Activin receptor IIB variants and methods of use thereof

Abstract

The present invention relates to activin receptor IIB type variants and methods of use thereof. The present invention features a polypeptide comprising an extracellular ActRIIB variant. In some embodiments, the polypeptides of the invention comprise extracellular ActRIIB variants fused to an Fc domain monomer or part. The invention also features pharmaceutical compositions and methods of using the polypeptides to treat diseases and conditions involving myasthenia and atrophy, bone injury, low red blood cell levels (e.g., anemia or blood loss), fibrosis, and / or pulmonary hypertension.

Description

[0001] This application is a divisional application of the patent application for invention titled "Activin Receptor Type IIB Variants and Methods of Use Thereof" with the international application number PCT / US2019 / 013329, an international filing date of January 11, 2019, and a Chinese application number 201980018774.8.

[0002] Technical Field and Background Art Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), and amyotrophic lateral sclerosis (ALS) are examples of muscle diseases that involve muscle weakness and atrophy and / or 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 calves. IBM is an inflammatory muscle disease that primarily affects the thigh muscles and the arm muscles that control finger and wrist flexion. ALS is a motor neuron disease characterized by generalized muscle stiffness, muscle twitching, and muscle atrophy due to motor neuron degeneration. Efforts to improve the treatment and survival of subjects with these devastating muscle diseases have not been successful.

[0003] Healthy bone undergoes continuous remodeling that involves both bone resorption and bone growth. Bone growth is mediated by osteoblast cell types, while osteoclasts resorb bone. Pathological conditions occur when these systems are out of balance through downregulation of anabolic programs, upregulation of catabolic systems, or a combination of both, resulting in net bone loss. Thus, controlling the balance of bone remodeling can be used to promote the healing of bone injuries and the treatment of conditions such as osteoporosis that are associated with bone mass loss and bone demineralization.

[0004] Bone injuries can result from a range of underlying causes, including age- or cancer-related bone loss, genetic conditions, or adverse side effects of drug therapies. 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 bone injury. In general, overall bone loss represents a pathological state for which there are few effective treatments. Instead, treatment focuses on immobilization, exercise, and dietary modulation rather than on agents that directly promote bone growth and increase bone density. In the case of osteoporosis, estrogen, calcitonin, osteocalcin with vitamin K, or high-dose dietary calcium can all be used as treatment interventions. Other treatment methods for osteoporosis include bisphosphonates, parathyroid hormone, parathyroid hormone-related protein (PTHrP), calcimimetics, statins, anabolic steroids, lanthanum and strontium salts, and sodium fluoride. However, such therapeutic agents are often associated with undesirable side effects.

[0005] Fibrosis is the formation of excessive connective tissue in an organ or tissue. Connective tissue that can form in response to injury (such as damage) or as part of an immune response (such as an inflammatory response) can disrupt the structure and function of the organ or tissue in which it forms, leading to an increase in tissue stiffness. Fibrosis can occur in many organs and tissues in the body, particularly including the lungs (such as pulmonary fibrosis, cystic fibrosis), liver (such as cirrhosis), heart (such as endomyocardial fibrosis or post-myocardial infarction fibrosis), brain (such as glial scar formation), skin (such as keloid formation), kidneys (such as renal fibrosis), and eyes (such as corneal fibrosis); and is known to be associated with certain medical treatments (such as chemotherapy, radiotherapy, and surgery). There are limited treatment options for patients with fibrosis, and most treatments focus on improving quality of life or temporarily slowing disease progression.

[0006] Anemia is a global health problem with health implications 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 cognitive decline, and children, pregnant women, women of childbearing age, and the elderly have been found to have the highest risk of developing anemia. The most common form of anemia is iron-deficiency anemia, but anemia can also be caused by chronic diseases, blood loss, and red blood cell destruction. While iron-deficiency anemia can be treated with iron supplementation, 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 the heart. PH can be classified into five main types: 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 blood clots), or miscellaneous (PH with an unclear or multifactorial mechanism), also known as WHO classes I-V. PAH is characterized by an increase in pulmonary vascular pressure due to scarring, obstruction, or narrowing of small blood vessels in the lungs. This results in increased resistance to blood flow through the lungs and forces the right side of the heart to work harder, which can lead to heart failure, reduced blood oxygenation, and shortened life expectancy. PAH can be idiopathic (such as without an identifiable cause), hereditary (such as familial, often due to genetic mutations), or may be associated with drug use (such as methamphetamine or cocaine use), infections (such as HIV infection or schistosomiasis), cirrhosis, congenital heart abnormalities, or connective tissue / autoimmune disorders (such as scleroderma or lupus). Treatments for PH include vasodilators, anticoagulants, and supplemental oxygen, but these treatments manage the symptoms of the disease rather than targeting the biological mechanisms that cause the disease.

[0008] There is a need for new and effective treatments for muscle diseases, bone diseases, anemia, fibrosis, and PH. Summary of the Invention The present invention features polypeptides comprising an extracellular activin receptor type IIB (ActRIIB) variant. In some embodiments, the polypeptides of the present invention comprise an extracellular ActRIIB variant fused to the N-terminus or C-terminus of an Fc domain monomer or another moiety. Such moieties can be attached by amino acid or other covalent bonds and can increase the stability of the polypeptide. Polypeptides comprising an extracellular ActRIIB variant fused to an Fc domain monomer can also form dimers (e.g., homodimers or heterodimers) through the interaction between two Fc domain monomers. The polypeptides of the present invention can be used to increase muscle weight and strength in a subject having a disease or condition or at risk of developing a disease or condition 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 also be used to increase bone mass or bone mineral density in a subject having a disease or condition or at risk of developing a disease or condition involving bone injury, such as primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, fractures, bone cancer, or bone loss associated with cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, obesity treatment-related bone loss, low gravity-related bone loss, or immobilization. Additionally, the polypeptides of the present invention can 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, such as a subject having anemia or blood loss or at risk of developing anemia or blood loss, to prevent or reduce fibrosis in a subject having fibrosis or at risk of developing fibrosis, or to treat, prevent, or delay the development or progression of pulmonary hypertension in a subject having pulmonary hypertension (e.g., arterial, venous, hypoxic, thromboembolic, or miscellaneous pulmonary hypertension) or at risk of developing pulmonary hypertension. Further, the polypeptides of the present invention can also be used to affect myostatin, activin, and / or bone morphogenetic protein 9 (BMP9) signaling in a subject having a risk of developing a disease or condition or having a disease or condition involving muscle weakness and atrophy, bone injury or bone 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 miscellaneous pulmonary hypertension).

[0010] In a first aspect, the invention features a polypeptide comprising an extracellular ActRIIB variant having one or more amino acid substitutions relative to the sequence of wherein the variant 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 the following: (a) the amino acid substitution E75K; (b) the amino acid substitutions Q69T and E70D; or (c) the amino acid substitutions Q69D and E70T.

[0011] In some embodiments, the one or more additional amino acid substitutions are selected from 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.

[0013] 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 are T74K, E76D, N77S, and Q79E.

[0014] In some embodiments, the variant further comprises one or more additional amino acid substitutions.

[0015] In some embodiments, the variant comprises the additional amino acid substitutions Y41F, R45K, and K56Q. In some embodiments, the variant further comprises the additional amino acid substitutions Y12F, L19K, E20D, R29P, E31Y, E33D, L38R, and F63I.

[0016] 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.

[0017] In some embodiments, the variant comprises the additional amino acid substitution R29P.

[0018] In some embodiments, the variant comprises the additional amino acid substitutions E31Y, E33D, and Q34K.

[0019] In some embodiments, the variant contains additional amino acid substitutions Y12F, L19K, and E20D.

[0020] In some embodiments, the variant contains additional amino acid substitutions E31Y, E33D, and L38R.

[0021] In some embodiments, the variant contains the amino acid substitutions Q69T and E70D, and additional amino acid substitutions I11L, L27V, Q34K, T50S, I51L, L53I, and F89M.

[0022] In some embodiments, the variant contains the amino acid substitutions Q69D and E70T, and additional amino acid substitutions I11L, L27V, Q34K, T50S, I51L, L53I, and F89M.

[0023] In some embodiments, the variant further contains the amino acid substitution E75K.

[0024] The following embodiments E1 to E59 describe other features of the present invention.

[0025] E1. A polypeptide comprising an ActRIIB variant having the following sequence: wherein X 1 is I or L; X 2 is F or Y; X 3 is L or K; X 4 is D or E; X 5 is T or S; X 6 is L or V; X 7 is P or R; X 8 is Y or E; X 9 is D or E; X 10 is K or Q; X 11 is 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 26is S or N; X 27 is E or Q; and X 28 is F or M, and wherein 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.

[0026] A variant of E2.E1, wherein X 1 is I.

[0027] A variant of E3.E1, wherein X 1 is L.

[0028] A variant of E4.E1 - any one of E3, wherein X 2 is F.

[0029] A variant of E5.E1 - any one of E3, wherein X 2 is Y.

[0030] A variant of E6.E1 - E5, wherein X 3 is L.

[0031] A variant of E7.E1 - E5, wherein X 3 is K.

[0032] A variant of E8.E1 - E7, wherein X 4 is D.

[0033] A variant of E9.E1 - E7, wherein X 4 is E.

[0034] A variant of E10.E1 - E9, wherein X 5 is T.

[0035] A variant of E11.E1 - E9, wherein X 5 is S.

[0036] A variant of E12.E1 - E11, wherein X 6 is L.

[0037] A variant of E13.E1 - E11, wherein X 6 is V.

[0038] A variant of E14.E1 - E13, wherein X 7 is P.

[0039] A variant of E15.E1 - E13, wherein X 7 is R.

[0040] A variant of any one of E16.E1 - E15, where X 8 is Y.

[0041] A variant of any one of E17.E1 - E15, where X 8 is E.

[0042] A variant of any one of E18.E1 - E17, where X 9 is D.

[0043] A variant of any one of E19.E1 - E17, where X 9 is E.

[0044] A variant of any one of E20.E1 - E19, where X 10 is K.

[0045] A variant of any one of E21.E1 - E19, where X 10 is Q.

[0046] A variant of any one of E22.E1 - E21, where X 11 is R.

[0047] A variant of any one of E23.E1 - E21, where X 11 is L.

[0048] A variant of any one of E24.E1 - E23, where X 12 is Y.

[0049] A variant of any one of E25.E1 - E23, where X 12 is F.

[0050] A variant of any one of E26.E1 - E25, where X 13 is R.

[0051] A variant of any one of E27.E1 - E25, where X 13 is K.

[0052] A variant of any one of E28.E1 - E27, where X 14 is S.

[0053] A variant of any one of E29.E1 - E27, where X 14 is I.

[0054] A variant of any one of E30.E1 - E29, where X 15 is S.

[0055] A variant of any one of E31.E1 - E29, where X 15 is T.

[0056] A variant of any one of E32.E1 - E31, where X 16 is S.

[0057] A variant of any one of E33.E1 - E31, where X 16 is T.

[0058] A variant of any one of E34.E1 - E33, where X 17 is I.

[0059] A variant of any one of E35.E1 - E33, where X 17 is L.

[0060] A variant of any one of E36.E1 - E35, where X 18 is I.

[0061] A variant of any one of E37.E1 - E35, where X 18 is L.

[0062] A variant of any one of E38.E1 - E37, where X 19 is K.

[0063] A variant of any one of E39.E1 - E37, where X 19 is Q.

[0064] A variant of any one of E40.E1 - E39, where X 20 is F.

[0065] A variant of any one of E41.E1 - E39, where X 20 is I.

[0066] A variant of any one of E42.E1 - E41, where X 21 is Q.

[0067] A variant of any one of E43.E1 - E41, where X 21 is T.

[0068] A variant of any one of E44.E1 - E41, where X 21 is D.

[0069] A variant of any one of E45.E1 - E42, where X 22 is E.

[0070] A variant of any one of E46.E1 - E41 and E43, where X 22It is D.

[0071] A variant of any one of E47.E1 - E41 and E44, where X 22 is T.

[0072] A variant of any one of E48.E1 - E47, where X 23 is K.

[0073] A variant of any one of E49.E1 - E47, where X 23 is T.

[0074] A variant of any one of E50.E1 - E49, where X 24 is K.

[0075] A variant of any one of E51.E1 - E41, E43, E44, E46 and E47 - E49, where X 24 is E.

[0076] A variant of any one of E52.E1 - E51, where X 25 is D.

[0077] A variant of any one of E53.E1 - E51, where X 25 is E.

[0078] A variant of any one of E54.E1 - E53, where X 26 is S.

[0079] A variant of any one of E55.E1 - E53, where X 26 is N.

[0080] A variant of any one of E56.E1 - E55, where X 27 is E.

[0081] A variant of any one of E57.E1 - E55, where X 27 is Q.

[0082] A variant of any one of E58.E1 - E57, where X 28 is F.

[0083] A variant of any one of E59.E1 - E57, where X 28 is M.

[0084] In some embodiments, the variant has a sequence of any one of SEQ ID NO: 2 - 15.

[0085] 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.

[0086] 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.

[0087] In some embodiments, the polypeptide further comprises an Fc domain having an amino acid substitution, the Fc domain being 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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).

[0092] 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), GSGSGSGS (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), GGSGGGSG (SEQ ID NO:47), GGAGGGAGGGAG (SEQ ID NO:48) and GGSGGGSGGGSG (SEQ ID NO:49), GGGGGAGGGGAGGGGA (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), GENLYFQSGG (SEQ ID NO:62), SACYCELS (SEQ ID NO:63), RSIAT (SEQ ID NO:64), RPACKIPNDLKQKVMNH (SEQ IDNO: 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).

[0093] In some embodiments, the polypeptide has a serum half-life of at least seven days.

[0094] In some embodiments, compared to wild-type ActRIIB, the polypeptide has increased or decreased binding to one or more ActRIIB ligands (such as activin, myostatin, GDF-11, or BMP9).

[0095] In some embodiments, the polypeptide binds to human bone morphogenetic protein 9 (BMP9) with a K D of 200 pM or higher.

[0096] In some embodiments, the polypeptide binds to activin and / or myostatin and has reduced or weak binding to human BMP9.

[0097] In some embodiments, the polypeptide binds substantially not to human BMP9.

[0098] In some embodiments, the polypeptide binds to human activin A with a K D of 800 pM or less.

[0099] In some embodiments, the polypeptide binds to human activin B with a K D of 800 pM or less.

[0100] In some embodiments, the polypeptide binds to human GDF-11 with a K D of 5 pM or higher.

[0101] In another aspect, the invention features a nucleic acid molecule encoding a polypeptide described herein (such as a polypeptide including an extracellular ActRIIB variant having any one of the sequences of SEQ ID NO: 1-15 (such as SEQ ID NO: 2-15)).

[0102] In another aspect, the invention features a vector comprising the nucleic acid molecule described herein.

[0103] In another aspect, the invention features a host cell expressing the polypeptide described herein, wherein the host cell comprises the nucleic acid molecule or vector described in the first two aspects, and the nucleic acid molecule or vector is expressed in the host cell.

[0104] In another aspect, the invention features a method of preparing the polypeptide described herein, the method comprising: a) providing a host cell comprising the nucleic acid molecule or vector described herein, and b) expressing the nucleic acid molecule or vector in the host cell under conditions that permit the formation of the polypeptide.

[0105] In another aspect, the invention features a pharmaceutical composition comprising the 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.

[0106] In another aspect, the invention features a construct (e.g., a homodimer) comprising two identical polypeptides, each polypeptide comprising an extracellular ActRIIB variant described herein (e.g., an ActRIIB variant having any one of the sequences 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.

[0107] In another aspect, the invention features a construct (e.g., a heterodimer) comprising two different polypeptides, each polypeptide comprising an extracellular ActRIIB variant described herein (e.g., an ActRIIB variant having any one of the sequences 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.

[0108] In another aspect, the invention features a method of increasing lean body mass in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or vector described herein, or the pharmaceutical composition described herein.

[0109] In another aspect, the invention features a method of increasing muscle mass in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of the polypeptide, nucleic acid molecule or vector described herein, or the pharmaceutical composition described herein.

[0110] In some embodiments of methods for increasing lean body mass or muscle mass in a subject, the subject has or is at risk of developing the following diseases: Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia.

[0111] In another aspect, the invention features methods of treating a subject having or at risk of developing a muscle disease. The method includes 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.

[0112] In another aspect, the invention features methods of 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 involving muscle weakness and atrophy, wherein the method includes 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.

[0113] In another aspect, the invention features a method of treating a subject having DMD 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.

[0114] In another aspect, the invention features a method of treating a subject having FSHD 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.

[0115] In another aspect, the invention features a method of treating a subject having IBM 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.

[0116] In another aspect, the invention features a method of treating a subject having ALS 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.

[0117] In another aspect, the invention features a method of treating a subject having sarcopenia 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.

[0118] In another aspect, the invention features a method of treating a subject having cancer cachexia 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.

[0119] In another aspect, the invention features a method of increasing bone mineral density in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0120] In another aspect, the invention features a method of reducing bone resorption in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0121] In another aspect, the invention features a method of increasing bone formation in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0122] In another aspect, the invention features a method of increasing bone strength in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0123] In another aspect, the invention features a method of reducing the risk of fracture in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0124] In some embodiments of any of the above aspects, the subject has or is at risk of developing: primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, fracture, bone cancer or bone loss associated with cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, obesity treatment-related bone loss, low gravity-related bone loss, or immobilization-related bone loss. In some embodiments, the subject has osteoporosis or is at risk of developing osteoporosis.

[0125] In another aspect, the invention features a method of treating a subject having or at risk of developing a bone disease. The method includes 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, osteosclerosis, fracture, bone cancer or bone loss associated with cancer metastasis, 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 immobilization-related bone loss. In some embodiments, the bone disease is osteoporosis.

[0126] In another aspect, the invention features a method of affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of myostatin, activin, and / or BMP9 to its endogenous receptor) in a subject having or at risk of developing a disease or condition involving bone injury, wherein the method includes 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, osteosclerosis, fracture, bone cancer or bone loss associated with cancer metastasis, 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 immobilization-related bone loss. In some embodiments, the bone disease or condition is osteoporosis.

[0127] In another aspect, the invention features 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.

[0128] In another aspect, the invention features 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.

[0129] In another aspect, the invention features 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.

[0130] In another aspect, the invention features a method of treating a subject having a fracture 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.

[0131] In another aspect, the invention features a method of treating a subject having bone cancer or bone loss associated with cancer metastasis or at risk of developing bone cancer or bone loss associated with cancer metastasis 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.

[0132] In another aspect, the invention features a method of treating a subject having Paget's disease 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.

[0133] In another aspect, the invention features a method of treating a subject having renal osteodystrophy 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.

[0134] In another aspect, the invention features a method of treating a subject having treatment-related bone loss 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.

[0135] In another aspect, the invention features a method of treating a subject having diet-related bone loss 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.

