Activin receptor type IIB variants and methods of use thereof

Abstract

The invention features polypeptides that include extracellular ActRIIB variants. In some embodiments, the polypeptides of the invention include extracellular ActRIIB variants fused to an Fc domain monomer or portion. The invention also features pharmaceutical compositions and methods of using the polypeptides to treat diseases and conditions involving muscle weakness and atrophy, bone damage, low red blood cell levels (e.g., anemia or blood loss), fibrosis, and / or pulmonary hypertension.

Description

Background of the Invention

[0002] 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 lower legs. 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 systemic muscle stiffness, muscle twitching and muscle atrophy due to motor neuron degeneration. Efforts to improve treatment and survival of subjects with these devastating muscle diseases have not been successful.

[0003] Healthy bones undergo constant remodeling, which involves both bone destruction and bone growth. Bone growth is mediated by osteoblastic cell types, while osteoclasts resorb bone. When these systems lose balance through downregulation of anabolic programs, upregulation of catabolic systems, or a combination of the two, pathological conditions occur, resulting in net bone loss. Therefore, controlling the balance of bone remodeling can be used to promote the healing of bone injuries and the treatment of conditions related to bone loss and bone demineralization, such as osteoporosis.

[0004] Bone damage can be caused by a range of underlying causes, including age- or cancer-related bone loss, genetic conditions, or adverse side effects of drug treatments. The World Health Organization estimates that osteoporosis alone affects 75 million people in the United States, Europe, and Japan, and is an important risk factor for bone damage. 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 agents that directly promote bone growth and increase bone density. For osteoporosis, estrogen, calcitonin, osteocalcin, and vitamin K or high doses of dietary calcium can all be used as therapeutic interventions. Other treatments 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 excess connective tissue in an organ or tissue. Connective tissue that can be formed in response to injury (e.g., damage) or as part of an immune response (e.g., inflammatory response) can destroy the structure and function of the organ or tissue in which it is formed, leading to an increase in tissue hardness. Fibrosis can occur in many organs and tissues in the body, including, in particular, the lungs (e.g., pulmonary fibrosis, cystic fibrosis), liver (e.g., cirrhosis), heart (e.g., endomyocardial fibrosis or post-myocardial infarction fibrosis), brain (e.g., glial scar formation), skin (e.g., keloid formation), kidneys (e.g., renal fibrosis), and eyes (e.g., corneal fibrosis); and is known to be associated with certain drug treatments (e.g., 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. Only in the U.S., the prevalence of anemia has almost doubled from 2003 to 2012. The symptoms of anemia include fatigue, weakness, shortness of breath, palpitations and cognitive decline, and it has been found that children, pregnant women, women of childbearing age and the elderly 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. Although iron deficiency anemia can be treated with iron supplements, many other forms of anemia, such as aplastic anemia, chronic disease anemia and hemolytic anemia may require blood transfusion.

[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), hypoxia (PH caused by lung disease), thromboembolic (PH caused by chronic arterial blockage such as blood clots), or miscellaneous (PH with unclear or multifactorial mechanisms), also known as WHO Class IV. PAH is characterized by increased pulmonary vascular pressure caused by obstruction or stenosis of small blood vessels in the lungs due to scarring. This leads to 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 (e.g., no identifiable cause), heritable (e.g., familial, often due to genetic mutations), or may be associated with drug use (e.g., methamphetamine or cocaine use), infection (e.g., HIV infection or schistosomiasis), cirrhosis, congenital heart abnormalities, or connective tissue / autoimmune disorders (e.g., scleroderma or lupus). Treatments for PH include vasodilators, anticoagulants, and supplemental oxygen, but these treatments manage disease symptoms 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

[0010] The present invention is characterized in that it includes a polypeptide of an extracellular activin receptor type IIB (ActRIIB) variant. In some embodiments, the polypeptide of the present invention includes an extracellular ActRIIB variant fused to the N-terminus or C-terminus of an Fc domain monomer or another part. Such a part can be attached by amino acids or other covalent bonds, and the stability of the polypeptide can be increased. The polypeptide including the extracellular ActRIIB variant fused to the Fc domain monomer can also form a dimer (e.g., a homodimer or a heterodimer) by interaction between two Fc domain monomers. The polypeptide of the present invention can be used to increase muscle weight and strength in a subject suffering from a disease or condition or at risk of developing a disease or condition, and the disease or condition involves 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 subjects suffering from a disease or condition or being at risk of developing a disease or condition, the disease or condition involving bone damage, such as primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone 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 fixation. In addition, 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 subjects in need thereof, such as subjects suffering from anemia or blood loss or being at risk of developing anemia or blood loss, to prevent or reduce fibrosis in subjects suffering from fibrosis or being at risk of developing fibrosis, or to treat, prevent or delay the development or progression of pulmonary hypertension in subjects suffering from pulmonary hypertension (e.g., arterial, venous, hypoxic, thromboembolic or miscellaneous pulmonary hypertension) or being at risk of developing pulmonary hypertension. Further, the polypeptides of the invention can also be used to affect myostatin, activin and / or bone morphogenetic protein 9 (BMP9) signaling in a subject at risk of developing or suffering from a disease or condition involving muscle weakness and atrophy, bone damage or bone demineralization, low blood cell levels (e.g., low hemoglobin levels, low hematocrit and / or low red blood cell counts), fibrosis, or pulmonary hypertension (e.g., arterial, venous, hypoxic, thromboembolic, or miscellaneous pulmonary hypertension).

[0011] In a first aspect, the invention features a polypeptide comprising an extracellular ActRIIB variant having a

[0012]

[0013] The variant comprises one or more amino acid substitutions in a sequence of a polypeptide comprising: (a) amino acid substitution E75K; (b) amino acid substitutions Q69T and E70D; or (c) amino acid substitutions Q69D and E70T.

[0014] 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.

[0015] In some embodiments, the variant contains the amino acid substitution E75K and the additional amino acid substitutions E20D and F63I.

[0016] In some embodiments, the variant contains the amino acid substitution E75K and an additional amino acid substitution that reduces BMP9 binding. In some embodiments of any of the above aspects, the additional amino acid substitution that reduces BMP9 binding is T74K, E76D, N77S, and Q79E.

[0017] In some embodiments, the variant further contains one or more additional amino acid substitutions.

[0018] In some embodiments, the variant contains additional amino acid substitutions Y41F, R45K and K56Q. In some embodiments, the variant further contains additional amino acid substitutions Y12F, L19K, E20D, R29P, E31Y, E33D, L38R and F63I.

[0019] In some embodiments, the variant contains the additional amino acid substitutions S25T and S47I. In some embodiments, the variant contains the additional amino acid substitution S48T.

[0020] In some embodiments, the variant contains the additional amino acid substitution R29P.

[0021] In some embodiments, the variant contains the additional amino acid substitutions E31Y, E33D, and Q34K.

[0022] In some embodiments, the variant contains the additional amino acid substitutions Y12F, L19K, and E20D.

[0023] In some embodiments, the variant contains the additional amino acid substitutions E31Y, E33D, and L38R.

[0024] 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.

[0025] 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.

[0026] In some embodiments, the variant further contains the amino acid substitution E75K.

[0027] Embodiments E1 to E59 below describe further features of the invention.

[0028] E1. A polypeptide comprising an ActRIIB variant having the following sequence:

[0029]

[0030] Where X1 is I or L; X2 is F or Y; X3 is L or K; X4 is D or E; X5 is T or S; X6 is L or V; X7 is P or R; X8 is Y or E; X9 is D or E; X 10 It 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 It is K or Q; X 20 Is F or I; X 21 is Q, T or D; X 22 is E, D or T; X 23 is K or T; X 24 Is K or E; X 25 Is D or E; X 26 is S or N; X 27is E or Q; and X 28 is F or M, and where X 24 Is E and / or X 21 is T and X 22 Is D or X 21 is D and X 22 It's T.

[0031] E2. A variant of E1, wherein X1 is I.

[0032] E3. A variant of E1, wherein X1 is L.

[0033] E4. A variant of any one of E1-E3, wherein X2 is F.

[0034] E5. A variant of any of E1-E3, wherein X2 is Y.

[0035] E6. A variant of any of E1-E5, wherein X3 is L.

[0036] E7. A variant of any of E1-E5, wherein X3 is K.

[0037] E8. A variant of any of E1-E7 wherein X4 is D.

[0038] E9. A variant of any of E1-E7 wherein X4 is E.

[0039] E10. A variant of any of E1-E9, wherein X5 is T.

[0040] E11. A variant of any of E1-E9 wherein X5 is S.

[0041] E12. A variant of any of E1-E11, wherein X6 is L.

[0042] E13. A variant of any of E1-E11 wherein X6 is V.

[0043] E14. A variant of any one of E1-E13, wherein X7 is P.

[0044] E15. A variant of any of E1-E13, wherein X7 is R.

[0045] E16. A variant of any one of E1-E15, wherein X8 is Y.

[0046] E17. A variant of any of E1-E15, wherein X8 is E.

[0047] E18. A variant of any of E1-E17, wherein X9 is D.

[0048] E19. A variant of any of E1-E17, wherein X9 is E.

[0049] E20. A variant of any of E1-E19, wherein X 10 It's K.

[0050] E21. A variant of any of E1-E19, wherein X 10 It's Q.

[0051] E22. A variant of any of E1-E21, wherein X 11 It's R.

[0052] E23. A variant of any of E1-E21, wherein X 11 It's L.

[0053] E24. A variant of any one of E1-E23, wherein X 12 It is Y.

[0054] E25. A variant of any one of E1-E23, wherein X 12 It's F.

[0055] E26. A variant of any of E1-E25, wherein X 13 It's R.

[0056] E27. A variant of any of E1-E25, wherein X 13 It's K.

[0057] E28. A variant of any of E1-E27, wherein X 14 It’s S.

[0058] E29. A variant of any of E1-E27, wherein X 14 It's I.

[0059] E30. A variant of any of E1-E29, wherein X 15 It’s S.

[0060] E31. A variant of any of E1-E29, wherein X 15 It's T.

[0061] E32. A variant of any of E1-E31, wherein X 16 It’s S.

[0062] E33. A variant of any of E1-E31, wherein X 16 It's T.

[0063] E34. A variant of any of E1-E33, wherein X17 It's I.

[0064] E35. A variant of any of E1-E33, wherein X 17 It's L.

[0065] E36. A variant of any of E1-E35, wherein X 18 It's I.

[0066] E37. A variant of any of E1-E35, wherein X 18 It's L.

[0067] E38. A variant of any of E1-E37, wherein X 19 It's K.

[0068] E39. A variant of any of E1-E37, wherein X 19 It's Q.

[0069] E40. A variant of any of E1-E39, wherein X 20 It's F.

[0070] E41. A variant of any one of E1-E39, wherein X 20 It's I.

[0071] E42. A variant of any of E1-E41, wherein X 21 It's Q.

[0072] E43. A variant of any of E1-E41, wherein X 21 It's T.

[0073] E44. A variant of any of E1-E41, wherein X 21 It is D.

[0074] E45. A variant of any of E1-E42, wherein X 22 It's E.

[0075] E46. A variant of any one of E1-E41 and E43, wherein X 22 It is D.

[0076] E47. A variant of any one of E1-E41 and E44, wherein X 22 It's T.

[0077] E48. A variant of any of E1-E47, wherein X 23 It's K.

[0078] E49. A variant of any of E1-E47, wherein X 23 It's T.

[0079] E50. A variant of any one of E1-E49, wherein X 24 It's K.

[0080] E51. A variant of any one of E1-E41, E43, E44, E46 and E47-E49, wherein X 24 It's E.

[0081] E52. A variant of any of E1-E51, wherein X 25 It is D.

[0082] E53. A variant of any of E1-E51, wherein X 25 It's E.

[0083] E54. A variant of any one of E1-E53, wherein X 26 It’s S.

[0084] E55. A variant of any one of E1-E53, wherein X 26 It is N.

[0085] E56. A variant of any of E1-E55, wherein X 27 It's E.

[0086] E57. A variant of any of E1-E55, wherein X 27 It's Q.

[0087] E58. A variant of any one of E1-E57, wherein X 28 It's F.

[0088] E59. A variant of any of E1-E57, wherein X 28 It’s M.

[0089] In some embodiments, the variant has the sequence of any one of SEQ ID NOs: 2-15.

[0090] In some embodiments, the polypeptide further comprises an Fc domain monomer fused to the C-terminus of the polypeptide (eg, 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.

[0091] In some embodiments, the polypeptide further comprises a wild-type Fc domain fused to the C-terminus of the polypeptide (eg, the C-terminus of the variant) via a linker. In some embodiments, the wild-type Fc domain has a sequence of SEQ ID NO: 71.

[0092] In some embodiments, the polypeptide further comprises an Fc domain having amino acid substitutions, wherein the Fc domain is fused to the C-terminus of the polypeptide (eg, the C-terminus of the variant) via a linker. In some embodiments, the Fc domain does not form a dimer.

[0093] In some embodiments, the polypeptide further comprises an albumin binding peptide fused to the C-terminus of the polypeptide (eg, the C-terminus of the variant) via a linker. In some embodiments, the albumin binding peptide has the sequence of SEQ ID NO:72.

[0094] In some embodiments, the polypeptide further comprises a fibronectin domain fused to the C-terminus of the polypeptide (eg, the C-terminus of the variant) via a linker. In some embodiments, the fibronectin domain has a sequence of SEQ ID NO:73.

[0095] In some embodiments, the polypeptide further comprises human serum albumin fused to the C-terminus of the polypeptide (eg, the C-terminus of the variant) via a linker. In some embodiments, the human serum albumin has the sequence of SEQ ID NO:74.

[0096] 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).

[0097] 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 (SEQID 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 (SEQID NO: 37), GAGAGAGAGAGA (SEQ ID NO: 38), GSGSGSGSGSGS (SEQ IDNO: 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 (SEQID NO: 62), SACYCELS (SEQ ID NO: 63), RSIAT (SEQ ID NO:64), RPACKIPNDLKQKVMNH (SEQ ID NO: 65), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 66), AAANSSIDLISVPVDSR (SEQ ID NO: 67), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 68), EAAAK (SEQ ID NO: 69), or PAPAP (SEQ ID NO: 70).

[0098] In some embodiments, the polypeptide has a serum half-life of at least seven days.

[0099] In some embodiments, the polypeptide has increased or decreased binding to one or more ActRIIB ligands (eg, activin, myostatin, GDF-11, or BMP9) compared to wild-type ActRIIB.

[0100] In some embodiments, the polypeptide has a K of 200 pM or higher. D Binds to human bone morphogenetic protein 9 (BMP9).

