Activin receptor IIA variants and methods of use thereof

Abstract

The present invention relates to activin receptor IIA type variants and methods of use thereof. The present invention features a polypeptide comprising an extracellular ActRIIa variant. In some embodiments, the polypeptides of the present invention comprise an extracellular ActRIIa variant fused to an Fc domain monomer or part. The invention also features the treatment of diseases and conditions involving muscle weakness and atrophy using said polypeptides, such as Duchenne muscular dystrophy, facial shoulder brachial muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia; or cancer cachexia; the present invention relates to pharmaceutical compositions and methods for treating a metabolic disease, such as obesity, type 1 diabetes mellitus or type 2 diabetes mellitus.

Description

[0001] This application is a divisional application of the international application PCT / US2017 / 060960 with an international application date of November 9, 2017, entering China, with application number 201780082790.4 and entitled “Activin receptor type IIA variants and methods of use thereof”.

[0002] Technical Field and Background Art Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM) and amyotrophic lateral sclerosis (ALS) are examples of muscle diseases involving weakness and atrophy of muscles 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 muscles of the thigh and the muscles of the arms that control the flexion of the fingers and wrists. ALS is a motor neuron disease characterized by systemic muscle stiffness, muscle twitching and muscle atrophy due to the degeneration of motor neurons. Efforts to improve the treatment and survival of subjects with these devastating muscle diseases have not been successful.

[0003] Being overweight is a growing problem in the U.S., with a prevalence of approximately 25% in the population. Increased visceral and subcutaneous fat causes dysfunction of various organs. Being overweight is a risk factor for a series of complications (including obesity, diabetes (e.g., type 1 and type 2 diabetes), cardiovascular disease, and several forms of cancer). Insulin resistance is also associated with obesity, and occurs when the pancreatic tissue requires an increased amount of insulin. Once pancreatic β cells cannot produce enough insulin to meet demand, hyperglycemia occurs and type 2 diabetes occurs. It is believed that the fat cells increased in obesity play a role in this process. Although obesity and metabolic diseases such as diabetes (e.g., type 1 and type 2 diabetes) and insulin resistance are ubiquitous, there are few treatment options.

[0004] Novel treatments for these muscle and metabolic diseases are needed. Summary of the invention The present invention is characterized in that it comprises a polypeptide of an extracellular activin receptor type IIa (ActRIIa) variant. In some embodiments, the polypeptide of the present invention comprises an extracellular ActRIIa variant fused to the N- or C-terminus of an Fc domain monomer or portion. Such a portion can be connected by amino acids or other covalent bonds and can increase the stability of the polypeptide. The polypeptide comprising an extracellular ActRIIa variant fused to an 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 mass and strength in a subject having a disease or condition involving muscle weakness and atrophy, such as Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia. The polypeptides of the present invention can also be used to reduce body weight, reduce body fat, increase glucose clearance, increase insulin sensitivity, or reduce fasting insulin levels in subjects with metabolic diseases, such as obesity, type 1 diabetes, or type 2 diabetes, or at risk of developing metabolic diseases, such as obesity, type 1 diabetes, or type 2 diabetes. Further, the polypeptides of the present invention can also be used to affect myostatin, activin, and / or bone morphogenetic protein 9 (BMP9) signaling in subjects at risk of developing a disease or condition or metabolic disease involving muscle weakness and atrophy or having a disease or condition or metabolic disease involving muscle weakness and atrophy.

[0006] In one aspect, the invention features a polypeptide comprising an extracellular activin receptor type IIa (ActRIIa) variant having the sequence: Where X1 is F or Y; X2 is F or Y; X3 is E or A; X4 is K or L; X5 is D or E; X6 is R or A; X7 is P or R; X8 is Y or E; X9 is D or E; X 10 It is K or Q; X 11 It is D or A; X 12 It is K or A; X 13 is R or A; X 14 is R or L; X 15 is F or Y; X 16 is K, R or A; X 17 is K, A, Y, F or I; X 18 Q or K; X 19 W or A; X 20 L or A; X 21 is D, K, R, A, F, G, M, N, or I; X 22 is I, F or A; X23 is K or T; X 24 Is K or E; X 25 Is D or E; X 26 is S or N; and X 27 is E or Q, and wherein the variant has at least one amino acid substitution relative to wild-type extracellular ActRIIa having the sequence of SEQ ID NO: 73 or extracellular ActRIIa having any one of the sequences of SEQ ID NOs: 76-96.

[0007] In some embodiments, the variant has the following sequence: Among them, X2, X3, X4, X5, X6, X7, X8, X9, X 11 , X 12 , X 13 , X 14 , X 15 , X 16 , X 17 , X 18 , X 19 , X 20 , X 21 , X 22 , X 23 , X 24 , X 25 , X 26 and X 27 As defined above.

[0008] In some embodiments, the variant has the following sequence: Among them, X2, X4, X5, X7, X8, X9, X 14 , X 15 , X 16 , X 18 , X 22 , X 23 , X 24 , X 25 , X 26 and X 27 As defined above.

[0009] In some embodiments, the variant has the following sequence: Among them, X2, X4, X7, X8, X9, X 14 , X 15 , X 16 , X 18 , X 22, X 23 , X 25 , X 26 and X 27 As defined above.

[0010] In some embodiments, the variant has the following sequence: Among them, X2, X4, X8, X9, X 14 , X 18 , X 23 , X 25 , X 26 and X 27 As defined above.

[0011] In any of the preceding embodiments, X1 is F or Y. In any of the preceding embodiments, X2 is F or Y. In any of the preceding embodiments, X3 is E or A. In any of the preceding embodiments, X4 is K or L. In any of the preceding embodiments, X5 is D or E. In any of the preceding embodiments, X6 is R or A. In any of the preceding embodiments, X7 is P or R. In any of the preceding embodiments, X8 is Y or E. In any of the preceding embodiments, X9 is D or E. In any of the preceding embodiments, X 10 is K or Q. In any of the foregoing embodiments, X 11 is D or A. In any of the foregoing embodiments, X 12 is K or A. In any of the preceding embodiments, X 13 is R or A. In any of the foregoing embodiments, X 14 is R or L. In any of the preceding embodiments, X 15 is F or Y. In any of the preceding embodiments, X 16 is K, R or A. In any of the foregoing embodiments, X 17 is K, A, Y, F or I. In any of the foregoing embodiments, X 18 is Q or K. In any of the foregoing embodiments, X 19 is W or A. In any of the preceding embodiments, X 20 is L or A. In any of the preceding embodiments, X 21 is D, K, R, A, F, G, M, N or I. In any of the foregoing embodiments, X 22 is I, F or A. In any of the foregoing embodiments, X 23 is K or T. In any of the preceding embodiments, X 24 is K or E. In any of the preceding embodiments, X 25 is D or E. In any of the preceding embodiments, X 26is S or N. In any of the foregoing embodiments, X 27 is E or Q. In any of the foregoing embodiments, X 23 It's T, X 24 Yes E, X 25 is E, and X 26 is N. In any of the preceding embodiments, X 23 It's T, X 24 It is K, X 25 is E, and X 26 is N. In any of the preceding embodiments, X 17 It's K.

[0012] In any of the preceding embodiments, the variant has the sequence of any one of SEQ ID NOs: 6-72.

[0013] In any of the preceding embodiments, position X 24 The amino acid at the position can be replaced by amino acid K.

[0014] In any of the preceding embodiments, position X 24 The amino acid at the position can be replaced by amino acid E.

[0015] In any of the foregoing embodiments, the polypeptides described herein can further comprise a C-terminal extension of one or more amino acids (e.g., 1, 2, 3, 4, 5, 6 or more amino acids). In some embodiments, the C-terminal extension is the amino acid sequence NP. In some embodiments, the C-terminal extension is the amino acid sequence NPVTPK (SEQ ID NO: 155).

[0016] In any of the foregoing embodiments, the polypeptides described herein may further comprise a moiety fused or covalently linked to the C-terminus of the polypeptide. In some embodiments, the moiety increases the stability of the polypeptide or improves the pharmacokinetics of the polypeptide. In some embodiments, the moiety is an Fc domain, an albumin binding peptide, a fibronectin domain, or human serum albumin.

[0017] In any of the foregoing embodiments, the polypeptide described herein may further comprise an Fc domain monomer fused to the C-terminus of the polypeptide by means of a linker. In some embodiments, a polypeptide comprising an extracellular ActRIIa variant described herein fused to an Fc domain monomer may form a dimer (e.g., a homodimer or a heterodimer) by interaction between two Fc domain monomers. In some embodiments, the Fc domain monomer has a sequence of SEQ ID NO: 97.

[0018] In any of the foregoing embodiments, the polypeptide described herein may further comprise an Fc domain fused to the C-terminus of the polypeptide by means of a linker. In some embodiments, the Fc domain is a wild-type Fc domain. In some embodiments, the wild-type Fc domain has a sequence of SEQ ID NO: 151. In some embodiments, the Fc domain contains one or more amino acid substitutions. In some embodiments, the Fc domain containing one or more amino acid substitutions does not form a dimer.