[0136] In another aspect, the invention features a method of treating a subject having low gravity-related bone loss or at risk of developing low gravity-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.

[0137] In another aspect, the invention features a method of treating a subject having immobilization-related bone loss or at risk of developing immobilization-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.

[0138] In some embodiments of any of the above aspects, primary osteoporosis is age-related osteoporosis or hormone-related osteoporosis.

[0139] In some embodiments of any of the above aspects, secondary osteoporosis is immobilization-induced osteoporosis or glucocorticoid-induced osteoporosis.

[0140] In some embodiments of any of the above aspects, the cancer is multiple myeloma.

[0141] In some embodiments of any of the above aspects, the treatment is FGF-21 treatment, GLP-1 treatment, cancer therapy, or treatment for obesity or type 2 diabetes.

[0142] In some embodiments of any of the above aspects, diet-related bone loss is rickets.

[0143] In some embodiments of any of the above aspects, the subject is at risk of fracture.

[0144] 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 generation. In some embodiments of any of the above aspects, the method reduces osteoclast activity or reduces osteoclast generation. 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.

[0145] 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.

[0146] In another aspect, the invention features 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.

[0147] In another aspect, the invention features 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.

[0148] In another aspect, the invention features 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.

[0149] In another aspect, the invention features a method of treating a subject having fibrosis 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.

[0150] In some embodiments of any of the above aspects, the fibrosis is chemotherapy drug-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis, myelofibrosis, mediastinal fibrosis, retroperitoneal fibrosis, articular fibrosis, osteoarticular fibrosis, tissue fibrosis, tumor stroma, fibroproliferative tumor, surgical adhesions, hypertrophic scar, or keloid.

[0151] In some embodiments of the above aspects, the fibrosis is fibrosis associated with: wound, 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 vitreoretinal disease, systemic or localized scleroderma, atherosclerosis, or restenosis. In some embodiments of any of the above aspects, the fibrosis results from chronic kidney disease.

[0152] In another aspect, the invention features a method of affecting myostatin, activin, and / or BMP9 signaling in a subject (e.g., reducing or inhibiting the binding of myostatin, activin, and / or BMP9 to their endogenous receptors), the subject having fibrosis or at risk of developing fibrosis, or having a disease or condition involving fibrosis or at risk of developing a disease or condition involving 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 this aspect, the disease or condition is chemotherapy drug-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, hepatic fibrosis, renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis, myelofibrosis, mediastinal fibrosis, retroperitoneal fibrosis, articular fibrosis, osteoarticular fibrosis, tissue fibrosis, tumor stroma, fibroproliferative tumor, surgical adhesions, hypertrophic scar, or keloid. In some embodiments of this aspect, the disease or condition is fibrosis associated with: wound, 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 vitreoretinal disease, systemic or localized scleroderma, atherosclerosis, or restenosis. In some embodiments of this aspect, the fibrosis results from chronic kidney disease.

[0153] In some embodiments of any of the above aspects, the tissue fibrosis is fibrosis affecting a tissue selected from: muscle tissue, skin epidermis, skin dermis, tendon, cartilage, pancreatic tissue, uterine tissue, nerve tissue, testis, ovary, adrenal gland, artery, vein, colon, small intestine, large intestine, biliary tract, and intestine.

[0154] In some embodiments of any of the above aspects, the method improves the function of 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 (e.g., reduces its frequency or severity) one or more symptoms of fibrosis.

[0155] In another aspect, the invention features a method of increasing the level of red blood cells (e.g., increasing hemoglobin level, 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.

[0156] In another aspect, the invention features 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.

[0157] In some embodiments of any of the above aspects, the subject has anemia or blood loss or is at risk of developing anemia or blood loss.

[0158] In another aspect, the invention features a method of affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of myostatin, activin, and / or BMP9 to its endogenous receptor) in a subject having a disease or condition or at risk of developing a disease or condition, wherein the disease or condition involves a low level of red blood cells (e.g., low hemoglobin level, low red blood cell count, or low hematocrit), and 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.

[0159] In some embodiments of the above aspects, the disease or condition is anemia or blood loss.

[0160] In some embodiments of any of the above aspects, anemia or blood loss is associated with: cancer, cancer treatment, kidney disease or failure (e.g., chronic kidney disease or acute kidney disease or failure), myelodysplastic syndromes, thalassemia, nutritional deficiencies, adverse reactions to drugs, inflammatory or autoimmune diseases, splenomegaly, porphyria, vasculitis, hemolysis, bone marrow defects, bone marrow transplantation, 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, Chus syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplantation, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schönlein purpura, Shwachman syndrome (e.g., Shwachman-Diamond syndrome), transfusion contraindications, surgery, trauma, wounds, ulcers, urinary tract bleeding, gastrointestinal bleeding, frequent blood donation or excessive menstrual bleeding.

[0161] In another aspect, the invention features a method of treating a subject having anemia 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, anemia is associated with: cancer, cancer treatment, kidney disease or failure (e.g., chronic kidney disease or acute kidney disease or failure), myelodysplastic syndromes, thalassemia, nutritional deficiencies, adverse reactions to drugs, inflammatory or autoimmune diseases, splenomegaly, porphyria, vasculitis, hemolysis, bone marrow defects, bone marrow transplantation, 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, Chus syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplantation, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schönlein purpura, Shwachman syndrome (e.g., Shwachman-Diamond syndrome), transfusion contraindications, surgery, trauma, wounds, ulcers, urinary tract bleeding, gastrointestinal bleeding, frequent blood donation or excessive menstrual bleeding.

[0162] In some embodiments of any of the above aspects, anemia results from chronic kidney disease.

[0163] In some embodiments of any of the above aspects, anemia is aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, anemia of chronic disease, anemia associated with bone marrow diseases, hemolytic anemia, sickle cell anemia, microcytic anemia, hypochromic anemia, sideroblastic anemia, Diamond-Blackfan anemia, Fanconi anemia, or refractory anemia with excess blasts.

[0164] In some embodiments of any of the above aspects, the subject does not respond well to treatment with erythropoietin (EPO) or is susceptible to adverse effects of EPO.

[0165] In some embodiments of any of the above aspects, the method increases erythropoiesis, red blood cell count, hemoglobin level, or hematocrit.

[0166] In some embodiments of any of the above aspects, the method reduces the need for blood transfusions in a subject.

[0167] In another aspect, the invention features a method for preventing 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.

[0168] In another aspect, the invention features a method for 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.

[0169] In another aspect, the invention features a method for 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.

[0170] In another aspect, the invention features a method for treating a subject having PH 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.

[0171] In another aspect, the invention features a method of 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 PH or at risk of developing PH, the disease or condition involving muscle weakness and atrophy, 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.

[0172] In another aspect, the invention features a method for reducing vascular remodeling in a subject having PH 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.

[0173] In another aspect, the invention features a method for reducing right ventricular hypertrophy in a subject having PH 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.

[0174] In another aspect, the invention features a method of reducing pulmonary vascular resistance in a subject having PH 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.

[0175] 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 heritable PAH. In some embodiments, 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 (such as scleroderma or lupus), or drug use or abuse (such as the use of cocaine or methamphetamine).

[0176] In some embodiments of any of the above aspects, the PH is venous PH. In some embodiments, 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.

[0177] 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 (such as emphysema), interstitial lung disease, sleep disordered breathing (such as sleep apnea), lung disease (such as pulmonary fibrosis), alveolar hypoventilation disorders, long-term exposure to high altitude, or developmental abnormalities.

[0178] 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 artery stenosis, or parasitic infection.

[0179] In some embodiments of any of the above aspects, the PH is miscellaneous PH. In some embodiments, the miscellaneous PH is associated with: hematologic disorders (such as chronic hemolytic anemia, sickle cell disease), systemic diseases (such as sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), metabolic disorders (such as glycogen storage disease, Gaucher disease, or thyroid disease), pulmonary tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension.

[0180] 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 frequency or severity of one or more of shortness of breath (dyspnea), fatigue, swelling in the legs, feet, abdomen (ascites), or neck (e.g., edema), chest pain or pressure, rapid or irregular heartbeat, bluish lips or skin (cyanosis), dizziness, or fainting).

[0181] In some embodiments of any of the above aspects, the method reduces pulmonary vascular remodeling.

[0182] In some embodiments of any of the above aspects, the method reduces vascular remodeling in the heart.

[0183] In some embodiments of any of the above aspects, the method reduces right ventricular hypertrophy.

[0184] In some embodiments of any of the above aspects, the method reduces pulmonary vascular resistance (e.g., reduces pulmonary vascular resistance as compared to measurements taken prior to treatment).

[0185] In some embodiments of any of the above aspects, the method improves performance in the 6-minute walk test (e.g., improves performance as compared to measurements taken prior to treatment).

[0186] In some embodiments of any of the above aspects, the method reduces or inhibits the binding of activin and / or myostatin to their receptors.

[0187] In some embodiments of any of the above aspects, the amount of polypeptide, nucleic acid, vector, or pharmaceutical composition administered is sufficient to increase muscle weight and / or strength, increase bone mineral density, reduce bone resorption, reduce bone loss, reduce the rate of bone resorption, increase bone formation, increase the rate of bone formation, reduce osteoclast activity, increase osteoblast activity, reduce the risk of fracture, increase bone strength, reduce fibrosis, prevent the development of fibrosis, delay the development of fibrosis, slow or inhibit the progression of fibrosis, reduce the risk of developing fibrosis, reduce one or more symptoms of fibrosis, improve the function of fibrotic tissue or organ, increase red blood cell levels, increase hemoglobin levels, increase hematocrit, reduce the need for transfusion, increase red blood cell formation, increase red blood cell count, treat anemia, increase prophylaxis against PH, reduce the risk of developing PH, reduce the severity or frequency of one or more symptoms of PH, delay the development of PH, slow 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 the 6-minute walk test, affect myostatin, activin, and / or BMP9 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 miscellaneous PH.

[0188] In some embodiments of any of the above aspects, the method does not cause vascular complications in a subject. In some embodiments, the method does not increase vascular permeability or leakage.

[0189] Definitions As used herein, the term "extracellular activin receptor type IIB (ActRIIB) variant" refers to a peptide comprising the soluble extracellular portion of the single transmembrane receptor ActRIIB, which has at least one amino acid substitution relative to wild-type extracellular ActRIIB (e.g., the bold 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), where the signal peptide is italicized and the extracellular portion is bold.

[0190] Wild-type human ActRIIB (SEQ ID NO: 18):

[0191] The extracellular ActRIIB variant can have the sequence of any one of SEQ ID NOs: 1-15. In certain embodiments, the extracellular ActRIIB variant has the sequence of any one of SEQ ID NOs: 2-15 (Table 2). In some embodiments, the extracellular ActRIIB variant can have at least 85% (such as at least 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or higher) amino acid sequence identity with the sequence of wild-type extracellular ActRIIB (SEQ ID NO: 17).

[0192] As used herein, the term "linker" refers to a connection between two elements (such as a peptide or protein domain). The polypeptides described herein can include an extracellular ActRIIB variant fused to a moiety (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)). The moiety can increase stability or improve the pharmacokinetic properties of the polypeptide. The moiety (e.g., an Fc domain monomer, wild-type Fc domain, Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), albumin-binding peptide, fibronectin domain, or human serum albumin) can be fused to the polypeptide by way of a linker. The linker can be a covalent bond or a spacer. The term "bond" refers to a chemical bond, such as an amide bond or a disulfide bond, or any kind of bond produced by a chemical reaction (e.g., chemical conjugation). The term "spacer" refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a 1-200 amino acid sequence) that exists between two elements (such as a peptide or protein domain) to provide space and / or flexibility between the two elements. The amino acid spacer is part of the primary sequence of the polypeptide (e.g., fused to the spaced peptide through 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.

[0193] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers. The Fc domain has at least 80% sequence identity with the human Fc domain comprising at least the C H 2 domain and the C H 3 domain (e.g., at least 85%, 90%, 95%, 97% or 100% sequence identity). The Fc domain monomer comprises the second and third antibody constant domains (C H 2 and C H3). In some embodiments, the Fc domain monomer further comprises a hinge domain. The Fc domain does not contain any part of an immunoglobulin that can act 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 dimerize through the interaction between two C H 3 antibody constant domains and one or more disulfide bonds formed between the hinge domains of two dimerized Fc domain monomers. In some embodiments, the Fc domain can be mutated to lack effector function, which is a typical "dead Fc domain". In certain embodiments, each Fc domain monomer in the Fc domain contains C H 2 antibody constant domains with amino acid substitutions that reduce the interaction or binding between the Fc domain and the Fcγ receptor. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. The Fc domain can be any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. Additionally, the Fc domain can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain can also be a non-naturally occurring Fc domain, such as a recombinant Fc domain.

[0194] As used herein, the term "albumin-binding peptide" refers to an amino acid sequence of 12 to 16 amino acids that has an affinity for serum albumin and the function of binding serum albumin. Albumin-binding peptides can be from different sources (e.g., human, mouse, or rat). In some embodiments, the albumin-binding peptide has the sequence DICLPRWGCLW (SEQ ID NO:72).

[0195] As used herein, the term "endogenous" describes a molecule (e.g., polypeptide, nucleic acid, or cofactor) that is naturally found in a particular organism (e.g., human) or a particular location within an organism (e.g., an organ, tissue, or cell, such as a human cell, such as a human hair cell).

[0196] As used herein, the term "fibronectin domain" refers to a high-molecular-weight glycoprotein of the extracellular matrix or a fragment thereof that binds, for example, transmembrane receptor proteins such as integrins and extracellular matrix components such as collagen and fibrin. In some embodiments, the fibronectin domain is the fibronectin type III domain of amino acids 610 - 702 of the sequence with UniProt ID NO:P02751 (SEQ ID NO:73). In other embodiments, the fibronectin domain is an adnectin protein.

[0197] As used herein, the term "human serum albumin" refers to albumin present in human plasma. Human serum albumin is the most abundant protein in blood. It constitutes approximately half of the serum proteins. In some embodiments, human serum albumin has the sequence of UniProt ID NO: P02768 (SEQ ID NO: 74).

[0198] As used herein, the term "fusion" is used to describe the combination or attachment 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 in a tandem series can be fused by means of chemical conjugation, chemical bonds, peptide linkers, or any other means of covalent attachment to form a continuous protein structure, such as 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: 1-15 (e.g., SEQ ID NOs: 2-15)) can be fused in a tandem series at the N- or C-terminus to 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 dimerization), 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)) via a linker. For example, an extracellular ActRIIB variant is fused to a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) via a peptide linker, wherein the N-terminus of the peptide linker is fused to the C-terminus of the extracellular ActRIIB variant by a chemical bond (e.g., a peptide bond), and the C-terminus of the peptide linker is fused to the moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) by a chemical bond (e.g., a peptide bond).

[0199] As used herein, the terms "bone mineral density (BMD)", "bone density", and "bone mass" refer to a measure of the amount of bone mineral (such as calcium) in bone tissue. BMD can be measured by well-established clinical techniques known to those of skill 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 per volume of bone, although clinically it is measured by proxy as the optical density per square centimeter of bone surface at the time of imaging. BMD measurements are used in clinical medicine as an indirect indicator of osteoporosis and fracture risk. In some embodiments, BMD test results are provided as a T-score, where the T-score 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 equal to the normal reference value for a healthy young adult. The difference between the measured BMD of the subject and the reference value for a healthy young adult is measured in standard deviation units (SD). Thus, a T-score between +1SD and -1SD can indicate normal BMD, a T-score between -1SD and -2.5SD can indicate low bone mass (e.g., osteopenia), and a T-score below -2.5SD can indicate osteoporosis or severe osteoporosis. In some embodiments, a polypeptide of the invention comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a 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, wherein the patient has low bone mass (e.g., a T-score between -1SD and -2.5SD). In some embodiments, a polypeptide of the invention comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a 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, wherein the patient has osteoporosis (e.g., a T-score less than -2.5SD). In some embodiments, administration of a polypeptide of the invention comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a 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 the subject by increasing their BMD.In some embodiments, administration of a polypeptide of the invention comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)), a nucleic acid encoding such polypeptide, or a vector containing such nucleic acid molecule increases the BMD of a subject, resulting in an increase in the T-score of the subject (e.g., resulting in an increase in the T-score of the subject by 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).

[0200] As used herein, the term "bone strength" refers to a measure of bone that is determined by bone quality in addition to bone mineral density. Bone quality is affected by bone geometry, microstructure, and properties of the constituent tissues. Bone strength can be used to evaluate the fracture risk of bone.

[0201] As used herein, the term "bone disease" refers to a condition characterized by bone damage (e.g., reduced bone mineral density, reduced bone strength, and / or bone loss). Such diseases or conditions may be caused by an imbalance in osteoblast and / or osteoclast activity (e.g., increased bone resorption or reduced bone formation). Bone diseases include primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, fractures, bone cancer or bone loss associated with 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 related to obesity treatment, low gravity-related bone loss, and immobilization-related bone loss.

[0202] As used herein, the term "bone remodeling" or "bone metabolism" refers to the process of maintaining bone strength and ion homeostasis by replacing discrete portions of old bone with newly synthesized protein matrix. Bone is resorbed by osteoclasts and deposited by osteoblasts in a process called ossification. Osteocyte activity plays a key role in this process. Conditions that result in reduced bone mass can be caused by increased resorption or reduced ossification. In healthy individuals, during childhood, bone formation exceeds resorption. As the aging process occurs, resorption exceeds formation. Due to estrogen deficiency associated with menopause, bone resorption rates are typically much higher in postmenopausal elderly women.

[0203] As used herein, the term "bone resorption" or "bone catabolic activity" refers to the process by which osteoclasts break down tissue in bone and release minerals, resulting in the transfer of minerals (e.g., calcium) from bone tissue to the blood. An increase in the rate of bone resorption is associated with aging (including postmenopausal women). A high rate of bone resorption, or a rate of bone resorption that exceeds the rate of ossification, is associated with skeletal disorders (such as reduced bone mineral density, including osteopenia and osteoporosis) and can lead to bone loss. In some embodiments, a polypeptide of the invention comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)), a nucleic acid encoding such polypeptide, or a vector containing such nucleic acid molecule is administered to a subject in need thereof to reduce bone resorption (e.g., reduce bone loss) (e.g., the amount or rate of bone resorption in the subject).

[0204] As used herein, the terms "bone formation", "ossification", "osteogenesis" or "bone anabolic activity" refer to the process by which osteoblasts form new bone tissue. In some embodiments, a polypeptide of the invention comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)), a nucleic acid encoding such polypeptide, or a vector containing such nucleic acid molecule is administered to a subject in need thereof to increase bone formation (e.g., increase the amount or rate of bone formation or osteogenesis in the subject). A reduced rate of bone formation or a rate of bone formation that is exceeded by the rate of bone resorption can lead to bone loss.