[0101] In some embodiments, the polypeptide binds to activin and / or myostatin and has reduced or weak binding to human BMP9.

[0102] In some embodiments, the polypeptide does not substantially bind to human BMP9.

[0103] In some embodiments, the polypeptide has a K of 800 pM or less. D Binds to human activin A.

[0104] In some embodiments, the polypeptide has a K of 800 pM or less. D Binds to human activin B.

[0105] In some embodiments, the polypeptide has a K of 5 pM or higher. D Binds to human GDF-11.

[0106] 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 a sequence of any one of SEQ ID NOs: 1-15 (eg, SEQ ID NOs: 2-15).

[0107] In another aspect, the invention features a vector including a nucleic acid molecule described herein.

[0108] In another aspect, the invention features a host cell expressing a polypeptide described herein, wherein the host cell comprises a nucleic acid molecule or vector as described in the first two aspects, wherein the nucleic acid molecule or vector is expressed in the host cell.

[0109] In another aspect, the invention features a method of making a polypeptide described herein, wherein the method includes: a) providing a host cell comprising a nucleic acid molecule or a vector described herein, and b) expressing the nucleic acid molecule or the vector in the host cell under conditions that allow formation of the polypeptide.

[0110] In another aspect, the invention features a pharmaceutical composition comprising a polypeptide, nucleic acid molecule or vector as 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 a therapeutically effective amount.

[0111] In another aspect, the invention features a construct (e.g., a homodimer) comprising two identical polypeptides, each of which comprises an extracellular ActRIIB variant described herein (e.g., an ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) fused to the N-terminus or C-terminus of an Fc domain monomer (e.g., a sequence of SEQ ID NO: 19). The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0112] In another aspect, the invention features a construct (e.g., a heterodimer) that includes two different polypeptides, each of which includes an extracellular ActRIIB variant described herein (e.g., an ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15)) fused to the N-terminus or C-terminus of an Fc domain monomer (e.g., a sequence of SEQ ID NO: 19). The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0113] 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 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 increasing muscle mass in a subject in need thereof. 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.

[0115] In some embodiments of the method of increasing lean body mass or muscle mass in a subject, the subject has or is at risk of developing Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia.

[0116] In another aspect, the invention features a method of treating a subject having a muscle disease 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 as described herein, or a pharmaceutical composition as described herein. In some embodiments, the muscle disease is DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia.

[0117] In another aspect, the invention features a method of affecting myostatin, activin and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin and / or BMP9 to their endogenous receptors) in a subject having a disease or condition or at risk of developing a 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. In some embodiments of this aspect, the disease or condition is DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia.

[0118] In another aspect, the invention features a method of treating a subject having or at risk of developing DMD by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0119] In another aspect, the invention features a method of treating a subject having or at risk of developing FSHD by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0120] In another aspect, the invention features a method of treating a subject having or at risk of developing IBM by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0121] 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.

[0122] In another aspect, the invention features a method of treating a subject having or at risk of developing sarcopenia by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0123] In another aspect, the invention features a method of treating a subject having or at risk of developing cancer cachexia by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] In another aspect, the invention features a method of reducing the risk of bone 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.

[0129] In some embodiments of any of the above aspects, the subject has or is at risk of developing primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone 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 or is at risk of developing osteoporosis.

[0130] In another aspect, the invention features a method for treating a subject suffering from a bone disease or being at risk of developing a bone disease. The method includes administering a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector as described herein, or a pharmaceutical composition as described herein to the subject. 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, obesity-related bone loss, low gravity-related bone loss, or fixation-related bone loss. In some embodiments, the bone disease is osteoporosis.

[0131] In another aspect, the invention features a method of affecting myostatin, activin and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin and / or BMP9 to their endogenous receptors) in a subject having a disease or condition or at risk of developing a disease or condition involving bone damage, 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 primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone 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 bone disease or condition is osteoporosis.

[0132] 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.

[0133] 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.

[0134] 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.

[0135] In another aspect, the invention features a method of treating a subject having or at risk of developing a bone 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.

[0136] In another aspect, the invention features a method of treating a subject having 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.

[0137] 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.

[0138] In another aspect, the invention features a method of treating a subject having or at risk of developing renal osteodystrophy by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0139] In another aspect, the invention features a method of treating a subject having or at risk of developing therapy-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.

[0140] In another aspect, the invention features a method of treating a subject having or at risk of developing diet-related bone loss by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0141] In another aspect, the invention features a method of treating a subject having or at risk of developing hypogravity-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.

[0142] In another aspect, the invention features a method of treating a subject having 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.

[0143] In some embodiments of any of the aforementioned aspects, the primary osteoporosis is age-related osteoporosis or hormone-related osteoporosis.

[0144] In some embodiments of any of the aforementioned aspects, the secondary osteoporosis is immobilization-induced osteoporosis or glucocorticoid-induced osteoporosis.

[0145] In some embodiments of any of the aforementioned aspects, the cancer is multiple myeloma.

[0146] In some embodiments of any of the aforementioned aspects, the therapy is FGF-21 therapy, GLP-1 therapy, cancer therapy, or a therapy for obesity or type 2 diabetes.

[0147] In some embodiments of any of the above aspects, the diet-related bone loss is rickets.

[0148] In some embodiments of any of the above aspects, the subject is at risk for bone fracture.

[0149] In some embodiments of any of the above aspects, the method increases bone formation in a subject. In some embodiments of any of the above aspects, the method reduces bone resorption in a subject. In some embodiments of any of the above aspects, the method reduces bone loss in a subject. In some embodiments of any of the above aspects, the method increases osteoblast activity or osteoblastogenesis. In some embodiments of any of the above aspects, the method reduces osteoclast activity or reduces osteoclastogenesis. In some embodiments of any of the above aspects, the method reduces the risk of bone fractures. In some embodiments of any of the above aspects, the method increases bone strength.

[0150] 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] In another aspect, the invention features a method of treating a subject having or at risk of developing fibrosis by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0155] In some embodiments of any of the aforementioned aspects, the fibrosis is chemotherapeutic drug-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis, myelofibrosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, osteoarthrotic fibrosis, tissue fibrosis, tumor stroma, desmoplastic tumors, surgical adhesions, hypertrophic scars, or keloids.

[0156] In some embodiments of the above aspects, the fibrosis is fibrosis associated with wounds, burns, hepatitis B or C infection, fatty liver disease, schistosomiasis 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.

[0157] In another aspect, the invention features a method of affecting myostatin, activin and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin and / or BMP9 to their endogenous receptors) in a subject having or at risk of developing fibrosis, or having 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 chemotherapeutic drug-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, liver fibrosis, renal fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis, myelofibrosis, mediastinal fibrosis, retroperitoneal fibrosis, arthrofibrosis, osteoarthrotic fibrosis, tissue fibrosis, tumor stroma, desmoplastic tumors, surgical adhesions, hypertrophic scars, or keloids. In some embodiments of this aspect, the disease or condition is fibrosis associated with wounds, burns, hepatitis B or C infection, fatty liver disease, schistosomiasis 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.

[0158] In some embodiments of any of the aforementioned aspects, the tissue fibrosis is fibrosis affecting a tissue selected from the group consisting of muscle tissue, skin epidermis, skin dermis, tendons, cartilage, pancreatic tissue, uterine tissue, neural tissue, testicles, ovaries, adrenal glands, arteries, veins, colon, small intestine, large intestine, bile duct, and intestine.

[0159] In some embodiments of any of the aforementioned aspects, the method improves the function of fibrotic tissue or organ. In some embodiments of any of the aforementioned aspects, the method slows or inhibits the progression of fibrosis. In some embodiments of any of the aforementioned aspects, the method reduces (e.g., reduces the frequency or severity of) one or more symptoms of fibrosis.

[0160] In another aspect, the invention features a method of increasing red blood cell levels (e.g., increasing hemoglobin levels, red blood cell count, or hematocrit) in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0161] 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.

[0162] In some embodiments of any of the above aspects, the subject has or is at risk of developing anemia or blood loss.

[0163] In another aspect, the invention features a method of affecting myostatin, activin and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin and / or BMP9 to their endogenous receptors) in a subject having a disease or condition or at risk of developing a disease or condition involving low red blood cell levels (e.g., low hemoglobin levels, low red blood cell counts, or low hematocrit), wherein the method comprises administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0164] In some embodiments of the above aspects, the disease or condition is anemia or blood loss.

[0165] In some embodiments of any of the aforementioned aspects, the anemia or blood loss is associated with cancer, cancer treatment, renal disease or failure (e.g., chronic renal disease or acute renal 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, diabetes, liver disease (e.g., acute liver disease or chronic liver disease), bleeding (e.g., acute or chronic bleeding), infection, hemoglobinopathy, drug use, alcohol abuse, advanced age, Churg-Fernandez syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplantation, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schonlein purpura, Schwabmann syndrome (e.g., Schwab-Fernandez syndrome), contraindications to transfusion, surgery, trauma, wound, ulcer, urinary tract bleeding, gastrointestinal bleeding, frequent blood donations, or excessive menstrual bleeding.

[0166] 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, renal disease or failure (e.g., chronic renal disease or acute renal 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, diabetes, liver disease (e.g., acute liver disease or chronic liver disease), bleeding (e.g., acute or chronic bleeding), infection, hemoglobinopathy, drug use, alcohol abuse, advanced age, Churg-Fernandez syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplantation, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schonlein purpura, Schwabmann syndrome (e.g., Schwab-Fernandez syndrome), contraindications to transfusion, surgery, trauma, wound, ulcer, urinary tract bleeding, gastrointestinal bleeding, frequent blood donations, or excessive menstrual bleeding.

[0167] In some embodiments of any of the aforementioned aspects, the anemia results from chronic kidney disease.

[0168] In some embodiments of any of the aforementioned aspects, the anemia is aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, anemia of chronic disease, anemia associated with bone marrow disease, hemolytic anemia, sickle cell anemia, microcytic anemia, hypochromic anemia, sideroblastic anemia, DeBouze-Schmidt anemia, Fanconi anemia, or refractory anemia with blastosis.

[0169] 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.

[0170] In some embodiments of any of the aforementioned aspects, the method increases red blood cell formation, red blood cell count, hemoglobin level, or hematocrit.

[0171] In some embodiments of any of the aforementioned aspects, the method reduces the subject's need for blood transfusions.

[0172] In another aspect, the invention features a method of 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.

[0173] In another aspect, the invention features a method of reducing the risk of developing PH in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule or vector described herein, or a pharmaceutical composition described herein.

[0174] In another aspect, the invention features a method of slowing or inhibiting the progression of PH in a subject in need thereof by administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule or vector described herein, or a pharmaceutical composition described herein.

[0175] In another aspect, the invention features a method of 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.

[0176] In another aspect, the invention features a method of affecting myostatin, activin and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin and / or BMP9 to their endogenous receptors) in a subject having 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.

[0177] In another aspect, the invention features a method of 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.

[0178] In another aspect, the invention features a method of 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.

[0179] 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.

[0180] In some embodiments of any of the above aspects, PH is pulmonary arterial hypertension (PAH). In some embodiments, PAH is idiopathic PAH. In some embodiments, PAH is heritable PAH. In some embodiments, PAH is associated with HIV infection, schistosomiasis, cirrhosis, congenital heart anomalies, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary angiomatosis, connective tissue disorders, autoimmune disorders (e.g., scleroderma or lupus), or drug use or abuse (e.g., cocaine or methamphetamine use).

[0181] In some embodiments of any of the aforementioned 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.

[0182] In some embodiments of any of the aforementioned aspects, the pH is a hypoxic pH. In some embodiments, the hypoxic pH is associated with chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), lung disease (e.g., pulmonary fibrosis), alveolar hypoventilation disorders, long-term exposure to high altitudes, or developmental abnormalities.

[0183] In some embodiments of any of the aforementioned aspects, PH is thromboembolic PH. In some embodiments, thromboembolic PH is associated with chronic thromboembolic pulmonary hypertension, pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or parasitic infection.

[0184] In some embodiments of any of the above aspects, the PH is miscellaneous PH. In some embodiments, the miscellaneous PH is associated with a hematologic disorder (e.g., chronic hemolytic anemia, sickle cell disease), a systemic disease (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), a metabolic disorder (e.g., glycogen storage disease, Gaucher disease, or thyroid disease), pulmonary tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension.

[0185] In some embodiments of any of the aforementioned 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 (e.g., edema) in the legs, feet, abdomen (ascites), or neck, chest pain or pressure, rapid pulse or palpitations, blue coloration of the lips or skin (cyanosis), dizziness, or syncope).

[0186] In some embodiments of any of the aforementioned aspects, the method reduces pulmonary vascular remodeling.

[0187] In some embodiments of any of the above aspects, the method reduces vascular remodeling in the heart.

[0188] In some embodiments of any of the above aspects, the method reduces right ventricular hypertrophy.

[0189] In some embodiments of any of the aforementioned aspects, the method reduces pulmonary vascular resistance (eg, reduces pulmonary vascular resistance as compared to a measurement taken before treatment).

[0190] In some embodiments of any of the above aspects, the method improves performance in a 6-minute walk test (eg, improves performance compared to a measurement taken before treatment).

[0191] In some embodiments of any of the aforementioned aspects, the method reduces or inhibits the binding of activin and / or myostatin to their receptors.

[0192] In some embodiments of any of the aforementioned aspects, the amount of the polypeptide, nucleic acid, vector, or pharmaceutical composition administered is sufficient to increase muscle mass 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 bone fractures, 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 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.

[0193] In some embodiments of any of the aforementioned aspects, the method does not cause vascular complications in the subject. In some embodiments, the method does not increase vascular permeability or leakage.

[0194] definition

[0195] As used herein, the term "extracellular activin receptor type IIB (ActRIIB) variant" refers to a peptide comprising a soluble extracellular portion of a single transmembrane receptor ActRIIB having 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), wherein the signal peptide is italicized and the extracellular portion is bold.

[0196] Wild-type human ActRIIB (SEQ ID NO: 18):

[0197]

[0198] The extracellular ActRIIB variant may have a sequence of any one of SEQ ID NOs: 1-15. In specific embodiments, the extracellular ActRIIB variant has a sequence of any one of SEQ ID NOs: 2-15 (Table 2). In some embodiments, the extracellular ActRIIB variant may have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity with the sequence of wild-type extracellular ActRIIB (SEQ ID NO: 17).

