as a therapeutic protein. Nucleic acids encoding the polypeptides described herein, or vectors containing the nucleic acids, can also be administered according to any of the methods described herein. In any of the methods described herein, the polypeptide, nucleic acid, or vector can be administered as part of a pharmaceutical composition. [Example]

[0250] The following examples are provided to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention, and by their illustrative nature it will be understood that other procedures, methodologies, or techniques known to those skilled in the art may be substituted.

[0251] Example 1: Effects of extracellular ActRIIB mutants on body and muscle weight C57Bl / 6 mice received a single hydrodynamic injection of a plasmid construct encoding one of the following 14 polypeptides and vehicle control (n=10 / group, see sequences provided in Figure 1).

[0252] (1) Vehicle; (2) extracellular ActRIIA fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 16); (3) extracellular ActRIIB fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 17); (4) extracellular ActRIIB mutant ActRIIB / A (SEQ ID NO: 2) fused to the N-terminus of hFc via a GGG linker; (5) extracellular ActRIIB mutant ActRIIBΔ9 (SEQ ID NO: 3) fused to the N-terminus of hFc via a GGG linker; (6) extracellular ActRIIB variant ActRIIB2.01 (SEQ ID NO: 4) fused to the N-terminus of hFc via a GGG linker; (7) extracellular ActRIIB variant ActRIIB2.02 (SEQ ID NO: 5) fused to the N-terminus of hFc via a GGG linker; (8) extracellular ActRIIB variant ActRIIB2.03 (SEQ ID NO: 6) fused to the N-terminus of hFc via a GGG linker; (9) extracellular ActRIIB variant ActRIIB2.04 (SEQ ID NO: 7) fused to the N-terminus of hFc via a GGG linker; (10) extracellular ActRIIB variant ActRIIB2.05 (SEQ ID NO: 8) fused to the N-terminus of hFc via a GGG linker; (11) extracellular ActRIIB variant ActRIIB2.06 (SEQ ID NO: 9) fused to the N-terminus of hFc via a GGG linker; (12) extracellular ActRIIB variant ActRIIB2.07 (SEQ ID NO: 10) fused to the N-terminus of hFc via a GGG linker; (13) extracellular ActRIIB variant ActRIIB2.08 (SEQ ID NO: 11) fused to the N-terminus of hFc via a GGG linker; (14) extracellular ActRIIB variant ActRIIB2.09 (SEQ ID NO: 12) fused to the N-terminus of hFc via a GGG linker; (15) Extracellular ActRIIB variant ActRIIB2.10 (SEQ ID NO: 13) fused to the N-terminus of hFc via a GGG linker.

[0253] 100 µg of the plasmid construct was delivered over 5–8 seconds at a volume of 10% of body weight. This high-volume, short-duration injection provided the necessary pressure to introduce the plasmid into hepatocytes, where it was expressed, specifically the protein of interest, under a strong and ubiquitous promoter (pLEV113). The protein of interest was secreted by endogenous hepatocyte machinery and circulated freely. Mice were weighed twice weekly for 28 days, and measurements were recorded as a percent change in body weight from baseline measurements (Figures 1 and 2). Muscle was also weighed at the end of the study, and measurements were recorded in milligrams (Figures 3A and 3B).

[0254] Example 2: Evaluation of ActRIIB mutant binding affinity by surface plasmon resonance (SPR) A GE Biacore 3000 was used to measure the kinetics of interactions between ActRIIB-Fc variants and their ligands, activin A, activin B, growth differentiation factor 11 (GDF11), and BMP-9. ActRIIA, ActRIIB, ActRIIB2.06, ActRIIB2.11, and ActRIIB2.12 were recombinant proteins. All other ActRIIB-Fc variants were transiently expressed in HEK293 cells and purified from conditioned medium using protein A-Sepharose chromatography. Flow cells 1–4 were immobilized with anti-human / anti-mouse capture antibodies from GE using an amine coupling kit. ActRII-Fc proteins were then captured onto the chip in flow cells 2–4, with flow cell 1 left empty as a reference cell to measure and subtract nonspecific binding. HBS-EP+ buffer from GE Healthcare™ was used as the running buffer. Each ligand was run in a duplicate concentration series at 40 μl / min to avoid mass transfer effects. All data were collected on a CM-5 chip, except for GDF-11, which was run on a CM-4. The K for each interaction was D Data were analyzed using Scrubber2 with BioLogic™ software to calculate (Table 3).

[0255] [Table 3]

[0256] Example 3: Effect of extracellular ActRIIB mutants on bone mineral density Adult male C57 / BL6 mice were sham-operated (SHAM) or castrated (ORX). Both surgical groups were allowed to recover for 14 days after surgery. All animals were housed in conventional cages with free access to food (normal chow) and water. SHAM and ORX animals were then assigned to either a vehicle-treated group (VEH) or an ActRII mutant-treated group and received biweekly systemic intraperitoneal administration of vehicle or ActRII mutant (10 mg / kg) for 71 days. Body weights were measured twice weekly during treatment. Body composition was analyzed using a MiniSpec LF50 NMR analyzer on day 0 of the study, and then on days 14, 28, 47, and 71 after the start of treatment. At the end of the study, tissues of interest (muscle, fat depots, and tibia) were surgically removed, weighed, and appropriately stored for further analysis. At this time, ORX animals were also examined to confirm complete removal of the testes. The cortical morphometry and trabecular structure of various bones were also assessed at the end of the experiment using micro-computed tomography.

[0257] Example 4: Effects of extracellular ActRIIB mutants on renal fibrosis The effects of extracellular ActRIIB mutants on renal fibrosis were determined using a unilateral ureteral obstruction (UUO) mouse model of renal fibrosis. The UUO model involves complete ligation of the left ureter without compromising right renal function. Briefly, UUO was performed on mice under anesthesia, whereby the left ureter was accessed through a flank incision, and two ligatures were placed 5 mm apart on the proximal third of the ureter using silk sutures. Sham surgery (sham) was performed in a similar manner, without placing ligatures on the ureter. In this model, severe fibrosis developed in the kidney within 14 days after UUO, which was assessed by measuring renal collagen by directly measuring the amount of hydroxyproline in the sample. After 14 days of UUO, dry kidney weight decreased as a result of parenchymal damage. 16-week-old male C57BL / 6 mice underwent either sham or UUO surgery, and the UUO-operated mice were divided into two groups. Each UUO group received subcutaneous injections of either an ActRllB mutant (10 mg / kg) that does not bind to a known mouse protein or vehicle (administered in the same amount as the body weight) on the day before surgery and on days 1, 3, 6, 8, 10, and 13 after surgery. Sham-operated mice received vehicle (sterile PBS) during this period, using the same schedule as the UUO group. All mice were sacrificed on day 14 after surgery. Kidney weights were measured, and kidneys were flash-frozen using liquid nitrogen. Hydroxyproline levels were measured to determine collagen content, and the kidneys were kept at -80°C until fibrosis was assessed.