[0205] As used herein, the terms "increase" and "decrease" refer to a modulation that results in a greater or lesser amount, respectively, of a function, expression, or activity relative to a reference. For example, after administration of a polypeptide of the invention comprising an extracellular ActRIIB variant by the methods described herein, the amount of a marker (e.g., lean body mass) as described herein can be increased or decreased relative to the amount of the marker prior to administration. Generally, the measurement of the metric is made at a time after administration, at which time the administration has achieved the effect, e.g., at least one week, one month, 3 months, or 6 months after the start of a treatment regimen.

[0206] As used herein, the term "fibrosis" refers to the pathological process of excessive formation of fibrous connective tissue. Fibrosis is characterized by an accumulation of fibroblasts and collagen deposition in excess of normal deposition in any given tissue. In response to inflammation or tissue damage, nearby fibroblasts can migrate into the wound, proliferate, and produce large amounts of collagen extracellular matrix. When fibrosis occurs in response to damage, the term "scar" can be used as a synonym. 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, nerve tissue, testis, ovary, adrenal gland, artery, vein, colon, small intestine and large intestine, biliary tract, and intestine.

[0207] As used herein, the term "pulmonary hypertension" or "PH" refers to a disease characterized by an increase in blood pressure between the lungs and the heart, which can include an increase in blood pressure in the pulmonary artery (pulmonary arterial hypertension), pulmonary vein, or pulmonary capillary. Pulmonary hypertension can have a variety of symptoms, including shortness of breath (dyspnea), fatigue, swelling in the legs, feet, abdomen (ascites), or neck (e.g., edema), chest pain or pressure, rapid pulse or palpitations, blue lips or skin (cyanosis), dizziness or fainting. PH is also characterized by a reduced exercise tolerance and can lead to heart failure.

[0208] As used herein, the term "pulmonary arterial hypertension" or "PAH" refers to a form of pulmonary hypertension characterized by stenosis or obstruction in small pulmonary arteries, often caused by scarring, and an increase in pulmonary artery blood pressure. PAH is also known as WHO Group I PH. PAH can be diagnosed based on an increase in mean pulmonary artery blood pressure above 25 mmHg at rest, accompanied by a normal pulmonary artery capillary wedge pressure. PAH can cause shortness of breath, dizziness, fainting, and other symptoms, all of which are exacerbated by exertion. PAH can be a severe disease with significantly reduced exercise tolerance and heart failure. The two main types of PAH include idiopathic PAH (e.g., PAH in which no causative factor has been identified) and heritable PAH (e.g., PAH associated with mutations in BMPR2, ALK1, SMAD9, caveolin 1, KCNK3, or EIF2AK4). In 70% of familial PAH cases, the mutation is located in the BMPR2 gene. Risk factors for PAH development include a family history of PAH, drug use (e.g., methamphetamine or cocaine use), infection (e.g., HIV infection or schistosomiasis), cirrhosis, congenital heart abnormalities, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, or connective tissue / autoimmune disorders (e.g., scleroderma or lupus).

[0209] As used herein, the terms "pulmonary venous hypertension" and "venous PH" refer to a form of pulmonary hypertension secondary to left heart disease. Venous PH is also known as WHO Group II PH. Venous PH may be associated with or caused by: left ventricular systolic dysfunction (such as left ventricular failure), left ventricular diastolic dysfunction, valvular heart disease (such as mitral or aortic valve disease), congenital cardiomyopathy, or congenital / acquired pulmonary vein stenosis.

[0210] As used herein, the terms "hypoxic pulmonary hypertension" and "hypoxic PH" refer to a form of pulmonary hypertension due to lung disease or chronic hypoxia. This form of PH is also known as WHO Group III PH. Hypoxic PH may be associated with or caused by: chronic obstructive pulmonary disease (such as emphysema), interstitial lung disease, sleep disordered breathing (such as sleep apnea), lung disease (such as pulmonary fibrosis), alveolar hypoventilation disorders, long-term exposure to high altitude, or developmental abnormalities.

[0211] As used herein, the terms "thromboembolic pulmonary hypertension" and "thromboembolic PH" refer to a form of pulmonary hypertension associated with chronic arterial obstruction (such as a blood clot). Thromboembolic PH is also known as WHO Group IV PH. Thromboembolic PH may be associated with or caused by: chronic thromboembolic pulmonary hypertension, or other pulmonary artery obstruction (such as pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or parasitic infection).

[0212] As used herein, the terms "miscellaneous pulmonary hypertension" and "miscellaneous PH" refer to a form of pulmonary hypertension with an unclear or multifactorial mechanism. This form of PH is classified as WHO Group V PH. Miscellaneous PH may be associated with or caused by: hematologic disorders (such as chronic hemolytic anemia, sickle cell disease), systemic diseases (such as sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), metabolic disorders (such as glycogen storage disease, Gaucher's disease, or thyroid disease), pulmonary tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension.

[0213] As used herein, the terms "increased red blood cell levels" and "promoted red blood cell formation" refer to clinically observable metrics such as hematocrit, red blood cell count, and hemoglobin measurement, and are expected 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 considered normal for the age and sex of the subject.

[0214] As used herein, the terms "erythropoiesis" and "erythrocyte production" refer to the production of red blood cells, such as the process of erythropoiesis in which red blood cells are produced in the bone marrow.

[0215] As used herein, the term "anemia" refers to any abnormality in hemoglobin or red blood cells that results in a decrease in the level of oxygen in the blood. Anemia can be associated with 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 the level of hemoglobin in the blood relative to normal blood levels.

[0216] 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 the amino acid (or nucleic acid) residues of a reference sequence after aligning the sequences and introducing gaps (if 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 the purpose of comparison). Alignment for the purpose of determining percent identity can be achieved in a variety of ways within the skill in the art, e.g., using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithms required to achieve maximum alignment over the full length of the sequences to be 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 (which can alternatively be expressed as a given candidate sequence having or comprising a specific percent amino acid (or nucleic acid) sequence identity to, with, or against a given reference sequence) is calculated as follows: 100 x (fraction of A / B) where A is the number of amino acid (or nucleic acid) residues scored as identical in the alignment of the candidate and reference sequences, and where B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments in which 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 is not equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.

[0217] In a specific embodiment, alignment of a reference sequence used for comparison with a candidate sequence can show that the candidate sequence exhibits 50% to 100% identity over the full length of the candidate sequence or a selected portion of contiguous amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes is at least 30% of the length of the reference sequence, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100%. A molecule is identical at a position when the position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence.

[0218] As used herein, in the case of administering a therapeutic protein to a subject, the term "serum half-life" refers to the time required for the plasma concentration of the protein in the subject to be reduced by half. The protein can redistribute or be cleared from the bloodstream, or degraded, i.e., by proteolysis. As described herein, polypeptides of extracellular ActRIIB variants comprising a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15) exhibit a serum half-life of 7 days in humans.

[0219] As used herein, the term "lean body mass" refers to a component of body composition, which includes, for example, lean body mass, body fat, and body fluids. Generally, lean body mass is calculated by subtracting the weight of body fat and body fluids from the total body weight. Generally, the lean body mass of a subject is 60% to 90% of the total body weight. In the present invention, administration of a polypeptide of an extracellular ActRIIB variant comprising a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)), or a vector containing such a nucleic acid molecule to a subject increases the lean body mass of the subject.

[0220] 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 sum of non-covalent interactions between a molecule and its binding partner (e.g., an extracellular ActRIIB variant and BMP9 or activin A). Unless otherwise specified, binding affinity refers to the intrinsic binding affinity, which reflects the 1:1 interaction between the members of the binding pair. The binding affinity between two molecules is typically described by the dissociation constant (K D ) or the association constant (K A ). Two molecules with low binding affinity for each other generally bind slowly, tend to dissociate readily, and exhibit a large K D。Two molecules with high affinity for each other generally bind easily, tend to remain bound longer, and exhibit a small K D 。Methods and techniques well known in the art, such as surface plasmon resonance, can be used to determine the K of two interacting molecules D 。K D is calculated as the ratio of k off / k on 。

[0221] As used herein, the term "muscle weight" refers to a component of body composition. Generally, muscle weight is calculated by subtracting the weights of body fat and body fluids from the total body weight. The percentage of muscle weight can vary greatly among individuals, depending on factors such as the subject's genetic makeup, age, race, and health status. Generally, the muscle weight of a subject may be 20% to 50% of the total body weight.

[0222] 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 (such as endogenous ActRIIB). In some embodiments, the polypeptides described herein that include extracellular ActRIIB variants reduce or inhibit the binding of myostatin, activin, and / or BMP9 to their receptors such as ActRIIA, ActRIIB, and BMPRII (such as endogenous ActRIIB). As described herein, the polypeptides of the invention that include extracellular ActRIIB variants having a sequence of any one of SEQ ID NOs: 1-15 (such as SEQ ID NOs: 2-15) may have a weak binding affinity for BMP9 (such as a K of 200 pM or higher D )。

[0223] As used herein, the term "vascular complication" refers to a vascular disorder or any damage to blood vessels, such as damage to the blood vessel wall. Damage to the blood vessel wall can cause an increase in vascular permeability or leakage. The term "vascular permeability or leakage" refers to the ability of the blood vessel wall to allow small molecules, proteins, and cells to flow into and out of the blood vessel. An increase in vascular permeability or leakage can be caused by an increase in the gaps between endothelial cells lining the blood vessel wall (e.g., an increase in the size and / or number of gaps) and / or thinning of the blood vessel wall.

[0224] As used herein, the term "polypeptide" describes a single polymer in which the monomers are amino acid residues covalently conjugated together by amide bonds. Polypeptides are intended to encompass any amino acid sequence that is naturally occurring, recombinant, or synthetically produced.

[0225] As used herein, the term "homodimer" refers to a molecular construct formed by two identical macromolecules such as proteins or nucleic acids. The two identical monomers can form a homodimer through covalent or non-covalent bonds. For example, if two Fc domain monomers contain the same sequence, the Fc domain can be a homodimer of the two Fc domain monomers. In another example, a polypeptide described herein that comprises an extracellular ActRIIB variant fused to an Fc domain monomer can form a homodimer through the interaction of two Fc domain monomers that form the Fc domain in the homodimer.

[0226] As used herein, the term "heterodimer" refers to a molecular construct formed by two different macromolecules such as proteins or nucleic acids. The two monomers can form a heterodimer through covalent or non-covalent bonds. For example, a polypeptide described herein that comprises 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) that form the Fc domain in the heterodimer.

[0227] As used herein, the term "host cell" refers to a vehicle that contains the essential cellular components (e.g., organelles) required for expressing a protein from its corresponding nucleic acid. The nucleic acid is typically included in a nucleic acid vector that can be introduced into the host cell by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). The host cell can be a prokaryotic cell, such as a bacterial cell, or a eukaryotic cell, such as a mammalian cell (e.g., a CHO cell or a HEK293 cell).

[0228] As used herein, the term "therapeutically effective amount" refers to the amount of a polypeptide, nucleic acid, or vector of the present invention, or a pharmaceutical composition containing the polypeptide, nucleic acid, or vector of the present invention, that effectively achieves the desired therapeutic effect of treating a patient suffering from a disease or at risk of developing a disease, such as a muscle disease, a condition 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, a disease or condition involving bone injury (e.g., osteoporosis or a condition involving bone injury, such as primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, fracture, bone cancer, or bone loss associated with cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, obesity treatment-related bone loss, low gravity-related bone loss, or immobilization-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 PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH). In particular, the therapeutically effective amount of the polypeptide, nucleic acid, or vector avoids adverse side effects.

[0229] As used herein, the term "pharmaceutical composition" refers to a pharmaceutical or medicinal preparation that contains an active ingredient, as well as excipients and diluents that render the active ingredient suitable for the method of administration. The pharmaceutical compositions of the present invention contain pharmaceutically acceptable components that are compatible with the polypeptide, nucleic acid, or vector. The pharmaceutical composition may be in the form of tablets or capsules for oral administration, or in an aqueous form for intravenous or subcutaneous administration.

[0230] As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to an excipient or diluent in a pharmaceutical composition. The pharmaceutically acceptable carrier must be compatible with the other components of the formulation and harmless to the recipient. In the present invention, the pharmaceutically acceptable carrier or excipient must provide sufficient pharmaceutical stability for a polypeptide containing an extracellular ActRIIB variant, a nucleic acid molecule encoding the polypeptide, or a vector containing such a nucleic acid molecule. The nature of the carrier or excipient varies with the mode of administration. For example, for intravenous administration, an aqueous solution carrier is usually used; for oral administration, a solid carrier is preferred.

[0231] As used herein, the term "treatment and / or prevention" refers to the treatment and / or prevention of diseases using the methods and compositions of the present invention, such diseases as muscle diseases (e.g., DMD, FSHD, IBM, and ALS), bone diseases (e.g., diseases or conditions involving bone injury, such as osteoporosis, osteopenia, osteosclerosis, fractures, bone cancer or bone loss associated with cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, obesity-treatment related bone loss, low gravity-related bone loss or immobilization-related bone loss), diseases involving low blood cell levels (e.g., anemia or blood loss), fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH). Generally, the treatment of muscle, bone, low blood cell, or fibrosis diseases or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH) occurs after a subject has developed a muscle, bone, low blood cell, or fibrosis disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), and / or has been diagnosed with a muscle, bone, low blood cell, or fibrosis disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH). The prevention of muscle, bone, low blood cell, or fibrosis diseases or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH) refers to the steps or procedures taken when a subject is at risk of developing a muscle, bone, low blood cell, or fibrosis disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH). A subject may exhibit signs or mild symptoms of an indication or risk factor that a doctor determines to be associated with the development of a muscle, bone, low blood cell, or fibrosis disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), have another disease or condition associated with the development of a muscle, bone, low blood cell, or fibrosis disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), be undergoing a treatment (e.g., surgery, chemotherapy, or radiation) that may cause anemia, fibrosis, or bone density loss, or have a family history or genetic predisposition to developing a muscle, bone, low blood cell, or fibrosis disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), but has not yet developed the disease.

[0232] 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, etc. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a sequence alignment showing the wild-type sequences of extracellular ActRIIA and ActRIIB and the amino acid substitutions in the ActRIIB variant.Figure 1 Also shown are the percentages of weight change resulting from treating mice with the respective variants.

[0234] Figure 2 Is a bar graph depicting the effect of extracellular ActRIIB variants on body weight at the end of 28 days. Mice received a single hydrodynamic injection of a plasmid construct encoding the indicated ActRIIB variant or a control plasmid. The legend lists the bars of 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 farthest from the y-axis).

[0235] Figure 3A and 3B Is a bar graph depicting the effect of extracellular ActRIIB variants on individual muscle weights by tissue analysis.

[0236] Figure 4 Is a graph depicting the effect of extracellular ActRIIB variants ActRIIB 2.11-Fc and ActRIIB 2.12-Fc on percentage body weight over a 28-day period.

[0237] Figures 5A - 5C Is a series of graphs depicting the effect of ActRIIB variants ActRIIB 2.11-Fc and ActRIIB 2.12-Fc on red blood cell count, hemoglobin level, and hematocrit.

[0238] Figures 6A - 6D Is a series of graphs depicting the effect of ActRIIB variant ActRIIB 2.12-Fc on trabecular bone in a mouse model of osteoporosis.

[0239] Figures 7A - 7C Is a series of graphs depicting the effect of ActRIIB variant ActRIIB 2.12-Fc on red blood cell mass parameters in wild-type rats.

[0240] Figures 8A - 8E Is a series of graphs depicting the effect of ActRIIB variant ActRIIB 2.12-Fc on trabecular bone in wild-type rats. DETAILED DESCRIPTION The present invention features polypeptides comprising an extracellular activin receptor type IIB (ActRIIB) variant. In some embodiments, the polypeptides of the present invention comprise an extracellular ActRIIB variant fused to a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain having an amino acid substitution (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin). A polypeptide comprising an extracellular ActRIIB variant fused to an Fc domain monomer can also form a dimer (e.g., a homodimer or a heterodimer) through the interaction between two Fc domain monomers. Relative to wild-type extracellular ActRIIB, the ActRIIB variants described herein may have reduced binding to bone morphogenetic protein 9 (BMP9), or have a weak or no binding affinity for BMP9 compared to the binding affinity for activin and myostatin. The present invention also includes methods of treating diseases and conditions involving muscle weakness and atrophy by increasing muscle weight and strength, methods of treating or preventing bone injury by increasing bone mineral density, increasing bone formation or reducing bone resorption, methods of treating or preventing fibrosis, methods of treating or preventing low blood cell levels (e.g., 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, methods of treating or preventing pulmonary hypertension (PH) (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), or methods of affecting myostatin, activin, and / or BMP9 signaling in a subject, by administering to the subject a polypeptide comprising an extracellular ActRIIB variant as described herein.

[0242] I. Extracellular activin receptor type IIB variants Activin type II receptors are single transmembrane domain receptors that regulate the signaling of ligands in the transforming growth factor-β (TGF-β) superfamily. Ligands in the TGF-β superfamily are involved in many physiological processes such as muscle growth, blood vessel growth, cell differentiation, homeostasis, and osteogenesis. Examples of ligands in the TGF-β superfamily include, for example, activin (e.g., activin A and activin B), inhibin, growth differentiation factor (GDF) (e.g., GDF8, also known as myostatin), and bone morphogenetic protein (BMP) (e.g., BMP9). Myostatin and activin are known to play roles in the regulation of skeletal muscle growth. For example, mice lacking myostatin show a substantial increase in skeletal muscle weight. Myostatin has also been implicated in promoting fibrosis. For example, mice lacking myostatin show a reduction in muscle fibrosis, while injection of myostatin-coated beads induces muscle fibrosis in mice. Mice overexpressing activin subunits also show fibrosis, which results in the production of diffusible activin A. In addition, activin is abundantly expressed in bone tissue and regulates bone formation by controlling both osteoblast and osteoclast function. Activin has been reported to be upregulated in bone diseases and to inhibit osteoblast activity. Myostatin is also involved in bone homeostasis by increasing osteogenesis and inhibiting osteoblast activity. An increase in activin A has also been observed in clinical and experimental pulmonary hypertension. Thus, methods for reducing or inhibiting activin or myostatin signaling can be used to treat diseases and conditions involving: muscle atrophy or weakness, fibrosis, bone injury, low red blood cell levels (e.g., anemia), or pulmonary hypertension (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH).