[0199] As used herein, the term "linker" refers to the connection between two elements (e.g., a peptide or protein domain). The polypeptide described herein may include an extracellular ActRIIB variant fused to a portion (e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NO: 1-15 (e.g., SEQ ID NO: 2-15)). The portion may increase stability or improve the pharmacokinetic properties of the polypeptide. The portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or human serum albumin) may be fused to the polypeptide by means of a linker. The linker may 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 type of bond produced by a chemical reaction (e.g., chemical conjugation). The term "spacer" refers to a portion (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 (e.g., peptides or protein domains) 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 spacer peptide via the polypeptide backbone). For example, the formation of a disulfide bond between the two hinge regions that form the Fc domain is not considered a linker.

[0200] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers. An Fc domain is a polypeptide having at least H 2 domains and C H The human Fc domain of the three domains has at least 80% sequence identity (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 comprise any portion of an immunoglobulin that can serve as an antigen recognition region, such as a variable domain or a complementarity determining region (CDR). In a wild-type Fc domain, two Fc domain monomers are connected by two C H 3 antibody constant domains and one or more disulfide bonds formed between the hinge domains of the two dimerized Fc domain monomers. In some embodiments, the Fc domain can be mutated to lack effector function, which is a typical "death Fc domain". In certain embodiments, each Fc domain monomer in the Fc domain comprises C H 2. Amino acid substitutions in the antibody constant domain 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. In addition, 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.

[0201] 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 a function of binding to serum albumin. The albumin binding peptide can be of different origins (e.g., human, mouse or rat). In some embodiments, the albumin binding peptide has the sequence DICLPRWGCLW (SEQ ID NO: 72).

[0202] As used herein, the term "endogenous" describes a molecule (e.g., a polypeptide, nucleic acid, or cofactor) that is naturally found in a particular organism (e.g., a human) or in a particular location within an organism (e.g., an organ, tissue, or cell, e.g., a human cell, e.g., a human hair cell).

[0203] 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 a fibronectin type III domain of amino acids 610-702 having a sequence of UniProt ID NO: P02751 (SEQ ID NO: 73). In other embodiments, the fibronectin domain is an adnectin protein.

[0204] 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 about half of serum proteins. In some embodiments, human serum albumin has the sequence of UniProt ID NO: P02768 (SEQ ID NO: 74).

[0205] 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 to form a continuous protein structure, such as a polypeptide, by chemical conjugation, chemical bonds, peptide linkers or any other means of covalent attachment. In some embodiments of the polypeptides described herein, 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 fused in a tandem series by means of a linker to the N- or C-terminus of a portion (e.g., an Fc domain monomer (e.g., a sequence of SEQ ID NO: 19), a wild-type Fc domain (e.g., a 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., a sequence of SEQ ID NO: 72), a fibronectin domain (e.g., a sequence of SEQ ID NO: 73), or human serum albumin (e.g., a sequence of SEQ ID NO: 74)). For example, the extracellular ActRIIB variant is fused to a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin-binding peptide, a fibronectin domain, or human serum albumin) by means of 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 with 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).

[0206] As used herein, the terms "bone mineral density (BMD)", "bone density" and "bone mass" refer to a measure of the amount of bone mineral (e.g., calcium) in bone tissue. BMD can be measured by well-established clinical techniques known to those skilled in the art (e.g., by single- or dual-energy photon or X-ray absorptiometry). The concept of BMD relates to the amount of minerals per volume of bone, although clinically it is measured by surrogate based on the optical density of each square centimeter of bone surface during imaging. BMD measurements are used as indirect indicators of osteoporosis and fracture risk in clinical medicine. In some embodiments, the BMD test results are provided as T-scores, wherein the T-scores represent the BMD of the subject compared to the ideal or peak bone mineral density of a healthy 30-year-old adult. A score of 0 indicates that BMD is equal to the normal reference value for a healthy young adult. The difference between the measured subject's BMD and the reference value for a healthy young adult is measured in standard deviation units (SD). Thus, a T-score between +1 SD and -1 SD can indicate normal BMD, a T-score between -1 SD and -2.5 SD can indicate low bone mass (e.g., osteopenia), and a T-score below -2.5 SD can indicate osteoporosis or severe osteoporosis. In some embodiments, 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)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule of the invention is administered to a subject in need thereof, wherein the patient has low bone mass (e.g., a T-score between -1 SD and -2.5 SD). In some embodiments, 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)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule of the invention is administered to a subject in need thereof, wherein the patient has osteoporosis (e.g., a T-score of less than -2.5 SD). In some embodiments, administration of 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)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule of the invention treats the subject by increasing its 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 a polypeptide, or a vector containing such a nucleic acid molecule increases BMD in a subject, resulting in an increase in the subject's T-score (e.g., resulting in an increase in the subject's T-score 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).

[0207] As used herein, the term "bone strength" refers to a measure of bone determined by bone mass in addition to bone mineral density. Bone mass is affected by bone geometry, microarchitecture, and the properties of the constituent tissues. Bone strength can be used to assess bone fracture risk.

[0208] As used herein, the term "bone disease" refers to a condition characterized by bone damage (e.g., decreased bone mineral density, decreased bone strength, and / or bone loss). Such diseases or conditions may be caused by an imbalance in osteoblast and / or osteoclast activity (e.g., increased bone resorption or decreased bone formation). Bone diseases include primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone 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, obesity treatment-related bone loss, low gravity-related bone loss, and immobilization-related bone loss.

[0209] 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 packets. 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 lead to decreased bone mass can be caused by an increase in resorption or a decrease in ossification. In healthy individuals, during childhood, bone formation exceeds resorption. As the aging process occurs, resorption exceeds formation. Bone resorption rates are also typically much higher in older women after menopause due to estrogen deficiency associated with menopause.

[0210] As used herein, the term "bone resorption" or "bone catabolic activity" refers to the process by which osteoclasts break down tissue in the bone and release minerals, resulting in the transfer of minerals (e.g., calcium) from the bone tissue to the blood. An increase in the rate of bone resorption is associated with aging (including postmenopausal women). A high bone resorption rate, or a bone resorption rate that exceeds the ossification rate, is associated with skeletal disorders (such as decreased bone mineral density, including osteopenia and osteoporosis) and can lead to bone loss. In some embodiments, a polypeptide comprising an extracellular ActRIIB variant of the invention (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 to reduce bone resorption (e.g., reduce bone loss) in the subject (e.g., the amount or rate of bone resorption in the subject).

[0211] As used herein, the terms "bone formation," "ossification," "osteogenesis," or "bone anabolic activity" refer to the process by which new bone tissue is formed by osteoblasts. In some embodiments, 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)), a nucleic acid encoding such a polypeptide, or a vector containing such a nucleic acid molecule of the invention is administered to a subject in need thereof to increase bone formation (e.g., to increase the amount or rate of bone formation or bone formation in a subject). A reduced rate of bone formation or a rate of bone formation that is exceeded by a rate of bone resorption can result in bone loss.

[0212] As used herein, the terms "increase" and "decrease" refer to modulation of a function, expression, or activity that results in a greater or lesser amount of a measure, respectively, relative to a reference. For example, following administration of a polypeptide of the invention including an extracellular ActRIIB variant as described herein, the amount of a marker of a measure as described herein (e.g., lean body mass) may be increased or decreased relative to the amount of the marker prior to administration. Typically, the measure is measured at a time after administration at which the administration has achieved the effect, such as at least one week, one month, three months, or six months after the treatment regimen has been initiated.

[0213] As used herein, the term "fibrosis" refers to the pathological process of excessive formation of fibrous connective tissue. Fibrosis is characterized by fibroblast accumulation and collagen deposition exceeding the normal deposition in any particular tissue. In response to inflammation or tissue damage, nearby fibroblasts can migrate into the wound, proliferate and produce a large amount 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, lungs, skin, liver, kidneys, heart, eyes, tendons, cartilage, pancreatic tissue, uterine tissue, neural tissue, testicles, ovaries, adrenal glands, arteries, veins, colon, small and large intestines, bile ducts and intestinal tracts.

[0214] As used herein, the term "pulmonary hypertension" or "PH" refers to a disease characterized by increased blood pressure between the lungs and the heart, which may include increased blood pressure in the pulmonary arteries (pulmonary hypertension), pulmonary veins, or pulmonary capillaries. Pulmonary hypertension can have a variety of symptoms, shortness of breath (dyspnea), fatigue, swelling (e.g., edema) in the legs, feet, abdomen (ascites), or neck, chest pain or pressure, rapid pulse or palpitations, blue coloration of the lips or skin (cyanosis), dizziness, or syncope. PH is also characterized by reduced exercise tolerance and may lead to heart failure.

[0215] As used herein, the term "pulmonary arterial hypertension" or "PAH" refers to a form of pulmonary hypertension characterized by stenosis or obstruction in the small pulmonary arteries, often caused by scarring, and an increase in pulmonary artery blood pressure. PAH is also known as WHO Class I PH. PAH can be diagnosed based on an increase in blood pressure in the pulmonary artery mean pulmonary artery pressure above 25 mmHg at rest, with a normal pulmonary artery capillary wedge pressure. PAH can cause shortness of breath, dizziness, syncope, and other symptoms, all of which are exacerbated by exertion. PAH can be a serious disease with significantly reduced exercise tolerance and heart failure. The two main types of PAH include idiopathic PAH (e.g., PAH in which no predisposing 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 the development of PAH include family history of PAH, drug use (such as methamphetamine or cocaine use), infection (such as HIV infection or schistosomiasis), cirrhosis, congenital heart anomalies, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary angiomatosis, or connective tissue / autoimmune disorders (such as scleroderma or lupus).

[0216] 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 Class II PH. Venous PH may be associated with or caused by left ventricular systolic dysfunction (e.g., left ventricular failure), left ventricular diastolic dysfunction, valvular heart disease (e.g., mitral or aortic valve disease), congenital cardiomyopathy, or congenital / acquired pulmonary vein stenosis.

[0217] 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 Class III PH. Hypoxic PH may be associated with or caused by chronic obstructive pulmonary disease (e.g., emphysema), interstitial lung disease, sleep-disordered breathing (e.g., sleep apnea), lung disease (e.g., pulmonary fibrosis), alveolar hypoventilation disorders, long-term exposure to high altitudes, or developmental abnormalities.

[0218] As used herein, the terms "thromboembolic pulmonary hypertension" and "thromboembolic PH" refer to a form of pulmonary hypertension associated with chronic arterial obstruction (e.g., blood clots). Thromboembolic PH is also known as WHO class IV PH. Thromboembolic PH may be associated with or caused by chronic thromboembolic pulmonary hypertension, or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or parasitic infection).

[0219] 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 class V PH. Miscellaneous PH may be associated with or caused by hematologic disease (e.g., chronic hemolytic anemia, sickle cell disease), systemic disease (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.

[0220] As used herein, the terms "increase red blood cell levels" and "promote red blood cell formation" refer to clinically observable metrics such as hematocrit, red blood cell count, and hemoglobin measurements, 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 of values ​​considered normal for the age and sex of the subject.

[0221] As used herein, the terms "erythropoiesis" and "erythropoiesis" 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.

[0222] As used herein, the term "anemia" refers to any abnormality in hemoglobin or red blood cells that results in a decrease in oxygen levels in the blood. Anemia can be associated with abnormal production, processing, or properties of red blood cells and / or hemoglobin. The term anemia refers to any decrease in the number of red blood cells and / or hemoglobin levels in the blood relative to normal blood levels.

[0223] As used herein, the term "identity percentage (%)" refers to the percentage of amino acid (or nucleic acid) residues identical with those of the reference sequence in the candidate sequence after aligning the sequences and introducing a gap (when necessary) to achieve the maximum identity percentage (that is, a gap can be introduced in one or both of the candidate sequence and the reference sequence for optimal alignment, and non-homologous sequences can be ignored for the purpose of comparison). The alignment for the purpose of determining the percentage identity can be achieved in various ways within the art, for example, 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 the alignment, including any algorithm required for achieving the maximum alignment within the full length of the sequence to be compared. In some embodiments, the percentage amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with or for a given reference sequence (which can be alternatively expressed as a given candidate sequence having or including a specific percentage amino acid (or nucleic acid) sequence identity to, with or for a given reference sequence) is calculated as follows:

[0224] 100 x (A / B score)

[0225] wherein A is the number of amino acid (or nucleic acid) residues that score as identical in the alignment of the candidate sequence and the reference sequence, and wherein B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments where 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.

[0226] In a specific embodiment, the reference sequence for comparison with the candidate sequence can be shown to be 50% to 100% identical to the candidate sequence over the full length of the candidate sequence or a selected portion of the consecutive amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence compared for comparison purposes is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90% or 100% of the length of the reference sequence. When a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence, the molecules are identical at that position.

[0227] As used herein, in the context of administering a therapeutic protein to a subject, the term "serum half-life" refers to the time required for the plasma concentration of a protein in a subject to be reduced by half. Proteins can be redistributed or cleared from the bloodstream, or degraded, i.e., by proteolysis. As described herein, polypeptides comprising an extracellular ActRIIB variant having 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.

[0228] As used herein, the term "lean body mass" refers to a component of body composition that includes, for example, lean body mass, body fat, and body fluid. Typically, lean body mass is calculated by subtracting the weight of body fat and body fluid from total body weight. Typically, a subject's lean body mass is 60% to 90% of total body weight. In the present invention, a polypeptide comprising an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15), 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 administration of a vector containing such a nucleic acid molecule to a subject increases the subject's lean body mass.

[0229] 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 intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair. The binding affinity between two molecules is often measured by the dissociation constant (K D ) or affinity constant (K A ). Two molecules with low binding affinity to each other generally bind slowly, tend to dissociate easily, and show a large K DTwo molecules that have a high affinity for each other generally bind easily, tend to remain bound longer, and exhibit a small K D The K of two interacting molecules can be determined using methods and techniques well known in the art, such as surface plasmon resonance. D . K D Calculated as k off / k on ratio.

[0230] As used herein, the term "muscle weight" refers to a component of body composition. Typically, muscle weight is calculated by subtracting the weight of body fat and body fluid from total body weight. The percentage of muscle weight may vary greatly among individuals, depending on the genetic makeup, age, race, and health status of the subject, etc. Typically, a subject's muscle weight may be 20% to 50% of total body weight.

[0231] As used herein, the phrase "affects myostatin, activin and / or BMP9 signaling" means altering the binding of myostatin, activin and / or BMP9 to its receptors, such as ActRIIA, ActRIIB and BMPRII (e.g., endogenous ActRIIB). In some embodiments, the polypeptides described herein including extracellular ActRIIB variants reduce or inhibit the binding of myostatin, activin and / or BMP9 to its receptors, such as ActRIIA, ActRIIB and BMPRII (e.g., endogenous ActRIIB). As described herein, polypeptides of the invention including extracellular ActRIIB variants having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15) may have a weak binding affinity (e.g., a K of 200 pM or more) to BMP9. D ).