[0019] In any of the foregoing embodiments, the polypeptide described herein may further comprise an albumin binding peptide fused to the C-terminus of the polypeptide by means of a linker. In some embodiments, the albumin binding peptide has the sequence of SEQ ID NO:152.

[0020] In any of the foregoing embodiments, the polypeptide described herein may further comprise a fibronectin domain fused to the C-terminus of the polypeptide by means of a linker.In some embodiments, the fibronectin domain peptide has the sequence of SEQ ID NO:153.

[0021] In any of the foregoing embodiments, the polypeptide described herein may further comprise human serum albumin fused to the C-terminus of the polypeptide by means of a linker. In some embodiments, the human serum albumin has a sequence of SEQ ID NO: 154.

[0022] In some embodiments, the linker is an amino acid spacer. In some embodiments, the amino acid spacer is GGG, GGGA (SEQ ID NO: 98), GGGG (SEQ ID NO: 100), GGGAG (SEQ ID NO: 130), GGGAGG (SEQ ID NO: 131), or GGGAGGG (SEQ ID NO: 132).

[0023] In some embodiments, the amino acid spacer is GGGS (SEQ ID NO:99), GGGGA (SEQ ID NO:101), GGGGS (SEQ ID NO:102), GGGGG (SEQ ID NO:103), GGAG (SEQ ID NO:104), GGSG (SEQ ID NO:105), AGGG (SEQ ID NO:106), SGGG (SEQ ID NO:107), GAGA (SEQ ID NO:108), GSGS (SEQ ID NO:109), GAGAGA (SEQ ID NO:110), GSGSGS (SEQ ID NO:111), GAGAGAGA (SEQ ID NO:112), GSGSGSGS (SEQ ID NO:113), GAGAGAGAGA (SEQ ID NO:114), GSGSGSGSGS (SEQ ID NO:115), GAGAGAGAGAGA (SEQ ID NO:116), and GSGSGSGSGSGS (SEQ ID NO:117), GGAGGA (SEQ ID NO:118), GGSGGS (SEQ ID NO:119), GGAGGAGGA (SEQ ID NO:120), GGSGGSGGS (SEQ ID NO:121), GGAGGAGGAGGA (SEQ ID NO:122), GGSGGSGGSGGS (SEQ ID NO:123), GGAGGGAG (SEQ ID NO:124), GGSGGGSG (SEQ ID NO:125), GGAGGGAGGGAG (SEQ ID NO:126), and GGSGGGSGGGSG (SEQ ID NO:127), GGGGGAGGGGAGGGGA (SEQ ID NO:128), GGGGSGGGGSGGGGS (SEQ ID NO:129), AAAL (SEQ ID NO:133), AAAK (SEQ ID NO:134), AAAR (SEQ ID NO:135), EGKSSGSGSESKST (SEQ ID NO:136), GSAGSAAGSGEF (SEQ ID NO:137), AEAAAKEAAAKA (SEQ ID NO:138), KESGSVSSEQLAQFRSLD (SEQ ID NO:139), GENLYFQSGG (SEQ ID NO:140), SACYCELS (SEQ ID NO:141), RSIAT (SEQ IDNO:142), RPACKIPNDLKQKVMNH (SEQ ID NO:143), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO:144), AAANSSIDLISVPVDSR (SEQ ID NO:145), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO:146), EAAAK (SEQ ID NO:146) NO:147) or PAPAP (SEQ ID NO:148).

[0024] In any of the preceding embodiments, the polypeptide described herein has a serum half-life of at least 7 days.

[0025] In any of the preceding embodiments, the polypeptides described herein have a K of 200 pM or more. D Binds to human bone morphogenetic protein 9 (BMP9). In some embodiments, the polypeptide binds to activin and / or myostatin and has reduced (eg, weak) binding to human BMP9. In some embodiments, the polypeptide does not substantially bind to human BMP9.

[0026] In any of the preceding embodiments, the polypeptides described herein have a K of 800 pM or less. D Binds human activin A.

[0027] In any of the preceding embodiments, the polypeptides described herein have a K of approximately 800 pM or less. D Binds human activin B.

[0028] In any of the preceding embodiments, the polypeptides described herein have a K of approximately 5 pM or higher. D Binds human GDF-11.

[0029] In another aspect, the invention features a nucleic acid molecule encoding a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72). In another aspect, the invention also features a vector comprising a nucleic acid molecule described herein.

[0030] In another aspect, the invention features a host cell expressing a polypeptide described herein, wherein the host cell comprises a nucleic acid molecule or a vector described in the first two aspects, wherein the nucleic acid molecule or vector is expressed in the host cell.

[0031] 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 vector in the host cell under conditions that allow formation of the polypeptide.

[0032] 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 in a therapeutically effective amount.

[0033] In another aspect, the invention also features a construct (e.g., a homodimer) comprising two identical polypeptides, each comprising an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72) fused to the N- or C-terminus of an Fc domain monomer (e.g., a sequence of SEQ ID NO: 97). The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0034] In another aspect, the invention also features a construct (e.g., a heterodimer) comprising two different polypeptides, each comprising an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72) fused to the N- or C-terminus of an Fc domain monomer (e.g., a sequence of SEQ ID NO: 97). The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0035] In another aspect, the invention features a method of increasing muscle mass 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.

[0036] In some embodiments of the method of increasing muscle mass in a subject, the subject has Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia.

[0037] In another aspect, the invention features a method of affecting myostatin, activin and / or BMP9 signaling (e.g., reducing or inhibiting the binding of myostatin, activin and / or BMP9 to its receptor) in a subject having a disease or condition involving weakness and atrophy of muscles, 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.

[0038] In another aspect, the invention features a method of treating a subject having 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.

[0039] In another aspect, the invention features a method of treating a subject having 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.

[0040] In another aspect, the invention features a method of treating a subject having 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.

[0041] In another aspect, the invention features a method of treating a subject having 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.

[0042] In another aspect, the invention features a method of reducing body fat 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.

[0043] In another aspect, the invention features a method of reducing body weight 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.

[0044] In another aspect, the invention features a method of reducing blood glucose 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.

[0045] In another aspect, the invention features a method of increasing insulin sensitivity 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.

[0046] In some embodiments of any of the above aspects, the subject has a metabolic disease or is at risk of developing a metabolic disease. In some embodiments, the metabolic disease is selected from obesity, type 1 diabetes, and type 2 diabetes.

[0047] 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 receptors) in a subject having a metabolic disease or at risk of developing a metabolic 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.

[0048] In another aspect, the invention features a method of treating and / or preventing a metabolic disease in a subject 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.

[0049] In some embodiments of any of the above aspects, the metabolic disease is selected from obesity, type 1 diabetes and type 2 diabetes. In some embodiments of any of the above aspects, the metabolic disease is obesity. In some embodiments of any of the above aspects, the metabolic disease is type 1 diabetes. In some embodiments of any of the above aspects, the metabolic disease is type 2 diabetes.

[0050] In some embodiments of any of the above aspects, the method reduces the subject's body weight and / or the percentage of body weight gain. In some embodiments of any of the above aspects, the method reduces the subject's body fat mass and / or the percentage of body fat. In some embodiments of any of the above aspects, the method does not affect the subject's appetite for food intake. In some embodiments of any of the above aspects, the method reduces the subject's obesity. In some embodiments of any of the above aspects, the method reduces the weight of the subject's epididymal and perirenal fat pads. In some embodiments of any of the above aspects, the method reduces the amount of subcutaneous and / or visceral fat in the subject. In some embodiments of any of the above aspects, the method reduces the subject's fasting insulin level. In some embodiments of any of the above aspects, the method reduces the subject's blood glucose level. In some embodiments of any of the above aspects, the method increases the subject's insulin sensitivity. In some embodiments of any of the above aspects, the method increases the subject's glucose clearance. In some embodiments of any of the above aspects, the method improves the subject's serum lipid profile. In some embodiments of any of the above aspects, the method does not reduce lean body mass.

[0051] In some embodiments of any of the above aspects, the method increases muscle mass.

[0052] In some embodiments of any of the above aspects, the method reduces or inhibits the binding of activin and / or myostatin to their receptors.

[0053] In some embodiments of any of the aforementioned aspects, the polypeptide, nucleic acid, vector or pharmaceutical composition is administered in an amount sufficient to increase muscle mass and / or strength, affect myostatin, activin and / or BMP9 signaling, or reduce or inhibit the binding of activin and / or myostatin to their receptors in a subject, In some embodiments of any of the aforementioned aspects, the polypeptide, nucleic acid, vector, or pharmaceutical composition is administered in an amount sufficient to reduce body fat, reduce subcutaneous fat mass, reduce visceral fat mass, reduce obesity, reduce the weight of epididymal and perirenal fat pads, reduce body fat percentage, reduce body weight, reduce percentage of weight gain, reduce fasting insulin levels, reduce blood glucose levels, increase insulin sensitivity, affect myostatin, activin and / or BMP9 signaling in a subject, reduce proliferation of adipocytes, reduce or inhibit binding of activin and / or myostatin to their receptors, reduce LDL, reduce triglycerides, improve serum lipid profile, modulate insulin biosynthesis and / or secretion from beta cells, delay, postpone or reduce the need for insulin, or increase glucose clearance.