[0243] There are two types of type II activin receptors: ActRIIA and ActRIIB. Studies have shown that BMP9 binds to ActRIIB with a binding affinity approximately 300-fold higher than that for ActRIIA (see, for example, Townson et al., J. Biol. Chem. 287:27313, 2012). ActRIIA is known to have a longer half-life compared to ActRIIB. The present invention describes extracellular ActRIIB variants that are 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. The amino acid substitutions can also confer beneficial physiological and pharmacokinetic properties to ActRIIA, such as a longer half-life or the ability to increase red blood cell levels. Optimal peptides confer a significant increase in lean body mass, muscle weight, bone mineral density, and / or red blood cell levels (such as an increase in red blood cell production), a decrease in fibrosis, or treatment of PH (such as PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), while having, for example, a reduced binding affinity for BMP9. Preferred ActRIIB variants also show similar or improved binding to activin and / or myostatin compared to wild-type ActRIIB, which allows 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 conditions in which activin receptor signaling is elevated, such as bone diseases (such as diseases or conditions involving bone injury), muscle diseases, fibrosis, PH (such as PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), and / or anemia, resulting in a decrease in bone resorption or osteoclast activity, an increase in bone formation or bone mineral density, an increase in muscle weight or strength, a decrease in fibrosis (such as a reduction in fibrosis or a slowing or stopping of fibrosis progression), an increase in red blood cell levels (such as an increase in hemoglobin level, hematocrit, or red blood cell count, such as an increase in red blood cell production), or a decrease in the symptoms or progression of PH (such as PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH). In some embodiments, amino acid substitutions can be introduced into the extracellular ActRIIB variants to reduce or eliminate the binding affinity of the variant for BMP9. The wild-type amino acid sequences of the extracellular portions of human ActRIIA and ActRIIB are shown below.

[0244] Human ActRIIA, extracellular portion (SEQ ID NO: 16): Human ActRIIB, extracellular portion (SEQ ID NO: 17):

[0245] The polypeptides described herein include extracellular ActRIIB variants having at least one amino acid substitution relative to wild-type extracellular ActRIIB having the sequence of SEQ ID NO: 17. Possible amino acid substitutions at 28 different positions can be introduced into the extracellular ActRIIB variants (Table 1). The extracellular ActRIIB variants 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, the extracellular ActRIIB variant (e.g., the extracellular ActRIIB variant having the sequence of SEQ ID NO: 1) can include amino acid substitutions at all 28 positions listed in Table 1. In some embodiments, the extracellular ActRIIB variant can include amino acid substitutions at a plurality of positions listed in Table 1, e.g., at 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 18, 20, 22, 24, 26, or 27 of the 28 positions. In some embodiments, the substitution is a substitution of an amino acid from ActRIIA within the same position in ActRIIB. In some embodiments, the substitution is a novel change (e.g., an amino acid substitution not in the corresponding position of ActRIIA, e.g., S48T, I51L, Q69D, or E70T).

[0246] Amino acid substitutions can worsen or improve the activity and / or binding affinity of the ActRIIB variants of the present invention (e.g., extracellular ActRIIB variants having any one of the sequences 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 variant for BMP9 (e.g., the variant has reduced binding to BMP9 relative to wild-type extracellular ActRIIB, or has lower binding to BMP9 compared to other ActRIIB ligands (e.g., activin A or B, myostatin, or GDF-11)). In some embodiments, the ActRIIB variant has reduced, weak, or substantially no binding to BMP9. In some embodiments, the amino acid substitution improves the binding affinity of ActRIIB for myostatin, activin A or B, and / or GDF-11 (e.g., the variant has improved binding affinity relative 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 for myostatin, activin A or B, and / or GDF-11 (e.g., the variant has reduced binding affinity relative to wild-type extracellular ActRIIB, or binds more weakly to myostatin, activin A or B, or GDF-11 than to BMP9). In some embodiments, the amino acid substitution substantially does not change the extracellular ActRIIB function (e.g., the ActRIIB variant increases lean body mass, muscle weight, or bone mineral density by an amount similar to 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 ActRIIA properties or activities on 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 increased binding to activin A or B, myostatin, and / or GDF-11, or reduced BMP9 binding, and a function equivalent to wild-type ActRIIB in increasing lean body mass, muscle weight, or bone mineral density, or reducing or preventing fibrosis).

[0247] The ActRIIB variants of the present invention (e.g., extracellular ActRIIB variants having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) 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 24 which is K in SEQ ID NO: 1). In some embodiments, the amino acid substitutions that reduce BMP9 binding are Q69T and E70D (e.g., X 21 which is T in SEQ ID NO: 1 and X 22 which is D). In some embodiments, the amino acid substitutions that reduce BMP9 binding are Q69D and E70T (e.g., X 21 which is D in SEQ ID NO: 1 and X 22 which is T). In some embodiments, the 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 28 which are K, K, D, S, and E, respectively, in SEQ ID NO: 1). In some embodiments, the ActRIIB variant has more than one of the above amino acid substitutions that reduce BMP9 binding (e.g., the substitution E75K and the substitutions Q69D and E70T, or the substitution E75K and the substitutions Q69T and E70D). In some embodiments, the ActRIIB variants of the present invention have 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, the amino acid substitutions T74K, E75K, E76D, N77S, and Q79E result in a decrease in ActRIIB variant activity (e.g., the variant has a reduced effect on lean body mass and muscle weight compared to wild-type extracellular ActRIIB), but including the additional substitutions S25T and S47I; E31Y, E33D, and Q34K; or Y41F, R45K, and K56Q improves the effect of the ActRIIB variant on lean body mass and / or muscle weight. The additional 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.

[0248] In some embodiments, the polypeptides described herein include an extracellular ActRIIB variant having the sequence of SEQ ID NO: 1.

[0249] Table 1. Amino acid substitutions in the extracellular ActRIIB variant having the sequence of SEQ ID NO: 1

[0250] In some embodiments, the polypeptides described herein include an extracellular ActRIIB variant having any one of the sequences of SEQ ID NOs: 2 - 15 (Table 2).

[0251] Table 2. Extracellular ActRIIB variants having the sequences of SEQ ID NOs: 2 - 15

[0252] In some embodiments, the polypeptides of the 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 having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin - binding peptide, a fibronectin domain, or human serum albumin), which moiety may 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 may form a dimer (e.g., a homodimer or a heterodimer) through the interaction between two Fc domain monomers, and the two Fc domain monomers combine in the dimer to form an Fc domain.

[0253] In addition, in some embodiments, the polypeptides described herein have a serum half - life of at least 7 days in humans. The polypeptide may have a K D binding to bone morphogenetic protein 9 (BMP9) at 200 pM or higher. The polypeptide may have a K D binding to activin A at 10 pM or higher. In some embodiments, the polypeptide does not bind BMP9 or activin A. In some embodiments, the polypeptide binds activin and / or myostatin and exhibits reduced (e.g., weak) binding to BMP9.

[0254] Additionally, in some embodiments, the polypeptide may have a K D (e.g., a K of about 200, 300, 400, 500, 600, 700, 800, or 900 pM or higher) D, for example, a K of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nM or higher D , for example, a K of about 200 pM to about 50 nM D ) binds to human BMP9. In some embodiments, the polypeptide substantially does not bind to human BMP9. In some embodiments, the polypeptide can have a K of about 800 pM or lower D (for example, 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 lower D , for example, a K of about 800 pM to about 200 pM D ) binds to human activin A. In some embodiments, the polypeptide can have a K of about 800 pM or lower D (for example, 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 lower D , for example, a K of about 800 pM to about 200 pM D ) binds to human activin B. The polypeptide can also have a K of about 5 pM or higher D (for example, 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 higher D ) binds to growth and differentiation factor 11 (GDF-11).

[0255] II. Fc domain In some embodiments, the polypeptides described herein can comprise an extracellular ActRIIB variant fused to a monomer of the Fc domain of an immunoglobulin or a fragment of the Fc domain to increase the serum half-life of the polypeptide. A polypeptide comprising an extracellular ActRIIB variant fused to a monomer of the Fc domain can form a dimer (e.g., a homodimer or a heterodimer) through the interaction between two monomers of the Fc domain, and the two monomers of the Fc domain form an Fc domain in the dimer. As is conventionally known in the art, the Fc domain is a protein structure found at the C-terminus of an immunoglobulin. The Fc domain comprises two monomers of the Fc domain, which are connected by C HDimerization occurs through the interaction between the constant domains of the 3 antibodies. The wild-type Fc domain forms the minimal structure that binds to Fc receptors, such as FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, FcγRIV. In some embodiments, the Fc domain can be mutated to lack effector function, which is a typical "dead" Fc domain. For example, the Fc domain can contain specific amino acid substitutions known to minimize the interaction between the Fc domain and Fcγ receptors. In some embodiments, the Fc domain is from an IgG1 antibody and contains the amino acid substitutions L234A, L235A, and G237A. In some embodiments, the Fc domain is from an IgG1 antibody and contains the amino acid substitutions D265A, K322A, and N434A. The above amino acid positions are defined according to Kabat (Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). By aligning the homologous regions of the antibody sequence with the "standard" Kabat numbering sequence, the Kabat number of the amino acid residues of a given antibody can be determined. Additionally, in some embodiments, the Fc domain does not induce any immune system-related responses. For example, the Fc domain in the dimer of a polypeptide comprising an extracellular ActRIIB variant fused to an Fc domain monomer can be modified to reduce the interaction or binding between the Fc domain and Fcγ receptors. The sequence of the Fc domain monomer that can be fused to the extracellular ActRIIB variant is shown below (SEQ ID NO:19):

[0256] In some embodiments, the Fc domain is from an IgG1 antibody and contains the amino acid substitutions L12A, L13A, and G15A relative to the sequence of SEQ ID NO:19. In some embodiments, the Fc domain is from an IgG1 antibody and contains the amino acid substitutions D43A, K100A, and N212A relative to the sequence of SEQ ID NO:19. In some embodiments, the extracellular ActRIIB variants described herein (e.g., extracellular ActRIIB variants having any of the sequences 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, such as 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., the C-terminus) of the extracellular ActRIIB variant.

[0257] In some embodiments, the polypeptides described herein can comprise an extracellular ActRIIB variant fused to an Fc domain. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit Fc domain dimerization. In some embodiments, the Fc domain contains a hinge domain. The Fc domain can be the Fc domain of an immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. Additionally, the Fc domain can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain can also be a non-naturally occurring Fc domain, such as a recombinant Fc domain.

[0258] Methods of engineering Fc domains with reduced dimerization are known in the art. In some embodiments, one or more amino acids with large side chains (e.g., tyrosine or tryptophan) can be introduced into the C H 3-C H 3 dimer interface to impede dimer formation due to steric clashes. In other embodiments, one or more amino acids with small side chains (e.g., alanine, valine, or threonine) can be introduced into the C H 3-C H 3 dimer interface to remove favorable interactions. At the C HMethods of introducing amino acids with large or small side chains into the 3 domain are described, for example, in Ying et al. (J Biol Chem. 287:19399-19408, 2012), US Patent Publication No. 2006 / 0074225, US Patent No. 8,216,805 and 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.

[0259] In still other embodiments, the C between the two Fc domains is constituted H 3-C H 3 interface of the C H One or more amino acid residues in the 3 domain are replaced with a positively charged amino acid residue (e.g., lysine, arginine, or histidine) or a negatively charged amino acid residue (e.g., aspartic acid or glutamic acid) such that the interaction becomes electrostatically unfavorable, depending on the particular charged amino acid introduced. In C H Methods of introducing charged amino acids into the 3 domain to disfavor or prevent dimer formation are described, for example, in Ying et al. (J Biol Chem. 287:19399-19408, 2012), US Patent Publication Nos. 2006 / 0074225, 2012 / 0244578, and 2014 / 0024111, all of which are incorporated herein by reference in their entirety.

[0260] In some embodiments of the present invention, relative to the sequence of human IgG1, the Fc domain comprises one or more of the following amino acid substitutions: 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 a specific embodiment, relative to the sequence of human IgG1, the Fc domain comprises the amino acid substitution T366W. The sequence of the wild-type Fc domain (SEQ ID NO:71) is:

[0261] III. Albumin-binding peptide 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 the protein drug.

[0262] As an example, albumin-binding peptides useful 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).

[0263] In the present invention, the albumin-binding peptide can be linked to the N- or C-terminus (e.g., the C-terminus) of the extracellular ActRIIB variant described herein (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 increase the serum half-life of the extracellular ActRIIB variant. In some embodiments, the albumin-binding peptide is linked directly or via a linker to the N- or C-terminus of the extracellular ActRIIB variant.

[0264] In some embodiments, the extracellular ActRIIB variants described herein (e.g., extracellular ActRIIB variants 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 NO: 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 expected that the inclusion of an albumin-binding peptide in the extracellular ActRIIB variants described herein can result in extended retention of the therapeutic protein through its binding to serum albumin.

[0265] IV. Fibronectin domain In some embodiments, the polypeptides described herein can comprise an extracellular ActRIIB variant fused to a fibronectin domain. Binding to the fibronectin domain can improve the pharmacokinetics of the protein drug.

[0266] The fibronectin domain is a high molecular weight glycoprotein of the extracellular matrix or a fragment thereof that binds, for example, transmembrane receptor proteins such as integrins and extracellular matrix components such as collagen and fibrin. In some embodiments of the invention, the fibronectin domain is linked to the N- or C-terminus (e.g., the C-terminus) of the 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)) to increase the serum half-life of the extracellular ActRIIB variant. The fibronectin domain can be linked directly or through a linker to the N- or C-terminus of the extracellular ActRIIB variant.

[0267] As an example, fibronectin domains useful in the methods and compositions described herein are generally known in the art. In one embodiment, the fibronectin domain is the fibronectin type III domain of amino acids 610-702 having the sequence of UniProt ID NO: P02751 (SEQ ID NO: 73):

[0268] In another embodiment, the fibronectin domain is an adnectin protein.

[0269] In some embodiments, the extracellular ActRIIB variants described herein (e.g., extracellular ActRIIB variants 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 NO: 73) 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 fibronectin domain. Without being bound by theory, it is expected that the inclusion of a fibronectin domain in the extracellular ActRIIB variants described herein can result in extended retention of the therapeutic protein through its binding to integrins and extracellular matrix components such as collagen and fibrin.

[0270] V. Serum albumin In some embodiments, the polypeptides described herein can comprise an extracellular ActRIIB variant fused to serum albumin. Binding to serum albumin can improve the pharmacokinetics of the protein drug.

[0271] Serum albumin is a globular protein that is the most abundant blood protein in mammals. Serum albumin is produced in the liver and accounts for approximately half of the serum proteins. It 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 the correct 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, human serum albumin is linked to the N- or C-terminus (e.g., the C-terminus) of the 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)) to increase the serum half-life of the extracellular ActRIIB variant. Human serum albumin can be linked directly or through a linker to the N- or C-terminus of the extracellular ActRIIB variant.

[0272] As an example, the 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).

[0273] In some embodiments, the extracellular ActRIIB variants described herein (e.g., extracellular ActRIIB variants having a 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 human serum albumin (e.g., SEQ ID NO: 74) 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 human serum albumin. Without being bound by theory, it is expected that the inclusion of human serum albumin in the extracellular ActRIIB variants described herein can result in extended retention of the therapeutic protein.

[0274] VI. Linker The polypeptides described herein can comprise an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a 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 having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin. In the present invention, the linker between the moiety (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 having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), 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 the extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) can be an amino acid spacer comprising 1-200 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 can contain motifs such as 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), e.g., multiple or repeating motifs.In some embodiments, the spacer may contain from 2 to 12 amino acids, including GA or GS, such as the motifs of 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 may contain from 3 to 12 amino acids, including GGA or GGS, such as the motifs of 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). In still other embodiments, the spacer may contain from 4 to 12 amino acids, including GGAG (SEQ ID NO:26), GGSG (SEQ ID NO:27), such as the motifs of 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 may contain GGGGA (SEQ ID NO:23) or GGGGS (SEQ ID NO:24), such as the motifs of GGGGGAGGGGAGGGGA (SEQ ID NO:50) and GGGGSGGGGSGGGGS (SEQ ID NO:51).In some embodiments of the present invention, the linker between a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) and an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) can 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).

[0275] In some embodiments, the spacer can also contain 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 can contain a motif of EAAAK (SEQ ID NO: 69), such as multiple or repeated motifs. In some embodiments, the spacer can contain a proline-rich sequence (such as (XP) n ) motif, such as multiple or repeated motifs, where X can be any amino acid (e.g., A, K, or E) and n is 1-5, and PAPAP (SEQ ID NO: 70).

[0276] The length of the peptide spacer and the amino acids used can be adjusted according to the two proteins involved and the desired degree of flexibility in the final protein fusion polypeptide. The length of the spacer can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0277] VII. Vectors, Host Cells, and Protein Production The polypeptides of the present invention can be produced from host cells. A host cell refers to an agent that contains the essential cellular components (e.g., organelles) required for expressing the polypeptides and fusion polypeptides described herein from their corresponding nucleic acids. The nucleic acid can be included in 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, injection, etc.). The choice of nucleic acid vector depends in part on the host cell to be used. Generally, preferred host cells are of eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origin.

[0278] Nucleic Acid Vector Construction and Host Cells The 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. The nucleic acid molecules encoding the polypeptides of the present invention can be obtained using standard techniques (e.g., gene synthesis). Alternatively, nucleic acid molecules encoding wild-type extracellular ActRIIB can be mutated using standard techniques in the art (e.g., QuikChange TM mutagenesis) to include specific amino acid substitutions. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR techniques.

[0279] The nucleic acid sequences encoding the polypeptides of the present invention can be inserted into vectors capable of replicating and expressing the nucleic acid molecules in prokaryotic or eukaryotic host cells. Many vectors are available in the art and can be used for the purposes of the present invention. Each vector can include various components that can be adjusted and optimized for compatibility with a particular host cell. For example, vector components can include, but are not limited to, an origin of replication, a selectable marker gene, a promoter, a ribosome binding site, a signal sequence, the nucleic acid sequence encoding the target protein, and a transcription termination sequence.

[0280] 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, HEK 293F), Chinese hamster ovary (CHO), HeLa, COS, PC3, Vero, MC3T3, NS0, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0 (a murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, and HsS78Bst cells. In some embodiments, Escherichia coli cells can also be used as host cells of the present invention. Examples of Escherichia coli strains include, but are not limited to, Escherichia coli 294 ( 31,446), Escherichia coli λ1776 ( 31,537), Escherichia coli BL21(DE3)( BAA-1025), and Escherichia coli RV308 ( 31,608). Different host cells have characteristics and specific mechanisms for post-transcriptional processing and modifying protein products (e.g., glycosylation). An appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed polypeptide. Conventional techniques in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection) can be used to introduce the above expression vectors into appropriate host cells. Once the vector is introduced into the host cell for protein production, the host cell is cultured in a suitably modified conventional nutrient medium for inducing the promoter, selecting transformants, or amplifying the gene encoding the desired sequence. Methods for expressing therapeutic proteins are known in the art, see, for example, 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.