[0258] Example 5: Effects of extracellular ActRIIB mutants on erythrocytes Ten male and ten female rats per group were administered two SC doses (on days 1 and 15) of vehicle or 6, 20, or 60 mg / kg of the ActRllB mutant. Hematological parameters were measured on day 29. Studies were also conducted to evaluate the time course and dose response for RBC, hemoglobin, and hematocrit induction. In the first study, the time course of erythropoiesis was examined in male and female rats administered subcutaneously (SC) at 10 mg / kg of the ActRllB mutant on days 1 and 8. Hematological parameters were assessed before administration and on days 3, 8, 15, 29, and 44 after administration. In the second study, the hematological dose response was examined in male and female rats administered 0.4, 2, 10, or 30 mg / kg of vehicle or the ActRllB mutant on days 1 and 15. Hematological parameters were assessed before treatment and on days 13 and 28 of treatment.

[0259] Example 6: Effects of extracellular ActRIIB mutants on PAH In one experiment, PAH was induced in male rats by a single subcutaneous injection of monocrotaline (MCT 40 mg / kg). To determine whether treatment with ActRIIB mutants could prevent the development of PAH, rats were randomized to vehicle or ActRIIB mutant treatment groups 24 h after PAH induction and treated twice weekly with ActRIIB mutants (5 or 15 mg / kg) or vehicle for 21 days. Rats were anesthetized with 1.5% isoflurane and electrocardiograms were used to examine pulmonary blood flow acceleration, right ventricular function and hypertrophy, and left ventricular function on day 14 using a small-animal high-frequency ultrasound probe with the animals held supine. Doppler imaging across the mitral and tricuspid valves was used to determine whether treatment with ActRIIB mutants induced significant regurgitation or lesions. On day 21, rats were anesthetized with pentobarbital, intubated via the trachea, and mechanically ventilated using a rodent ventilator. Hemodynamics was assessed using a fluid-filled catheter inserted through the right ventricular apex. Rats were perfused with PBS followed by 1% formaldehyde. To measure right ventricular hypertrophy (RVH), hearts were removed, and the right ventricular free wall was dissected from the left ventricle plus septum (LV+S) and weighted separately. The degree of RVH was determined from the ratio RV / (LV+S).

[0260] In the second experiment, male rats were given a single subcutaneous injection of monocrotaline (40 mg / kg) to induce PAH. To determine whether treatment with ActRIIB mutants could slow or reduce the progression of PAH, rats were reinjected with MCT on day 18 and randomized to vehicle or ActRIIB mutant treatment. Rats were injected with ActRIIB mutants (15 mg / kg) or vehicle three times weekly. Hemodynamics and RVH were examined on day 35 as described above.

[0261] Example 7: Evaluation of ActRIIB mutants using a genetic luciferase reporter assay C2C12-BRE-luciferase and HEK293-SBE-luciferase cells were plated onto 96-well plates in DMEM supplemented with 2% FBS and placed in an incubator for at least 3 hours to acclimate to the plate surface. For each ActRIIB / A-Fc variant or positive control (ActRIIA-Fc and ActRIIB-Fc), a dilution series was prepared in 2% DMEM and incubated with GDF-11, activin A, activin B, and BMP-9 for 30 minutes at 37°C. ActRIIA, ActRIIB, ActRIIB2.06, ActRIIB2.11, and ActRIIB2.12 were recombinant proteins. All others were conditioned medium from transiently transfected cells. The medium in the plate was aspirated, and the ActRIIB / A / ligand mixture was added to the plate as a medium exchange. The remaining wells were used for positive control and background replicates. Plates were incubated overnight and then read using Promega Steady-Glo® and Molecular Devices Spectramax® M5e. Cell-based assays demonstrate the ability of mutants to inhibit signaling at endogenous cell surface receptors. As shown in Table 4, mutants except ActRIIB / A and ActRIIB2.11 have comparable inhibition of activin A, activin B, and GDF-11, but reduced BMP9 inhibition compared to ActRIIB-Fc.

[0262] [Table 4]

[0263] Example 8: Effects of extracellular ActRIIB mutants on body weight and red blood cell mass Eight-week-old male C57BL / 6 mice were divided into three groups (n=10 / group). Groups were administered 5 ml / kg of vehicle (Tris-buffered saline, pH 7.4) or either ActRllB2.11 (SEQ ID NO: 14)-Fc or ActRllB2.12 (SEQ ID NO: 15)-Fc (20 mg / kg). Treatment was administered intraperitoneally (IP) twice weekly for 4 weeks (8 doses), and the study was terminated on study day 28. Body weights were recorded on each administration day during the study. In addition, red blood cell mass parameters were hematologically evaluated at the end of the study. As shown in Figure 4, both ActRllB2.11-Fc and ActRllB2.12-Fc increased body weight in wild-type mice ( * =p<0.05; **** = p<0.01). ActRIIB2.12-Fc also significantly reduced red blood cell mass ( ), including red blood cell count, hemoglobin level, and hematocrit (Figures 5A-5C). *** =p<0.001; **** =p<0.0001) increased the parameter.

[0264] Example 9: Effects of ActRllB mutants in a mouse model of osteoporosis C57BL / 6 mice underwent orchiectomy (ORX) or sham surgery at 9 weeks of age. During a 6-week recovery period, ORX mice developed an osteoporotic phenotype, after which they received either vehicle or ActRIIB2.12 (SEQ ID NO: 15)-Fc (10 mg / kg) intraperitoneally twice weekly. MicroCT (Perkin Elmer Quantum Fx) imaging was performed 11 weeks after the start of treatment. ASBMR bone morphometry parameters for each dataset were calculated using AnalyzePro software (AnalyzeDirect, Overland Park, KS) with the bone morphometry analysis add-on. A 50-slice region of the CT volume just distal to the proximal tibial growth plate was selected to assess changes in trabecular bone parameters. As shown in Figures 6A-6D, treatment with ActRIIB2.12-Fc increased bone mass, trabecular bone volume fraction, and trabecular number relative to orchiectomy (* =p<0.05; ** =p<0.01). These data indicate that treatment of osteoporotic mice with ActRIIB2.12-Fc increases trabecular bone mass as a result of increased bone formation.

[0265] Example 10: Effects of ActRIIB variant 2.12 on red blood cell mass and trabecular bone in rats Hydrodynamic injection of ActRIIB2.12 (SEQ ID NO: 15)-Fc plasmid DNA was performed in 4-week-old Sprague-Dawley rats via lateral tail vein injection. Four weeks after injection, blood was analyzed for hematological parameters and ActRIIB2.12-Fc levels. MicroCT imaging (Perkin Elmer Quantum Fx) was performed ex vivo on the tibia. ASBMR bone morphometry parameters for each dataset were calculated using AnalyzePro software (AnalyzeDirect, Overland Park, KS) with the bone morphometry analysis add-on. A 150-slice region of the CT volume just distal to the proximal tibial growth plate was selected to evaluate changes in trabecular bone parameters. As shown in Figures 7A-7C, ActRIIB2.12-Fc increased red blood cell mass parameters, including red blood cell count, hemoglobin level, and hematocrit, in wild-type rats (Figure 7B). * =p<0.05; ** = p < 0.01). ActRIIB2.12-Fc also increased trabecular bone (trabecular bone volume, trabecular bone fraction, and trabecular bone thickness) in wild-type rats (Figures 8A-8E; * =p<0.05; ** =p<0.01). These data demonstrate that treatment with ActRIIB2.12-Fc in wild-type rats increases trabecular bone mass.