[0054] In some embodiments of any of the methods described herein, the method does not cause vascular complications in the subject (eg, increased vascular permeability or leakage). In some embodiments of any of the methods described herein, the method increases bone mineral density in the subject.

[0055] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 69. In some embodiments, the variant having a sequence of SEQ ID NO: 69 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 TEEN or TKEN at position X, and / or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids at the C-terminus, e.g., amino acids NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method comprises administering to the subject a therapeutically effective amount of a sequence having SEQ ID NO: 69 (optionally having position X 17 The amino acid K at position X23 , X 24 , X 25 and X 26 The invention relates to a pharmaceutical composition comprising a variant of an amino acid sequence TEEN or TKEN at the ...

[0056] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 58. In some embodiments, the variant having a sequence of SEQ ID NO: 58 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 TEEN or TKEN at position X, and / or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids at the C-terminus, e.g., amino acids NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a sequence having SEQ ID NO: 58 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26The invention relates to a pharmaceutical composition comprising a variant of an amino acid sequence TEEN or TKEN at the ...

[0057] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 6. In some embodiments, the variant having a sequence of SEQ ID NO: 6 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 TEEN or TKEN at position X, and / or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids at the C-terminus, e.g., amino acids NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a sequence having SEQ ID NO: 6 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26The invention relates to a pharmaceutical composition comprising a variant of an amino acid sequence TEEN or TKEN at the ...

[0058] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 38. In some embodiments, the variant having a sequence of SEQ ID NO: 38 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 TEEN or TKEN at position X, and / or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids at the C-terminus, e.g., amino acids NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method comprises administering to the subject a therapeutically effective amount of a sequence having SEQ ID NO: 38 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26The invention relates to a pharmaceutical composition comprising a variant of an amino acid sequence TEEN or TKEN at the ...

[0059] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 41. In some embodiments, the variant having a sequence of SEQ ID NO: 41 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 TEEN or TKEN at position X, and / or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids at the C-terminus, e.g., amino acids NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a sequence having SEQ ID NO: 41 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26The invention relates to a pharmaceutical composition comprising a variant of an amino acid sequence TEEN or TKEN at the ...

[0060] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 44. In some embodiments, the variant having a sequence of SEQ ID NO: 44 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 TEEN or TKEN at position X, and / or a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids at the C-terminus, e.g., amino acids NP or NPVTPK (SEQ ID NO: 155)). In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a sequence having SEQ ID NO: 44 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26The invention relates to a pharmaceutical composition comprising a variant of an amino acid sequence TEEN or TKEN at the ...

[0061] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 70. In some embodiments, the variant having a sequence of SEQ ID NO: 70 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 In some embodiments of any of the above aspects, the method comprises administering to the subject a therapeutically effective amount of a sequence having SEQ ID NO: 70 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26The invention relates to a pharmaceutical composition comprising a variant of an amino acid sequence TEEN or TKEN at the ...

[0062] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 71. In some embodiments, the variant having a sequence of SEQ ID NO: 71 has a position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26 In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a sequence having SEQ ID NO: 71 (optionally having position X 17 The amino acid K at position X 23 , X 24 , X 25 and X 26The invention relates to a pharmaceutical composition comprising a variant of an amino acid sequence TEEN or TKEN at the ...

[0063] In some embodiments of any of the above aspects, the variant has a sequence of SEQ ID NO: 72. In some embodiments, the variant having a sequence of SEQ ID NO: 72 has a position X 17 The amino acid K at position, and / or position X 23 , X 24 , X 25 and X 26 In some embodiments of any of the above aspects, the method comprises administering to the subject a therapeutically effective amount of a sequence having SEQ ID NO: 72 (optionally having an amino acid sequence at position X) 17 The amino acid K at position, and / or position X 23 , X 24 , X 25 and X 26The invention relates to a method for increasing muscle mass or treating a muscle disorder in a subject in need thereof (e.g., a subject with DMD, FSHD, IBM, ALS, sarcopenia, or cancer cachexia), affecting myostatin, activin, and / or BMP9 signaling in a subject (e.g., a subject with DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity, type 1 diabetes or type 2 diabetes or at risk of developing DMD, FSHD, IBM, ALS, sarcopenia, cancer cachexia, obesity, type 1 diabetes or type 2 diabetes), reducing body fat or weight in a subject (e.g., a subject with obesity, type 1 diabetes or type 2 diabetes), or treating and / or preventing a metabolic disease in a subject (e.g., a subject with obesity, type 1 diabetes or type 2 diabetes or at risk of developing obesity, type 1 diabetes or type 2 diabetes).

[0064] definition As used herein, the term "extracellular activin receptor type IIa (ActRIIa) variant" refers to a peptide comprising a soluble, extracellular portion of a single transmembrane receptor ActRIIa having at least one amino acid substitution relative to wild-type extracellular ActRIIa (e.g., the bold portion of the sequence of SEQ ID NO: 75 shown below) or an extracellular ActRIIa having any of the sequences of SEQ ID NOs: 76-96. The sequence of the wild-type, human ActRIIa precursor protein is shown below (SEQ ID NO: 75), wherein the signal peptide is italicized and the extracellular portion is in bold.

[0065] Wild-type, human ActRIIa precursor protein (SEQ ID NO: 75):

[0066] The extracellular ActRIIa variant may have a sequence of any one of SEQ ID NOs: 1-72. In specific embodiments, the extracellular ActRIIa variant has a sequence of any one of SEQ ID NOs: 6-72 (Table 2). In some embodiments, the extracellular ActRIIa variant may have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 97% or more) amino acid sequence identity with the sequence of wild-type extracellular ActRIIa (SEQ ID NO: 73).

[0067] As used herein, the term "extracellular ActRIIb variant" refers to a peptide comprising a soluble, extracellular portion of ActRIIb of a single transmembrane receptor ActRIIb having at least one amino acid substitution relative to wild-type extracellular ActRIIb (e.g., the sequence of SEQ ID NO: 74). The extracellular ActRIIb variant may have the sequence of SEQ ID NO: 149 as shown below: Extracellular ActRIIb variant (SEQ ID NO: 149):

[0068] As used herein, the term "linker" refers to a connection between two elements (e.g., a peptide or protein domain). The polypeptide described herein may include an extracellular ActRIIa variant fused to a portion (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72)). 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.

[0069] 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 C 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 H 3). 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 CH 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.

[0070] 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: 152).

[0071] 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 (SEQ ID NO: 153) having amino acids 610-702 of the sequence of UniProt ID NO: P02751. In other embodiments, the fibronectin domain is an adnectin protein.

[0072] 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: 154).

[0073] 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 ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N- or C-terminus of a portion (e.g., an Fc domain monomer (e.g., a sequence of SEQ ID NO: 97), a wild-type Fc domain (e.g., a sequence of SEQ ID NO: 151), 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: 152), a fibronectin domain (e.g., a sequence of SEQ ID NO: 153), or human serum albumin (e.g., a sequence of SEQ ID NO: 154)) in a tandem series by means of a linker. For example, the extracellular ActRIIa 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 ActRIIa 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).

[0074] As used herein, the term "C-terminal extension" refers to the addition of one or more amino acids to a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)). The C-terminal extension can be 1-6 amino acids (e.g., 1, 2, 3, 4, 5, 6 or more amino acids). Exemplary C-terminal extensions are the amino acid sequence NP (a two amino acid C-terminal extension) and the amino acid sequence NPVTPK (SEQ ID NO: 155) (a six amino acid C-terminal extension). Any amino acid sequence that does not destroy the activity of the polypeptide can be used. SEQ ID NO: 71 (which is the sequence of SEQ ID NO: 69 with a C-terminal extension of NP) and SEQ ID NO: 72 (which is the sequence of SEQ ID NO: 69 with a C-terminal extension of NPVTPK) represent two possible ways in which a polypeptide of the invention can be modified to include a C-terminal extension.

[0075] As used herein, the term "percent (%) identity" refers to the percentage of amino acid (or nucleic acid) residues of a candidate sequence (e.g., an extracellular ActRIIa variant) that are identical to amino acid (or nucleic acid) residues of a reference sequence (e.g., wild-type extracellular ActRIIa (e.g., SEQ ID NO: 73)), after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate and reference sequences for optimal alignment, and nonhomologous sequences can be ignored for comparison purposes). Alignment for the purpose of determining percent identity can be achieved in various ways within the skill in the art, for example, using publicly available computer software such as BLAST, ALIGN or Megalign (DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm needed to achieve maximum alignment over the full length of the sequences being compared. In some embodiments, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence (which may alternatively be expressed as a given candidate sequence having or comprising a particular percent amino acid (or nucleic acid) sequence identity to, with, or against a given reference sequence) is calculated as follows: 100 x (fraction of A / B) 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.