[0232] As used herein, the term "vascular complication" refers to a vascular disorder or any damage to a blood vessel, such as damage to a 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 may 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 the gaps) and / or thinning of the blood vessel wall.

[0233] As used herein, the term "polypeptide" describes a single polymer in which monomers are amino acid residues covalently joined together by amide bonds. Polypeptide is intended to encompass any amino acid sequence occurring naturally, recombinantly, or synthetically produced.

[0234] As used herein, the term "homodimer" refers to a molecular construct formed by two identical macromolecules (such as proteins or nucleic acids). Two identical monomers can form homodimers by covalent or non-covalent bonds. For example, if two Fc domain monomers contain the same sequence, the Fc domain can be a homodimer of two Fc domain monomers. In another example, a polypeptide comprising an extracellular ActRIIB variant fused to an Fc domain monomer as described herein can form a homodimer by the interaction of two Fc domain monomers, and the Fc domain monomers form an Fc domain in a homodimer.

[0235] As used herein, the term "heterodimer" refers to a molecular construct formed by two different macromolecules (such as proteins or nucleic acids). Two monomers can form a heterodimer by covalent or non-covalent bonds. For example, a polypeptide comprising an extracellular ActRIIB variant fused to an Fc domain monomer as described herein can form a heterodimer by the interaction of two Fc domain monomers (each of which is fused to a different ActRIIB variant), and the Fc domain monomers form an Fc domain in the heterodimer.

[0236] As used herein, the term "host cell" refers to a vehicle containing the necessary cellular components (e.g., organelles) required for expressing a protein from its corresponding nucleic acid. Nucleic acids are typically included in nucleic acid vectors, which can be introduced into host cells by conventional techniques known in the art (transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). Host cells can be prokaryotic cells, such as bacterial cells, or eukaryotic cells, such as mammalian cells (e.g., CHO cells or HEK293 cells).

[0237] As used herein, the term "therapeutically effective amount" refers to an amount of a polypeptide, nucleic acid or vector of the invention, or a pharmaceutical composition comprising a polypeptide, nucleic acid or vector of the invention, effective to achieve the desired therapeutic effect in treating a patient suffering from 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 damage (e.g. osteoporosis), Diseases or conditions involving bone damage, such as primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone 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 or conditions involving low red blood cell levels (e.g., anemia or blood loss), diseases or conditions 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.

[0238] As used herein, the term "pharmaceutical composition" refers to a medicine or pharmaceutical preparation comprising an active ingredient and excipients and diluents that enable the active ingredient to be suitable for a method of administration. The pharmaceutical composition of the present invention comprises pharmaceutically acceptable components that are compatible with the polypeptide, nucleic acid or vector. The pharmaceutical composition can be in the form of tablets or capsules for oral administration, or in an aqueous form for intravenous or subcutaneous administration.

[0239] As used herein, the term "pharmaceutically acceptable carrier or excipient" refers to an excipient or diluent in a pharmaceutical composition. A pharmaceutically acceptable carrier must be compatible with the other ingredients of the formulation and not harmful to the recipient. In the present invention, a pharmaceutically acceptable carrier or excipient must provide sufficient pharmaceutical stability for a polypeptide comprising 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 carrier is generally used; for oral administration, a solid carrier is preferred.

[0240] As used herein, the term "treatment and / or prevention" refers to the treatment and / or prevention of a disease, such as a muscle disease (e.g., DMD, FSHD, IBM, and ALS), a bone disease (e.g., a disease or condition involving bone damage, such as osteoporosis, osteopenia, osteopetrosis, bone fractures, bone cancer or bone loss associated with cancer metastasis, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, obesity treatment-related bone loss, low gravity-related bone loss, or immobilization-related bone loss), a disease 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) using the methods and compositions of the invention. Generally, treatment of a muscular, skeletal, hypocytic or fibrotic disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH) occurs after a subject has developed a muscular, skeletal, hypocytic or fibrotic disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), and / or has been diagnosed with a muscular, skeletal, hypocytic or fibrotic disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH). Prevention of a muscular, skeletal, hypocytic or fibrotic disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH) refers to steps or procedures taken when a subject is at risk of developing a muscular, skeletal, hypocytic or fibrotic disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH). A subject may display signs or mild symptoms judged by a physician to be indicative of or risk factors for developing a muscular, skeletal, hypocytic, or fibrotic 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 muscular, skeletal, hypocytic, or fibrotic disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), be undergoing treatment (e.g., surgery, chemotherapy, or radiation) that may cause anemia, fibrosis, or loss of bone density, or have a family history or genetic predisposition to develop a muscular, skeletal, hypocytic, or fibrotic disease or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), but have not yet developed the disease.

[0241] 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

[0243] Figure 1is a sequence alignment showing the amino acid substitutions in the wild-type sequences of extracellular ActRIIA and ActRIIB and in ActRIIB variants. Figure 1 Also shown are the percent changes in body weight resulting from treatment of mice with the corresponding variants.

[0244] 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).

[0245] Figure 3A and 3B is a bar graph depicting the effect of extracellular ActRIIB variants on individual muscle weights by tissue analysis.

[0246] Figure 4 is a graph depicting the effect of extracellular ActRIIB variants ActRIIB 2.11-Fc and ActRIIB2.12-Fc on percent body weight over the course of 28 days.

[0247] Figures 5A-5C is a series of graphs depicting the effects of the ActRIIB variants ActRIIB 2.11-Fc and ActRIIB 2.12-Fc on red blood cell counts, hemoglobin levels, and hematocrit.

[0248] Figures 6A-6D is a series of graphs depicting the effects of the ActRIIB variant ActRIIB 2.12-Fc on trabecular bone in a mouse model of osteoporosis.

[0249] Figures 7A-7C is a series of graphs depicting the effects of the ActRIIB variant ActRIIB 2.12-Fc on red blood cell mass parameters in wild-type rats.

[0250] Figures 8A-8E is a series of graphs depicting the effects of the ActRIIB variant ActRIIB 2.12-Fc on trabecular bone in wild-type rats. DETAILED DESCRIPTION OF THE INVENTION

[0252] The invention features polypeptides comprising extracellular activin receptor type IIB (ActRIIB) variants. In some embodiments, the polypeptides of the invention include an extracellular ActRIIB variant fused to a portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or human serum albumin). Polypeptides comprising an extracellular ActRIIB variant fused to an Fc domain monomer can also form dimers (e.g., homodimers or heterodimers) through interactions between two Fc domain monomers. The ActRIIB variants described herein may have reduced binding to bone morphogenetic protein 9 (BMP9) relative to wild-type extracellular ActRIIB, or have weak or no binding affinity to BMP9 compared to the binding affinity to activin and myostatin. The invention also includes methods of treating diseases and conditions involving muscle weakness and atrophy by increasing muscle mass and strength, methods of treating or preventing bone damage 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 a subject a polypeptide comprising an extracellular ActRIIB variant described herein.

[0253] I. Extracellular activin receptor type IIB variant

[0254] 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, activins (e.g., activin A and activin B), inhibins, growth differentiation factors (GDFs) (e.g., GDF8, also known as myostatin), and bone morphogenetic proteins (BMPs) (e.g., BMP9). Myostatin and activin are known to play a role in the regulation of skeletal muscle growth. For example, mice without myostatin show a large increase in skeletal muscle weight. Myostatin has also been implicated in promoting fibrosis. For example, mice lacking myostatin show a decrease 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 inhibit osteoblast activity. Myostatin is also involved in bone homeostasis by increasing osteogenesis and inhibiting osteoblast activity. Elevated activin A has also been observed in clinical and experimental pulmonary hypertension. Therefore, methods of reducing or inhibiting activin or myostatin signaling can be used to treat diseases and conditions involving muscle atrophy or weakness, fibrosis, bone damage, low red blood cell levels (e.g., anemia), or pulmonary hypertension (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH).

[0255] There are two types of type II activin receptors: ActRIIA and ActRIIB. Studies have shown that BMP9 binds to ActRIIB with approximately 300-fold higher affinity than ActRIIA (see, e.g., Townson et al., J. Biol. Chem.287:27313, 2012). ActRIIA is known to have a longer half-life than ActRIIB. The present invention describes an extracellular ActRIIB variant, which is constructed by introducing amino acid residues of ActRIIA into ActRIIB, or by introducing novel amino acid substitutions, with the aim of reducing BMP9 binding to prevent or reduce disruption of endogenous BMP9 signaling. Amino acid substitutions 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. The optimal peptide confer a significant increase in lean body mass, muscle mass, bone mineral density, and / or red blood cell levels (e.g., increased red blood cell production), a decrease in fibrosis, or treatment of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), while, for example, having a reduced binding affinity to BMP9. Preferred ActRIIB variants also exhibit 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 disease (e.g., a disease or condition involving bone damage), muscle disease, fibrosis, PH (e.g., 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 (e.g., reduced fibrosis or a slowing or cessation of fibrosis progression), an increase in red blood cell levels (e.g., an increase in hemoglobin levels, hematocrit, or red blood cell count, such as an increase in red blood cell production), or a decrease in symptoms or progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH). In some embodiments, amino acid substitutions can be introduced into the extracellular ActRIIB variant 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.

[0256] Human ActRIIA, extracellular portion (SEQ ID NO: 16):

[0257]

[0258] Human ActRIIB, extracellular portion (SEQ ID NO: 17):

[0259]

[0260] The polypeptides described herein include extracellular ActRIIB variants having at least one amino acid substitution relative to the 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, an extracellular ActRIIB variant (e.g., an extracellular ActRIIB variant having a sequence of SEQ ID NO: 1) may include amino acid substitutions at all 28 positions as listed in Table 1. In some embodiments, an extracellular ActRIIB variant may include amino acid substitutions at multiple positions as listed in Table 1, such as 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 that is not in the corresponding position of ActRIIA, such as S48T, I51L, Q69D, or E70T).

[0261] Amino acid substitutions may worsen or improve the activity and / or binding affinity of an ActRIIB variant of the invention (eg, an extracellular ActRIIB variant having the sequence of any one of SEQ ID NOs: 1-15 (eg, SEQ ID NOs: 2-15)). In some embodiments, the amino acid substitutions worsen the binding affinity of the ActRIIB variant to BMP9 (e.g., the variant has reduced binding to BMP9 relative to wild-type extracellular ActRIIB, or has lower binding to BMP9 than other ActRIIB ligands (e.g., activin A or B, myostatin, or GDF-11). In some embodiments, the ActRIIB variant has reduced binding, weak binding, or substantially no binding to BMP9. In some embodiments, the amino acid substitutions improve the binding affinity of ActRIIB to 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 to myostatin, activin A or B, or GDF-11 more strongly than to BMP9). In some embodiments, the amino acid substitutions reduce the binding affinity of ActRIIB to 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 to myostatin , activin A or B or GDF-11 binds more weakly than BMP9). In some embodiments, the amino acid substitution does not substantially alter the extracellular ActRIIB function (e.g., the ActRIIB variant increases lean body mass, muscle mass, or bone mineral density by an amount similar to that of 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 to the ActRIIB variant (e.g., the ActRIIB variant can increase red blood cell levels, or has a longer half-life than WT extracellular ActRIIB). Preferably, the ActRIIB variant has one or more, two or more, or three or more of the above properties (e.g., reduced BMP9 binding and increased binding to activin A or B, myostatin and / or GDF-11, or reduced BMP9 binding, and equivalent functions to wild-type ActRIIB in increasing lean body mass, muscle mass, or bone mineral density, or reducing or preventing fibrosis).

[0262] The ActRIIB variants of the invention (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)) 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 In SEQ ID NO: 1, it is K). In some embodiments, the amino acid substitutions that reduce BMP9 binding are Q69T and E70D (eg, X 21 In SEQ ID NO: 1, T and X 22 In some embodiments, the amino acid substitutions that reduce BMP9 binding are Q69D and E70T (eg, X 21 In SEQ ID NO: 1, D and X 22 is T). In some embodiments, the amino acid substitutions that reduce BMP9 binding are T74K, E75K, E76D, N77S, and Q79E (eg, X 23 , X 24 , X 25 , X 26 and X 28 In some embodiments, the ActRIIB variant has more than one of the above amino acid substitutions that reduce BMP9 binding (e.g., substitution E75K and substitutions Q69D and E70T, or substitution E75K and substitutions Q69T and E70D). In some embodiments, the ActRIIB variants of the 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 or improved activity to activin or myostatin. For example, amino acid substitutions T74K, E75K, E76D, N77S, and Q79E result in a reduction in the activity of the ActRIIB variant (e.g., the variant has a reduced effect on lean body mass and muscle weight compared to wild-type extracellular ActRIIB), but inclusion of 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. Additional amino acid substitutions may include substitutions of one or more of 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.

[0263] In some embodiments, a polypeptide described herein includes an extracellular ActRIIB variant having the sequence of SEQ ID NO:1.

[0264] Table 1. Amino acid substitutions in extracellular ActRIIB variants having the sequence of SEQ ID NO: 1

[0265] .

[0266] In some embodiments, the polypeptides described herein include an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 2-15 (Table 2).

[0267] Table 2. Extracellular ActRIIB variants having the sequences of SEQ ID NOs: 2-15

[0268]

[0269] .

[0270] In some embodiments, a polypeptide 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 with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or human serum albumin) that may be fused to the N-terminus or C-terminus (e.g., 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) by interaction between two Fc domain monomers that combine in the dimer to form an Fc domain.

[0271] In addition, in some embodiments, the polypeptides described herein have a serum half-life of at least 7 days in humans. The polypeptides can be expressed as D Binds to bone morphogenetic protein 9 (BMP9). The polypeptide can bind to bone morphogenetic protein 9 (BMP9) with a K of 10 pM or higher. D Binds to activin A. In some embodiments, the polypeptide does not bind to BMP9 or activin A. In some embodiments, the polypeptide binds to activin and / or myostatin and exhibits reduced (eg, weak) binding to BMP9.

[0272] Additionally, in some embodiments, the polypeptide can be expressed as a polypeptide having a K of about 200 pM or more. D (e.g., a K of about 200, 300, 400, 500, 600, 700, 800, or 900 pM or moreD , for example, a K of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nM or more D , for example, a K of about 200 pM to about 50 nM D ) binds to human BMP9. In some embodiments, the polypeptide does not substantially bind to human BMP9. In some embodiments, the polypeptide can bind to human BMP9 with a K of about 800 pM or less. D (e.g., a K of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less) D , for example, a K of about 800 pM to about 200 pM D ) binds to human activin A. In some embodiments, the polypeptide can bind to human activin A with a K of about 800 pM or less. D (e.g., a K of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less) D , for example, a K of about 800 pM to about 200 pM D ) binds to human activin B. The polypeptide may also bind to human activin B with a K of about 5 pM or more D (e.g., a K of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or more) D ) binds to growth and differentiation factor 11 (GDF-11).