[0266] Example 11: Treatment of muscle diseases by administration of extracellular ActRIIB mutants According to the methods disclosed herein, one skilled in the art can treat a subject, such as a human patient with a muscle disease (e.g., DMD, ALS, or inclusion body myositis), to increase muscle mass or maintain or improve muscle strength (e.g., reduce muscle weakness). Treatment methods can include diagnosing or identifying a subject as a candidate for treatment based on standard clinical tests for muscle disease (e.g., blood tests, muscle biopsy, genetic testing, and / or electromyography). To treat a subject, one skilled in the art can administer a composition containing an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) to the subject. The composition containing the extracellular ActRIIB variant can be administered to the subject, for example, by parenteral injection (e.g., intravenous injection) or local administration (e.g., injection into a muscle) to treat the muscle disease. The extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) is administered in a therapeutically effective amount, for example, 0.01-500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRIIB variant is administered every other month, once a month, once every two weeks, or at least once a week or more (e.g., 1, 2, 3, 4, 5, 6, or 7 or more times a week). The extracellular ActRIIB variant is administered in an amount sufficient to increase muscle mass or maintain or improve muscle strength (eg, reduce muscle weakness).

[0267] After administering the composition to the patient, those skilled in the art can monitor the patient's improvement in response to treatment by various methods. For example, a doctor can monitor the patient's muscle mass, muscle strength, and motor function. If the patient shows an increase in muscle mass or maintains or improves muscle strength after administering the composition compared to the test results before administering the composition, it indicates that the patient is responding favorably to treatment. Subsequent doses can be determined and administered as needed.

[0268] Example 12: Treatment of bone disease by administration of extracellular ActRIIB mutants According to the methods disclosed herein, one skilled in the art can treat a subject, such as a human patient, with a bone disease (e.g., osteoporosis or osteopenia) to increase bone mineral density, increase bone formation, reduce bone resorption, reduce bone loss, or reduce the risk of fracture. Treatment methods can include diagnosing or identifying a subject as a candidate for treatment based on a standard clinical test for bone mineral density (e.g., dual-energy X-ray absorptiometry). To treat a subject, one skilled in the art can administer a composition containing an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) to the subject. The composition containing the extracellular ActRIIB variant can be administered to the subject, for example, by parenteral injection (e.g., intravenous injection) to treat the bone disease. The extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) is administered in a therapeutically effective amount, for example, 0.01-500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRIIB variant is administered every other month, once a month, once every two weeks, or at least once a week or more (e.g., 1, 2, 3, 4, 5, 6, or 7 or more times a week). The extracellular ActRIIB variant is administered in an amount sufficient to increase bone mineral density, increase bone formation, reduce bone resorption, reduce bone loss, or reduce the risk of fracture.

[0269] After administering the composition to patients, those skilled in the art can monitor the improvement of patients in response to treatment by various methods.For example, doctors can monitor the bone mineral density of patients by performing dual X-ray absorptiometry.Compared with the test results before administering the composition, after administering the composition, the patient shows an increase in bone mineral density, an increase in bone formation, a decrease in bone resorption, a decrease in bone loss, or a decrease in the risk of fracture, indicating that the patient responds favorably to treatment.Subsequent doses can be determined and administered as needed.

[0270] Example 13: Treatment of anemia by administration of extracellular ActRIIB mutants According to the methods disclosed herein, one skilled in the art can treat a subject, such as a human patient, with anemia (e.g., vitamin deficiency anemia or anemia associated with chronic kidney disease) to increase parameters of red blood cell mass, such as red blood cell count, hemoglobin level, or hematocrit. Treatment methods can include diagnosing or identifying a subject as a candidate for treatment based on a blood test measuring a hematological parameter. To treat a subject, one skilled in the art can administer a composition containing an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) to the subject. The composition containing the extracellular ActRIIB variant can be administered to the subject, for example, by parenteral injection (e.g., intravenous injection), to treat anemia. The extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) is administered in a therapeutically effective amount, for example, 0.01-500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRIIB variant is administered every other month, once a month, once every two weeks, or at least once a week or more (e.g., 1, 2, 3, 4, 5, 6, or 7 or more times a week). The extracellular ActRIIB variant is administered in an amount sufficient to increase hemoglobin levels, increase red blood cell count, or increase hematocrit.

[0271] After administering the composition to a patient, those skilled in the art can monitor the improvement of the patient in response to treatment by various methods.For example, a doctor can monitor the patient's hemoglobin level, red blood cell count, or hematocrit by performing blood tests.Compared with the test results before administering the composition, if the patient improves his hemoglobin level, red blood cell count, or hematocrit after administering the composition, this indicates that the patient is responding favorably to treatment.Subsequent doses can be determined and administered as needed.

[0272] Example 14: Treatment of fibrosis by administration of extracellular ActRIIB mutants According to the methods disclosed herein, a skilled artisan can treat a subject, such as a human patient, with fibrosis (e.g., pulmonary fibrosis or fibrosis associated with chronic kidney disease) to reduce the symptoms of fibrosis or slow or halt the progression of fibrosis. Treatment methods can include diagnosing or identifying a subject as a candidate for treatment based on a clinical test for fibrosis (e.g., an imaging test such as an X-ray or CT scan). To treat a subject, a skilled artisan can administer a composition containing an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) to the subject. The composition containing the extracellular ActRIIB variant can be administered to the subject, for example, by parenteral injection (e.g., intravenous injection) to treat fibrosis, or can be administered locally (e.g., injected) into a fibrotic tissue or organ. The extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) is administered in a therapeutically effective amount, for example, 0.01-500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRIIB variant is administered every other month, once a month, once every two weeks, or at least once a week or more (e.g., 1, 2, 3, 4, 5, 6, or 7 or more times a week). The extracellular ActRIIB variant is administered in an amount sufficient to reduce the symptoms of fibrosis or to slow or stop the progression of fibrosis.

[0273] After administering the composition to the patient, those skilled in the art can monitor the improvement of the patient in response to treatment by various methods.For example, the doctor can carry out imaging tests to monitor the patient's fibrosis, and use standard clinical tests to monitor the patient's symptoms.Compared with the test results before administering the composition, the discovery that the patient's symptoms are reduced or the progression of the patient's fibrosis is delayed or stopped after administering the composition indicates that the patient is responding favorably to treatment.Subsequent doses can be determined and administered as needed.