[0076] 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 continuous 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.

[0077] As used herein, 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 in the context of administering a therapeutic protein to a subject. The protein may be redistributed or cleared from the bloodstream, or, for example, degraded by proteolysis. As described herein, polypeptides comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) exhibit a serum half-life of 7 days in humans.

[0078] As used herein, the term "metabolic disease" refers to a disease, disorder or syndrome associated with the metabolism of a subject, such as the breakdown of carbohydrates, proteins and fats in food to release energy, as well as the conversion of chemicals into other substances and their transport within cells for energy utilization and / or storage. Some symptoms of metabolic diseases include high serum triglycerides, high low-density cholesterol (LDL), low high-density cholesterol (HDL) and / or high fasting insulin levels, elevated fasting blood glucose, abdominal (central) obesity and elevated blood pressure. Metabolic diseases increase the risk of developing other diseases, such as cardiovascular disease. In the present invention, metabolic diseases include but are not limited to obesity, type 1 diabetes and type 2 diabetes.

[0079] As used herein, the term "percentage of weight gain" refers to the percentage of weight gained compared to the subject's previous weight at a previous time. The percentage of weight gain can be calculated as follows: 100X [(weight at a later time - weight at a previous time) / (weight at a previous time)] In the present invention, administering a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), or a vector containing such a nucleic acid to a subject reduces the percentage of weight gain in the subject.

[0080] As used herein, the term "appetite for food intake" refers to a subject's natural desire or need for food. The subject's appetite for food intake can be monitored by measuring the amount of food consumed after administration of a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). In the present invention, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), or a vector containing such a nucleic acid is administered to a subject without affecting the subject's appetite for food intake.

[0081] As used herein, the term "obesity" refers to fat stored in adipose tissue of a subject. In the present invention, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), or a vector containing such a nucleic acid is administered to a subject to reduce obesity in the subject without affecting lean body mass.

[0082] As used herein, the term "lean body mass" refers to a component of body composition, which includes, for example, lean body mass, body fat, and body fluid. Lean body mass is usually calculated by subtracting the weight of body fat and body fluid from total body weight. Typically, the lean body mass of a subject is between 60% and 90% of total body weight. In the present invention, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72)), or a vector containing such a nucleic acid is administered to a subject to reduce the subject's adiposity (i.e., fat) without affecting lean body mass.

[0083] As used herein, the term "epididymal and perirenal fat pads" refers to the densely packed fat cells in the epididymis and around the kidney. In the present invention, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), or a vector containing such a nucleic acid is administered to a subject to reduce the weight of the epididymal and perirenal fat pads of the subject.

[0084] As used herein, the term "fasting insulin" refers to the insulin level of a subject, while the subject does not have any food intake for a period of time (i.e., 12-24 hours). Fasting insulin levels are used to diagnose metabolic diseases. Fasting insulin levels are also used as an indicator of whether a subject is at risk of developing metabolic diseases. Typically, in subjects with type 1 diabetes, the fasting insulin level of the subject is lower than the fasting insulin level of healthy subjects. In subjects with insulin resistance (i.e., type 2 diabetes), the fasting insulin level of the subject is higher than that of healthy subjects. In the present invention, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72)) or a vector containing such a nucleic acid is administered to a subject to reduce the fasting insulin level of the subject.

[0085] As used herein, the term "glucose clearance rate" refers to the rate at which glucose is cleared from the blood. The glucose clearance rate can be measured in a glucose tolerance test (GTT). In a GTT, a subject is given a specific amount of glucose, and then a blood sample is collected to determine how quickly it is cleared from the blood. The glucose clearance rate can be used as a parameter for diagnosing and / or determining the risk of developing metabolic diseases such as obesity, diabetes, and insulin resistance.

[0086] As used herein, the term "serum lipid profile" refers to the measurement of the distribution of different types of lipids and lipoproteins in the serum of a subject. This measurement can be accomplished by a set of blood tests. The types of lipids and lipoproteins in the serum of a subject include, but are not limited to, cholesterol (e.g., high-density lipoprotein (HDL) and low-density lipoprotein (LDL)), triglycerides, and free fatty acids (FFA). The distribution of different types of lipids and lipoproteins can be used as a parameter for diagnosing and / or determining the risk of developing metabolic diseases (such as obesity, diabetes, and insulin resistance). High levels of cholesterol, especially low-density lipoprotein, are generally considered to be signs or risk factors for the development of certain metabolic diseases, or in some serious medical cases, cardiovascular disease. In the present invention, administering a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)), or a vector containing such a nucleic acid to a subject improves the subject's serum lipid profile, such that the levels of cholesterol (particularly low-density lipoprotein) and triglycerides are reduced.

[0087] As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. Typically, binding affinity refers to the strength of the sum of non-covalent interactions between a molecule and its binding partner (such as an extracellular ActRIIa 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 typically measured by the dissociation constant (K D ) or affinity constant (K A ). Two molecules with low binding affinity for each other typically associate slowly, tend to dissociate easily, and exhibit a large K D Two molecules that have a high affinity for each other generally bind easily, tend to remain bound longer, and exhibit a small K DThe 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.

[0088] As used herein, the term "muscle mass" refers to a component of body composition. Muscle mass is usually calculated by subtracting the weight of body fat and body fluid from total body weight. The percentage of muscle mass can vary greatly between individuals, depending on the subject's genetic makeup, age, race, and health status, etc. Typically, a subject's muscle mass can be 20% to 50% of total body weight.

[0089] As used herein, the phrase "affecting myostatin, activin and / or BMP9 signaling" refers to altering the binding of myostatin, activin and / or BMP9 to its receptors, e.g., ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa). In some embodiments, a polypeptide comprising an extracellular ActRIIa variant described herein reduces or inhibits the binding of myostatin, activin and / or BMP9 to its receptors, e.g., ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa). As described herein, a polypeptide of the invention comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may have a weak binding affinity for BMP9 (e.g., a K of 200 pM or more). D ).

[0090] 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.

[0091] 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.

[0092] 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 a homodimer 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 described herein comprising an extracellular ActRIIa variant fused to an Fc domain monomer can form a homodimer by the interaction of two Fc domain monomers, which form an Fc domain in a homodimer.

[0093] 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 described herein comprising an extracellular ActRIIa variant fused to an Fc domain monomer can form a heterodimer by the interaction of two Fc domain monomers (each of which is fused to a different ActRIIa variant), wherein the Fc domain monomers form an Fc domain in the heterodimer.

[0094] As used herein, the term "host cell" refers to a medium containing the necessary cellular components (e.g., organelles) required for expressing proteins from its corresponding nucleic acid. Nucleic acids are generally 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).

[0095] As used herein, the term "therapeutically effective amount" refers to the amount of a polypeptide, nucleic acid or vector of the invention, or a pharmaceutical composition containing the polypeptide, nucleic acid or vector of the invention, which is effective in achieving the desired therapeutic effect in treating a patient with a disease, such as a muscle disease, or a disease or condition involving weakness and atrophy of muscles, such as Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia. The term "therapeutically effective amount" also refers to the amount of a polypeptide, nucleic acid or vector of the invention, or a pharmaceutical composition containing the polypeptide, nucleic acid or vector of the invention, which is effective in achieving the desired therapeutic effect in treating a patient with a disease, such as a metabolic disease, or a condition involving excess weight, excess body fat, high blood glucose, high fasting insulin levels, or insulin resistance, such as obesity, type 1 diabetes, or type 2 diabetes. Specifically, a therapeutically effective amount of a polypeptide, nucleic acid or vector avoids adverse side effects.

[0096] 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 the 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.

[0097] 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 ActRIIa 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.

[0098] As used herein, the term "treating and / or preventing" refers to the use of the methods and compositions of the present invention to treat and / or prevent a disease, such as a metabolic disease (e.g., obesity, type 1 and type 2 diabetes) or a muscle disease (e.g., DMD, FSHD, IBM, and ALS). Typically, treatment of a metabolic or muscle disease occurs after the subject has developed a metabolic or muscle disease and / or has been diagnosed with a metabolic or muscle disease. Prevention of a metabolic or muscle disease refers to steps or procedures taken when a subject is at risk of developing a metabolic or muscle disease. A subject may show signs or mild symptoms that are judged by a physician to be signs or risk factors for developing a metabolic or muscle disease, or may have a family history or genetic predisposition to developing a metabolic or muscle disease, but has not yet developed the disease.