[0281] Protein production, recovery, and purification The host cells used to produce the polypeptides of the present invention can grow 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 Medium (DMEM), Expi293 TM Expression Medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria Broth (LB) plus essential supplements, such as selection agents, e.g., ampicillin. The host cells are cultured at a suitable temperature such as about 20°C to about 39°C, e.g., 25°C to about 37°C, preferably 37°C, and CO 2 levels, such as 5 to 10%. The pH of the medium is typically about 6.8 to 7.4, e.g., 7.0, which mainly depends on the host organism. If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activating the promoter.

[0282] 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 involve filtering the cell culture medium to remove cell debris. The protein can be further purified. The polypeptides of the present invention can be purified by any method known in the art of protein purification, e.g., by chromatography (e.g., ion exchange, affinity, and size exclusion column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. For example, the protein can be separated and purified by appropriately selecting and combining affinity columns, such as Protein A columns (e.g., POROS Protein A chromatography), and chromatography columns (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, salting out, and dialysis procedures.

[0283] In other embodiments, the host cells can be disrupted, e.g., by osmotic shock, sonication, or lysis, to recover the expressed protein. Once the cells are disrupted, the cell debris can be removed by centrifugation or filtration. In some cases, the polypeptide can be conjugated with a tag sequence, such as a peptide, to facilitate purification. Examples of tagged amino acid sequences are hexahistidine peptides (His-tags), which bind to nickel-functionalized agarose affinity columns with micromolar affinity. Other peptide tags that can be used 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).

[0284] Alternatively, the polypeptides of the invention can be produced by the cells of a subject (e.g., a human), e.g., in the context of gene therapy, by administering a vector (such as a viral vector (e.g., a retroviral vector, an adenoviral vector, a poxviral vector (e.g., a vaccinia viral vector, such as modified vaccinia virus Ankara (MVA)), an adeno-associated viral vector, and an alphaviral vector)) containing a nucleic acid molecule encoding the polypeptide of the invention. The vector, once inside the cells of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), will promote the expression of the polypeptide, which is then secreted from the cell. If the treatment of a disease or disorder is the desired outcome, no further action may be required. If collection of the protein is desired, blood can be collected from the subject and the protein purified from the blood by methods known in the art.

[0285] VIII. Pharmaceutical Compositions and Formulations The invention features pharmaceutical compositions that comprise a polypeptide described herein (e.g., a polypeptide comprising 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))). In some embodiments, the pharmaceutical compositions of the invention comprise a polypeptide comprising 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)) fused to a moiety (e.g., an Fc domain monomer or its dimer, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) as a therapeutic protein. In some embodiments, the pharmaceutical compositions of the invention that comprise a polypeptide of the invention can be used in combination with other agents (e.g., therapeutic biologics and / or small molecules) or compositions for therapy. In addition to a therapeutically effective amount of the polypeptide, the pharmaceutical composition can further comprise one or more pharmaceutically acceptable carriers or excipients, which can be formulated by methods known to those of skill in the art. In some embodiments, the pharmaceutical compositions of the invention comprise a nucleic acid molecule (DNA or RNA, e.g., mRNA) encoding the polypeptide of the invention, or a vector containing such a nucleic acid molecule.

[0286] The acceptable carriers and excipients in the pharmaceutical composition are non-toxic to the recipient at the dosages and concentrations used. Acceptable carriers and excipients can include buffering agents such as phosphates, citrates, HEPES and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyl dimethyl benzyl ammonium 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 injectable preparation. A sterile solution or any pharmaceutically acceptable liquid can be used as a vehicle to formulate the pharmaceutical composition for injection. Pharmaceutically acceptable vehicles include but are not limited to sterile water, physiological saline and cell culture media (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-Modified Eagle Medium (α-MEM), F-12 medium). The 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).

[0287] The pharmaceutical composition of the present invention can be prepared in microcapsules such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methyl methacrylate) microcapsules. The pharmaceutical composition of the present invention can also be prepared 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). The pharmaceutical composition to be used for in vivo administration must be sterile. This can be easily accomplished by filtration through a sterile filtration membrane.

[0288] The pharmaceutical composition of the present invention can also be prepared as a sustained release formulation. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptide of the present invention. Examples of sustained release matrices include polyesters, hydrogels, poly(lactide), copolymers of L-glutamic acid and γ-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT TM and poly-D-(-)-3-hydroxybutyric acid. Some sustained release formulations enable the release of molecules over several months, for example one to six months, while other formulations release the pharmaceutical composition of the present invention over a shorter period, for example several days to several weeks.

[0289] The pharmaceutical composition can be formed in unit dosage forms as needed. The amount of the active ingredient (e.g., the polypeptide of the present invention) included in the pharmaceutical preparation is such that a suitable dose within a specified range (e.g., a dose within the range of 0.01 - 100 mg / kg body weight) is provided.

[0290] The pharmaceutical composition for gene therapy can be in an acceptable diluent or can comprise a sustained-release matrix in which a gene delivery vector is embedded. If hydrodynamic injection is used as the delivery method, the pharmaceutical composition containing the nucleic acid molecule encoding the polypeptide described herein or a vector (e.g., a viral vector) containing the nucleic acid molecule is rapidly delivered intravenously in a large fluid volume. Vectors that can be used as in vivo gene delivery vehicles include, but are not limited to, retroviral vectors, adenoviral vectors, poxviral vectors (e.g., vaccinia virus vectors such as modified vaccinia virus Ankara), adeno-associated viral vectors, and alphavirus vectors.

[0291] IX. Routes, Doses, and Administrations The pharmaceutical composition containing the polypeptide of the present invention as a therapeutic protein can be formulated for, e.g., intravenous administration, parenteral administration, subcutaneous administration, intramuscular administration, intra-arterial administration, intrathecal administration, or intraperitoneal administration. The pharmaceutical composition can also be formulated for oral, nasal, spray, aerosol, rectal, or vaginal administration or administered via oral, nasal, spray, aerosol, rectal, or vaginal administration. For injectable preparations, various effective pharmaceutical carriers are known in the art. See, e.g., ASHP Handbook on Injectable Drugs, Toissel, 18th Edition (2014).

[0292] In some embodiments, the pharmaceutical composition containing the nucleic acid molecule encoding the polypeptide of the present invention or a vector containing such a nucleic acid molecule can be administered by means of 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, poxviral vectors (e.g., vaccinia virus vectors such as modified vaccinia virus Ankara (MVA)), adeno-associated viral vectors, and alphavirus vectors. In some embodiments, the mRNA molecule encoding the polypeptide of the present invention can be directly administered to a subject.

[0293] In some embodiments of the present invention, a nucleic acid molecule encoding a polypeptide described herein or a vector containing such a nucleic acid molecule can be administered using a hydrodynamic injection platform. In the hydrodynamic injection method, the nucleic acid molecule encoding the polypeptide described herein is placed under the control of a strong promoter in an engineered plasmid (e.g., a viral plasmid). The plasmid is often rapidly delivered intravenously in a large fluid volume. Hydrodynamic injection uses the controlled hydrodynamic pressure in the vein to enhance cell permeability such that the elevated pressure from the rapid injection of a large fluid volume causes fluid and plasmid to extravasate from the vein. Expression of the nucleic acid molecule is mainly driven by the liver. In mice, hydrodynamic injection is often performed by injecting the plasmid into the tail vein. In certain embodiments, an mRNA molecule encoding a polypeptide described herein can be administered using hydrodynamic injection.

[0294] The dosage of the pharmaceutical composition of the present invention depends on factors including the route of administration, the disease to be treated, and the physical characteristics of the subject, such as age, weight, and general health status. The pharmaceutical composition of the present invention can include a dosage of the polypeptide of the present invention in the range of: 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), and in a more specific embodiment, about 0.1 to about 30 mg / kg, and in a more specific embodiment, about 0.3 to about 30 mg / kg. The dosage can be adjusted by a physician according to conventional factors such as the degree of the disease and different parameters of the subject.

[0295] The pharmaceutical composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount that results in improvement or remediation of the symptoms. The pharmaceutical composition is administered in various dosage forms, such as intravenous dosage forms, subcutaneous dosage forms, 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. The pharmaceutical composition containing the polypeptide of the present invention can be administered to a subject in need thereof, e.g., daily, weekly, bi-weekly, monthly, bi-monthly, quarterly, biennially, annually, once or multiple times (e.g., 1 - 10 times or more), or when medically necessary. In some embodiments, the pharmaceutical composition containing the polypeptide of the present invention can be administered to a subject in need thereof weekly, bi-weekly, monthly, bi-monthly, or quarterly. The dosage can be provided in a single-dose or multi-dose regimen. As the health of the patient deteriorates, as the medical condition improves or increases, the timing between administrations can be reduced.

[0296] X. Methods of Treatment The present invention is based on the following discovery: substituting amino acids from the extracellular portion of ActRIIA into the extracellular portion of ActRIIB produces ActRIIB variants with improved properties. ActRIIB variants generated by introducing residues from ActRIIA into ActRIIB can retain the beneficial properties of ActRIIB, such as the ability to increase muscle mass and high binding affinity for activin A and B, and acquire some beneficial properties of ActRIIA, such as reduced binding affinity for BMP9 or the ability to increase red blood cell levels. Since the ActRIIB variants contain the extracellular portion of the receptor, they will be soluble and able to compete with endogenous activin receptors by binding and chelating ligands (such as activin A and B, myostatin, GDF11) without activating the intracellular signaling pathway. Thus, the extracellular ActRIIB variants described herein can be used to treat diseases or conditions in which elevated activin signaling has been implicated in the pathogenesis (such as 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. Myostatin has also been implicated in promoting fibrosis, as loss of myostatin has been shown to reduce fibrosis, while increased myostatin or activin induces fibrosis. Additionally, activin has been found to be upregulated in bone diseases and is known to inhibit osteoblast activity, suggesting that increased activin levels contribute to bone diseases. Activin A has also been found to be elevated in clinical and experimental pulmonary hypertension. In another example, the activin receptor ligand GDF11 is overexpressed in a murine 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 (such as GDF11, myostatin, and / or activin) and reduce their binding 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 (such as PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH).

[0297] The compositions and methods described herein can be used to treat and / or prevent (e.g., prevent the development thereof or treat a subject diagnosed therewith) medical conditions such as muscle diseases, bone diseases, low red blood cell levels (e.g., low hemoglobin levels or low red blood cell counts, e.g., anemia), fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH). In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a 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 muscle weight and strength. In some embodiments, a polypeptide described herein can be administered to increase lean body mass. The polypeptides described herein can increase muscle weight or lean body mass compared to measurements obtained prior to treatment. In some embodiments, the subject may have a disease or be at risk of developing a disease that results in muscle weakness or atrophy (e.g., skeletal muscle weakness or atrophy). In some embodiments, the methods described herein involve affecting myostatin, activin, and / or BMP9 signaling in a subject having a disease or condition involving muscle weakness and atrophy (e.g., reducing or inhibiting the binding of activin, myostatin, and / or BMP9 to their endogenous receptors).

[0298] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a 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, increase bone strength, reduce the risk of fractures, or reduce bone resorption. The polypeptides described herein can increase bone mineral density, increase bone formation, or reduce bone resorption compared to measurements obtained prior to treatment. In some embodiments, the subject may have a disease or be at risk of developing a disease that results in bone damage (e.g., osteoporosis or osteopenia). In some embodiments, the methods described herein involve affecting myostatin, activin, and / or BMP9 signaling in a subject having a disease or condition involving bone damage (e.g., reducing or inhibiting the binding of activin, myostatin, and / or BMP9 to their endogenous receptors).

[0299] In some embodiments, the polypeptides described herein (e.g., polypeptides comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15), such as an effective amount of an ActRIIB variant)) can be administered to a subject in need thereof 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). Compared to the measurements obtained prior to treatment, the polypeptides described herein can increase red blood cell levels (e.g., increase hemoglobin levels, red blood cell count, hematocrit, or red blood cell formation). In some embodiments, the subject may have a disease or condition associated with low red blood cell levels (e.g., anemia or blood loss). In some embodiments, the subject may have anemia or blood loss or be at risk of developing anemia or blood loss (e.g., due to other diseases or conditions, or due to medical treatment (e.g., chemotherapy, radiotherapy, or surgery), the subject may be at risk of developing anemia, the other diseases or conditions such as chronic kidney disease, rheumatoid arthritis, cancer, or inflammatory diseases (e.g., Crohn's disease, SLE, or ulcerative colitis)). In some embodiments, the methods described herein involve affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of activin, myostatin, and / or BMP9 to their endogenous receptors) in a subject having a disease or condition involving low red blood cell levels (e.g., anemia or blood loss).

[0300] In some embodiments, a polypeptide as described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15), such as an effective amount of an ActRIIB variant)) can be administered to a subject in need thereof to prevent or reduce fibrosis. In some embodiments, a polypeptide as described herein can be administered to slow or stop the progression of fibrosis, to reduce the risk of developing fibrosis, or to reduce one or more symptoms of fibrosis (e.g., reduce its frequency or severity). The polypeptides described herein can reduce fibrosis or slow the progression of fibrosis by at least a certain amount compared to the progression of fibrosis prior to treatment or compared to the progression of fibrosis in an untreated subject. In some embodiments, the subject may have fibrosis or be at risk of 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 or surgery). In some embodiments, the polypeptides described herein prevent or delay the development of fibrosis in a subject at risk of developing fibrosis (e.g., a subject undergoing chemotherapy, radiation or surgical treatment, or a subject having 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 involve affecting myostatin, activin and / or BMP9 signaling in a subject having fibrosis or a disease or condition associated with fibrosis (e.g., reducing or inhibiting the binding of activin, myostatin and / or BMP9 to their endogenous receptors).

[0301] In some embodiments, the polypeptides described herein (e.g., polypeptides comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15), such as 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, abdomen (ascites) or neck (e.g., edema), chest pain or pressure, rapid pulse or palpitations, bluish lips or skin (cyanosis), dizziness or fainting), prevent PH (e.g., prevent its development), reduce the risk of developing PH, or slow or stop the progression of PH. Compared to the symptoms or progression observed prior to treatment, or compared to the PH symptoms or progression in untreated subjects, the polypeptides described herein can reduce the symptoms of PH (e.g., reduce the severity or frequency of one or more symptoms, such as shortness of breath (dyspnea), fatigue, swelling in the legs, feet, abdomen (ascites) or neck (e.g., edema), chest pain or pressure, rapid pulse or palpitations, bluish lips or skin (cyanosis), dizziness or fainting), or slow the progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH or miscellaneous PH).In some embodiments, a subject may have PH or be at risk of 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 increases the risk of developing PAH), such as HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, cirrhosis, congenital heart anomalies, connective tissue / autoimmune disorders (e.g., scleroderma or lupus), or drug use or abuse (e.g., methamphetamine or cocaine use); the subject may have a family history of PH (e.g., heritable PAH); the subject may have a disease or condition associated with venous PH (e.g., a disease or condition that increases the 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 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 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, long-term exposure to high altitude, or developmental abnormalities; 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 disease or condition associated with miscellaneous PH (e.g., a disease or condition that increases the risk of developing miscellaneous PH), such as hematologic diseases (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 tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension (pulmonary hypertension limited to one or more lung lobes)).In some embodiments, the polypeptides described herein prevent or delay the development of PH in a subject at risk of developing PH (e.g., a subject with a family history of PH (e.g., heritable PAH), or a disease or condition that increases the risk of developing PAH (e.g., HIV infection, schistosomiasis, cirrhosis, congenital heart anomalies, connective tissue / autoimmune disorders (e.g., scleroderma or 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), lung disease (e.g., pulmonary fibrosis), alveolar hypoventilation disorders, long-term exposure to high altitude or developmental anomalies), thromboembolic PH (e.g., chronic thromboembolic pulmonary hypertension, or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis or parasitic infection)), or miscellaneous PH (e.g., hematologic diseases (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 tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure or segmental pulmonary hypertension)). In some embodiments, the methods described herein involve affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of activin, myostatin, and / or BMP9 to their 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 miscellaneous PH.

[0302] In some embodiments, a polypeptide comprising an extracellular ActRIIB variant as described herein reduces or inhibits the binding of myostatin, activin, and / or BMP9 to their endogenous receptors such as ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIB). Compared to the binding of myostatin, activin, and / or BMP9 to their endogenous receptors in the absence of the polypeptide of the invention, the polypeptides described herein can reduce the binding of myostatin, activin, and / or BMP9 to their endogenous receptors. In some embodiments, affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of myostatin, activin, and / or BMP9 to their endogenous receptors such as ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIB)) results in an increase in muscle weight 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 in a subject (e.g., hemoglobin level, hematocrit, or red blood cell count, e.g., promoting or increasing red blood cell formation or production), a decrease in fibrosis or risk of developing fibrosis in a subject, a delay in fibrosis development, a decrease in fibrosis progression (e.g., slowing or inhibiting), a decrease in PH symptoms (e.g., shortness of breath (dyspnea), fatigue, swelling in the legs, feet, abdomen (ascites), or neck (e.g., edema), chest pain or pressure, rapid pulse or palpitations, blue lips or skin (cyanosis), dizziness or fainting), a decrease in the risk of developing PH, a delay in PH development, and / or a decrease in PH progression (e.g., slowing or inhibiting). PH can be PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH.

[0303] In some embodiments, the polypeptides described herein (e.g., polypeptides comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a 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 to increase muscle weight 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 (e.g., increase hemoglobin levels, increase hematocrit, increase red blood cell count, or induce or increase red blood cell formation), prevent or reduce fibrosis (e.g., reduce fibrosis, prevent or delay the development of fibrosis, or slow or stop the progression of fibrosis), prevent or treat PH (e.g., reduce the symptoms of PH, prevent or delay the development of PH, or slow or stop the progression of PH, where the PH is, for example, PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), or affect myostatin, activin, and / or BMP9 signaling in the subject. Compared to measurements obtained prior to treatment, or compared to measurements obtained from untreated subjects having the same disease or condition, an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a 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 weight 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. In some embodiments, the methods described herein do not cause any vascular complications in the subject, such as increased vascular permeability or leakage. In some embodiments of the methods described herein, the subject has a disease or condition or is at risk of developing a disease or condition that involves muscle weakness and atrophy (e.g., Duchenne muscular dystrophy (DMD), facioscapulohumeral 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 a disease or condition or is at risk of developing a disease or condition that involves bone injury (e.g., primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, fracture, bone cancer, or bone loss associated with cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, obesity treatment-related bone loss, low gravity-related bone loss, or immobilization-related bone loss).In some embodiments of the methods described herein, the subject has a disease or condition or is at risk of developing a disease or condition that involves 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 radiotherapy), myelodysplastic syndromes, chronic or acute kidney disease or failure (e.g., chronic kidney disease), inflammatory or autoimmune diseases (e.g., rheumatoid arthritis, inflammatory bowel disease such as Crohn's disease or ulcerative colitis, SLE) or surgery). In some embodiments of the methods described herein, the subject has a disease or condition or is at risk of developing a disease or condition that involves fibrosis (e.g., chemotherapy drug-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, osteoarticular fibrosis, tissue fibrosis, tumor stroma, fibroproliferative 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 vitreoretinal diseases, systemic or localized scleroderma, atherosclerosis or restenosis).In some embodiments of the methods described herein, a subject has a disease or condition or is at risk of developing a disease or condition that involves PH (such as PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH, such as idiopathic PAH; heritable PAH; PAH associated with or caused by: HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, cirrhosis, congenital heart anomalies, connective tissue / autoimmune disorders (such as scleroderma or lupus), or drug use or abuse (such as methamphetamine or cocaine use); venous PH associated with or caused by: left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital / acquired pulmonary vein stenosis; hypoxic PH associated with or caused by: chronic obstructive pulmonary disease (such as emphysema), interstitial lung disease, sleep disordered breathing (such as sleep apnea), lung disease (such as pulmonary fibrosis), alveolar hypoventilation disorders, long-term exposure to high altitude, or developmental abnormalities; thromboembolic PH associated with or caused by: chronic thromboembolic pulmonary hypertension, or other pulmonary artery obstruction (such as pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or parasitic infection); miscellaneous PH associated with or caused by: hematologic diseases (such as chronic hemolytic anemia, sickle cell disease), systemic diseases (such as sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), metabolic disorders (such as glycogen storage disease, Gaucher's disease, or thyroid disease), pulmonary tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension).