[0274] Example 15: Treatment of pulmonary hypertension by administration of extracellular ActRIIB mutants According to the methods disclosed herein, a skilled artisan can treat a subject, such as a human patient, with pulmonary hypertension (PH, e.g., PAH) to reduce the symptoms of PH or slow or stop the progression of PH. Treatment methods can include diagnosing or identifying a subject as a candidate for treatment based on standard clinical tests for PH (e.g., echocardiogram, electrocardiogram, chest X-ray, right heart catheterization). To treat a subject, a skilled artisan can administer a composition containing an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOS: 1-15 (e.g., SEQ ID NOS: 2-15)) to the subject. The composition containing the extracellular ActRIIB variant can be administered to the subject, for example, by parenteral injection (e.g., intravenous injection) to treat PH. The extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) is administered in a therapeutically effective amount, for example, 0.01-500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg). In some embodiments, the extracellular ActRIIB variant is administered every other month, once a month, once every two weeks, or at least once a week or more (e.g., 1, 2, 3, 4, 5, 6, or 7 or more times a week). The extracellular ActRIIB variant is administered in an amount sufficient to reduce the symptoms of PH or to slow or stop the progression of PH.

[0275] After administering the composition to the patient, those skilled in the art can monitor the patient's improvement in response to treatment using various methods.For example, doctors can monitor the patient's symptoms using standard clinical tests and patient self-reports.Compared to the test results before administering the composition, the discovery that the patient's PH symptoms are reduced or the progression of the patient's PH is delayed or stopped after administering the composition indicates that the patient is responding favorably to treatment.Subsequent doses can be determined and administered as needed.

[0276] Other embodiments While the invention has been described in terms of particular embodiments thereof, it will be understood that further modifications are possible, and that this application is intended to cover any variations, uses, or adaptations of the invention which generally follow from the principles of the invention, including such departures from the present disclosure as are within known or related practice within the art to which this invention pertains and which may fall within the essential characteristics set forth above.

[0277] The entire contents of all publications, patents, and patent applications are incorporated herein by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0278] Other embodiments are within the scope of the following claims. The technical concepts that can be understood from the above-described embodiment will be described below as supplementary notes. [Appendix 1] A polypeptide comprising an extracellular activin type IIB receptor (ActRIIB) mutant, the mutant comprising: GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT (SEQ ID NO: 17) wherein the variant comprises one or more amino acid substitutions that confer reduced BMP9 binding to wild-type extracellular ActRIIB, and one or more further amino acid substitutions, wherein the substitutions that reduce BMP9 binding are a) amino acid substitution E75K; b) the amino acid substitutions Q69T and E70D; or c) amino acid substitutions Q69D and E70T; A polypeptide comprising one or more of:

[0279] [Appendix 2] The polypeptide of Appendix 1, wherein the variant comprises one or more amino acid substitutions selected from the group consisting of I11L, Y12F, L19K, E20D, S25T, L27V, R29P, E31Y, E33D, Q34K, L38R, Y41F, R45K, S47I, S48T, T50S, I51L, L53I, K56Q, F63I, T74K, E76D, N77S, Q79E, and F89M.

[0280] [Appendix 3] The polypeptide of Appendix 1 or Appendix 2, wherein the variant comprises the amino acid substitutions E75K, E20D, and F63I. [Appendix 4] The polypeptide of Appendix 1 or Appendix 2, wherein the variant comprises the amino acid substitution E75K.

[0281] [Appendix 5] The polypeptide of Appendix 4, wherein the variant comprises the amino acid substitutions T74K, E76D, N77S, and Q79E. [Appendix 6] The polypeptide of Appendix 5, wherein the variant further comprises one or more additional amino acid substitutions.

[0282] [Appendix 7] The polypeptide of Appendix 6, wherein the variant comprises the amino acid substitutions Y41F, R45K, and K56Q. [Appendix 8] The polypeptide of Appendix 7, wherein the variant further comprises the amino acid substitutions Y12F, L19K, E20D, R29P, E31Y, E33D, L38R, and F63I.

[0283] [Appendix 9] The polypeptide of Appendix 6, wherein the variant comprises the amino acid substitutions S25T and S47I. [Appendix 10] The polypeptide of Appendix 9, wherein the variant comprises the amino acid substitution S48T.

[0284] [Appendix 11] The polypeptide of Appendix 6, wherein the variant comprises the amino acid substitution R29P. [Appendix 12] The polypeptide of Appendix 6, wherein the variant comprises the amino acid substitutions E31Y, E33D, and Q34K.

[0285] [Appendix 13] The polypeptide of Appendix 6, wherein the variant comprises the amino acid substitutions Y12F, L19K, and E20D. [Appendix 14] The polypeptide of Appendix 6, wherein the variant comprises the amino acid substitutions E31Y, E33D, and L38R.

[0286] [Appendix 15] The polypeptide of Appendix 1 or Appendix 2, wherein the variant comprises the amino acid substitutions Q69T and E70D and the additional amino acid substitutions I11L, L27V, Q34K, T50S, I51L, L53I, and F89M.

[0287] [Appendix 16] The polypeptide of Appendix 1 or Appendix 2, wherein the variant comprises the amino acid substitutions Q69D and E70T and the additional amino acid substitutions I11L, L27V, Q34K, T50S, I51L, L53I, and F89M.

[0288] [Appendix 17] The polypeptide of Appendix 15 or Appendix 16, wherein the variant comprises the amino acid substitution E75K. [Appendix 18] The polypeptide according to Appendix 1 or Appendix 2, wherein the mutant has any one of the sequences of SEQ ID NOs: 2 to 15.

[0289] [Appendix 19] The polypeptide according to any one of Appendices 1 to 18, further comprising an Fc domain monomer fused to the C-terminus of the polypeptide via a linker. [Appendix 20] The polypeptide described in Appendix 19, wherein the Fc domain monomer comprises the sequence of SEQ ID NO: 19.

[0290] [Appendix 21] The polypeptide of Appendix 19 or Appendix 20, wherein the polypeptide forms a dimer. [Appendix 22] The polypeptide according to any one of Appendices 1 to 18, comprising a wild-type Fc domain fused to the C-terminus of the polypeptide via a linker.

[0291] [Appendix 23] The polypeptide described in Appendix 22, wherein the wild-type Fc domain comprises the sequence of SEQ ID NO: 71. [Appendix 24] The polypeptide according to any one of Appendices 1 to 18, comprising an Fc domain having an amino acid substitution fused to the C-terminus of the polypeptide via a linker.

[0292] [Appendix 25] The polypeptide described in Appendix 24, wherein the Fc domain does not form a dimer. [Appendix 26] The polypeptide according to any one of Appendices 1 to 18, comprising an albumin-binding peptide fused to the C-terminus of the polypeptide via a linker.

[0293] [Appendix 27] The polypeptide described in Appendix 26, wherein the albumin-binding peptide comprises the sequence of SEQ ID NO: 72. [Appendix 28] A polypeptide according to any one of Appendices 1 to 18, comprising a fibronectin domain fused to the C-terminus of the polypeptide via a linker.

[0294] [Appendix 29] The polypeptide described in Appendix 28, wherein the fibronectin domain comprises the sequence of SEQ ID NO: 73. [Appendix 30] The polypeptide according to any one of Appendices 1 to 18, comprising human serum albumin fused to the C-terminus of the polypeptide via a linker.