[0099] As used herein, the term "subject" refers to a mammal, such as preferably a human. Mammals include, but are not limited to, humans and domestic and farm animals, such as monkeys (e.g., cynomolgus monkeys), mice, dogs, cats, horses, and cows, etc. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a sequence alignment showing the wild-type sequences of extracellular ActRIIa and ActRIIb and the amino acid substitutions in the ActRIIa variants.

[0101] Figure 2A and 2B is a scatter plot showing the effect of extracellular ActRIIa variants on body weight. Mice received a single hydrodynamic injection of plasmid constructs encoding the indicated ActRIIa variants or a control plasmid.

[0102] Figure 3A and 3Bis a bar graph showing the effect of extracellular ActRIIa variants on muscle mass.

[0103] Figure 4A is a scatter plot showing the effect of extracellular ActRIIa variants on body weight. Mice received intraperitoneal injections of the indicated purified recombinant ActRIIa variants or vehicle control twice a week for four weeks.

[0104] Figure 4B is a bar graph showing the effect of extracellular ActRIIa variants on individual muscle weights by tissue analysis.

[0105] Figure 5A is a scatter plot showing the effect of extracellular ActRIIa variants on body weight during the course of the study. Mice received a single hydrodynamic injection of a plasmid construct encoding the indicated ActRIIa variant or a control plasmid.

[0106] Figure 5B is a bar graph showing the effect of extracellular ActRIIa variants on body weight at the end of 28 days.

[0107] Fig. 6A and 6B is a bar graph showing the effect of extracellular ActRIIa variants on body weight by tissue analysis.

[0108] Fig. 7A and 7B is a scatter plot showing the effect of different doses of extracellular ActRIIa variants on body weight. Mice received intraperitoneal injections of the indicated purified recombinant ActRIIa variants or vehicle control twice a week for four weeks.

[0109] Fig. 8A and 8B is to show the effect of different doses of extracellular ActRIIa variants on muscle mass ( Fig. 8A ) and fat mass ( Figure 8B )’s bar graph.

[0110] Fig.9A and 9B is a bar graph showing the effect of different doses of extracellular ActRIIa variants on muscle weight by tissue analysis. DETAILED DESCRIPTION The invention features polypeptides comprising extracellular activin receptor type IIa (ActRIIa) variants. In some embodiments, the polypeptides of the invention comprise an extracellular ActRIIa 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 ActRIIa variant fused to an Fc domain monomer can also form dimers (e.g., homodimers or heterodimers) through interactions between two Fc domain monomers. The ActRIIa variants described herein have weak or no binding affinity for bone morphogenetic protein 9 (BMP9) compared 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 metabolic diseases, or methods of affecting myostatin, activin and / or BMP9 signaling in a subject by administering to the subject a polypeptide comprising an extracellular ActRIIa variant described herein.

[0112] I. Extracellular activin receptor type IIa (ActRIIa) variants Activin type II receptors are single transmembrane domain receptors that regulate the signaling of ligands in the transforming growth factor β (TGF-β) superfamily. Ligands in the TGF-β superfamily are involved in many physiological processes, such as muscle growth, angiogenesis, cell differentiation, homeostasis, and osteogenesis. Examples of ligands in the TGF-β superfamily include, for example, activins, inhibins, growth differentiation factors (GDFs) (e.g., GDF8, also known as myostatin), and bone morphogenetic proteins (BMPs) (e.g., BMP9). Myostatin and activins are known to play a role in the regulation of skeletal muscle growth. For example, mice without myostatin show a substantial increase in skeletal muscle mass.

[0113] Activins are also highly expressed in adipose tissue, and increased myostatin levels and activin receptor levels have been observed in subcutaneous and visceral fat of obese mice. In addition, myostatin has been shown to be elevated in skeletal muscle and plasma of obese and insulin-resistant females, and both type I and type II activin receptors are associated with pancreatic function and diabetes. These data suggest that increased activin receptor signaling, due to increased expression of activin ligands (e.g., activin, myostatin) or increased expression of the activin receptors themselves, may lead to obesity and metabolic disorders, such as type 1 and type 2 diabetes. Therefore, methods of reducing or inhibiting this signaling may be useful in treating obesity and metabolic disorders.

[0114] There are two types of activin type II receptors: ActRIIa and ActRIIb. Studies have shown that BMP9 binds to ActRIIb with a binding affinity about 300 times higher 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 ActRIIa variant constructed by introducing the amino acid residues of ActRIIb into ActRIIa, the purpose of which is to confer the physiological properties conferred by ActRIIb while also maintaining the beneficial physiological and pharmacokinetic properties of ActRIIa. For example, the best peptide confer a significant increase in muscle mass while maintaining a longer serum half-life and a low binding affinity to BMP9. Preferred ActRIIa variants also exhibit improved binding to activin and / or myostatin compared to wild-type ActRIIa, 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 metabolic conditions, resulting in reduced body fat, body weight, or insulin resistance (e.g., increased insulin sensitivity). In some embodiments, amino acid substitutions can be introduced into the extracellular ActRIIa variants to reduce or remove the binding affinity of the variant to BMP9. The wild-type amino acid sequences of the extracellular portions of human ActRIIa and ActRIIb are shown below.

[0115] Human ActRIIa, extracellular portion (SEQ ID NO: 73): Human ActRIIb, extracellular portion (SEQ ID NO: 74):

[0116] The polypeptides described herein include extracellular ActRIIa variants having at least one amino acid substitution relative to wild-type extracellular ActRIIa having a sequence of SEQ ID NO: 73 or an extracellular ActRIIa having a sequence of any one of SEQ ID NOs: 76-96. Possible amino acid substitutions at 27 different positions can be introduced into the extracellular ActRIIa variants (Table 1). In some embodiments, the extracellular ActRIIa variants can have at least 85% (e.g., at least 85%, 87%, 90%, 92%, 95%, 97% or more) amino acid sequence identity with the sequence of wild-type extracellular ActRIIa (SEQ ID NO: 73). The extracellular ActRIIa variant can have one or more (e.g., 1-27, 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 ActRIIa (SEQ ID NO: 73). In some embodiments, the extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of SEQ ID NO: 1) can comprise amino acid substitutions at all 27 positions as listed in Table 1. In some embodiments, the extracellular ActRIIb variants can comprise amino acid substitutions at a number of positions, for example, at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26 of the 27 positions as listed in Table 1.

[0117] Amino acid substitutions may deteriorate or improve the activity and / or binding affinity of the ActRIIa variants of the invention. To maintain polypeptide function, it is important to retain position X in the sequences shown in Tables 1 and 2 (SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). 17 Substitution at this position may result in loss of activity. For example, ActRIIa variants have reduced in vivo activity, indicating that alanine (A) substitution at position X is not tolerated 17 Thus, the ActRIIa variants of the invention, including the variants in Tables 1 and 2 (e.g., SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) have a lysine (K) at position X. 17 The amino acid K was retained.

[0118] The ActRIIa variants of the present invention preferably have reduced, weak or substantially no binding to BMP9. 23 , X 24 , X 25 and X 26 and maintaining the amino acid K at position X24 and having the amino acid K at position X24. 23 , X 24 , X 25 and X 26 In variants of the amino acid sequence TKEN at TEEN, BMP9 binding is reduced. The sequences TEEN and TKEN can be used interchangeably in ActRIIa variants of the invention (e.g., variants in Tables 1 and 2, e.g., SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) to provide reduced BMP9 binding.

[0119] The ActRIIa variants of the present invention may further comprise a C-terminal extension (e.g., additional amino acids at the C-terminus). The C-terminal extension may add one to six additional amino acids (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids) at the C-terminus to any of the variants shown in Tables 1 and 2 (e.g., SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)). One potential C-terminal extension that may be included in the ActRIIa variants of the present invention is the amino acid sequence NP. For example, a sequence comprising a C-terminal extension is SEQ ID NO: 71 (e.g., SEQ ID NO: 69 with a C-terminal extension of NP). Another exemplary C-terminal extension that may be included in the ActRIIa variants of the present invention is the amino acid sequence NPVTPK (SEQ ID NO: 155). For example, a sequence comprising a C-terminal extension is SEQ ID NO: 72 (e.g., SEQ ID NO: 69 with a C-terminal extension of NPVTPK). Table 1. Amino acid substitutions in extracellular ActRIIa variants having the sequence of any one of SEQ ID NOs: 1-5

[0120] In some embodiments of the extracellular ActRIIa variant having the sequence of SEQ ID NO: 2, X3 is E, X6 is R, X 11 It is D, X 12 It is K, X 13 Yes R, X 16 Is K or R, X 17 It is K, X 19 It is W, X 20 Yes L, X21 is D, and X 22 is I or F. In some embodiments of the extracellular ActRIIa variant having the sequence of SEQ ID NO: 1 or 2, X 17 is K. In some embodiments of the extracellular ActRIIa variants having the sequence of SEQ ID NOs: 1-3, X 17 It is K, X 23 It's T, X 24 Yes E, X 25 is E, and X 26 is N. In some embodiments of the extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1-5, X 17 It is K, X 23 It's T, X 24 It is K, X 25 is E, and X 26 It is N.