[0273] II. Fc domain

[0274] In some embodiments, the polypeptides described herein may include an extracellular ActRIIB variant fused to an Fc domain monomer or a fragment of an Fc domain of an immunoglobulin to increase the serum half-life of the polypeptide. A polypeptide comprising an extracellular ActRIIB variant fused to an Fc domain monomer may form a dimer (e.g., a homodimer or a heterodimer) by interaction between two Fc domain monomers, wherein the two Fc domain monomers form an Fc domain in the dimer. As is conventionally known in the art, an Fc domain is a protein structure found at the C-terminus of an immunoglobulin. The Fc domain comprises two Fc domain monomers, which are bound by a C H 3 antibody constant domains and dimerize. Wild-type Fc domains form the smallest 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 functions, which is a typical "death" Fc domain. For example, the Fc domain can include specific amino acid substitutions known to minimize the interaction between the Fc domain and the Fcγ receptor. In some embodiments, the Fc domain is from an IgG1 antibody and includes amino acid substitutions L234A, L235A and G237A. In some embodiments, the Fc domain is from an IgG1 antibody and includes 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 comparing the homology region of the antibody sequence with the "standard" Kabat numbering sequence, the Kabat numbering of the amino acid residues of a given antibody can be determined. In addition, in some embodiments, the Fc domain does not induce any immune system-related response. For example, the Fc domain in a 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 the Fcγ receptor. The sequence of the Fc domain monomer that can be fused to the extracellular ActRIIB variant is shown below (SEQ ID NO: 19):

[0275]

[0276] In some embodiments, the Fc domain is from an IgG1 antibody and comprises 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 comprises 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 a sequence of any one of SEQ ID NO: 1-15 (e.g., SEQ ID NO: 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., C-terminus) of the extracellular ActRIIB variant.

[0277] In some embodiments, the polypeptide described herein may include 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 an Fc domain of an immunoglobulin antibody isotype IgG, IgE, IgM, IgA or IgD. In addition, 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.

[0278] Methods for 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 hinder dimer formation due to steric collision. 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. H Methods for introducing amino acids with large or small side chains into the 3 domain are described, for example, by Ying et al. ( J Biol Chem. 287:19399-19408, 2012), U.S. Patent Publication No. 2006 / 0074225, U.S. Patent Nos. 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.

[0279] In yet other embodiments, the C segment between the two Fc domains H 3-C H 3. Interface C H One or more amino acid residues in the C3 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 specific charged amino acid introduced. H Methods for 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), U.S. Patent Publication Nos. 2006 / 0074225, 2012 / 0244578 and 2014 / 0024111, all of which are incorporated herein by reference in their entirety.

[0280] In some embodiments of the invention, the Fc domain comprises one or more of the following amino acid substitutions relative to the sequence of human IgG1: T366W, T366Y, T394W, F405W, Y349T, Y349E, Y349V, L351T, L351H, L351N, L352K, P353S, S354D, D356K, D356R, D356S, E357K, E357R, E357Q, S364A, T366E, L368T, L368Y, L368E, K370E, K370D, K370Q, K392E, K392D, T394N, P395N, P396T, V397T, V397Q, L398T, D399K, D399R, D399N, F405T, F405H, F405R, Y407T, Y407H, Y407I, K409E, K409D, K409T and K409I. In a specific embodiment, the Fc domain comprises the amino acid substitution T366W relative to the sequence of human IgG1. The sequence of the wild-type Fc domain (SEQ ID NO: 71) is:

[0281]

[0282] III. Albumin Binding Peptides

[0283] In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIB variant fused to a serum protein binding peptide. Binding to a serum protein peptide may improve the pharmacokinetics of a protein drug.

[0284] 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).

[0285] In the present invention, the albumin binding peptide can be linked to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRIIB variant described herein (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)) 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.

[0286] In some embodiments, the extracellular ActRIIB variants described herein (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 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 prolonged retention of the therapeutic protein through its binding to serum albumin.

[0287] IV. Fibronectin Domain

[0288] In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIB variant fused to a fibronectin domain. Binding to the fibronectin domain may improve the pharmacokinetics of protein drugs.

[0289] The fibronectin domain is a high molecular weight glycoprotein or fragment thereof of the extracellular matrix, which binds, for example, transmembrane receptor proteins such as integrins and extracellular matrix components such as collagen and fibrin. In some embodiments of the present invention, the fibronectin domain is connected to an extracellular ActRIIB variant described herein (e.g., an extracellular ActRIIB variant with a sequence of any one of SEQ ID NO: 1-15 (e.g., SEQ ID NO: 2-15)) N- or C- terminal (e.g., C- terminal) to increase the serum half-life of the extracellular ActRIIB variant. The fibronectin domain can be directly or via a linker connected to the N- or C- terminal of an extracellular ActRIIB variant.

[0290] As an example, fibronectin domains that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the fibronectin domain is a fibronectin type III domain of amino acids 610-702 having the sequence of UniProt ID NO: P02751 (SEQ ID NO: 73):

[0291]

[0292] In another embodiment, the fibronectin domain is an adnectin protein.

[0293] In some embodiments, the extracellular ActRIIB variants described herein (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 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 prolonged retention of the therapeutic protein through its binding to integrins and extracellular matrix components (such as collagen and fibrin).

[0294] V. Serum albumin

[0295] In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIB variant fused to serum albumin. Binding to serum albumin may improve the pharmacokinetics of protein drugs.

[0296] 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 about half of 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, serum albumin is human serum albumin. In some embodiments of the present invention, human serum albumin is connected to the extracellular ActRIIB variants described herein (e.g., extracellular ActRIIB variants with a sequence of any one of SEQ ID NO: 1-15 (e.g., SEQ ID NO: 2-15)) N- or C-terminal (e.g., C-terminal) to increase the serum half-life of extracellular ActRIIB variants. Human serum albumin can be directly or via a linker connected to the N- or C-terminal of an extracellular ActRIIB variant.

[0297] As an example, serum albumin that can be used in the methods and compositions described herein is generally known in the art. In one embodiment, the serum albumin comprises the sequence of UniProt ID NO: P02768 (SEQ ID NO: 74). :

[0298]

[0299] In some embodiments, the extracellular ActRIIB variants described herein (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 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 the 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 prolonged retention of the therapeutic protein.

[0300] VI. Connectors

[0301] The polypeptides described herein may 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 by means of 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 a portion (e.g., an Fc domain monomer (e.g., a 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 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 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 region may contain a motif, such as a multiple or repeated motif, of 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).In some embodiments, the spacer region can contain 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 region can contain 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 can contain 4 to 12 amino acids, including a motif of GGAG (SEQ ID NO:26), GGSG (SEQ ID NO:27), e.g., GGAG (SEQ ID NO:26), GGSG (SEQ ID NO:27), GGAGGGAG (SEQ ID NO:46), GGSGGGSG (SEQ ID NO:47), GGAGGGAGGGAG (SEQ ID NO:48), and GGSGGGSGGGSG (SEQ ID NO:49). In some embodiments, the spacer can contain a motif of GGGGA (SEQ ID NO:23) or GGGGS (SEQ ID NO:24), e.g., GGGGAGGGGAGGGGA (SEQ ID NO:50), and GGGGSGGGGSGGGGS (SEQ ID NO:51).In some embodiments of the invention, the linker between the portion (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 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 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).

[0302] In some embodiments, the spacer region 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 may contain a motif of EAAAK (SEQ ID NO: 69), such as multiple or repeated motifs. In some embodiments, the spacer may contain a proline-rich sequence (such as (XP) n ), such as a multiple or repeating motif, wherein X can be any amino acid (e.g., A, K or E) and n is 1-5, and PAPAP (SEQ ID NO: 70).

[0303] The length of the peptide spacer and the amino acids used can be adjusted depending on the two proteins involved and the degree of flexibility desired in the final protein fusion polypeptide. The length of the spacer can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0304] VII. Vectors, Host Cells, and Protein Production

[0305] The polypeptide of the present invention can be produced from a host cell. A host cell refers to a medium comprising essential cellular components (e.g., organelles) required for expressing a polypeptide described herein and a fusion polypeptide from its corresponding nucleic acid. Nucleic acid can be included in a nucleic acid vector, which can be introduced into a host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, injection, etc.). The selection of nucleic acid vectors depends in part on the host cell to be used. Typically, a preferred host cell is a eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) source.

[0306] Nucleic acid vector construction and host cells

[0307] The nucleic acid sequence encoding the amino acid sequence of the polypeptide of the present invention can be prepared by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. Nucleic acid molecules encoding the polypeptide of the present invention can be obtained using standard techniques (e.g., gene synthesis). Alternatively, standard techniques in the art (e.g., QuikChange TM Mutagenesis) mutates a nucleic acid molecule encoding wild-type extracellular ActRIIB to include a specific amino acid substitution. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR technology.

[0308] The nucleotide sequence encoding the polypeptide of the present invention can be inserted into a vector that can replicate and express nucleic acid molecules in a prokaryotic or eukaryotic host cell. Many vectors are available in the art and can be used for purposes of the present invention. Every kind of vector can include various components that can be adjusted and optimized for compatibility with a specific 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, a nucleotide sequence encoding a target protein, and a transcription termination sequence.

[0309] 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, NSO, Sp2 / 0, VERY, BHK, MDCK, W138, BT483, Hs578T, HTB2, BT20, T47D, NSO (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7030, and HsS78Bst cells. In some embodiments, E. coli cells can also be used as host cells of the present invention. Examples of E. coli strains include, but are not limited to, E. coli 294 (ATCC ® 31,446), Escherichia coli λ1776 (ATCC ® 31,537, Escherichia coli BL21 (DE3) (ATCC ® BAA-1025), and Escherichia coli RV308 (ATCC ® 31,608). Different host cells have characteristics and specific mechanisms for post-transcriptional processing and modification of protein products (e.g., glycosylation). Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the expressed polypeptide. The above-mentioned expression vectors can be introduced into appropriate host cells using conventional techniques in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection). Once the vector is introduced into the host cell for protein production, the host cell is cultured in a conventional nutrient medium appropriately modified for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequence. Methods for expressing therapeutic proteins are known in the art, see, for example, Paulina Balbas, Argelia Lorence (ed.) 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.

[0310] Protein production, recovery and purification

[0311] The host cell for producing the polypeptide of the present invention can be grown in a culture medium known in the art and suitable for cultivating the selected host cell. Examples of suitable culture medium for mammalian host cells include minimal essential medium (MEM), Dulbecco's modified Eagle's medium (DMEM), Expi293™ expression medium, DMEM and RPMI-1640 supplemented with fetal bovine serum (FBS). Examples of suitable culture medium for bacterial host cells include Luria broth (LB) plus necessary supplements, such as selection agents, such as ampicillin. Host cells are cultured at a suitable temperature such as about 20°C to about 39°C, for example 25°C to about 37°C, preferably 37°C, and CO2 levels, such as 5 to 10%. The pH of the culture medium is generally about 6.8 to 7.4, for example 7.0, which depends primarily 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.

[0312] 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 polypeptide of the present invention can be purified by any method known in the field of protein purification, for example, 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 chromatographic columns (e.g., POROS HS-50 cation exchange chromatography), filtering, ultrafiltration, salting out and dialysis procedures.

[0313] In other embodiments, the host cells can be destroyed, for example, by osmotic shock, sonication, or lysis, to recover the expressed protein. Once the cells are destroyed, the cell debris can be removed by centrifugation or filtration. In some cases, the polypeptide can be conjugated to a marker sequence (such as a peptide) to facilitate purification. An example of a marker amino acid sequence is a hexa-histidine peptide (His-tag), which binds to a nickel-functionalized agarose affinity column 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., 2001). Cell 37:767, 1984).

[0314] Alternatively, the polypeptides of the invention can be produced by cells of a subject (e.g., a human), for example, 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 virus vector, such as modified vaccinia virus Ankara (MVA)), an adeno-associated virus vector, and an alphavirus vector)) containing a nucleic acid molecule encoding a polypeptide of the invention. The vector, once within the cells of the subject (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), will promote expression of the polypeptide, which is then secreted from the cells. If treatment of the disease or condition is the desired outcome, no further action may be required. If it is desired to collect the protein, blood can be collected from the subject and the protein purified from the blood by methods known in the art.

[0315] VIII. Pharmaceutical compositions and formulations

[0316] The invention features a pharmaceutical composition comprising 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)). In some embodiments, a pharmaceutical composition of the invention comprises 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)) fused to a portion (e.g., an Fc domain monomer or dimer thereof, 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, a pharmaceutical composition of the invention comprising a polypeptide of the invention can be used in therapy in combination with other agents (e.g., therapeutic biologics and / or small molecules) or compositions. In addition to a therapeutically effective amount of polypeptide, the pharmaceutical composition may also include one or more pharmaceutically acceptable carriers or excipients, which may be formulated by methods known to those skilled in the art. In some embodiments, the pharmaceutical composition of the present invention includes a nucleic acid molecule (DNA or RNA, such as mRNA) encoding a polypeptide of the present invention, or a vector containing such a nucleic acid molecule.

[0317] Acceptable carrier and excipient in the pharmaceutical composition are nontoxic to receptors at dosage and concentration used.Acceptable carrier and excipient can include buffer, such as phosphate, citrate, HEPES and TAE, antioxidant, such as ascorbic acid and methionine, preservative, such as hexamethylammonium chloride, octadecyl dimethyl benzyl ammonium chloride, resorcinol and benzalkonium chloride, protein, such as human serum albumin, gelatin, dextran and immunoglobulin, hydrophilic polymer, such as polyvinyl pyrrolidone, amino acid, such as glycine, glutamine, histidine and lysine, and carbohydrate, such as glucose, mannose, sucrose and sorbitol.The pharmaceutical composition of the present invention can be administered parenterally in the form of injectable preparations.A sterile solution or any pharmaceutically acceptable liquid can be used to prepare a pharmaceutical composition for injection as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture medium (e.g., Dulbecco's modified Eagle medium (DMEM), α-modified Eagles medium (α-MEM), F-12 medium). Formulation methods are known in the art, see, e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (3rd ed.) Taylor&Francis Group, CRC Press (2015).

[0318] 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 technology is 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 filtering through a sterile filtration membrane.

[0319] 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, polylactides, 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 Some sustained release formulations enable release of the molecule over several months, for example one to six months, while other formulations release the pharmaceutical composition of the invention for shorter periods of time, for example days to weeks.