[0304] The invention also includes methods of treating a subject having or at risk of developing: Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, cancer cachexia, primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, fractures, bone cancer, or bone loss associated with cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, obesity treatment-related bone loss, low gravity-related bone loss, immobilization-related bone loss, anemia, blood loss, fibrosis, or PH (such as PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH) by administering to the subject an effective amount of a polypeptide described herein (such as a polypeptide comprising an extracellular ActRIIB variant (such as an extracellular ActRIIB variant polypeptide having any one of the sequences of SEQ ID NOs: 1-15 (such as SEQ ID NOs: 2-15))).

[0305] In any of the methods described herein, a subject having a bone disease (such as a bone injury) or at risk of developing a bone disease (such as a bone injury) has a disease or condition or is at risk of developing a disease or condition, said disease or condition including primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, fractures, bone cancer or bone loss associated with cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss related to obesity treatment, low gravity-related bone loss, or immobilization-related bone loss. In some embodiments, primary osteoporosis is age-related or hormone-related (such as related to a decline in estrogen). In some embodiments, secondary osteoporosis is immobilization-induced or glucocorticoid-induced osteoporosis. In some embodiments, bone cancer is multiple myeloma, or bone loss associated with cancer metastasis is caused by multiple myeloma. In some embodiments, treatment-related bone loss occurs due to treatment with FGF-21 or GLP-1, due to treatment with a therapeutic agent containing FGF-21 or GLP-1, due to treatment of type 2 diabetes and / or obesity, or due to cancer therapies (such as chemotherapy or radiation). In some embodiments, diet-related bone loss is rickets (such as vitamin D deficiency). In some embodiments, low gravity-related bone loss is a lack of load-related bone loss. In some embodiments, the methods described herein increase bone mineral density (such as increasing bone mass), such as increasing bone mineral density, compared to a measurement obtained prior to treatment or compared to the bone mineral density typically observed in untreated subjects. In some embodiments, the methods described herein reduce bone resorption (such as reducing bone catabolic activity), such as reducing bone resorption, compared to a measurement obtained prior to treatment or compared to the bone resorption typically observed in untreated subjects. In some embodiments, the methods described herein increase bone formation (such as increasing bone anabolic activity or increasing osteogenesis), such as increasing bone formation, compared to a measurement obtained prior to treatment or compared to the bone formation typically observed in untreated subjects. In some embodiments, the methods described herein increase osteoblast activity or osteoblastogenesis, such as increasing osteoblast activity or osteoblastogenesis, compared to a measurement obtained prior to treatment or compared to the osteoblast activity or osteoblastogenesis typically observed in untreated subjects. In some embodiments, the methods described herein reduce osteoclast activity or osteoclastogenesis, such as reducing osteoclast activity or osteoclastogenesis, compared to a measurement obtained prior to treatment or compared to the osteoclast activity or osteoclastogenesis typically observed in untreated subjects. In some embodiments, the bone is cortical bone or trabecular bone.

[0306] The present invention also includes a method of treating a subject having anemia or blood loss or at risk of developing anemia or blood loss by administering to the subject an effective amount of a polypeptide as described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15))). In any of the methods described herein, a subject having a low level of red blood cells (e.g., a low hemoglobin level, a low hematocrit, or a low red blood cell count) or at risk of developing a low level of red blood cells has anemia or blood loss or is at risk of developing anemia or blood loss. In some embodiments, anemia is associated with: nutritional deficiencies (e.g., vitamin deficiencies), bone marrow defects (e.g., paroxysmal nocturnal hemoglobinuria), adverse reactions to drug therapy (e.g., antiretroviral HIV drugs), myelodysplastic syndromes, bone marrow transplantation, cancer (e.g., solid tumors, such as breast cancer, lung cancer, colon cancer; lymphatic system tumors, such as chronic lymphocytic leukemia, non-Hodgkin lymphoma, Hodgkin lymphoma; or hematopoietic system tumors, such as leukemia or multiple myeloma), cancer treatment (e.g., radiation or chemotherapy, such as chemotherapy with platinum-containing agents), inflammatory or autoimmune diseases (e.g., rheumatoid arthritis, other inflammatory arthritides, systemic lupus erythematosus (SLE), acute or chronic skin diseases (e.g., psoriasis), or inflammatory bowel disease (e.g., Crohn's disease or ulcerative colitis), cystitis, gastritis), acute or chronic kidney disease or failure (e.g., chronic kidney disease) including idiopathic or congenital conditions, acute or chronic liver disease, acute or chronic bleeding, infections (e.g., malaria, osteomyelitis), splenomegaly, porphyria, vasculitis, hemolysis, urinary tract infections, hemoglobinopathies (e.g., sickle cell disease), thalassemia, Chusid syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplantation, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schönlein purpura, Shwachman syndrome (e.g., Shwachman-Diamond syndrome), drug use or abuse (e.g., alcohol abuse), or transfusion contraindications (e.g., elderly patients, patients with allo- or autoantibodies, pediatric patients, patients with cardio-pulmonary diseases, patients who object to transfusion 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 respond well to erythropoietin (EPO), or are susceptible to the adverse effects of EPO (such as hypertension, headache, vascular thrombosis, flu-like syndrome, shunt occlusion, and myocardial infarction). In some embodiments, blood loss is due to surgery, trauma, wounds, ulcers, urinary tract bleeding, gastrointestinal bleeding, frequent blood donation, or excessive menstrual bleeding (such as menorrhagia). In some embodiments, the methods described herein increase red blood cell levels (such as hemoglobin levels, hematocrit, or red blood cell count) compared to measurements obtained prior to treatment. In some embodiments, the methods described herein increase or induce red blood cell formation compared to measurements obtained prior to treatment. In some embodiments, the compositions and methods described herein reduce the need for transfusion in a subject (such as the subject no longer requires transfusion, or the subject requires less frequent transfusion compared to prior to treatment with the compositions and methods described herein). Subjects with normal red blood cell levels can also be treated with the methods and compositions described herein to increase red blood cell levels such that blood can be drawn and stored for use in later transfusions.

[0307] The present invention also includes a method of treating a subject having fibrosis or at risk of developing fibrosis by administering to the subject an effective amount of a polypeptide as described herein (e.g., a polypeptide comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a 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 (e.g., cirrhosis, biliary atresia), renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis (e.g., endomyocardial fibrosis or fibrosis associated with myocardial infarction), myelofibrosis, mediastinal fibrosis, retroperitoneal fibrosis, joint fibrosis, osteoarticular fibrosis, tissue fibrosis (e.g., fibrosis affecting muscle tissue, skin epidermis, skin dermis, tendon, cartilage, pancreatic tissue, uterine tissue, nerve tissue, testis, ovary, adrenal gland, artery, vein, colon, small intestine, large intestine, biliary tract, or intestine), tumor stroma, fibroproliferative tumor, surgical adhesion, hypertrophic scar, or keloid. In some embodiments, the fibrosis is associated with: wound, burn, hepatitis B or C infection, fatty liver disease, schistosome infection, kidney disease (e.g., chronic kidney disease), heart disease, macular degeneration, retinal or vitreoretinal disease, Crohn's disease, systemic or localized scleroderma, atherosclerosis, or restenosis. In some embodiments, the subject is at risk of developing fibrosis associated with: cancer treatment (chemotherapy or radiation), 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 vitreoretinal disease, Crohn's disease, systemic or localized scleroderma, atherosclerosis, restenosis), surgery, wound, or burn. In some embodiments, the methods described herein reduce fibrosis compared to a measurement obtained prior to treatment or compared to fibrosis in an untreated subject. In some embodiments, the methods described herein prevent the development of fibrosis or reduce the risk of developing fibrosis (e.g., reduce the risk of developing fibrosis compared to the development of fibrosis in an untreated subject). In some embodiments, the methods described herein slow or stop the progression of fibrosis (e.g., slow the progression of fibrosis compared to the progression prior to treatment or compared to the progression in a non-treated or 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 having fibrosis) as compared to the organ or tissue function prior to treatment. Tissue and organ function can be evaluated using any standard clinical tests commonly used to assess tissue and organ function.

[0308] The present invention also includes a method of treating a subject having PH (such as PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH) or at risk of developing PH by administering to the subject an effective amount of a polypeptide described herein (such as a polypeptide comprising an extracellular ActRIIB variant (such as an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (such as SEQ ID NOs: 2-15))). In any of the methods described herein, the subject may have PH (such as PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH) or be at risk of developing PH. In some embodiments, the PH is PAH. In some embodiments, the PAH is idiopathic PAH. In some embodiments, the PAH is heritable PAH. In some embodiments, the PAH is PAH associated with (such as caused by or related to) the following: HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, cirrhosis, congenital heart anomalies, connective tissue / autoimmune disorders (such as scleroderma or lupus), or drug use or abuse (such as methamphetamine or cocaine use). In some embodiments, the PH is venous PH. In some embodiments, the venous PH is venous PH associated with (such as caused by or related to) the following: left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, 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 (such as caused by or related to) the following: chronic obstructive pulmonary disease (such as emphysema), interstitial lung disease, sleep disordered breathing (such as sleep apnea), lung disease (such as pulmonary fibrosis), alveolar hypoventilation disorders, long-term exposure to high altitude, or developmental abnormalities. In some embodiments, the PH is thromboembolic PH. In some embodiments, the thromboembolic PH is thromboembolic PH associated with (such as caused by or related to) the following: chronic thromboembolic pulmonary hypertension, or other pulmonary artery obstruction (such as pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or parasitic infection). In some embodiments, the PH is miscellaneous PH. In some embodiments, the miscellaneous PH is miscellaneous PH associated with (such as caused by or related to) the following: hematologic disorders (such as chronic hemolytic anemia, sickle cell disease), systemic diseases (such as sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), metabolic disorders (such as glycogen storage disease, Gaucher disease, or thyroid disease), pulmonary tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension).In some embodiments, the methods described herein reduce the symptoms of PH (e.g., reduce the severity or frequency of symptoms such as shortness of breath (dyspnea), fatigue, swelling in the legs, feet, abdomen (ascites), or neck (e.g., edema), chest pain or pressure, rapid or irregular pulse, bluish coloration of the lips or skin (cyanosis), dizziness or fainting) compared to the frequency or severity of symptoms prior to treatment. In some embodiments, the methods described herein prevent the development 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 miscellaneous PH) compared to the development of PH in untreated subjects). In some embodiments, the methods described herein slow or stop the progression of PH (e.g., slow the progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH) compared to the progression prior to treatment or compared to the progression in untreated or un-treated subjects). In some embodiments, the methods described herein reduce pulmonary vascular remodeling or vascular remodeling in the heart of the subject (e.g., the onset or progression of vascular remodeling in the heart or lungs) compared to the vascular remodeling prior to treatment or compared to the vascular remodeling in untreated subjects. In some embodiments, the methods described herein reduce right ventricular hypertrophy (e.g., reduce right ventricular hypertrophy or the progression of right ventricular hypertrophy) compared to the right ventricular hypertrophy prior to treatment or compared to the right ventricular hypertrophy in untreated subjects. The symptoms of PH can be evaluated using standard clinical tests before and after treatment. Commonly used tests for evaluating PH include electrocardiogram, pulmonary function tests, 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 the pulmonary vascular resistance prior to treatment). In some embodiments, the methods described herein improve performance in the 6-minute walk test compared to the performance in the 6-minute walk test prior to treatment).

[0309] In any of the methods described herein, a dimer (e.g., a homodimer or heterodimer) formed by the interaction of two Fc domain monomers can be used as a therapeutic protein, wherein each of the two Fc domain monomers is fused to a polypeptide comprising 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)). In any of the methods described herein, a polypeptide comprising 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)) fused to a moiety (e.g., a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or serum albumin) can be used as a therapeutic protein. A nucleic acid encoding a polypeptide described herein, or a vector containing the nucleic acid, 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. Examples

[0310] The following examples are provided to further illustrate some embodiments of the invention, but are not intended to limit the scope of the invention; it is understood by way of its exemplary nature, and other procedures, methods, or techniques known to those skilled in the art can alternatively be used.

[0311] Example 1 – Effect of Extracellular ActRIIB Variants on Body Weight and Muscle Weight C57Bl / 6 mice received a single hydrodynamic injection of a plasmid construct encoding one of the following fourteen polypeptides or a vehicle control (n = 10 / group, see Figure 1 the sequences provided in): (1) Vehicle, (2) Extracellular ActRIIA (SEQ ID NO: 16) fused to the N-terminus of hFc via a GGG linker; (3) Extracellular ActRIIB (SEQ ID NO: 17) fused to the N-terminus of hFc via a GGG linker; (4) Extracellular ActRIIB variant ActRIIB / A (SEQ ID NO: 2) fused to the N-terminus of hFc via a GGG linker; (5) Extracellular ActRIIB variant ActRIIBΔ9 (SEQ ID NO: 3) fused to the N-terminus of hFc via a GGG linker; (6) The extracellular ActRIIB variant ActRIIB 2.01 (SEQ ID NO: 4) fused to the N-terminus of hFc via a GGG linker; (7) The extracellular ActRIIB variant ActRIIB 2.02 (SEQ ID NO: 5) fused to the N-terminus of hFc via a GGG linker; (8) The extracellular ActRIIB variant ActRIIB 2.03 (SEQ ID NO: 6) fused to the N-terminus of hFc via a GGG linker; (9) The extracellular ActRIIB variant ActRIIB 2.04 (SEQ ID NO: 7) fused to the N-terminus of hFc via a GGG linker; (10) The extracellular ActRIIB variant ActRIIB 2.05 (SEQ ID NO: 8) fused to the N-terminus of hFc via a GGG linker; (11) The extracellular ActRIIB variant ActRIIB 2.06 (SEQ ID NO: 9) fused to the N-terminus of hFc via a GGG linker; (12) The extracellular ActRIIB variant ActRIIB 2.07 (SEQ ID NO: 10) fused to the N-terminus of hFc via a GGG linker; (13) The extracellular ActRIIB variant ActRIIB 2.08 (SEQ ID NO: 11) fused to the N-terminus of hFc via a GGG linker; (14) The extracellular ActRIIB variant ActRIIB 2.09 (SEQ ID NO: 12) fused to the N-terminus of hFc via a GGG linker; and (15) The extracellular ActRIIB variant ActRIIB 2.10 (SEQ ID NO: 13) fused to the N-terminus of hFc via a GGG linker.

[0312] After 5 - 8 seconds, 100 μg of the plasmid construct was delivered at a volume of 10% body weight. The high volume and short injection period provided the pressure required to introduce the plasmid into the hepatocytes in which the plasmid was expressed. In particular, the target protein was expressed under a strong and ubiquitous promoter (pLEV113). The target protein was secreted and freely circulated under the endogenous mechanism of the hepatocytes. The mice were weighed twice a week for a total of 28 days, and the measurements were recorded as the percentage change in body weight from the baseline measurement ( Figure 1 、 Figure 2 ). The muscles were also weighed at the end of the study, and the measurements were recorded in milligrams ( Figure 3A and 3B ).

[0313] Example 2 - Evaluation of the binding affinity of ActRIIB variants by surface plasmon resonance (SPR) The Biacore 3000 was used to measure the kinetics of the interaction between ActRIIB-Fc variants and the ligands activin A, activin B, growth differentiation factor 11 (GDF11), and BMP-9. ActRIIA, ActRIIB, ActRIIB 2.06, ActRIIB 2.11, and ActRIIB 2.12 are recombinant proteins. All other ActRIIB-Fc variants were expressed by transient expression in HEK293 cells and purified from the 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. The 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 any non-specific binding. HBS-EP+ buffer from GE Healthcare TM was used as the running buffer. Each ligand was run in 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 used a CM-4. The data were analyzed using Scrubber2 software by BioLogic TM to calculate the K D (Table 3).

[0314] Table 3: Comparison of the binding affinities (K D ) of ActRIIB variants with various ligands

[0315] Example 3 - Effect of extracellular ActRIIB variants on bone mineral density Adult male C57 / BL6 mice received sham operation (SHAM) or orchiectomy (ORX). Both surgical groups were allowed to recover for 14 days after surgery. All animals were placed in conventional cages with free access to food (regular chow) and water. Then, SHAM and ORX animals were assigned to either the vehicle treatment group (VEH) or the ActRII variant treatment group and received systemic intraperitoneal administration of vehicle or ActRII variant (10 mg / kg) once every two weeks for 71 days. Body weight was measured twice a week during treatment. Body composition was analyzed using a MiniSpec LF50 NMR analyzer on day 0 of treatment and then on days 14, 28, 47, and 71 after the start of treatment. At the study termination date, the target tissues (muscle, fat depot, and tibia) were surgically excised, weighed, and stored appropriately for further analysis. At this time, ORX animals were also examined to confirm complete removal of the testes. After the experiment was terminated, the cortical morphology and trabecular structure of each bone were also evaluated using micro-computed tomography.