[0121] In some embodiments, a polypeptide described herein comprises an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 6-72 (Table 2). Table 2. Extracellular ActRIIa variants having the sequences of SEQ ID NOs: 6-72

[0122] In some embodiments, a polypeptide of the invention comprising an extracellular ActRIIa variant (eg, any one of SEQ ID NOs: 1-72 (eg, SEQ ID NOs: 6-72)) has position X 17 Amino acid K at position X 17 The amino acids at the position of ActRIIa variants have reduced in vivo activity, suggesting that substitution of position X by A is not tolerated 17 K at.

[0123] In some embodiments, comprising a 23 , X 24 , X 25 and X 26The polypeptide of the invention having an extracellular ActRIIa variant of the sequence TEEN at position (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be substituted with amino acid K at position X 24 In some embodiments, the amino acid E at position X 23 , X 24 , X 25 and X 26 The polypeptide of the invention having an extracellular ActRIIa variant of the sequence TKEN at position (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) may be substituted with amino acid E at position X 24 The amino acid K at position X 23 , X 24 , X 25 and X 26 Peptides with the sequences TEEN or TKEN at the position of the nucleotide sequence have reduced or weak binding to BMP9.

[0124] In some embodiments, a polypeptide of the invention comprising an extracellular ActRIIa variant (e.g., any one of SEQ ID NOs: 1-70 (e.g., SEQ ID NOs: 6-70)) may further comprise a C-terminal extension (e.g., additional amino acids at the C-terminus). In some embodiments, the C-terminal extension is the amino acid sequence NP. For example, the sequence comprising the C-terminal extension is SEQ ID NO: 71 (e.g., SEQ ID NO: 69 with a C-terminal extension of NP). In some embodiments, the C-terminal extension is the amino acid sequence NPVTPK (SEQ ID NO: 155). For example, the sequence comprising the C-terminal extension is SEQ ID NO: 72 (e.g., SEQ ID NO: 69 with a C-terminal extension of NPVTPK). The C-terminal extension may add one to six additional amino acids (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) to the C-terminus.

[0125] In some embodiments, a polypeptide of the invention comprising an extracellular ActRIIa variant may further comprise a 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) fused to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRIIa variant by means of a linker or other covalent bond. A polypeptide comprising an extracellular ActRIIa 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 to form an Fc domain in a dimer.

[0126] In some embodiments, the extracellular ActRIIb variants described herein do not have the sequence of any of SEQ ID NOs: 76-96 shown in Table 3 below. Table 3. Excluded extracellular ActRIIa variants.

[0127] 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 (e.g., weak) binding to BMP9. In some embodiments, the polypeptide having reduced or weak binding to BMP9 is at position X. 23 , X 24 , X 25 and X 26 The sequence TEEN or TKEN is present at the position.

[0128] Additionally, in some embodiments, the polypeptide can be expressed at 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 more D , 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 higher 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).

[0129] II. Fc domain In some embodiments, the polypeptides described herein may include an extracellular ActRIIa 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 ActRIIa 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, which 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. An Fc domain comprises two Fc domain monomers, which are bound by a C H3 antibody constant domains and dimerize. The wild-type Fc domain forms 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)). The Kabat numbering of amino acid residues of a given antibody can be determined by aligning the homology regions of the antibody sequence with the "standard" Kabat numbering sequence. 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 ActRIIa 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 ActRIIa variant is shown below (SEQ ID NO: 97):

[0130] 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: 97. 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: 97. In some embodiments, the extracellular ActRIIa variants described herein (e.g., extracellular ActRIIa variants having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72))) can be fused to the N- or C-terminus of an Fc domain monomer (e.g., SEQ ID NO: 97) by conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIa 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 ActRIIa variant.

[0131] In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIa variant fused to an Fc domain. In some embodiments, the Fc domain contains one or more amino acid substitutions that reduce or inhibit dimerization of the Fc domain. In some embodiments, the Fc domain contains a hinge domain. The Fc domain may be an Fc domain of an immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain may be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain may also be a non-naturally occurring Fc domain, such as a recombinant Fc domain.

[0132] 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. HMethods for introducing amino acids with large or small side chains into the 3 domains are described, for example, in 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.

[0133] 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.

[0134] 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 is shown in SEQ ID NO: 151.

[0135] III. Albumin Binding Peptides In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIa variant fused to a serum protein binding peptide. Binding to a serum protein peptide may improve the pharmacokinetics of a protein drug.

[0136] 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: 152).

[0137] In the present invention, the albumin binding peptide can be linked to the N- or C-terminus (e.g., C-terminus) of the extracellular ActRIIa variant described herein (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72)) to increase the serum half-life of the extracellular ActRIIa variant. In some embodiments, the albumin binding peptide is linked directly or through a linker to the N- or C-terminus of the extracellular ActRIIa variant.

[0138] In some embodiments, the extracellular ActRIIa variants described herein (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N- or C-terminus of an albumin binding peptide (e.g., SEQ ID NO: 152) by conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIa 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 ActRIIa variants described herein can result in prolonged retention of the therapeutic protein through its binding to serum albumin.

[0139] IV. Fibronectin Domain In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIa variant fused to a fibronectin domain. Binding to the fibronectin domain may improve the pharmacokinetics of protein drugs.

[0140] The fibronectin domain is a high molecular weight glycoprotein or fragment thereof of the extracellular matrix that 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 the N- or C-terminus (e.g., C-terminus) of an extracellular ActRIIa variant described herein (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72)) to increase the serum half-life of the extracellular ActRIIa variant. The fibronectin domain can be connected directly or via a linker to the N- or C-terminus of an extracellular ActRIIa variant.

[0141] 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 (SEQ ID NO: 153) having amino acids 610-702 of the sequence of UniProt ID NO: P02751. In another embodiment, the fibronectin domain is an adnectin protein.

[0142] In some embodiments, the extracellular ActRIIa variants described herein (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N- or C-terminus of a fibronectin domain (e.g., SEQ ID NO: 153) by conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIa variant and the fibronectin domain. Without being bound by theory, it is expected that the inclusion of a fibronectin domain in the extracellular ActRIIa 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).

[0143] V. Serum albumin In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIa variant fused to serum albumin. Binding to serum albumin may improve the pharmacokinetics of protein drugs.

[0144] 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 ActRIIa variant described herein (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NO: 1-72 (e.g., SEQ ID NO: 6-72)) N- or C-terminus (e.g., C-terminus) to increase the serum half-life of the extracellular ActRIIa variant. Human serum albumin can be directly or via a linker connected to the N- or C-terminus of the extracellular ActRIIa variant.

[0145] As an example, serum albumin useful 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: 154).

[0146] In some embodiments, the extracellular ActRIIa variants described herein (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N- or C-terminus of human serum albumin (e.g., SEQ ID NO: 154) by conventional genetic or chemical means, such as chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIa variant and the human serum albumin. Without being bound by theory, it is expected that the inclusion of human serum albumin in the extracellular ActRIIa variants described herein can result in prolonged retention of the therapeutic protein.

[0147] VI. Connectors The polypeptides described herein may comprise an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) 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 moiety (e.g., an Fc domain monomer (e.g., a sequence of SEQ ID NO: 97), a wild-type Fc domain (e.g., SEQ ID NO: 151), 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: 152), a fibronectin domain (e.g., SEQ ID NO: 153, or human serum albumin (e.g., SEQ ID NO: 154)) and an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) 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:98), GGGS (SEQ ID NO:99), GGGG (SEQ ID NO:100), GGGGA (SEQ ID NO:101), GGGGS (SEQ ID NO:102), GGGGG (SEQ ID NO:103), GGAG (SEQ ID NO:104), GGSG (SEQ ID NO:105), AGGG (SEQ ID NO:106), or SGGG (SEQ ID NO:107).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: 108), GSGS (SEQ ID NO: 109), GAGAGA (SEQ ID NO: 110), GSGSGS (SEQ ID NO: 111), GAGAGAGA (SEQ ID NO: 112), GSGSGSGS (SEQ ID NO: 113), GAGAGAGAGA (SEQ ID NO: 114), GSGSGSGSGS (SEQ ID NO: 115), GAGAGAGAGAGA (SEQ ID NO: 116), and GSGSGSGSGSGS (SEQ ID NO: 117). 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: 118), GGSGGS (SEQ ID NO: 119), GGAGGAGGA (SEQ ID NO: 120), GGSGGSGGS (SEQ ID NO: 121), GGAGGAGGAGGA (SEQ ID NO: 122), and GGSGGSGGSGGS (SEQ ID NO: 123). In still other embodiments, the spacer can contain 4 to 12 amino acids, including a motif of GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), e.g., GGAG (SEQ ID NO: 104), GGSG (SEQ ID NO: 105), GGAGGGAG (SEQ ID NO: 124), GGSGGGSG (SEQ ID NO: 125), GGAGGGAGGGAG (SEQ ID NO: 126), and GGSGGGSGGGSG (SEQ ID NO: 127). In some embodiments, the spacer can contain a motif of GGGGA (SEQ ID NO: 101) or GGGGS (SEQ ID NO: 102), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 128), and GGGGSGGGGSGGGGS (SEQ ID NO: 129).In some embodiments of the invention, the linker between the moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or human serum albumin) and the extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be GGG, GGGA (SEQ ID NO: 98), GGGG (SEQ ID NO: 100), GGGAG (SEQ ID NO: 130), GGGAGG (SEQ ID NO: 131), or GGGAGGG (SEQ ID NO: 132).