[0320] The pharmaceutical composition can be formed in unit dosage form as desired. The amount of active ingredient (eg, polypeptide of the present invention) included in the pharmaceutical preparation is such as to provide a suitable dosage within the specified range (eg, a dosage within the range of 0.01-100 mg / kg body weight).

[0321] The pharmaceutical composition for gene therapy can be in an acceptable diluent, or can include a sustained-release matrix wherein a gene delivery vector is embedded. If hydrodynamic injection is used as a delivery method, the pharmaceutical composition containing the nucleic acid molecules encoding polypeptides as described herein or the carrier (e.g., viral vector) containing the nucleic acid molecules containing the nucleic acid molecules is delivered quickly intravenously with a large fluid volume. The carrier that can be used as a gene delivery vehicle in vivo includes but is not limited to retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors, such as modified vaccinia virus Ankara), adeno-associated virus vectors and alphavirus vectors.

[0322] IX. Route, Dosage, and Administration

[0323] Pharmaceutical compositions comprising polypeptides of the present invention as therapeutic proteins can be formulated for, for example, intravenous administration, parenteral administration, subcutaneous administration, intramuscular administration, intraarterial administration, intrathecal administration, or intraperitoneal administration. The pharmaceutical compositions 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 formulations, various effective drug carriers are known in the art. See, for example, ASHP Handbook on Injectable Drugs, Toissel, 18th edition (2014).

[0324] In some embodiments, the pharmaceutical composition comprising a nucleic acid molecule encoding a polypeptide of the present invention or a vector containing such a nucleic acid molecule can be administered by gene delivery. The method of gene delivery is well known to those skilled in the art. Vectors that can be used for in vivo gene delivery and expression include, but are not limited to, retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors, such as modified vaccinia virus Ankara (MVA)), adeno-associated virus vectors, and alphavirus vectors. In some embodiments, the mRNA molecule encoding a polypeptide of the present invention can be directly administered to a subject.

[0325] In some embodiments of the present invention, the nucleic acid molecules encoding polypeptides described herein or the carriers containing such nucleic acid molecules can be administered using a fluid dynamic injection platform. In the fluid dynamic injection method, the nucleic acid molecules encoding polypeptides described herein are placed under the control of a strong promoter in an engineered plasmid (e.g., a viral plasmid). Plasmids are often delivered intravenously and rapidly with a large fluid volume. The fluid dynamic injection uses the controlled fluid dynamic pressure in the vein to enhance cell permeability, so that the pressure from the rise of the rapid injection of a large fluid volume causes fluid and plasmid to extravasate from the vein. The expression of nucleic acid molecules is mainly driven by the liver. In mice, fluid dynamic injection is often performed by injecting plasmids into the tail vein. In certain embodiments, the mRNA molecules encoding polypeptides described herein can be administered using fluid dynamic injection.

[0326] The dosage of the pharmaceutical composition of the present invention depends on factors, including route of administration, disease to be treated and physical characteristics of the subject, such as age, weight, general health. The pharmaceutical composition of the present invention can include a dosage of the polypeptide of the present invention ranging from 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450 or 500 mg / kg), 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.

[0327] The pharmaceutical composition is administered in a manner compatible with the dosage formulation and in a therapeutically effective amount that results in the improvement or remedy of 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). Typically, therapeutic proteins are administered at 0.1-100 mg / kg, such as 1-50 mg / kg. The pharmaceutical composition comprising the polypeptide of the present invention can be administered to a subject in need thereof, for example, every day, every week, every two weeks, every month, every two months, every quarter, every two years, once or multiple times per year (e.g., 1-10 times or more), or when medically necessary. In some embodiments, the pharmaceutical composition comprising the polypeptide of the present invention can be administered to a subject in need thereof weekly, every two weeks, every month, every two months, or every quarter. The dosage can be provided in a single dose or multiple dose regimen. As the patient's health condition declines, as medical conditions improve or increase, the timing between administrations can be reduced.

[0328] X. Treatment

[0329] The present invention is based on the discovery that amino acids from the extracellular portion of ActRIIA are substituted into the extracellular portion of ActRIIB to produce 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 to activin A and B, and gain some of the beneficial properties of ActRIIA, such as reduced binding affinity to BMP9 or the ability to increase red blood cell levels. Since 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 (e.g., activin A and B, myostatin, GDF11) without activating intracellular signaling pathways. Therefore, the extracellular ActRIIB variants described herein can be used to treat diseases or conditions in which elevated activin signaling has been implicated in pathogenesis (e.g., diseases or conditions in which increased expression of activin receptors or activin receptor ligands has been observed). For example, loss of myostatin has been shown to increase skeletal muscle weight, 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 disease, and is known to inhibit osteoblast activity, suggesting that increased activin levels contribute to bone disease. Activin A has also been found to be elevated in clinical and experimental pulmonary hypertension. In another example, the activin receptor ligand GDF11 is overexpressed in a mouse model of hemolytic anemia and is associated with defects in red blood cell production. Without wishing to be bound by theory, therapeutic agents that bind to activin receptor ligands (e.g., GDF11, myostatin and / or activin) and reduce their binding or interaction with endogenous activin receptors may have therapeutic utility for treating or preventing a variety of diseases or conditions, such as muscle disease, bone disease, fibrosis, anemia, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH).

[0330] The compositions and methods described herein can be used to treat and / or prevent (e.g., prevent its development or treat a subject diagnosed with it) a medical condition, such as a muscle disease, a bone disease, low red blood cell levels (e.g., low hemoglobin levels or low red blood cell counts, such as 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 increase muscle mass and strength in a subject in need thereof. In some embodiments, a polypeptide described herein can be administered to increase lean body mass. A polypeptide described herein can increase muscle mass or lean body mass as compared to a measurement obtained prior to treatment. In some embodiments, a 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 relate to affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of activin, myostatin, and / or BMP9 to their endogenous receptors) in a subject having a disease or condition involving muscle weakness and atrophy.

[0331] 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 bone fracture, or reduce bone resorption. The polypeptides described herein can increase bone mineral density, increase bone formation, or reduce bone resorption as 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 causes bone damage (e.g., osteoporosis or osteopenia). In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of activin, myostatin, and / or BMP9 to their endogenous receptors) in a subject having a disease or condition that causes bone damage.

[0332] 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 red blood cell levels (e.g., increase hemoglobin levels, increase red blood cell counts, increase hematocrit, or increase red blood cell formation or production). A polypeptide described herein can increase red blood cell levels (e.g., increase hemoglobin levels, red blood cell counts, hematocrit, or red blood cell formation) as compared to a measurement obtained prior to treatment. 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 a treatment A subject may be at risk of developing anemia (e.g., due to other diseases or conditions, such as chronic kidney disease, rheumatoid arthritis, cancer, or inflammatory diseases (e.g., Crohn's disease, SLE, or ulcerative colitis), or due to medical treatment (e.g., chemotherapy, radiation therapy, or surgery) in a subject who is at risk of developing anemia or blood loss. In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting 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).

[0333] 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 prevent or reduce fibrosis. In some embodiments, a polypeptide described herein can be administered to slow or stop the progression of fibrosis, to reduce the risk of developing fibrosis, or to reduce (e.g., reduce the frequency or severity of) one or more symptoms of fibrosis. A polypeptide described herein can at least reduce fibrosis or slow the progression of fibrosis compared to the progression of fibrosis prior to treatment, or compared to the progression of fibrosis in an untreated subject. In some embodiments, a subject may have fibrosis or be at risk of developing fibrosis (e.g., a 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), In some embodiments, the polypeptides described herein prevent or delay the development of fibrosis in a subject at risk for developing fibrosis (e.g., a subject undergoing chemotherapy, radiation, or surgery, or a subject suffering from 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 (e.g., reducing or inhibiting the binding of activin, myostatin, and / or BMP9 to their endogenous receptors) in a subject suffering from fibrosis or a disease or condition associated with fibrosis.

[0334] 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 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 (e.g., edema) in the legs, feet, abdomen (ascites), or neck, chest pain or pressure, rapid pulse or palpitations, blue lips or skin (cyanosis), dizziness or syncope), prevent PH (e.g., prevent its development), reduce the risk of developing PH, or slow or stop the onset of PH The polypeptides described herein can reduce symptoms of PH (e.g., reduce the severity or frequency of one or more symptoms such as shortness of breath (dyspnea), fatigue, swelling (e.g., edema) in the legs, feet, abdomen (ascites), or neck, chest pain or pressure, rapid pulse or palpitations, blue coloration of the lips or skin (cyanosis), dizziness or syncope), or slow the progression of PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), compared to symptoms or progression observed prior to treatment, or compared to symptoms or progression of PH in an untreated subject.In some embodiments, the subject may have PH or be at risk for developing PH (e.g., the subject may have idiopathic PAH; the subject may have a disease or condition associated with PAH (e.g., a disease or condition that results in an increased risk of developing PAH), such as HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary angiomatosis, 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 results in an increased risk of developing venous PH), such as left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital / acquired pulmonary vein stenosis; the subject may have a disease or condition associated with hypoxic PH (e.g., a disease or condition that results in an increased 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 disorder, chronic 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 miscellaneous PH-related Diseases or conditions that increase the risk of developing miscellaneous PH, such as hematologic disorders (e.g., chronic hemolytic anemia, sickle cell disease), systemic disorders (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 lobes of the lung)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 subject with a condition that causes the development of 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 abnormalities), thromboembolic PH (e.g., chronic thromboembolic pulmonary hypertension, or other pulmonary artery obstruction (e.g., pulmonary embolism, vascular In some embodiments, the methods described herein involve affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting 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.

[0335] In some embodiments, polypeptides including extracellular ActRIIB variants described herein reduce or inhibit the binding of myostatin, activin, and / or BMP9 to their endogenous receptors, such as ActRIIA, ActRIIB, and BMPRII (e.g., ActRIIB). The polypeptides described herein can reduce the binding of myostatin, activin, and / or BMP9 to their endogenous receptors as compared to the binding of myostatin, activin, and / or BMP9 to their endogenous receptors in the absence of the polypeptides of the invention. 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, red blood cell levels (e.g., hemoglobin levels, hematocrit, or red blood cell count, e.g., promoting or increasing The invention relates to an increase in PH (e.g., ...

[0336] In some embodiments, a polypeptide described herein (e.g., including an extracellular ActRIIB variant (e.g., having SEQ ID NOs: 1-15 (e.g., SEQ ID A polypeptide of an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 2-15), such as an effective amount of an ActRIIB variant, can be administered to a subject to increase muscle mass or strength, increase bone mineral density, increase bone formation, increase bone strength, reduce the risk of fractures, reduce bone resorption, increase red blood cell levels (e.g., increase hemoglobin levels, increase hematocrit, increase red blood cell counts, 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 symptoms of PH, prevent or delay the development of PH, or slow or stop the progression of PH, such as PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), or affect myostatin, activin, and / or BMP9 signaling in a subject. An extracellular ActRIIB variant (e.g., having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 1-15)) can be administered to a subject to increase muscle mass or strength, increase bone mineral density, increase bone formation, increase bone strength, reduce the risk of fractures, reduce bone resorption, increase red blood cell levels (e.g., increase hemoglobin levels, increase hematocrit, increase red blood cell counts, 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 symptoms of PH, prevent or delay the development of PH, or slow or stop the progression of PH, such as PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH), or affect myostatin, activin, and / or BMP9 signaling in a subject. An extra NO: 2-15), such as 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 fractures, 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). 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 involving bone damage (e.g., primary osteoporosis, secondary osteoporosis, osteopenia, osteosclerosis, bone 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 or is at risk of developing a disease or condition involving low red blood cell levels (e.g., anemia or blood loss, such as anemia associated with cancer (e.g., multiple myeloma, leukemia, breast cancer, lung cancer, colon cancer), cancer treatment (e.g., chemotherapy or radiation therapy), myelodysplastic syndrome, chronic or acute kidney disease or failure (e.g., chronic kidney disease), inflammatory or autoimmune disease (e.g., rheumatoid arthritis, inflammatory bowel disease such as Crohn's disease or ulcerative colitis, SLE), or surgery). In some embodiments of the methods described herein, the subject has or is at risk of developing a disease or condition involving fibrosis (e.g., chemotherapeutic drug-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis, liver fibrosis (e.g., cirrhosis), kidney fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis, osteoarticular fibrosis, tissue fibrosis, tumor stroma, desmoplastic tumors, surgical adhesions, hypertrophic scars, keloids, or fibrosis associated with wounds, burns, hepatitis B or C infection, fatty liver disease, schistosomiasis infection, kidney disease, heart disease, macular degeneration, retino- or vitreoretinopathy, systemic or localized scleroderma, atherosclerosis, or restenosis).In some embodiments of the methods described herein, the subject has or is at risk for developing a disease or condition involving PH (e.g., 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 angiomatosis, 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 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; PH associated with or caused by: chronic thromboembolic pulmonary hypertension, or other pulmonary artery obstruction (e.g., pulmonary embolism, angiosarcoma, arteritis, congenital pulmonary artery stenosis, or parasitic infection); miscellaneous PH associated with or caused by: hematologic disorders (e.g., chronic hemolytic anemia, sickle cell disease), systemic disorders (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).

[0337] 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, bone fractures, bone cancer or bone loss associated with cancer metastasis, Paget's disease, renal osteodystrophy, therapy-related bone loss, diet-related bone loss, obesity therapy-related bone loss, low gravity-related bone loss, immobilization-related bone loss, anemia, blood loss, fibrosis, or PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH) by administering to the subject an effective amount of a polypeptide described herein, e.g., a polypeptide comprising an extracellular ActRIIB variant, e.g., an extracellular ActRIIB variant having a sequence of any one of SEQ ID NOs: 1-15 (e.g., SEQ ID NOs: 2-15).