[0295] [Appendix 31] The polypeptide described in Appendix 30, wherein the human serum albumin comprises the sequence of SEQ ID NO: 74. [Appendix 32] The polypeptide according to any one of Appendices 19 to 31, wherein the linker is an amino acid spacer.

[0296] [Appendix 33] The polypeptide described in Appendix 32, wherein the amino acid spacer is GGG, GGGA (SEQ ID NO: 20), GGGG (SEQ ID NO: 22), GGGAG (SEQ ID NO: 52), GGGAGG (SEQ ID NO: 53), or GGGAGGG (SEQ ID NO: 54).

[0297] [Appendix 34] The polypeptide according to any one of Appendices 1 to 33, wherein the polypeptide has a serum half-life of at least 7 days. [Appendix 35] The polypeptide has a K D 35. The polypeptide of any one of appendices 1 to 34, which binds to human bone morphogenetic protein 9 (BMP9) at

[0298] [Appendix 36] A polypeptide according to any one of Appendices 1 to 35, which binds to at least one of activin and myostatin, and has low or weak binding to human BMP9.

[0299] [Appendix 37] The polypeptide of Appendix 35 or Appendix 36, wherein the polypeptide does not substantially bind to human BMP9. [Appendix 38] The polypeptide has a K D 38. The polypeptide of any one of appendices 1 to 37, which binds to human activin A at

[0300] [Appendix 39] The polypeptide has a K D 39. The polypeptide of any one of appendices 1 to 38, which binds to human activin B at [Appendix 40] The polypeptide has a K D 40. The polypeptide of any one of appendices 1 to 39, which binds to human GDF-11 at

[0301] [Appendix 41] A nucleic acid molecule encoding the polypeptide according to any one of Appendices 1 to 40. [Appendix 42] A vector comprising the nucleic acid molecule described in Appendix 41.

[0302] [Appendix 43] A host cell expressing a polypeptide according to any one of Appendices 1 to 40, the host cell comprising a nucleic acid molecule according to Appendices 41 or a vector according to Appendices 42, the nucleic acid molecule or vector being expressed in the host cell.

[0303] [Appendix 44] A method for producing a polypeptide according to any one of Appendices 1 to 43, the method comprising: a) providing a host cell comprising the nucleic acid molecule of Appendix 41 or the vector of Appendix 42; b) expressing the nucleic acid molecule or vector in the host cell under conditions that allow the formation of the polypeptide; A method comprising:

[0304] [Appendix 45] A pharmaceutical composition comprising the polypeptide of any one of Appendices 1 to 40, the nucleic acid molecule of Appendices 41, or the vector of Appendices 42, and one or more pharmaceutically acceptable carriers or excipients.

[0305] [Appendix 46] The pharmaceutical composition of Appendices 45, wherein the polypeptide is in a therapeutically effective amount. [Appendix 47] A method of increasing lean mass in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of Appendices 1 to 40, a nucleic acid molecule according to Appendix 41, a vector according to Appendix 42, or a pharmaceutical composition according to Appendix 45 or Appendix 46.

[0306] [Appendix 48] A method of increasing muscle mass in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of Appendices 1 to 40, a nucleic acid molecule according to Appendix 41, a vector according to Appendix 42, or a pharmaceutical composition according to Appendix 45 or Appendix 46.

[0307] [Appendix 49] The method of Appendices 48, wherein the subject has Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia, or cancer cachexia.

[0308] [Appendix 50] A method for affecting at least one of myostatin, activin, and BMP9 signaling in a subject having a disease or condition involving muscle weakness and muscle atrophy, the method comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1-40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0309] [Appendix 51] The method of Appendices 50, wherein the disease or condition is Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia, or cancer cachexia.

[0310] [Appendix 52] A method of treating a subject having or at risk of developing Duchenne muscular dystrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0311] [Appendix 53] A method of treating a subject having or at risk of developing facioscapulohumeral muscular dystrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0312] [Appendix 54] A method of treating a subject having or at risk of developing inclusion body myositis, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0313] [Appendix 55] A method of treating a subject having or at risk of developing amyotrophic lateral sclerosis, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0314] [Appendix 56] A method of treating a subject having or at risk of developing sarcopenia, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0315] [Appendix 57] A method of treating a subject having or at risk of developing cancer cachexia, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0316] [Appendix 58] A method of increasing bone mineral density in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of Appendices 1 to 40, a nucleic acid molecule according to Appendix 41, a vector according to Appendix 42, or a pharmaceutical composition according to Appendix 45 or Appendix 46.

[0317] [Appendix 59] A method of reducing bone resorption in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of Appendices 1 to 40, a nucleic acid molecule according to Appendix 41, a vector according to Appendix 42, or a pharmaceutical composition according to Appendix 45 or Appendix 46.

[0318] [Appendix 60] A method of increasing bone formation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of Appendices 1 to 40, a nucleic acid molecule according to Appendix 41, a vector according to Appendix 42, or a pharmaceutical composition according to Appendix 45 or Appendix 46.

[0319] [Appendix 61] A method of increasing bone strength in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of Appendices 1 to 40, a nucleic acid molecule according to Appendix 41, a vector according to Appendix 42, or a pharmaceutical composition according to Appendix 45 or Appendix 46.

[0320] [Appendix 62] A method for reducing the risk of fracture in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of Appendices 1 to 40, a nucleic acid molecule according to Appendix 41, a vector according to Appendix 42, or a pharmaceutical composition according to Appendix 45 or Appendix 46.

[0321] [Appendix 63] The method of any one of Appendices 58 to 62, wherein the subject is suffering from primary osteoporosis, secondary osteoporosis, osteopenia, osteopetrosis, fracture, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with treatment of obesity, low-gravity-related bone loss, or immobility-related bone loss.

[0322] [Appendix 64] A method for affecting at least one of myostatin, activin, and BMP9 signaling in a subject having a disease or condition associated with bone damage, the method comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0323] [Appendix 65] The method of Appendices 64, wherein the disease or condition is primary osteoporosis, secondary osteoporosis, osteopenia, osteopetrosis, fracture, bone cancer or cancer metastasis-associated bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with treatment of obesity, low-gravity-associated bone loss, or immobility-associated bone loss.

[0324] [Appendix 66] A method of treating a subject having or at risk of developing a bone disease, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of Appendices 1 to 40, a nucleic acid molecule of Appendices 41, a vector of Appendices 42, or a pharmaceutical composition of Appendices 45 or 46.

[0325] [Appendix 67] The method of Appendices 66, wherein the bone disease is primary osteoporosis, secondary osteoporosis, osteopenia, osteopetrosis, fracture, bone loss associated with bone cancer or cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with treatment for obesity, low-gravity-related bone loss, or immobility-related bone loss.

[0326] [Appendix 68] A method of treating a subject having primary osteoporosis, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0327] [Appendix 69] A method of treating a subject having secondary osteoporosis, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0328] [Appendix 70] A method of treating a subject having osteopenia, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0329] [Appendix 71] A method of treating a subject with bone cancer or cancer metastasis-associated bone loss, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0330] [Appendix 72] A method of treating a subject having Paget's disease, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0331] [Appendix 73] A method of treating a subject having renal osteodystrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0332] [Appendix 74] A method of treating a subject having treatment-related bone loss, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0333] [Appendix 75] A method of treating a subject having diet-related bone loss, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of Appendices 1-40, a nucleic acid molecule of Appendices 41, a vector of Appendices 42, or a pharmaceutical composition of Appendices 45 or 46.