[0148] In some embodiments, the spacer can also contain amino acids other than glycine, alanine and serine, such as AAAL (SEQ ID NO: 133), AAAK (SEQ ID NO: 134), AAAR (SEQ ID NO: 135), EGKSSGSGSESKST (SEQ ID NO: 136), GSAGSAAGSGEF (SEQ ID NO: 137), AEAAAKEAAAKA (SEQ ID NO: 138), KESGSVSSEQLAQFRSLD (SEQ ID NO: 139), GENLYFQSGG (SEQ ID NO: 140), SACYCELS (SEQ ID NO: 141), RSIAT (SEQ ID NO: 142), RPACKIPNDLKQKVMNH (SEQ ID NO: 143), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 144), AAANSSIDLISVPVDSR (SEQ ID NO: 145), In some embodiments, the spacer may contain a motif, such as multiple or repeated motifs, of EAAAK (SEQ ID NO: 147). 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: 148).

[0149] 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.

[0150] VII. Vectors, Host Cells and Protein Production 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.

[0151] Nucleic acid vector construction and host cells 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 ActRIIa to include a specific amino acid substitution. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR technology.

[0152] 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.

[0153] 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 ( 31,446), Escherichia coli λ1776( 31,537, Escherichia coli BL21(DE3) ( BAA-1025), and Escherichia coli RV308 ( 31,608). Different host cells have characteristics and specific mechanisms for post-transcriptional processing and 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 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 (eds.) Recombinant Gene Expression: Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd edition 2004 and Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins: Methods and Protocols (Methods in Molecular Biology) Humana Press; 2nd edition 2012.

[0154] Protein production, recovery and purification The host cells used to produce the polypeptides of the present invention can be grown in a medium known in the art and suitable for culturing the selected host cells. Examples of suitable culture media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Expi293 TM Expression medium, DMEM and RPMI-1640 supplemented with fetal bovine serum (FBS). Examples of suitable culture media 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, depending 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.

[0155] 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.

[0156] In other embodiments, the host cells can be destroyed, for example, by osmotic shock, ultrasonic treatment or lysis to destroy the host cells 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 six-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, hemagglutinin "HA" tags, which correspond to epitopes derived from influenza hemagglutinin proteins (Wilson et al., Cell 37:767, 1984).

[0157] 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 the polypeptides 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.

[0158] VIII. Pharmaceutical Compositions and Formulations The invention features a pharmaceutical composition comprising a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)). In some embodiments, a pharmaceutical composition of the invention comprises a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) having a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) as a therapeutic protein. In some embodiments, the pharmaceutical composition of the present invention comprises a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) 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, the pharmaceutical composition of the present invention comprising a polypeptide of the present invention can be used in combination with other agents (e.g., therapeutic biologics and / or small molecules) or compositions for therapy. In addition to a therapeutically effective amount of the polypeptide, the pharmaceutical composition may also comprise 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 comprises 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.

[0159] 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 edition) Taylor & Francis Group, CRC Press (2015).

[0160] 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.

[0161] 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.

[0162] 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).

[0163] 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 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 intravenously and rapidly 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.

[0164] IX. Routes, Dosages, and Administration 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).

[0165] In some embodiments, a 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 the polypeptide of the present invention can be directly administered to a subject.

[0166] 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.

[0167] 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.

[0168] 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.

[0169] X. Treatment Methods The present invention is based on the discovery that amino acids from the extracellular portion of ActRIIb are substituted into the extracellular portion of ActRIIa to produce ActRIIa variants with improved properties. ActRIIa variants generated by introducing residues from ActRIIb into ActRIIa retain the beneficial properties of ActRIIa, such as longer serum half-life and low binding affinity to BMP9, and gain some of the beneficial properties of ActRIIb, such as increased binding to activin A and B (see Table 4) and the ability to increase muscle mass (see Examples 1-3 and 5-6). These ActRIIa variant properties produce useful therapeutic agents that can compete with endogenous activin receptors for ligand binding. Because ActRIIa variants contain the extracellular portion of the receptor, they will be soluble and able to bind and sequester ligands (e.g., activin A and B, myostatin, GDF11) without activating intracellular signaling pathways. Thus, extracellular ActRIIa variants can be used to treat diseases or conditions in which elevated activin signaling has been implicated (e.g., associated with increased expression of an activin receptor or an activin receptor ligand). For example, loss of myostatin has been shown to increase skeletal muscle mass, suggesting that myostatin inhibits skeletal muscle growth. Therefore, treatment with therapeutic agents that bind myostatin and reduce its interaction with endogenous receptors may be a viable approach to increase muscle mass. Indeed, the extracellular ActRIIa variants of the present invention increase muscle mass in mice (see Examples 1-3 and 5-6). These data suggest that the extracellular ActRIIa variants described herein can be used to increase muscle mass and treat subjects with diseases or conditions that result in weakness or atrophy of muscle.

[0170] Moreover, these data provide a compelling reason to use the extracellular ActRIIa variants of the invention to treat other diseases or conditions associated with elevated expression of activin receptors or activin receptor ligands, such as metabolic diseases (e.g., obesity, type 1 diabetes, and type 2 diabetes). Many studies have shown that increasing muscle mass is a method of reducing body fat and / or body weight, suggesting that the extracellular ActRIIa variants described herein may be used to treat metabolic diseases (e.g., obesity, type 1 diabetes, and type 2 diabetes) indirectly by increasing muscle mass. However, since activin receptors and activin receptor ligands have been shown to be increased in obese mice and humans, the extracellular ActRIIa variants described herein may be used to treat obesity by reducing elevated activin receptor signaling (e.g., by binding and sequestering endogenous activin receptor ligands, e.g., activin and myostatin).

[0171] The present invention provides compositions and methods of treating muscle mass and strength that can be used to increase subjects in need thereof. In some embodiments, the subject may have a disease that causes 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 in subjects with diseases or conditions involving muscle weakness and atrophy. In some embodiments, a polypeptide comprising an extracellular ActRIIa variant as described herein reduces or inhibits myostatin, activin, and / or BMP9 binding to its receptors, such as ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa). In some embodiments, affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting myostatin, activin, and / or BMP9 binding to its receptors, such as ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa)) results in an increase in muscle mass in the subject.

[0172] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72))) can be administered to a subject to increase muscle mass, or to affect myostatin, activin, and / or BMP9 signaling in the subject. In some embodiments, the methods described herein increase bone mineral density in the subject. 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 involving weakness and atrophy of muscle (e.g., Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia).

[0173] The present invention also includes methods of treating a subject having Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia by administering to the subject a polypeptide described herein, e.g., a polypeptide comprising an extracellular ActRIIa variant, e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72).

[0174] The compositions and methods described herein can also be used to treat and / or prevent medical conditions, such as metabolic diseases, e.g., obesity and diabetes (type 1 and type 2 diabetes). In some embodiments, the subject may have a disease that causes obesity. In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling in subjects with obesity, diabetes (type 1 and type 2 diabetes), or a disease or condition that causes obesity. In some embodiments, polypeptides comprising extracellular ActRIIa variants described herein reduce or inhibit the binding of myostatin, activin, and / or BMP9 to its receptors, e.g., ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa). In some embodiments, affecting myostatin, activin and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin and / or BMP9 to their receptors, e.g., ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa)) results in a decrease in body fat (e.g., body fat mass or body fat percentage) in the subject, a decrease in body weight or weight gain in the subject, a decrease in fasting insulin levels, an increase in glucose clearance, or an increase in insulin sensitivity (e.g., a decrease in insulin resistance).

[0175] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72))) can be administered to a subject to prevent the development of obesity (e.g., in a patient at risk of developing obesity, e.g., an overweight patient, a patient with a family history of obesity, or a patient with other medical conditions or genetic risk factors associated with an increased risk of obesity), and / or to treat a patient already diagnosed with obesity. For example, administering an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) to a subject can help reduce the subject's weight by reducing fat mass. In some embodiments, the extracellular ActRIIa variant reduces fat mass while maintaining or increasing lean body mass.