[0338] In any of the methods described herein, a subject suffering from a bone disease (e.g., bone injury) or at risk of developing a bone disease (e.g., bone injury) suffers from a disease or condition or is at risk of developing a 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, obesity treatment-related bone loss, low gravity-related bone loss, or immobilization-related bone loss. In some embodiments, primary osteoporosis is age-related or hormone-related (e.g., associated with a decline in estrogen) osteoporosis. In some embodiments, secondary osteoporosis is immobilization-induced or glucocorticoid-induced osteoporosis. In some embodiments, bone cancer is multiple myeloma, or the 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 therapy (e.g., chemotherapy or radiation). In some embodiments, diet-related bone loss is rickets (e.g., vitamin D deficiency). In some embodiments, low gravity-related bone loss is a lack of load-related bone loss. In some embodiments, compared to measurements obtained before treatment, or compared to bone mineral density typically observed in untreated subjects, the methods described herein increase bone mineral density (e.g., increase bone mass), such as increasing bone mineral density. In some embodiments, compared to measurements obtained before treatment, or compared to bone resorption typically observed in untreated subjects, the methods described herein reduce bone resorption (e.g., reduce bone decomposition activity), such as reducing bone resorption. In some embodiments, compared to measurements obtained before treatment, or compared to bone formation typically observed in untreated subjects, the methods described herein increase bone formation (e.g., increase bone anabolic activity or increase osteogenesis), such as increasing bone formation. In some embodiments, the methods described herein increase osteoblast activity or osteoblast production, such as increase osteoblast activity or osteoblast production, compared to measurements obtained before treatment or compared to osteoblast activity or osteoblast production typically observed in untreated subjects. In some embodiments, the methods described herein reduce osteoclast activity or osteoclast production, such as reduce osteoclast activity or osteoclast production, compared to measurements obtained before treatment or compared to osteoclast activity or osteoclast production typically observed in untreated subjects. In some embodiments, the bone is cortical bone or trabecular bone.

[0339] The invention also includes methods 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 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 having low red blood cell levels (e.g., low hemoglobin levels, low hematocrit, or low red blood cell count) or at risk of developing low red blood cell levels has anemia or blood loss or is at risk of developing anemia or blood loss. In some embodiments, the anemia is associated with nutritional deficiencies (e.g., vitamin deficiencies), bone marrow defects (e.g., paroxysmal nocturnal hemoglobinuria), response to medication, or other adverse reactions. adverse reactions to antiretroviral drugs (e.g., HIV drugs), myelodysplastic syndrome, bone marrow transplantation, cancer (e.g., solid tumors, such as breast, lung, 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 arthritis, systemic lupus erythematosus (SLE), acute or chronic skin diseases (e.g., psoriasis), or inflammatory bowel disease (e.g., Crohn's disease), Enn's disease or ulcerative colitis), cystitis, gastritis), acute or chronic renal disease or failure (e.g., chronic renal disease) including idiopathic or congenital conditions, acute or chronic liver disease, acute or chronic bleeding, infection (e.g., malaria, osteomyelitis), splenomegaly, porphyria, vasculitis, hemolysis, urinary tract infection, hemoglobinopathies (e.g., sickle cell disease), thalassemia, Churg-Fernandez syndrome, Felty syndrome, graft-versus-host disease, hematopoietic stem cell transplantation, myelofibrosis, pancytopenia, pure red cell aplasia, Henoch-Schonlein purpura, Schwabmann syndrome (e.g., Schwartz-Diesel syndrome), drug use or abuse (e.g., alcohol abuse), or contraindications to transfusion (e.g., elderly patients, patients with allo- or autoantibodies, pediatric patients, patients with cardiopulmonary disease, patients who object to transfusions for religious reasons (e.g., some Jehovah's Witnesses). In some embodiments, the anemia is aplastic anemia, iron deficiency anemia, vitamin deficiency anemia, anemia of chronic disease, anemia associated with bone marrow disease, hemolytic anemia, sickle cell anemia, microcytic anemia, hypochromic anemia, sideroblastic anemia, DeBouze anemia, Fanconi anemia, or refractory anemia with blastosis.The compositions and methods described herein can also be used to treat subjects who do not respond well to erythropoietin (EPO), or who are susceptible to the adverse effects of EPO (e.g., hypertension, headache, vascular thrombosis, flu-like syndrome, shunt obstruction, and myocardial infarction). In some embodiments, blood loss is due to surgery, trauma, wounds, ulcers, urinary tract bleeding, gastrointestinal bleeding, frequent blood donations, or excessive menstrual bleeding (e.g., menorrhagia). In some embodiments, the methods described herein increase red blood cell levels (e.g., hemoglobin levels, hematocrit, or red blood cell counts) compared to measurements obtained before treatment. In some embodiments, the methods described herein increase or induce red blood cell formation compared to measurements obtained before treatment. In some embodiments, the compositions and methods described herein reduce the subject's need for blood transfusions (e.g., the subject no longer needs blood transfusions, or the subject needs less frequent blood transfusions than before treatment with the compositions and methods described herein). Subjects with normal red blood cell levels can also be treated using the methods and compositions described herein to increase red blood cell levels so that blood can be drawn and stored for use in later transfusions.

[0340] The invention also includes methods of treating a subject having fibrosis or at risk of developing fibrosis by administering to the subject an effective amount of a polypeptide described herein, e.g., a polypeptide comprising an extracellular ActRIIB 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 or is at risk of developing fibrosis. In some embodiments, the fibrosis is chemotherapeutic drug-induced fibrosis, radiation-induced fibrosis, pulmonary fibrosis (e.g., cystic fibrosis, idiopathic fibrosis, or fibrosis associated with tuberculosis, pneumonia, or coal dust), liver fibrosis (e.g., cirrhosis, biliary atresia), kidney fibrosis (e.g., fibrosis associated with chronic kidney disease), corneal fibrosis, cardiac fibrosis (e.g., In some embodiments, fibrosis is associated with wounds, burns, hepatitis B or C infection, fatty liver disease, schistosomiasis infection, kidney disease (e.g., chronic kidney disease), kidney disease (e.g., kidney failure), kidney failure ... Disease), heart disease, macular degeneration, retinal or vitreoretinopathy, Crohn's disease, systemic or localized scleroderma, atherosclerosis, or restenosis. In some embodiments, the subject is at risk for developing fibrosis associated with cancer treatment (chemotherapy or radiation), disease or infection (e.g., tuberculosis, pneumonia, myocardial infarction, hepatitis B or C infection, fatty liver disease, schistosomiasis infection, kidney disease (e.g., chronic kidney disease), heart disease, macular degeneration, retinal or vitreoretinopathy, Crohn's disease, systemic or localized scleroderma, atherosclerosis, restenosis), surgery, wounds, or burns. In some embodiments In some embodiments, the methods described herein reduce fibrosis compared to measurements 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., reducing 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., slowing the progression of fibrosis compared to progression prior to treatment, or compared to 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., function of an organ or tissue with fibrosis) compared to organ or tissue function prior to treatment. Tissue and organ function can be evaluated using any standard clinical test commonly used to assess tissue and organ function.

[0341] The present invention also includes methods of treating a subject having PH (e.g., 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, 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 may have PH (e.g., PAH, venous PH, hypoxic PH, thromboembolic PH, or miscellaneous PH) or at risk of developing PH. In some embodiments, PH is PAH. In some embodiments, PAH is idiopathic PAH. In some embodiments, PAH is heritable PAH. In some embodiments, PAH In some embodiments, the PH is PAH associated with (e.g., caused by or associated with) HIV infection, schistosomiasis, portal hypertension, pulmonary veno-occlusive disease, pulmonary capillary angiomatosis, cirrhosis, congenital heart anomalies, connective tissue / autoimmune disorders (e.g., scleroderma or lupus), or drug use or abuse (e.g., methamphetamine or cocaine use). In some embodiments, the PH is venous PH. In some embodiments, the venous PH is venous PH associated with (e.g., caused by or associated with) left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular heart disease, congenital cardiomyopathy, or congenital / acquired pulmonary venous dysfunction. Stenosis. In some embodiments, PH is hypoxic PH. In some embodiments, hypoxic PH is hypoxic PH associated with (e.g., caused by or related to) 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 altitudes, or developmental abnormalities. In some embodiments, PH is thromboembolic PH. In some embodiments, thromboembolic PH is thromboembolic PH associated with (e.g., caused by or related to) chronic thromboembolic pulmonary hypertension, or other pulmonary arteries. In some embodiments, the PH is a miscellaneous PH. In some embodiments, the miscellaneous PH is a miscellaneous PH associated with (e.g., caused by or related to) a hematologic disease (e.g., chronic hemolytic anemia, sickle cell disease), a systemic disease (e.g., sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, or vasculitis), a metabolic disorder (e.g., glycogen storage disease, Gaucher disease, or thyroid disease), pulmonary tumor thrombotic microangiopathy, fibrosing mediastinitis, chronic renal failure, or segmental pulmonary hypertension).In some embodiments, the methods described herein reduce symptoms of PH (e.g., reduce the severity or frequency of symptoms such as shortness of breath (dyspnea), fatigue, swelling (e.g., edema) of the legs, feet, abdomen (ascites), or neck, chest pain or pressure, rapid pulse or palpitations, blue tint to the lips or skin (cyanosis), dizziness, or syncope, 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 an untreated subject). 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 an untreated or untreated subject). In some embodiments, the methods described herein reduce pulmonary vascular remodeling or vascular remodeling in the heart of a subject (e.g., the initiation or progression of vascular remodeling in the heart or lungs) compared to vascular remodeling prior to treatment or compared to vascular remodeling in an untreated subject. In some embodiments, the methods described herein reduce right ventricular hypertrophy (e.g., reduce right ventricular hypertrophy or the progression of right ventricular hypertrophy) compared to right ventricular hypertrophy prior to treatment or compared to right ventricular hypertrophy in an untreated subject. Symptoms of PH can be assessed using standard clinical tests before and after treatment. Common tests used to assess PH include electrocardiograms, pulmonary function tests, echocardiograms, right heart catheterization, computed tomography scans, measurements of pulmonary vascular resistance, and a 6-minute walk test. In some embodiments, the methods described herein reduce pulmonary vascular resistance (e.g., result in a decrease in pulmonary vascular resistance compared to pulmonary vascular resistance prior to treatment). In some embodiments, the methods described herein improve performance in a 6-minute walk test compared to performance in a 6-minute walk test prior to treatment.

[0342] In any of the methods described herein, a dimer (e.g., a homodimer or a heterodimer) formed by the interaction of two Fc domain monomers, each fused to 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)) can be used as a therapeutic protein. In any of the methods described herein, a polypeptide fused to a portion (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), 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)) can be used as a therapeutic protein. Nucleic acids encoding polypeptides described herein, or vectors containing the nucleic acids, can also be administered according to any of the methods described herein. In any of the methods described herein, the polypeptides, nucleic acids, or vectors can be administered as part of a pharmaceutical composition. Example

[0343] The following examples are provided to further illustrate some embodiments of the present invention, but are not intended to limit the scope of the invention; it is understood by their exemplary nature that other procedures, methods or techniques known to those skilled in the art may alternatively be used.

[0344] Example 1 - Effects of extracellular ActRIIB variants on body and muscle mass

[0345] C57Bl / 6 mice received a single hydrodynamic injection of a plasmid construct encoding one of the fourteen polypeptides described below or a vehicle control (n = 10 / group, see Figure 1 : The sequence provided in

[0346] (1) medium,

[0347] (2) extracellular ActRIIA (SEQ ID NO: 16) fused to the N-terminus of hFc via a GGG linker;

[0348] (3) extracellular ActRIIB (SEQ ID NO: 17) fused to the N-terminus of hFc via a GGG linker;

[0349] (4) an extracellular ActRIIB variant ActRIIB / A (SEQ ID NO: 2) fused to the N-terminus of hFc via a GGG linker;

[0350] (5) an extracellular ActRIIB variant ActRIIBΔ9 (SEQ ID NO: 3) fused to the N-terminus of hFc via a GGG linker;

[0351] (6) an extracellular ActRIIB variant ActRIIB 2.01 (SEQ ID NO: 4) fused to the N-terminus of hFc via a GGG linker;

[0352] (7) an extracellular ActRIIB variant ActRIIB 2.02 (SEQ ID NO: 5) fused to the N-terminus of hFc via a GGG linker;

[0353] (8) an extracellular ActRIIB variant ActRIIB 2.03 (SEQ ID NO: 6) fused to the N-terminus of hFc via a GGG linker;

[0354] (9) an extracellular ActRIIB variant ActRIIB 2.04 (SEQ ID NO: 7) fused to the N-terminus of hFc via a GGG linker;

[0355] (10) an extracellular ActRIIB variant ActRIIB 2.05 (SEQ ID NO: 8) fused to the N-terminus of hFc via a GGG linker;

[0356] (11) an extracellular ActRIIB variant ActRIIB 2.06 (SEQ ID NO: 9) fused to the N-terminus of hFc via a GGG linker;

[0357] (12) an extracellular ActRIIB variant ActRIIB 2.07 (SEQ ID NO: 10) fused to the N-terminus of hFc via a GGG linker;

[0358] (13) an extracellular ActRIIB variant ActRIIB 2.08 (SEQ ID NO: 11) fused to the N-terminus of hFc via a GGG linker;

[0359] (14) an extracellular ActRIIB variant ActRIIB 2.09 (SEQ ID NO: 12) fused to the N-terminus of hFc via a GGG linker; and

[0360] (15) Extracellular ActRIIB variant ActRIIB 2.10 (SEQ ID NO: 13) fused to the N-terminus of hFc via a GGG linker.

[0361] 100 μg of plasmid construct was delivered in a volume of 10% body weight over 5-8 seconds. The high volume and short injection period provided the pressure required to introduce the plasmid into the hepatocytes where the plasmid was expressed, particularly the protein of interest was expressed under a strong and ubiquitous promoter (pLEV113). The protein of interest was secreted and freely circulated under the endogenous machinery of the hepatocytes. Mice were weighed twice a week for 28 days and measurements were recorded as the percentage of body weight change from baseline measurement ( Figure 1 , Figure 2 Muscles were also weighed at the end of the study and measurements were recorded in milligrams ( Figure 3A and 3B ).

[0362] Example 2 - Evaluation of Binding Affinity of ActRIIB Variants by Surface Plasmon Resonance (SPR)

[0363] The GE 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, ActRIIB2.11, and ActRIIB 2.12 are recombinant proteins. All other ActRIIB-Fc variants were expressed by transient expression in HEK293 cells and purified from conditioned media 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 protein was then captured onto the chip in flow cells 2-4, with flow cell 1 left empty as a reference cell to measure and deduct any nonspecific binding. HBS-EP+ buffer from GE Healthcare™ was used as the running buffer. Each ligand was run in duplicate concentration series at 40µl / min to avoid mass transfer effects. All data were collected on CM-5 chips, except for GDF-11, which used CM-4. Data were analyzed by BioLogic™ software using Scrubber2 to calculate the K for each interaction. D (Table 3).

[0364] Table 3: Binding affinities (K) of ActRIIB variants to various ligands D )

[0365] .

[0366] Example 3 - Effects of Extracellular ActRIIB Variants on Bone Mineral Density

[0367] Adult male C57 / BL6 mice received either sham surgery (SHAM) or castration surgery (ORX). Both surgical groups were allowed to recover for 14 days after surgery. All animals were housed in regular cages with free access to food (regular chow) and water. SHAM and ORX animals were then assigned to vehicle-treated groups (VEH) or ActRII variant-treated groups 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 while on 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. On the study termination date, tissues of interest (muscle, fat depots, and tibia) were surgically removed, weighed, and stored appropriately for further analysis. At this time, ORX animals were also examined to confirm complete removal of the testes. After termination of the experiment, the cortical morphology and trabecular architecture of each bone were also evaluated using microcomputed tomography.