[0334] [Appendix 76] A method of treating a subject having low-gravity associated bone loss, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1-40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0335] [Appendix 77] A method of treating a subject having immobility-associated bone loss, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0336] [Appendix 78] The method of any one of Appendices 63, 65, 67, or 68, wherein the primary osteoporosis is age-related osteoporosis or hormone-related osteoporosis. [Appendix 79] The method of any one of Appendices 63, 65, 67, or 69, wherein the secondary osteoporosis is immobility-induced osteoporosis or glucocorticoid-induced osteoporosis.

[0337] [Appendix 80] The method of any one of Appendices 63, 65, 67, or 71, wherein the cancer is multiple myeloma. [Appendix 81] The method of any one of Appendices 63, 65, 67, or 74, wherein the treatment is FGF-21 treatment, GLP-1 treatment, cancer treatment, or treatment for obesity or treatment for type 2 diabetes.

[0338] [Appendix 82] The method of any one of Appendices 63, 65, 67, or 75, wherein the diet-related bone loss is rickets. [Appendix 83] The method of any one of Appendices 58 to 82, wherein the subject is at risk of fracture.

[0339] [Appendix 84] The method of any one of Appendices 58 to 83, wherein the method increases bone formation in the subject. [Appendix 85] The method of any one of Appendices 58 to 84, wherein the method reduces bone resorption in the subject.

[0340] [Appendix 86] The method of any one of Appendices 58 to 85, wherein the method increases osteoblast activity or osteoblast formation. [Appendix 87] The method of any one of Appendices 58 to 86, wherein the method reduces osteoclast activity or reduces osteoclast formation.

[0341] [Appendix 88] The method of any one of Appendices 58 to 87, wherein the method reduces the risk of fracture. [Appendix 89] The method of any one of Appendices 58 to 88, wherein the method increases bone strength.

[0342] [Appendix 90] The method of any one of Appendices 58 to 89, wherein the bone is cortical bone. [Appendix 91] The method of any one of Appendices 58 to 89, wherein the bone is trabecular bone. [Appendix 92] A method of reducing or preventing fibrosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of Appendices 1 to 40, a nucleic acid molecule according to Appendices 41, a vector according to Appendices 42, or a pharmaceutical composition according to Appendices 45 or 46.

[0343] [Appendix 93] A method of treating a subject having or at risk of developing fibrosis, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0344] [Appendix 94] A method of delaying or inhibiting the progression of fibrosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of Appendices 1 to 40, a nucleic acid molecule according to Appendices 41, a vector according to Appendices 42, or a pharmaceutical composition according to Appendices 45 or 46.

[0345] [Appendix 95] A method for affecting at least one of myostatin, activin, and BMP9 signaling in a subject having or at risk of developing fibrosis, or a disease or condition associated with fibrosis, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1-40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0346] [Appendix 96] The method of any one of Appendices 92 to 95, wherein the fibrosis is chemotherapy-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, corneal fibrosis, cardiac fibrosis, bone marrow fibrosis, mediastinal fibrosis, retroperitoneal fibrosis, osteoarthrofibrosis, arthrofibrosis, tissue fibrosis, tumor stroma, desmoplastic tumor, surgical adhesion, hypertrophic scar, or keloid.

[0347] [Appendix 97] The method described in Appendices 96, wherein the tissue fibrosis is fibrosis affecting a tissue selected from the group consisting of muscle tissue, skin epidermis, skin dermis, tendon, cartilage, pancreatic tissue, uterine tissue, nervous tissue, testis, ovary, adrenal gland, artery, vein, colon, small intestine, large intestine, biliary tract, and intestine.

[0348] [Appendix 98] The method of any one of Appendices 92 to 95, wherein the fibrosis is fibrosis associated with a wound, a burn, hepatitis B or C infection, fatty liver disease, schistosome infection, kidney disease, chronic kidney disease, heart disease, macular degeneration, retinal or vitreoretinopathy, Crohn's disease, systemic or localized scleroderma, atherosclerosis, or restenosis.

[0349] [Appendix 99] The method according to any one of Appendices 92 to 98, wherein the method improves the function of fibrotic tissue or organ. [Appendix 100] A method of increasing red blood cell levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0350] [Appendix 101] A method of promoting or increasing red blood cell formation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0351] [Appendix 102] The method of Appendices 100 or 101, wherein the subject has or is at risk of developing anemia or blood loss. [Appendix 103] A method for affecting the signaling of at least one of myostatin, activin, and BMP9 in a subject having or at risk of developing a disease or condition involving low red blood cell levels or low hemoglobin levels, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0352] [Appendix 104] The method of Appendices 103, wherein the disease or condition is anemia or blood loss. [Appendix 105] The method of Appendices 102 and 104, wherein the anemia or blood loss is associated with cancer, cancer treatment, chronic kidney disease, acute kidney disease or renal failure, chronic kidney disease or renal failure, myelodysplastic syndrome, thalassemia, nutritional deficiency, adverse drug reaction, inflammatory or autoimmune disease, splenomegaly, porphyria, vasculitis, hemolysis, bone marrow deficiency, bone marrow transplant, acute liver disease, chronic liver disease, acute bleeding, chronic bleeding, infection, hemoglobinopathies, drug use, alcohol abuse, Churg-Strauss syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplant, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schönlein purpura, Schwachman syndrome, advanced age, contraindications to blood transfusion, surgery, trauma, wound, ulcer, urinary tract bleeding, gastrointestinal bleeding, frequent blood donations, or heavy menstrual bleeding.

[0353] [Appendix 106] The method of any one of Appendices 102, 104, or 105, wherein the anemia is aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, anemia of chronic disease, anemia associated with bone marrow disease, hemolytic anemia, sickle cell anemia, microcytic anemia, hypochromic anemia, sideroblastic anemia, Diamond-Blackfan anemia, Fanconi anemia, or refractory anemia with excess blasts.

[0354] [Appendix 107] A method of treating a subject having or at risk of developing anemia, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0355] [Appendix 108] The method of Appendices 107, wherein the anemia is associated with cancer, cancer treatment, chronic kidney disease, acute kidney disease or renal failure, chronic kidney disease or renal failure, myelodysplastic syndrome, thalassemia, nutritional deficiency, adverse drug reaction, inflammatory or autoimmune disease, splenomegaly, porphyria, vasculitis, hemolysis, bone marrow deficiency, bone marrow transplant, acute liver disease, chronic liver disease, acute bleeding, chronic bleeding, infection, hemoglobinopathies, drug use, alcohol abuse, Churg-Strauss syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplant, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schönlein purpura, Schwachman syndrome, advanced age, contraindications to blood transfusion, surgery, trauma, wound, ulcer, urinary tract bleeding, gastrointestinal bleeding, frequent blood donations, or heavy menstrual bleeding.