[0316] Example 4 - Effect of Extracellular ActRIIB Variant on Renal Fibrosis A unilateral ureteral obstruction (UUO) mouse model of renal fibrosis was used to determine the effect of extracellular ActRIIB variant on renal fibrosis. The UUO model involves complete ligation of the left ureter while keeping the right renal function intact. Briefly, UUO was performed on mice under anesthesia, whereby the left ureter was accessed via a flank incision and two ligatures were placed on the proximal third of the ureter using silk sutures spaced 5 mm apart. Sham surgery was performed in a similar manner without placing any ligatures on the ureter. In this model, severe fibrosis developed in the kidney was evaluated by measuring renal collagen by directly measuring the amount of hydroxyproline in the samples within 14 days after UUO. Fourteen days after UUO, the renal dry weight decreased due to renal parenchymal injury. Sham operation or UUO surgery was performed on 16-week-old male C57BL / 6 mice, and the UUO-operated mice were divided into two groups. Starting from the day before surgery and on days 1, 3, 6, 8, 10, and 13 after surgery, each UUO group received subcutaneous injection of ActRIIB variant (10 mg / kg) or vehicle (weight / volume of administration), which did not bind to any known mouse proteins. Sham-operated mice received vehicle (sterile PBS) using the same schedule as the UUO groups during this period. All mice were sacrificed on day 14 after surgery. Renal weight was measured, and the kidneys were quickly frozen using liquid nitrogen and kept at -80 °C until the collagen content was measured by measuring the amount of hydroxyproline to evaluate fibrosis.

[0317] Example 5 - Effect of Extracellular ActRIIB Variant on Red Blood Cells Groups of ten male and ten female rats received two SC doses of vehicle (days 1 and 15), or ActRllB variants at 6, 20 or 60 mg / kg. Hematological parameters were measured on day 29. Studies were also conducted to evaluate the time course and dose response induced on RBC, hemoglobin, and hematocrit. In the first study, the time course of erythropoiesis was investigated in male and female rats that received a subcutaneous (SC) dose of ActRllB variant (10 mg / kg) on days 1 and 8. Hematological parameters were evaluated before dosing and on days 3, 8, 15, 29, and 44. In the second study, the hematological dose response was investigated in male and female rats that received an SC dose of vehicle on days 1 and 15, or ActRllB variants at 0.4, 2, 10 or 30 mg / kg. Hematological parameters were evaluated before dosing and on days 13 and 28.

[0318] Example 6 - Effect of Extracellular ActRIIB Variants on PAH In one experiment, pulmonary arterial hypertension (PAH) was induced in male rats using a single subcutaneous injection of monocrotaline (MCT, 40 mg / kg). To determine whether treatment with ActRIIB variants could prevent the development of PAH, 24 hours after PAH induction, the rats were randomly assigned to either vehicle or ActRIIB variant treatment groups and treated twice weekly with ActRIIB variants (5 or 15 mg / kg) or vehicle for 21 days. On day 14, ventricular function and right ventricular (RV) remodeling were examined by electrocardiogram by anesthetizing the rats with 1.5% isoflurane and using a small animal high-frequency ultrasound probe to detect pulmonary blood flow acceleration, right ventricular function and hypertrophy, and left ventricular function while keeping the animals in the supine position. Doppler across the mitral and tricuspid valves was used to determine whether treatment with ActRIIB variants induced any significant regurgitation or lesions. On day 21, the rats were anesthetized with pentobarbital, intubated via the trachea, and mechanically ventilated using a rodent ventilator. Hemodynamics were evaluated through the RV apex using a fluid-filled catheter. The rats were perfused with PBS followed by 1% formaldehyde. To measure right ventricular hypertrophy (RVH), the heart was excised and the RV wall was dissected free from the left ventricle plus septum (LV+S) and weighed separately. The degree of RVH was determined by the ratio RV / (LV+S).

[0319] In the second experiment, pulmonary arterial hypertension (PAH) was induced in male rats by a single subcutaneous injection of monocrotaline (MCT, 40 mg / kg). To determine whether treatment with the ActRIIB variants could slow or reduce the progression of PAH, the rats were reinjected with MCT on day 18 and randomly assigned to receive either vehicle or ActRIIB variant treatment. The rats were injected three times per week with either the ActRIIB variant (15 mg / kg) or vehicle. Hemodynamics and RVH were examined on day 35 as described above.

[0320] Example 7 – Evaluation of ActRIIB Variants Using a Gene Luciferase Reporter Assay C2C12-BRE-luciferase and HEK293-SBE-luciferase cells were plated in 96-well plates in DMEM supplemented with 2% FBS and placed in an incubator for at least three hours to adapt 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 at 37 °C for 30 minutes. ActRIIA, ActRIIB, ActRIIB 2.06, ActRIIB 2.11, and ActRIIB 2.12 are recombinant proteins. All others are conditioned media from transiently transfected cells. The media in the plates was aspirated and the ActRIIB / A / ligand mixture was added to the plates as a media replacement. The remaining wells were used for repeats of the positive control and background. The plates were incubated overnight and then read using Promega Steady Glo and a Molecular Devices Spectramax M5e. The cell-based assays confirmed the ability of the variants to inhibit signaling at endogenous cell surface receptors. As shown in Table 4, with the exception of ActRIIB / A and ActRIIB 2.11, the variants had comparable inhibition of activin A, activin B, and GDF-11 but reduced BMP9 inhibition compared to ActRIIB-Fc.

[0321] Table 4: Results from the Luciferase Reporter Assay

[0322] Example 8 – Effects of ActRIIB Variants on Body Weight and Red Blood Cell Mass Eight-week-old male C57BL / 6 mice were assigned to three groups (n = 10 / group). Each group was administered with 5 ml / kg of vehicle (Tris-buffered saline, pH 7.4) or ActRllB 2.11 (SEQ ID NO: 14)-Fc or ActRllB 2.12 (SEQ ID NO: 15)-Fc (20 mg / kg). Treatments were administered intraperitoneally (IP) twice a week for four weeks (eight doses), and the study was terminated on day 28 of the study. Body weight was recorded on the dosing days throughout the study. Additionally, erythrocyte volume parameters were evaluated by hematology at the end of the study. As Figure 4 shown, both ActRllB 2.11-Fc and ActRllB 2.12-Fc increased body weight in wild-type mice (* = p < 0.05; **** = p < 0.01). ActRIIB 2.12-Fc also increased parameters of erythrocyte volume (*** = p < 0.001; **** = p < 0.0001), including red blood cell count, hemoglobin level, and hematocrit ( Figures 5A - 5C ).

[0323] Example 9 – Effect of ActRIIB Variants in a Mouse Model of Osteoporosis C57BL / 6 mice were subjected to orchiectomy (ORX) or sham surgery at 9 weeks of age. After a six-week recovery period during which ORX mice developed an osteoporosis phenotype, ORX mice received intraperitoneal injections of vehicle or ActRIIB 2.12 (SEQ ID NO: 15)-Fc (10 mg / kg) twice a week. Micro-CT (PerkinElmer Quantum Fx) imaging was performed at week 11 after the start of dosing. ASBMR bone morphometry parameters for each dataset were calculated using the Bone Morphometry Analysis Add-on with AnalyzePro software (AnalyzeDirect, Overland Park KS). A 50-slice region of the CT volume immediately adjacent to the distal to proximal tibial growth plate was selected to evaluate changes in trabecular bone parameters. As Figures 6A - 6D shown, treatment with ActRIIB 2.12-Fc increased bone volume, increased bone volume fraction, and increased trabecular number associated with orchiectomy (* = p < 0.05; ** = p < 0.01). These data indicate that treatment of osteoporotic mice with ActRIIB 2.12-Fc increased trabecular bone mass due to increased bone formation.

[0324] Example 10 - Effects of ActRIIB Variant 2.12 on Red Blood Cell Volume and Trabecular Bone in Rats Hydrodynamic injection of ActRIIB 2.12 (SEQ ID NO: 15)-Fc plasmid DNA was delivered to four-week-old Sprague Dawley rats via the lateral tail vein. Four weeks after injection, hematological parameters of the blood and ActRIIB 2.12-Fc levels were analyzed. MicroCT imaging (Perkin Elmer Quantum Fx) was performed ex vivo on the tibiae. ASBMR bone morphometry parameters for each dataset were calculated using the Bone Morphometry Analysis Add-on with AnalyzePro software (AnalyzeDirect, Overland Park KS). A 150-slice region of the CT volume immediately adjacent to the distal to proximal tibial growth plate was selected to evaluate changes in trabecular bone parameters. As Figures 7A - 7C shown, ActRIIB 2.12-Fc increased parameters of red blood cell volume in wild-type rats (* = p < 0.05; ** = p < 0.01), including red blood cell count, hemoglobin level, and hematocrit. ActRIIB 2.12-Fc also increased trabecular bone (trabecular bone volume, trabecular bone fraction, and trabecular thickness) in wild-type rats ( Figures 8A - 8E ; * = p < 0.05; ** = p < 0.01). These data indicate that treatment with ActRIIB 2.12-Fc in wild-type rats increased trabecular bone mass.

[0325] Example 11 - Treatment of Muscle Diseases by Administering Extracellular ActRIIB Variants According to the methods disclosed herein, a physician in the art can treat a subject, such as a human patient, suffering from a muscle disease (e.g., DMD, ALS, or inclusion body myositis) to increase muscle weight or maintain or improve muscle strength (e.g., reduce muscle weakness). The treatment method can include diagnosing or identifying the subject as a candidate for treatment based on standard clinical tests for muscle diseases (e.g., blood tests, muscle biopsies, genetic tests, and / or electromyograms). To treat the subject, a physician in the art can administer to the subject a composition containing an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)). The composition containing the extracellular ActRIIB variant can be administered to the subject, for example, by parenteral injection (e.g., intravenous injection) or by topical administration (e.g., injection into the muscle) to treat the muscle disease. The extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) is administered in a therapeutically effective amount, such as 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). In some embodiments, the extracellular ActRIIB variant is administered once every two months, 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 times a week or more). The extracellular ActRIIB variant is administered in an amount sufficient to increase muscle weight or maintain or improve muscle strength (e.g., reduce muscle weakness).

[0326] After the composition is administered to the patient, those skilled in the art can monitor the improvement of the patient's response to the therapy by various methods. For example, a physician can monitor the patient's muscle weight, muscle strength, and motor function. The finding that the patient shows an increased muscle weight or maintains or improves muscle strength after the composition is administered, as compared to the test results before the composition is administered, indicates that the patient responds well to the treatment. Subsequent doses can be determined and administered as needed.

[0327] Example 12 - Treating Bone Diseases by Administering Extracellular ActRIIB Variants According to the methods disclosed herein, a physician in the art can treat a subject, such as a human patient, suffering from a bone disease (e.g., osteoporosis or osteopenia) in order to increase bone mineral density, increase bone formation, reduce bone resorption, reduce bone loss, or reduce the risk of fracture. The treatment method can include diagnosing or identifying the subject as a candidate for treatment based on standard clinical tests for bone mineral density (e.g., dual X-ray absorptiometry). To treat the subject, a physician in the art can administer to the subject a composition comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)). The composition comprising 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 a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) is administered in a therapeutically effective amount, such as 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). In some embodiments, the extracellular ActRIIB variant is administered once every two months, 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 times a week or more). 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.

[0328] After the composition is administered to the patient, those skilled in the art can monitor the improvement of the patient's response to the therapy by various methods. For example, a physician can monitor the patient's bone mineral density by performing dual X-ray absorptiometry. The finding that the patient shows increased bone mineral density, increased bone formation, reduced bone resorption, reduced bone loss, or reduced risk of fracture after the composition is administered, as compared to the test results before the composition is administered, indicates that the patient responds well to the treatment. Subsequent doses can be determined and administered as needed.

[0329] Example 13 – Treatment of anemia by administering an extracellular ActRIIB variant According to the methods disclosed herein, a physician in the art can treat a subject, such as a human patient, suffering from anemia (e.g., vitamin deficiency anemia or anemia associated with chronic kidney disease) in order to increase parameters of red blood cell mass, such as red blood cell count, hemoglobin level, or hematocrit. The treatment method can include a blood test based on measuring hematological parameters to diagnose or identify the subject as a candidate for treatment. To treat the subject, a physician in the art can administer to the subject a composition comprising an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)). The composition comprising the extracellular ActRIIB variant can be administered to the subject, for example, by parenteral injection (e.g., intravenous injection) to treat the anemia disorder. The extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) is administered in a therapeutically effective amount, such as 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). In some embodiments, the extracellular ActRIIB variant is administered once every two months, 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 times a week or more). The extracellular ActRIIB variant is administered in an amount sufficient to increase the hemoglobin level, increase the red blood cell count, or increase the hematocrit.

[0330] After the composition is administered to the patient, a person skilled in the art can monitor the improvement of the patient's response to the therapy by various methods. For example, a physician can monitor the patient's hemoglobin level, red blood cell count, or hematocrit by performing a blood test. The finding that the patient shows an improved hemoglobin level, red blood cell count, or hematocrit after the composition is administered, compared to the test results before the composition is administered, indicates that the patient responds well to the treatment. Subsequent doses can be determined and administered as needed.

[0331] Example 14 - Treatment of fibrosis by administering an extracellular ActRIIB variant According to the methods disclosed herein, a physician in the art can treat a subject, such as a human patient, suffering from fibrosis (such as pulmonary fibrosis or fibrosis associated with chronic kidney disease) in order to reduce the symptoms of fibrosis or slow or stop the progression of fibrosis. The treatment method can include diagnosing or identifying a subject as a candidate for treatment based on clinical tests for fibrosis (such as imaging tests, such as X-rays or CT scans). To treat a subject, a physician in the art can administer to the subject a composition containing an extracellular ActRIIB variant (such as an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (such as SEQ ID NOs: 2-15)). The composition containing the extracellular ActRIIB variant can be administered to the subject, for example, by parenteral injection (such as intravenous injection) to treat fibrosis, or can be administered locally (such as by injection) into the fibrotic tissue or organ. The extracellular ActRIIB variant (such as an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (such as SEQ ID NOs: 2-15)) is administered in a therapeutically effective amount, such as 0.01 to 500 mg / kg (such as 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 once every two months, once a month, once every two weeks, or at least once a week or more frequently (such as 1, 2, 3, 4, 5, 6, or 7 times a week or more frequently). The extracellular ActRIIB variant is administered in an amount sufficient to reduce the symptoms of fibrosis or slow or stop the progression of fibrosis.

[0332] After the composition is administered to the patient, those skilled in the art can monitor the improvement of the patient's response to the therapy by various methods. For example, a physician can monitor the patient's fibrosis by performing imaging tests and can use standard clinical tests to monitor the patient's symptoms. The finding that the patient's symptoms are reduced or the progression of the patient's fibrosis is slowed or stopped after the composition is administered, compared with the test results before the composition is administered, indicates that the patient responds well to the treatment. Subsequent doses can be determined and administered as needed.

[0333] Example 15 - Treatment of Pulmonary Hypertension by Administration of Extracellular ActRIIB Variants According to the methods disclosed herein, a physician skilled in the art can treat a subject, such as a human patient, suffering from pulmonary hypertension (PH, such as PAH) in order to reduce the symptoms of PH or slow or stop the progression of PH. The treatment method can include diagnosing or identifying a subject as a candidate for treatment based on standard clinical tests for PH (such as echocardiogram, electrocardiogram, chest X-ray, or right heart catheterization). To treat a subject, a physician skilled in the art can administer to the subject a composition comprising an extracellular ActRIIB variant (such as an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 1-15 (such as SEQ ID NOs: 2-15)). The composition comprising the extracellular ActRIIB variant can be administered to the subject, for example, by parenteral injection (such as intravenous injection) to treat PH. The extracellular ActRIIB variant (such as an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 1-15 (such as SEQ ID NOs: 2-15)) is administered in a therapeutically effective amount, such as 0.01 to 500 mg / kg (such as 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 once every two months, once a month, once every two weeks, or at least once a week or more often (such as 1, 2, 3, 4, 5, 6, or 7 times a week or more often). The extracellular ActRIIB variant is administered in an amount sufficient to reduce the symptoms of PH or slow or stop the progression of PH.

[0334] After the composition is administered to the patient, a person skilled in the art can monitor the improvement of the patient's response to the therapy by various methods. For example, a physician can use standard clinical tests and patient self-reporting to monitor the patient's symptoms. The finding that the patient's PH symptoms are reduced or the progression of the patient's PH is slowed or stopped after the composition is administered, compared to the test results before the composition was administered, indicates that the patient is responding well to the treatment. Subsequent doses can be determined and administered as needed.

[0335] Other Embodiments Although the invention has been described in connection with specific embodiments of the invention, it is to be understood that it is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention that follow generally the principles of the invention and include such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains and can be applied to the essential features described above.

[0336] All publications, patents, and patent applications are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0337] Other embodiments are within the following claims.

[0338] The present invention relates to: 1. A polypeptide comprising a variant of activin receptor type IIB (ActRIIB) extracellular, said variant having one or more amino acid substitutions relative to the sequence, wherein said variant 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 comprise one or more of the following: a) the amino acid substitution E75K; b) the amino acid substitutions Q69T and E70D; or c) the amino acid substitutions Q69D and E70T.

[0339] 2. The polypeptide of item 1, wherein said variant comprises one or more amino acid substitutions selected from the following: 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.

[0340] 3. The polypeptide of item 1 or 2, wherein said variant comprises the amino acid substitutions E75K, E20D, and F63I.

[0341] 4. The polypeptide of item 1 or 2, wherein said variant comprises the amino acid substitution E75K.

[0342] 5. The polypeptide of item 4, wherein said variant comprises the amino acid substitutions T74K, E76D, N77S, and Q79E.

[0343] 6. The polypeptide of item 5, wherein said variant further comprises one or more additional amino acid substitutions.

[0344] 7. The polypeptide of item 6, wherein said variant comprises the amino acid substitutions Y41F, R45K, and K56Q.

[0345] 8. The polypeptide of item 7, wherein the variant further comprises the amino acid substitutions Y12F, L19K, E20D, R29P, E31Y, E33D, L38R, and F63I.

[0346] 9. The polypeptide of item 6, wherein the variant comprises the amino acid substitutions S25T and S47I.

[0347] 10. The polypeptide of item 9, wherein the variant comprises the amino acid substitution S48T.

[0348] 11. The polypeptide of item 6, wherein the variant comprises the amino acid substitution R29P.

[0349] 12. The polypeptide of item 6, wherein the variant comprises the amino acid substitutions E31Y, E33D, and Q34K.

[0350] 13. The polypeptide of item 6, wherein the variant comprises the amino acid substitutions Y12F, L19K, and E20D.

[0351] 14. The polypeptide of item 6, wherein the variant comprises the amino acid substitutions E31Y, E33D, and L38R.

[0352] 15. The polypeptide of item 1 or 2, wherein the variant comprises the amino acid substitutions Q69T and E70D, and additional amino acid substitutions I11L, L27V, Q34K, T50S, I51L, L53I, and F89M.

[0353] 16. The polypeptide of item 1 or 2, wherein the variant comprises the amino acid substitutions Q69D and E70T, and additional amino acid substitutions I11L, L27V, Q34K, T50S, I51L, L53I, and F89M.

[0354] 17. The polypeptide of item 15 or 16, wherein the variant comprises the amino acid substitution E75K.