[0368] Example 4 - Effects of extracellular ActRIIB variants on renal fibrosis

[0369] The unilateral ureteral obstruction (UUO) mouse model of renal fibrosis was used to determine the effect of extracellular ActRllB variants on renal fibrosis. The UUO model involves complete ligation of the left ureter while maintaining intact right kidney function. In brief, UUO was performed on mice under anesthesia, whereby the left ureter was approached via a flank incision, and two ligatures were placed on the proximal third of the ureter using silk threads 5 mm apart. Sham surgery was performed in a similar manner without placing any ligatures on the ureter. In this model, within 14 days after UUO, renal collagen was measured by directly measuring the amount of hydroxyproline in the sample to evaluate the development of severe fibrosis in the kidney. Fourteen days after UUO, renal dry weight was reduced due to renal parenchymal damage. Sham 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 at 1, 3, 6, 8, 10 and 13 days after surgery, each UUO group received subcutaneous injections of ActR11B variants (10 mg / kg) or vehicle (body weight volume administered), which does not bind to any known mouse protein. Sham-operated mice received vehicle (sterile PBS) during this period using the same schedule as the UUO group. All mice were sacrificed on the 14th day after surgery. Kidney weights were measured, and kidneys were snap-frozen using liquid nitrogen and kept at -80°C until collagen content was measured by measuring the amount of hydroxyproline to evaluate fibrosis.

[0370] Example 5 - Effects of extracellular ActRIIB variants on red blood cells

[0371] Ten male and ten female rats per group received two SC doses of vehicle (day 1 and day 15), or 6, 20, or 60 mg / kg of ActRllB variants. Hematological parameters were measured on day 29. Studies were also conducted to evaluate the time course and dose response for RBC, hemoglobin, and hematocrit induction. In the first study, the time course of erythropoiesis was investigated in male and female rats that received subcutaneous (SC) doses of ActRllB variants (10 mg / kg) on ​​days 1 and 8. Hematological parameters were assessed before dosing and on days 3, 8, 15, 29, and 44. In the second study, hematological dose responses were investigated in male and female rats that received SC doses of vehicle on days 1 and 15, or 0.4, 2, 10, or 30 mg / kg of ActRllB variants. Hematological parameters were assessed before dosing and on days 13 and 28.

[0372] Example 6 - Effects of extracellular ActRIIB variants on PAH

[0373] In one experiment, 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, rats were randomized to vehicle or ActRIIB variant treatment groups 24 hours after PAH induction and treated with ActRIIB variants (5 or 15 mg / kg) or vehicle twice a week for 21 days. On day 14, ventricular function and right ventricular (RV) remodeling were examined by electrocardiography, which was performed 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 the animals were maintained in a 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 through the trachea, and mechanically ventilated using a rodent ventilator. Hemodynamics were assessed using a fluid-filled catheter through the RV apex. Rats were perfused with PBS followed by 1% formaldehyde. To measure RV hypertrophy (RVH), the heart was excised and dissected from the left ventricle plus septum (LV+S) without the RV wall and weighed separately. The extent of RVH was determined by the ratio RV / (LV+S).

[0374] In a second experiment, PAH was induced in male rats using a single subcutaneous injection of monocrotaline (MCT, 40 mg / kg). To determine whether treatment with the ActRIIB variant could slow or reduce the progression of PAH, rats were injected again with MCT on day 18 and randomized to vehicle or ActRIIB variant treatment groups. Rats were injected with ActRIIB variant (15 mg / kg) or vehicle three times per week. Hemodynamics and RVH were examined on day 35 as described above.

[0375] Example 7 - Evaluation of ActRIIB variants using a luciferase reporter assay

[0376] C2C12-BRE-luciferase and HEK293-SBE-luciferase cells were plated in DMEM supplemented with 2% FBS on 96-well plates and placed in an incubator for no less than 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 medium from transiently transfected cells. The medium in the plate was aspirated and the ActRIIB / A / ligand mixture was added to the plate as a medium replacement. The remaining wells were used for positive control and background duplication. The plates were incubated overnight and then read using Promega SteadyGlo and Molecular Devices Spectramax M5e. 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.

[0377] Table 4: Results from luciferase reporter assay

[0378] .

[0379] Example 8 - Effects of ActRIIB variants on body weight and red blood cell mass

[0380] Eight-week-old male C57BL / 6 mice were assigned to three groups (n=10 / group). Each group was dosed 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). Treatment was administered intraperitoneally (IP) twice a week for four weeks (eight doses), and the study was terminated on study day 28. Body weight was recorded on dosing days throughout the study. In addition, red blood cell mass parameters were assessed by hematology at the termination of the study. Figure 4 As shown in Figure 2, both ActRllB 2.11-Fc and ActRllB 2.12-Fc increased body weight in wild-type mice (*=p<0.05; ****=p<0.01). ActRllB 2.12-Fc also increased parameters of erythrocyte mass (***=p<0.001; ****=p<0.0001), including red blood cell count, hemoglobin level, and hematocrit ( Figures 5A-5C ).

[0381] Example 9 - Effects of ActRIIB variants in a mouse model of osteoporosis

[0382] C57BL / 6 mice underwent orchiectomy (ORX) or sham surgery at 9 weeks of age. After a six-week recovery period, during which time ORX mice developed an osteoporotic phenotype, ORX mice received twice-weekly intraperitoneal injections of vehicle or ActRIIB 2.12 (SEQ ID NO: 15)-Fc (10 mg / kg). Micro-CT (PerkinElmerQuantum Fx) imaging was performed at week 11 after the start of dosing. ASBMR bone morphometric 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. Figures 6A-6D As shown in, treatment with ActRIIB 2.12-Fc increased bone volume, increased bone volume fraction, and increased trabecular number relative to orchiectomy (*=p<0.05; **=p<0.01). These data indicate that treatment of osteoporotic mice with ActRIIB2.12-Fc increased trabecular bone mass due to increased bone formation.

[0383] Example 10 - Effects of ActRIIB variant 2.12 on red blood cell mass and trabecular bone in rats

[0384] Four-week-old Sprague Dawley rats were delivered hydrodynamic injections of ActRIIB 2.12 (SEQ ID NO: 15)-Fc plasmid DNA via lateral tail vein injection. Four weeks after injection, blood was analyzed for hematological parameters and ActRIIB2.12-Fc levels. MicroCT imaging (Perkin Elmer Quantum Fx) was performed on tibiae ex vivo. ASBMR bone morphometric 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. Figures 7A-7C As shown in Figure 2, ActRIIB 2.12-Fc increased parameters of red blood cell mass 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 increased trabecular bone mass in wild-type rats.

[0385] Example 11 - Treatment of muscle diseases by administering extracellular ActRIIB variants

[0386] According to the methods disclosed herein, a physician skilled in the art can treat a subject, such as a human patient, with a muscle disease (e.g., DMD, ALS, or inclusion body myositis) to increase muscle weight or maintain or improve muscle strength (e.g., reduce muscle weakness). Treatment methods may 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 electromyography). To treat the subject, a physician skilled 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 local administration (e.g., injection into a 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, e.g., 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 mass or maintain or improve muscle strength (eg, reduce muscle weakness).

[0387] After the composition is applied to the patient, those skilled in the art can monitor the improvement of the patient's response therapy by a variety of methods. For example, the doctor can monitor the patient's muscle weight, muscle strength and motor function. Compared with the test result before the composition is used, the patient shows an increased muscle weight or maintains or improves the discovery of muscle strength after the composition is used, indicating that the patient responds well to the treatment. Subsequent doses can be determined and applied as needed.

[0388] Example 12 - Treatment of bone disease by administration of extracellular ActRIIB variants

[0389] According to the methods disclosed herein, a physician in the art can treat a subject, such as a human patient, with a bone disease (e.g., osteoporosis or osteopenia) to increase bone mineral density, increase bone formation, reduce bone resorption, reduce bone loss, or reduce the risk of fracture. Treatment methods may 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 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) 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, e.g., 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 bone fracture.

[0390] After the composition is applied to the patient, those skilled in the art can monitor the improvement of the patient's response therapy by various methods. For example, the doctor can monitor the patient's bone mineral density by performing dual X-ray absorptiometry. Compared with the test results before the composition is applied, the patient shows an increased bone mineral density, increased bone formation, reduced bone resorption, reduced bone loss or a reduced fracture risk after the composition is applied, indicating that the patient responds well to the treatment. Subsequent doses can be determined and applied as needed.

[0391] Example 13 - Treatment of anemia by administration of extracellular ActRIIB variants

[0392] According to the methods disclosed herein, a physician in the art can treat a subject, such as a human patient, with anemia (e.g., vitamin deficiency anemia or anemia associated with chronic kidney disease) to increase a parameter of red blood cell mass, such as red blood cell count, hemoglobin level, or hematocrit. The treatment method may include diagnosing or identifying the subject as a candidate for treatment based on a blood test that measures a hematological parameter. In order to treat the 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 (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) to treat anemic diseases. 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, e.g., 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 hemoglobin levels, increase red blood cell counts, or increase hematocrit.

[0393] After the composition is applied to the patient, those skilled in the art can monitor the improvement of the patient's response therapy by various methods. For example, the doctor can monitor the patient's hemoglobin level, red blood cell count or hematocrit by performing a blood test. Compared with the test results before the composition is applied, the patient shows an improved hemoglobin level, red blood cell count or hematocrit after the composition is applied, indicating that the patient responds well to the treatment. Subsequent doses can be determined and applied as needed.

[0394] Example 14 - Treatment of fibrosis by administration of extracellular ActRIIB variants

[0395] According to the methods disclosed herein, a physician skilled in the art can treat a subject, such as a human patient, with fibrosis (e.g., 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. Treatment methods may include diagnosing or identifying the subject as a candidate for treatment based on a clinical test for fibrosis (e.g., an imaging test, such as an X-ray or CT scan). To treat the subject, a physician skilled 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) to treat fibrosis, or can be administered locally (e.g., injected) to a fibrotic tissue or organ. 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, e.g., 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 reduce the symptoms of fibrosis or slow or stop the progression of fibrosis.

[0396] After the composition is applied to the patient, those skilled in the art can monitor the improvement of the patient's response therapy by various methods. For example, the doctor can monitor the patient's fibrosis by performing an imaging test, and can use standard clinical tests to monitor the patient's symptoms. Compared with the test results before the composition is applied, the discovery that the patient's symptoms are reduced or the progress of the patient's fibrosis is slowed or stopped after the composition is applied indicates that the patient responds well to the treatment. Subsequent doses can be determined and applied as needed.

[0397] Example 15 - Treatment of pulmonary hypertension by administration of extracellular ActRIIB variants

[0398] According to the methods disclosed herein, a physician skilled in the art can treat a subject, such as a human patient, with pulmonary hypertension (PH, such as PAH) to reduce the symptoms of PH or slow or stop the progression of PH. Treatment methods may include diagnosing or identifying the subject as a candidate for treatment based on standard clinical tests for PH (e.g., echocardiogram, electrocardiogram, chest X-ray, or right heart catheterization). To treat the subject, a physician skilled 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 (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) to treat PH. 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, e.g., 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 reduce the symptoms of PH or slow or stop the progression of PH.

[0399] After the composition is applied to the patient, those skilled in the art can monitor the improvement of the patient's response to therapy by various methods. For example, the doctor can use standard clinical tests and patient self-reports to monitor the patient's symptoms. Compared with the test results before the composition is applied, the finding that the patient's PH symptoms are reduced or the patient's PH progress is slowed or stopped after the composition is applied indicates that the patient responds well to the treatment. Subsequent doses can be determined and applied as needed.

[0400] Other Implementations

[0401] Although the invention has been described in conjunction with specific embodiments thereof, it will be understood that it is capable of further modifications, and this application is intended to cover any variations, uses, or adaptations of the invention which follow from the principles of the invention in general and include such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains, and which may be applied to the basic features herein above described.

[0402] All publications, patents, and patent applications are herein 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.

[0403] Other implementations are within the following claims.

Claims

1. A polypeptide comprising an extracellular activin receptor type IIB variant, wherein the sequence of the extracellular activin receptor type IIB variant is SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO:

15.

2. The polypeptide of claim 1, wherein the sequence of the extracellular activin receptor type IIB variant is SEQ ID NO:

12.

3. The polypeptide of claim 1, wherein the sequence of the extracellular activin receptor type IIB variant is SEQ ID NO:

13.

4. The polypeptide of claim 1, wherein the sequence of the extracellular activin receptor type IIB variant is SEQ ID NO:

14.

5. The polypeptide of claim 1, wherein the sequence of the extracellular activin receptor type IIB variant is SEQ ID NO:

15.

6. The polypeptide of any one of claims 1 to 5, further comprising an Fc domain monomer fused to the C-terminus of the polypeptide via a linker. The polypeptide of claim 6 , wherein the Fc domain monomer is a human IgG1 Fc domain monomer.

8. The polypeptide of claim 6, wherein the polypeptide is in the form of a homodimer.

9. The polypeptide of any one of claims 1 to 5, further comprising an Fc domain fused to the C-terminus of the polypeptide via a linker.

10. The polypeptide of any one of claims 1 to 5, further comprising an albumin binding peptide fused to the C-terminus of the polypeptide via a linker.

11. The polypeptide of any one of claims 1 to 5, further comprising a fibronectin domain fused to the C-terminus of the polypeptide via a linker.

12. The polypeptide of any one of claims 1 to 5, further comprising human serum albumin fused to the C-terminus of the polypeptide via a linker.

13. The polypeptide of claim 6, wherein the linker is an amino acid spacer.

14. The polypeptide of claim 13, 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).

15. A nucleic acid molecule encoding the polypeptide of any one of claims 1-14.

16. A vector comprising the nucleic acid molecule of claim 15.

17. A host cell expressing the polypeptide of any one of claims 1 to 14, wherein the host cell comprises the nucleic acid molecule of claim 15 or the vector of claim 16, wherein the nucleic acid molecule or the vector is expressed in the host cell.

18. A method for preparing the polypeptide of any one of claims 1 to 14, the method comprising: a) providing a host cell comprising the nucleic acid molecule of claim 15 or the vector of claim 16, and b) expressing the nucleic acid molecule or the vector in said host cell under conditions allowing the formation of said polypeptide.

19. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 14, the nucleic acid molecule of claim 15 or the vector of claim 16, and one or more pharmaceutically acceptable carriers or excipients.

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