[0356] [Appendix 109] The method of Appendices 107 and 108, wherein the anemia is aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, anemia of chronic disease, anemia associated with bone marrow disease, hemolytic anemia, sickle cell anemia, microcytic anemia, hypochromic anemia, sideroblastic anemia, Diamond-Blackfan anemia, Fanconi anemia, or refractory anemia with excess blasts.

[0357] [Appendix 110] The method of any one of Appendices 100 to 109, wherein the subject does not respond well to treatment with erythropoietin (EPO) or is sensitive to the side effects of EPO.

[0358] [Appendix 111] The method of any one of Appendices 100 to 110, wherein the method increases erythropoiesis, red blood cell count, hematocrit, or hemoglobin levels. [Appendix 112] The method of any one of Appendices 100 to 111, wherein the method reduces the subject's need for blood transfusions.

[0359] [Appendix 113] A method of preventing pulmonary hypertension (PH) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1 to 40, a nucleic acid molecule described in Appendix 41, a vector described in Appendix 42, or a pharmaceutical composition described in Appendix 45 or Appendix 46.

[0360] [Appendix 114] A method of slowing or inhibiting the progression of PH in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide according to any one of Appendices 1 to 40, a nucleic acid molecule according to Appendices 41, a vector according to Appendices 42, or a pharmaceutical composition according to Appendices 45 or 46.

[0361] [Appendix 115] A method of treating a subject having or at risk of developing PH, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of Appendices 1-40, a nucleic acid molecule of Appendices 41, a vector of Appendices 42, or a pharmaceutical composition of Appendices 45 or 46.

[0362] [Appendix 116] A method of affecting at least one of myostatin, activin, and BMP9 signaling in a subject having or at risk of developing PH, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 1-40, a nucleic acid molecule described in Appendices 41, a vector described in Appendices 42, or a pharmaceutical composition described in Appendices 45 or 46.

[0363] [Appendix 117] The method of any one of Appendices 113 to 116, wherein the PH is pulmonary arterial hypertension (PAH). [Appendix 118] The method described in Appendix 117, wherein the PAH is idiopathic PAH.

[0364] [Appendix 119] The method described in Appendix 117, wherein the PAH is hereditary PAH. [Appendix 120] The method of Appendices 117, wherein the PAH is associated with HIV infection, schistosomiasis, cirrhosis, congenital heart abnormalities, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, connective tissue disorders, autoimmune disorders, or drug use or abuse.

[0365] [Appendix 121] The method according to any one of Appendices 113 to 116, wherein the PH is venous PH. [Appendix 122] The method of Appendices 121, wherein the venous PH is associated with left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital or acquired pulmonary vein stenosis.

[0366] [Appendix 123] The method described in any one of Appendices 113 to 116, wherein the PH is hypoxic PH. [Appendix 124] The method of Appendices 123, wherein the hypoxic PH is associated with chronic obstructive pulmonary disease, interstitial lung disease, sleep-disordered breathing, pulmonary fibrosis, alveolar hypoventilation disorder, chronic exposure to high altitude, or a developmental abnormality.

[0367] [Appendix 125] The method according to any one of Appendices 113 to 116, wherein the PH is thromboembolic PH. [Appendix 126] The method of Appendices 125, wherein the thromboembolic PH is associated with chronic thromboembolic pulmonary hypertension, pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary stenosis, or parasitic infection.

[0368] [Appendix 127] The method described in any one of Appendices 113 to 116, wherein the PH is another PH. [Appendix 128] The method of Appendices 127, wherein the other PH is associated with a blood disorder, a systemic disease, a metabolic disorder, pulmonary tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension.

[0369] [Appendix 129] The method of any one of Appendices 113-128, wherein the method reduces the frequency or severity of one or more symptoms of PH. [Appendix 130] The method of any one of Appendices 113 to 129, wherein the method reduces pulmonary vascular remodeling or vascular remodeling in the subject's heart.

[0370] [Appendix 131] The method of any one of Appendices 113 to 130, wherein the method reduces right ventricular hypertrophy. [Appendix 132] The method of any one of Appendices 113 to 131, wherein the method reduces pulmonary vascular resistance.

[0371] [Appendix 133] The method of any one of Appendices 113 to 132, wherein the method improves performance in a 6-minute walk test. [Appendix 134] The method of any one of Appendices 47 to 133, wherein the method reduces or inhibits binding of at least one of activin and myostatin to their receptors.

[0372] [Appendix 135] The method of any one of Appendices 47-57 and 134, wherein the polypeptide, nucleic acid, vector, or pharmaceutical composition is administered in an amount sufficient to increase at least one of muscle mass and muscle strength, to affect signaling of at least one of myostatin, activin, and BMP9 in a subject, or to reduce or inhibit binding of at least one of activin and myostatin to their receptors.

[0373] [Appendix 136] The method of any one of Appendices 58-91 and 134, wherein the polypeptide, nucleic acid, vector, or pharmaceutical composition is administered in an amount sufficient to increase bone mineral density, reduce bone resorption, reduce the rate of bone resorption, increase bone formation, increase the rate of bone formation, reduce osteoclast activity, increase osteoblast activity, increase bone strength, reduce the risk of fracture, affect signaling of at least one of myostatin, activin, and BMP9 in a subject, or reduce or inhibit binding of at least one of activin and myostatin to their receptors.

[0374] [Appendix 137] The method of any one of Appendices 92-99 and 134, wherein the polypeptide, nucleic acid, vector, or pharmaceutical composition is administered in an amount sufficient to reduce fibrosis, prevent fibrosis, reduce the risk of developing fibrosis, delay the onset of fibrosis, delay or inhibit the progression of fibrosis, treat fibrosis, reduce one or more symptoms of fibrosis, improve fibrotic tissue or organ function, affect signaling of at least one of myostatin, activin, and BMP9 in a subject, or reduce or inhibit binding of at least one of activin and myostatin to their receptors.

[0375] [Appendix 138] The method of any one of Appendices 100-112 and 134, wherein the polypeptide, nucleic acid, vector, or pharmaceutical composition is administered in an amount sufficient to increase red blood cell levels, increase hemoglobin levels, increase erythropoiesis, increase red blood cell count, increase hematocrit, reduce the need for blood transfusions, treat anemia, affect signaling of at least one of myostatin, activin, and BMP9 in the subject, or reduce or inhibit binding of at least one of activin and myostatin to their receptors.

[0376] [Appendix 139] The method of any one of Appendices 113-134, wherein the polypeptide, nucleic acid, vector, or pharmaceutical composition is administered in an amount sufficient to prevent PH, reduce the risk of developing PH, reduce the severity or frequency of one or more symptoms of PH, delay the onset of PH, delay or inhibit the progression of PH, treat PH, reduce pulmonary vascular remodeling, reduce cardiac vascular remodeling, reduce right ventricular hypertrophy, reduce pulmonary vascular resistance, improve performance in a 6-minute walk test, affect signaling of at least one of myostatin, activin, and BMP9 in a subject, or reduce or inhibit binding of at least one of activin and myostatin to their receptors.