[0355] 18. The polypeptide of item 1 or 2, wherein the variant has the sequence of any one of SEQ ID NOs: 2 - 15.

[0356] 19. The polypeptide of any one of items 1 - 18, which further comprises an Fc domain monomer fused to the C-terminus of the polypeptide via a linker.

[0357] 20. The polypeptide of item 19, wherein the Fc domain monomer comprises the sequence of SEQ ID NO: 19.

[0358] 21. The polypeptide of item 19 or 20, wherein the polypeptide forms a dimer.

[0359] 22. A polypeptide according to any one of items 1 - 18, which comprises a wild - type Fc domain fused to the C - terminus of the polypeptide via a linker.

[0360] 23. The polypeptide of item 22, wherein the wild - type Fc domain comprises the sequence of SEQ ID NO: 71.

[0361] 24. A polypeptide according to any one of items 1 - 18, which comprises an Fc domain having an amino acid substitution, and the Fc domain is fused to the C - terminus of the polypeptide via a linker.

[0362] 25. The polypeptide of item 24, wherein the Fc domain does not form a dimer.

[0363] 26. A polypeptide according to any one of items 1 to 18, which comprises an albumin - binding peptide fused to the C - terminus of the polypeptide via a linker.

[0364] 27. The polypeptide of item 26, wherein the albumin - binding peptide comprises the sequence of SEQ ID NO: 72.

[0365] 28. A polypeptide according to any one of items 1 to 18, which comprises a fibronectin domain fused to the C - terminus of the polypeptide via a linker.

[0366] 29. The polypeptide of item 28, wherein the fibronectin domain comprises the sequence of SEQ ID NO: 73.

[0367] 30. A polypeptide according to any one of items 1 - 18, which comprises human serum albumin fused to the C - terminus of the polypeptide via a linker.

[0368] 31. The polypeptide of item 30, wherein the human serum albumin comprises the sequence of SEQ ID NO: 74.

[0369] 32. The polypeptide of any one of items 19 - 31, wherein the linker is an amino acid spacer.

[0370] 33. The polypeptide of item 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).

[0371] 34. The polypeptide of any one of items 1 - 33, wherein the polypeptide has a serum half - life of at least seven days.

[0372] 35. The polypeptide of any one of items 1 - 34, wherein the polypeptide has a K of 200 pM or higher DBinds to human bone morphogenetic protein 9 (BMP9).

[0373] 36. A polypeptide according to any one of items 1 - 35, wherein said polypeptide binds to activin and / or myostatin and has reduced or weak binding to human BMP9.

[0374] 37. A polypeptide according to item 35 or 36, wherein said polypeptide binds substantially not to human BMP9.

[0375] 38. A polypeptide according to any one of items 1 - 37, wherein said polypeptide has a K of 800 pM or less D Binds to human activin A.

[0376] 39. A polypeptide according to any one of items 1 - 38, wherein said polypeptide has a K of 800 pM or less D Binds to human activin B.

[0377] 40. A polypeptide according to any one of items 1 - 39, wherein said polypeptide has a K of 5 pM or higher D Binds to human GDF - 11.

[0378] 41. A nucleic acid molecule encoding a polypeptide according to any one of items 1 - 40.

[0379] 42. A vector comprising the nucleic acid molecule of item 41.

[0380] 43. A host cell expressing a polypeptide according to any one of items 1 - 40, wherein said host cell comprises the nucleic acid molecule of item 41 or the vector of item 42, and wherein the nucleic acid molecule or vector is expressed in the host cell.

[0381] 44. A method for preparing a polypeptide according to any one of items 1 - 43, said method comprising: a) providing a host cell comprising the nucleic acid molecule of item 41 or the vector of item 42, and b) expressing the nucleic acid molecule or vector in the host cell under conditions allowing the formation of said polypeptide.

[0382] 45. A pharmaceutical composition comprising a polypeptide according to any one of items 1 - 40, the nucleic acid molecule of item 41 or the vector of item 42, and one or more pharmaceutically acceptable carriers or excipients.

[0383] 46. The pharmaceutical composition of item 45, wherein the polypeptide is in a therapeutically effective amount.

[0384] 47. A method for increasing lean body mass in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0385] 48. A method for increasing muscle weight in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0386] 49. The method of item 48, wherein the subject has Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia, or cancer cachexia.

[0387] 50. A method of affecting myostatin, activin, and / or BMP9 signaling in a subject having a disease or condition involving muscle weakness and atrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0388] 51. The method of item 50, wherein the disease or condition is Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia, or cancer cachexia.

[0389] 52. A method of treating a subject having Duchenne muscular dystrophy or at risk of developing Duchenne muscular dystrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0390] 53. A method of treating a subject having facioscapulohumeral muscular dystrophy or at risk of developing facioscapulohumeral muscular dystrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0391] 54. A method of treating a subject having inclusion body myositis or at risk of developing inclusion body myositis, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0392] 55. A method for treating a subject suffering from amyotrophic lateral sclerosis or at risk of developing amyotrophic lateral sclerosis, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0393] 56. A method for treating a subject suffering from sarcopenia or at risk of developing sarcopenia, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0394] 57. A method for treating a subject suffering from cancer cachexia or at risk of developing cancer cachexia, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0395] 58. A method for increasing bone mineral density in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0396] 59. A method for reducing bone resorption in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0397] 60. A method for increasing bone formation in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0398] 61. A method for increasing bone strength in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0399] 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 of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0400] 63. The method of any one of items 58 - 62, wherein the subject has primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, fracture, bone cancer or bone loss associated with cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss related to obesity treatment, low gravity-related bone loss or immobilization-related bone loss.

[0401] 64. A method of modulating myostatin, activin and / or BMP9 signaling in a subject having a disease or condition involving bone injury, comprising administering to the subject a therapeutically effective amount of the polypeptide of any one of items 1 - 40, the nucleic acid molecule of item 41, the vector of item 42, or the pharmaceutical composition of item 45 or 46.

[0402] 65. The method of item 64, wherein the disease or condition is primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, fracture, bone cancer or bone loss associated with cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss related to obesity treatment, low gravity-related bone loss or immobilization-related bone loss.

[0403] 66. A method of treating a subject having a bone disease or at risk of developing a bone disease, comprising administering to the subject a therapeutically effective amount of the polypeptide of any one of items 1 - 40, the nucleic acid molecule of item 41, the vector of item 42, or the pharmaceutical composition of item 45 or 46.

[0404] 67. The method of item 66, wherein the bone disease is primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, fracture, bone cancer or bone loss associated with cancer metastasis, Paget's disease, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss related to obesity treatment, low gravity-related bone loss or immobilization-related bone loss.

[0405] 68. A method of treating a subject having primary osteoporosis, comprising administering to the subject a therapeutically effective amount of the polypeptide of any one of items 1 - 40, the nucleic acid molecule of item 41, the vector of item 42, or the pharmaceutical composition of item 45 or 46.

[0406] 69. A method of treating a subject having secondary osteoporosis, comprising administering to the subject a therapeutically effective amount of the polypeptide of any one of items 1 - 40, the nucleic acid molecule of item 41, the vector of item 42, or the pharmaceutical composition of item 45 or 46.

[0407] 70. A method of treating a subject having osteopenia, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0408] 71. A method of treating a subject having bone cancer or bone loss associated with cancer metastasis, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0409] 72. A method of treating a subject having Paget's disease, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0410] 73. A method of treating a subject having renal osteodystrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0411] 74. A method of treating a subject having treatment-related bone loss, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0412] 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 items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0413] 76. A method of treating a subject having low gravity-related bone loss, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0414] 77. A method of treating a subject having immobilization-related bone loss, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0415] 78. The method of any one of items 63, 65, 67 or 68, wherein the primary osteoporosis is age-related osteoporosis or hormone-related osteoporosis.

[0416] The method according to any one of items 63, 65, 67 or 69, wherein the secondary osteoporosis is immobilization-induced osteoporosis or glucocorticoid-induced osteoporosis.

[0417] The method according to any one of items 63, 65, 67 or 71, wherein the cancer is multiple myeloma.

[0418] The method according to any one of items 63, 65, 67 or 74, wherein the treatment is FGF-21 treatment, GLP-1 treatment, cancer therapy, or treatment for obesity or type 2 diabetes.

[0419] The method according to any one of items 63, 65, 67 or 75, wherein the diet-related bone loss is rickets.

[0420] The method according to any one of items 58-82, wherein the subject is at risk of fracture.

[0421] The method according to any one of items 58-83, wherein the method increases bone formation in the subject.

[0422] The method according to any one of items 58-84, wherein the method reduces bone resorption in the subject.

[0423] The method according to any one of items 58-85, wherein the method increases osteoblast activity or osteoblast generation.

[0424] The method according to any one of items 58-86, wherein the method reduces osteoclast activity or reduces osteoclast generation.

[0425] The method according to any one of items 58-87, wherein the method reduces the risk of fracture.

[0426] The method according to any one of items 58-88, wherein the method increases bone strength.

[0427] The method according to any one of items 58-89, wherein the bone is cortical bone.

[0428] The method according to any one of items 58-89, wherein the bone is trabecular bone.

[0429] A method for reducing or preventing fibrosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polypeptide according to any one of items 1-40, the nucleic acid molecule according to item 41, the vector according to item 42, or the pharmaceutical composition according to item 45 or 46.

[0430] 93. A method of treating a subject having fibrosis or at risk of developing fibrosis, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0431] 94. A method of slowing or inhibiting the progression of fibrosis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0432] 95. A method of affecting myostatin, activin, and / or BMP9 signaling in a subject having fibrosis or at risk of developing fibrosis, or having a disease or condition involving fibrosis or at risk of developing a disease or condition involving fibrosis, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0433] 96. The method of any one of items 92-95, wherein the fibrosis is chemotherapy drug-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis, corneal fibrosis, cardiac fibrosis, myelofibrosis, mediastinal fibrosis, retroperitoneal fibrosis, osteoarticular fibrosis, joint fibrosis, tissue fibrosis, tumor stroma, fibroproliferative tumor, surgical adhesion, hypertrophic scar, or keloid.

[0434] 97. The method of item 96, wherein the tissue fibrosis is fibrosis affecting a tissue selected from: muscle tissue, skin epidermis, skin dermis, tendon, cartilage, pancreatic tissue, uterine tissue, nerve tissue, testis, ovary, adrenal gland, artery, vein, colon, small intestine, large intestine, biliary tract, and intestine.

[0435] 98. The method of any one of items 92-95, wherein the fibrosis is fibrosis associated with: wound, burn, hepatitis B or C infection, fatty liver disease, schistosome infection, kidney disease, chronic kidney disease, heart disease, macular degeneration, retinal or vitreoretinal disease, Crohn's disease, systemic or localized scleroderma, atherosclerosis, or restenosis.

[0436] 99. The method of any one of items 92-98, wherein the method improves the function of the fibrotic tissue or organ.

[0437] 100. A method for increasing red blood cell levels in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0438] 101. A method for promoting or increasing erythropoiesis in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0439] 102. The method of item 100 or 101, wherein the subject has anemia or blood loss or is at risk of developing anemia or blood loss.

[0440] 103. A method of modulating myostatin, activin, and / or BMP9 signaling in a subject having a disease or condition involving low red blood cell or low hemoglobin levels, or at risk of developing a disease or condition involving low red blood cell or low hemoglobin levels, the method comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0441] 104. The method of item 103, wherein the disease or condition is anemia or blood loss.

[0442] 105. The method of item 102 or 104, wherein the anemia or blood loss is associated with: cancer, cancer treatment, chronic kidney disease, acute kidney disease or failure, chronic kidney disease or failure, myelodysplastic syndrome, thalassemia, nutritional deficiency, adverse drug reactions, inflammatory or autoimmune diseases, splenomegaly, porphyria, vasculitis, hemolysis, bone marrow defect, bone marrow transplantation, acute liver disease, chronic liver disease, acute bleeding, chronic bleeding, infection, hemoglobinopathy, drug use, alcohol abuse, Chusid syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplantation, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schönlein purpura, Shwachman syndrome, advanced age, transfusion contraindications, surgery, trauma, wound, ulcer, urinary tract bleeding, gastrointestinal bleeding, frequent blood donation or excessive menstrual bleeding.

[0443] 106. The method of item 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.

[0444] 107. A method for treating a subject suffering from anemia or at risk of developing anemia, which comprises administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0445] 108. The method of item 107, wherein the anemia is associated with: cancer, cancer treatment, chronic kidney disease, acute kidney disease or failure, chronic kidney disease or failure, myelodysplastic syndrome, thalassemia, nutritional deficiency, adverse reaction to a drug, inflammatory or autoimmune disease, splenomegaly, porphyria, vasculitis, hemolysis, bone marrow defect, bone marrow transplantation, acute liver disease, chronic liver disease, acute hemorrhage, chronic hemorrhage, infection, hemoglobinopathy, drug use, alcohol abuse, Chus syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplantation, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schönlein purpura, Shwachman syndrome, advanced age, transfusion contraindication, surgery, trauma, wound, ulcer, urinary tract hemorrhage, gastrointestinal hemorrhage, frequent blood donation or excessive menstrual bleeding.

[0446] 109. The method of item 107 or 108, wherein the anemia is aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, anemia of chronic disease, anemia associated with a 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.

[0447] 110. The method of any one of items 100-109, wherein the subject does not respond well to treatment with erythropoietin (EPO), or is susceptible to the adverse effects of EPO.

[0448] 111. The method of any one of items 100-110, wherein the method increases erythropoiesis, red blood cell count, hematocrit or hemoglobin level.

[0449] 112. The method of any one of items 100-111, wherein the method reduces the subject's need for blood transfusion.

[0450] 113. A method for preventing pulmonary hypertension (PH) in a subject in need thereof, which comprises administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0451] 114. A method of slowing or inhibiting PH progression in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0452] 115. A method of treating a subject having PH or at risk of developing PH, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0453] 116. A method of affecting myostatin, activin, and / or BMP9 signaling in a subject having PH or at risk of developing PH, comprising administering to the subject a therapeutically effective amount of a polypeptide of any one of items 1-40, a nucleic acid molecule of item 41, a vector of item 42, or a pharmaceutical composition of item 45 or 46.

[0454] 117. The method of any one of items 113-116, wherein the PH is pulmonary arterial hypertension (PAH).

[0455] 118. The method of item 117, wherein the PAH is idiopathic PAH.

[0456] 119. The method of item 117, wherein the PAH is heritable PAH.

[0457] 120. The method of item 117, wherein the PAH is associated with: HIV infection, schistosomiasis, cirrhosis, congenital heart anomalies, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, connective tissue disorders, autoimmune disorders, or drug use or abuse.

[0458] 121. The method of any one of items 113-116, wherein the PH is venous PH.

[0459] 122. The method of item 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.

[0460] 123. The method of any one of items 113-116, wherein the PH is hypoxic PH.

[0461] 124. The method of item 123, wherein the hypoxic PH is associated with: chronic obstructive pulmonary disease, interstitial lung disease, sleep disordered breathing, pulmonary fibrosis, alveolar hypoventilation disorders, long-term exposure to high altitude, or developmental abnormalities.

[0462] 125. The method of any one of items 113 - 116, wherein the PH is thromboembolic PH.

[0463] 126. The method of item 125, wherein the thromboembolic PH is associated with: chronic thromboembolic pulmonary hypertension, pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or parasitic infection.

[0464] 127. The method of any one of items 113 - 116, wherein the PH is miscellaneous PH.

[0465] 128. The method of item 127, wherein the miscellaneous PH is associated with: hematologic diseases, systemic diseases, metabolic disorders, pulmonary tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension.

[0466] 129. The method of any one of items 113 - 128, wherein the method reduces the frequency or severity of one or more symptoms of PH.

[0467] 130. The method of any one of items 113 - 129, wherein the method reduces pulmonary vascular remodeling or vascular remodeling in the heart of the subject.

[0468] 131. The method of any one of items 113 - 130, wherein the method reduces right ventricular hypertrophy.

[0469] 132. The method of any one of items 113 - 131, wherein the method reduces pulmonary vascular resistance.

[0470] 133. The method of any one of items 113 - 132, wherein the method improves performance in the 6 - minute walk test.

[0471] 134. The method of any one of items 47 - 133, wherein the method reduces or inhibits the binding of activin and / or myostatin to their receptors.

[0472] 135. The method of any one of items 47 - 57 or 134, wherein the amount of the polypeptide, nucleic acid, vector, or pharmaceutical composition administered is sufficient to increase muscle weight and / or strength, affect myostatin, activin, and / or BMP9 signaling in the subject, or reduce or inhibit the binding of activin and / or myostatin to their receptors.

[0473] The method of any one of items 58 - 91 or 134, wherein the amount of the polypeptide, nucleic acid, vector or pharmaceutical composition administered is sufficient to increase mineral bone 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 fractures, affect myostatin, activin and / or BMP9 signaling in the subject, or reduce or inhibit the binding of activin and / or myostatin to their receptors.

[0474] The method of any one of items 92 - 99 or 134, wherein the amount of the polypeptide, nucleic acid, vector or pharmaceutical composition administered is sufficient to reduce fibrosis, prevent fibrosis, reduce the risk of developing fibrosis, delay the development of fibrosis, slow or inhibit the progression of fibrosis, treat fibrosis, reduce one or more symptoms of fibrosis, improve the function of fibrotic tissue or organ, affect myostatin, activin and / or BMP9 signaling in the subject, or reduce or inhibit the binding of activin and / or myostatin to their receptors.

[0475] The method of any one of items 100 - 112 or 134, wherein the amount of the polypeptide, nucleic acid, vector or pharmaceutical composition administered is sufficient to increase red blood cell levels, increase hemoglobin levels, increase red blood cell formation, increase red blood cell count, increase hematocrit, reduce the need for blood transfusion, treat anemia, affect myostatin, activin and / or BMP9 signaling in the subject, or reduce or inhibit the binding of activin and / or myostatin to their receptors.

[0476] The method of any one of items 113 - 134, wherein the amount of the polypeptide, nucleic acid, vector or pharmaceutical composition administered is sufficient to prevent PH, reduce the risk of developing PH, reduce the severity or frequency of one or more symptoms of PH, delay the development of PH, slow 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 the 6 - minute walk test, affect myostatin, activin and / or BMP9 signaling in the subject, or reduce or inhibit the binding of activin and / or myostatin to their endogenous receptors.

[0477] The method of any one of items 47 - 139, wherein the method does not cause vascular complications in the subject.

[0478] The method of item 140, wherein the method does not increase vascular permeability or leakage.

Claims

1. A polypeptide comprising an extracellular activin receptor type IIB (ActRIIB) variant, said variant having relative to one or more amino acid substitutions of the sequence, wherein the variant 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 comprise one or more of the following: a) the amino acid substitution E75K; b) the amino acid substitutions Q69T and E70D; or c) the amino acid substitutions Q69D and E70T.

Images