[0377] [Appendix 140] The method of any one of Appendices 47 to 139, wherein the method does not cause vascular complications in the subject. [Appendix 141] The method of Appendices 140, wherein the method does not increase vascular permeability or leakage.

Claims

1. Sequence of sequence number 17: GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT A polypeptide comprising an extracellular activin IIB receptor (ActRIIB) variant having one or more amino acid substitutions compared to the above, wherein the variant comprises amino acid substitutions I11L, L27V, Q34K, T50S, L53I, Q69D, E70T, E75K, and F89M.

2. The polypeptide according to claim 1, wherein the variant further comprises the amino acid substitution I51L.

3. The polypeptide according to claim 1 or 2, further comprising an Fc domain monomer, an Fc domain, an albumin-binding peptide, a fibronectin domain, or human serum albumin, wherein these are fused to the C-terminus of the polypeptide via a linker.

4. The polypeptide according to claim 3, wherein the polypeptide comprises an Fc domain monomer fused to the C-terminus of the polypeptide via a linker.

5. The polypeptide according to claim 4, wherein the Fc domain monomer is a human IgG1 Fc domain monomer.

6. The polypeptide according to claim 4 or 5, wherein the polypeptide is in the form of a homodimer.

7. The linker is GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 20), GGGS (SEQ ID NO: 21), GGGG (SEQ ID NO: 22), GGGGA (SEQ ID NO: 23), GGGGS (SEQ ID NO: 24), GGGGG (SEQ ID NO: 25), GGAG (SEQ ID NO: 26), GGSG (SEQ ID NO: 27), AGGG (SEQ ID NO: 28), SGGG (SEQ ID NO: 29), GAGA (SEQ ID NO: 30), GSGS (SEQ ID NO: 31), GAGAGA (SEQ ID NO: 32), GSGSGS (SEQ ID NO: 33), GAGAGAGA (SEQ ID NO: 34), GSGSGSGS (SEQ ID NO: 30) 35) GAGAGAGAGA (SEQ ID NO: 36), GSGSGSGSGS (SEQ ID NO: 37), GAGAGAGAGAGA (SEQ ID NO: 38), GSGSGSGSGSGS (SEQ ID NO: 39), GGAGGA (SEQ ID NO: 40), GGGSGS (SEQ ID NO: 41), GGAGGAGGA (SEQ ID NO: 42), GGGSGGGSGS (SEQ ID NO: 43), GGAGGAGGGAGGA (SEQ ID NO: 44), GGGSGGGSGSGS (SEQ ID NO: 45), GGAGGGAG (SEQ ID NO: 46), GGGSGGGSGS (SEQ ID NO: 47), GGAGGGGAGGGGAG (SEQ ID NO: 48), GG SGGGGGGGSG (SEQ ID NO: 49), GGGGAGGGGGAGGGGA (SEQ ID NO: 50), GGGGGSGGGGGGGGGS (SEQ ID NO: 51), GGGAG (SEQ ID NO: 52), GGGAGG (SEQ ID NO: 53), GGGAGGG (SEQ ID NO: 54), AAAL (SEQ ID NO: 55), AAAK (SEQ ID NO: 56), AAAR (SEQ ID NO: 57), EGKSSGGSESKST (SEQ ID NO: 58), GSAGSAAGSGEF (SEQ ID NO: 59), AEAAAAKEAAAKA (SEQ ID NO: 60), KESGSVSSEQLAQFRSLD (SEQ ID NO: 61), GENLY A polypeptide according to any one of claims 3 to 6, having the sequence FQSGG (SEQ ID NO: 62), SACYCELS (SEQ ID NO: 63), RSIAT (SEQ ID NO: 64), RPACKIPNDLKQKVMNH (SEQ ID NO: 65), GGSAGGSGSGSGSGSGSGSAGASGTGTTAGGTGGSGSGSGTGS (SEQ ID NO: 66), AAANSSIDLISVPVDSR (SEQ ID NO: 67), GGSGGGGSEGGGGSEGGGGSEGGGGSEGGGGSEGGGGGS (SEQ ID NO: 68), EAAAK (SEQ ID NO: 69), or PAPAP (SEQ ID NO: 70).

8. A nucleic acid molecule encoding the polypeptide according to any one of claims 1 to 7.

9. A vector comprising the nucleic acid molecule described in Claim 8.

10. A host cell expressing the nucleic acid molecule described in Claim 8.

11. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 7, a nucleic acid molecule according to claim 8, or a vector according to claim 9, and one or more pharmaceutically acceptable carriers or excipients.

12. A pharmaceutical composition for treating a subject having or at risk of developing a bone disease, comprising a therapeutically effective amount of a polypeptide according to any one of claims 1 to 7, a nucleic acid molecule according to claim 8, or a vector according to claim 9.

13. The pharmaceutical composition according to claim 12, wherein the bone disease is osteoporosis, osteopenia, osteopetrosis, fracture, bone cancer or cancer metastasis-related bone loss, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, treatment-related bone loss for obesity, low gravity-related bone loss, or immobility-related bone loss.

14. A pharmaceutical composition for treating a subject having or at risk of developing pulmonary hypertension (PH), comprising a therapeutically effective amount of a polypeptide according to any one of claims 1 to 7, a nucleic acid molecule according to claim 8, or a vector according to claim 9.

15. The pharmaceutical composition according to claim 14, wherein the PH is pulmonary hypertension (PAH), venous PH, hypoxic PH, thromboembolic PH, or other PH.

16. A pharmaceutical composition for treating a subject having fibrosis or being at risk of developing it, comprising a therapeutically effective amount of a polypeptide according to any one of claims 1 to 7, a nucleic acid molecule according to claim 8, or a vector according to claim 9.

17. The pharmaceutical composition according to claim 16, wherein the fibrosis is chemotherapy-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis, corneal fibrosis, cardiac fibrosis, myelofibrosis, mediastinal fibrosis, retroperitoneal fibrosis, osteoarthritis fibrosis, articular fibrosis, histological fibrosis, tumor stroma, fibroplastic tumor, surgical adhesions, hypertrophic scars, keloids, or fibrosis associated with wounds, burns, hepatitis B or C infection, fatty liver disease, schistosomiasis infection, kidney disease, chronic kidney disease, heart disease, macular degeneration, retinal or vitreoretinopathy, Crohn's disease, systemic or focal scleroderma, atherosclerosis, or restenosis.

18. The pharmaceutical composition according to claim 17, wherein the tissue fibrosis is a fibrosis that acts on tissue selected from the group consisting of muscle tissue, epidermis, dermis, tendons, cartilage, pancreatic tissue, uterine tissue, nerve tissue, testes, ovaries, adrenal glands, arteries, veins, colon, small intestine, large intestine, biliary tract, and intestine.

19. A pharmaceutical composition for treating a subject having or at risk of developing a disease or condition accompanied by muscle weakness or muscle atrophy, comprising a therapeutically effective amount of a polypeptide according to any one of claims 1 to 7, a nucleic acid molecule according to claim 8, or a vector according to claim 9.

20. The pharmaceutical composition according to claim 19, wherein the subject has Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia, or cancer cachexia.

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