[0176] In some embodiments, polypeptides described herein (e.g., polypeptides comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72))) can be used to prevent the development of diabetes (e.g., type 1 and type 2 diabetes) and / or treat patients already diagnosed with diabetes. Polypeptides described herein (e.g., polypeptides comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72))) can be prophylactically administered to patients who may develop diabetes, such as individuals with a genetic predisposition to diabetes, a family history, prediabetes, association with other autoimmune diseases or other metabolic diseases, such that the extracellular ActRIIa polypeptide can maintain the normal function and health of β-cells and prevent or delay autoimmune inflammatory damage to β-cells. In other embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72))) can be administered to an individual before the individual is diagnosed with diabetes (e.g., type 1 and type 2 diabetes) or develops clinical symptoms of diabetes, such as high blood glucose levels, high fasting insulin levels, insulin resistance, polyuria, polydipsia, and polyphagia. In some embodiments, an extracellular ActRIIa polypeptide can be administered to a patient before the patient requires insulin. In still other embodiments, administration of an extracellular ActRIIa polypeptide can delay or postpone the need for insulin therapy in a diabetic patient. For example, administration of an extracellular ActRIIa polypeptide of the invention to a subject can help increase the rate of glucose clearance from the blood.

[0177] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72))) can be administered to a subject to prevent the development of obesity or diabetes (e.g., type 1 and type 2 diabetes) and / or treat a patient with obesity or diabetes (e.g., type 1 and type 2 diabetes), or to affect myostatin, activin, and / or BMP9 signaling in a subject (e.g., reducing or inhibiting the binding of activin, myostatin, and / or BMP9 to its receptor). In some embodiments, the methods described herein reduce body fat (e.g., reducing the amount of subcutaneous and / or visceral fat, reducing obesity, reducing the weight of epididymal and perirenal fat pads, or reducing body fat percentage). In some embodiments, the methods described herein reduce body weight or reduce weight gain (e.g., reducing the percentage of weight gain). In some embodiments, the methods described herein reduce the proliferation of adipocytes. In some embodiments, the methods described herein reduce LDL. In some embodiments, the methods described herein reduce triglycerides. In some embodiments, the methods described herein improve the serum lipid profile of the subject. In some embodiments, the methods described herein reduce body fat without reducing lean body mass (e.g., not affecting lean body mass or increasing lean body mass). In some embodiments, the methods described herein reduce body fat and increase muscle mass. In some embodiments, the methods described herein reduce blood glucose levels (e.g., fasting glucose levels) or / and increase glucose clearance. In some embodiments, the methods described herein reduce fasting insulin levels and / or improve insulin sensitivity (e.g., reduce insulin resistance). In some embodiments, the methods described herein regulate insulin biosynthesis and / or secretion from β-cells. In some embodiments, the methods described herein do not affect the appetite of food intake. In some embodiments, the methods described herein do not cause any vascular complications in the subject, such as increased vascular permeability or leakage.

[0178] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72))) reduces body fat, reduces body weight, or increases insulin sensitivity and / or glucose clearance by increasing muscle mass.

[0179] In any of the methods described herein, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-71 (e.g., SEQ ID NOs: 6-71)) further comprising a C-terminal extension of one to six amino acids (e.g., 1, 2, 3, 4, 5, 6, or more amino acids) can be used as a therapeutic protein. In any of the methods described herein, a dimer (e.g., a homodimer or heterodimer) of a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having a sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a 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) 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

[0180] Example 1 - Effects of extracellular ActRIIa variants on body weight C57B1 / 6 mice received a single hydrodynamic injection of a plasmid construct encoding one of the following six polypeptides (n=10 / group): (1) human Fc (hFc), (2) extracellular ActRIIa (SEQ ID NO: 73) fused to the N-terminus of hFc via a GGG linker; (3) extracellular ActRIIb (SEQ ID NO: 74) fused to the N-terminus of hFc via a GGG linker; (4) an extracellular ActRIIa variant fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 69); and (5) Extracellular ActRIIb variant fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 149).

[0181] 100 μg of plasmid construct was delivered in a volume of 10% body weight over 5-8 seconds. The high volume and short duration injection provides the pressure required to introduce the plasmid into the hepatocytes where the plasmid will be expressed (especially the target protein is expressed under a strong and ubiquitous promoter). The target protein is secreted and circulates freely under the endogenous mechanism of the hepatocytes. The mice were weighed twice a week for 30 days, and the measurements were recorded as absolute body weight (BW) in grams and the percentage change in body weight from the baseline measurement (respectively). Figure 2A and 2B ).

[0182] Example 2 - Effects of extracellular ActRIIa variants on muscle mass Mice received a single hydrodynamic injection of a plasmid construct encoding one of the following six polypeptides (n=10 / group): (1) human Fc (hFc), (2) extracellular ActRIIa (SEQ ID NO: 73) fused to the N-terminus of hFc via a GGG linker; (3) extracellular ActRIIb (SEQ ID NO: 74) fused to the N-terminus of hFc via a GGG linker; (4) an extracellular ActRIIa variant fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 69); and (5) Extracellular ActRIIb variant fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 149).

[0183] 100 μg of plasmid construct was delivered in a volume of 10% body weight over 5-8 seconds. High volume and short period injection provides the pressure required for introducing plasmid into hepatocytes where the plasmid will be expressed (especially the target protein is expressed under a strong and ubiquitous promoter). The target protein is secreted and circulates freely under the endogenous mechanism of the hepatocyte. On study day 0 (baseline), day 14 and day 28, mice were subjected to NMR analysis to determine lean body mass using a MiniSpec LF90 NMR analyzer (Bruker, Woodlands, TX). The percent change in lean body mass from baseline was recorded on day 14 and day 28 ( Figure 3A and 3B ).

[0184] Example 3 - Effect of extracellular ActRIIa variants on body weight when administered as purified recombinant proteins Female C57Bl / 6 mice (Taconic Biosciences, Hudson NY) received intraperitoneal injections of tris-buffered saline vehicle or one of the following five purified recombinant polypeptides at a dose of 10 mg / kg twice a week for four weeks (n=10 / group): (1) tris-buffered saline vehicle, (2) extracellular ActRIIa (SEQ ID NO: 73) fused to the N-terminus of hFc via a GGG linker; (3) extracellular ActRIIb (SEQ ID NO: 74) fused to the N-terminus of hFc via a GGG linker; (4) an extracellular ActRIIa / b variant fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 69); (5) an extracellular ActRIIa / bΔ9 variant fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 58); and (6) Extracellular ActRII a / bΔ9min variant fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 6).

[0185] Purified recombinant protein was prepared by transient expression in HEK293 cells and purified from conditioned media using Protein-A Sepharose chromatography.

[0186] After four weeks of dosing, mice were humanely sacrificed and autopsied. The autopsy included collection of the weight of the whole body as well as the gastrocnemius, pectoralis, and quadriceps muscles. Statistical analysis of muscle / body weight data was performed in GraphPad Prism 7 (GraphPad Software, La Jolla CA) (respectively Figure 4A and 4B ).

[0187] Example 4 - Evaluation of ActRIIa variant binding affinity by surface plasmon resonance (SPR) Biacore3000 was used to measure the kinetics of the interaction between ActRIIa variants and ligands activin A, activin B, growth differentiation factor 11 (GDF11) and BMP-9. ActRIIa variants were expressed and purified according to the methods described in Example 3. ActRIIa variants were immobilized on the chip (CM4 or CM5) in flow chambers 2-4 with capture antibodies (anti-mouse from GEGE) to ensure correct orientation. Flow chamber 1 was used as a reference chamber to subtract any nonspecific binding and volume effects. From GE HealthcareTM HBS-EP+ buffer was used as running buffer. Each ligand was run in a concentration series at 40 μl / min to avoid mass transfer effects. TM The Scrubber2 software analyzed the data to calculate the K for each interaction. D (Table 4). Table 4: Binding affinity (K) of ActRIIa variants to various ligands D ) *Not done in HDI, but recombinant protein showed similar BW results to ActRIIa / b

[0188] Example 5 - Effects of extracellular ActRIIa variants on body and muscle weight C57B1 / 6 mice received a single hydrodynamic injection of a plasmid construct encoding one of the following twelve polypeptides (n=10 / group): (1) Vehicle; (2) μLEV113-ActRIIa(19-127) fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 73); (3) pLEV113 - ActRIIb (41-155) fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 74); (4) pLEV113—ActRIIa / b fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 69); (5) pLEV113—ActRIIb / a fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 149); (6) pLEV113—ActRIIa / b+ fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 150); (7) pLEV113—ActRIIa / b-delta 9m2 (SEQ ID NO: 38) fused to the N-terminus of hFc via a GGG linker; (8) pLEV113—ActRIIa / b-delta 9m3 (SEQ ID NO: 41) fused to the N-terminus of hFc via a GGG linker; (9) pLEV113—ActRIIa / b-delta 9m4 (SEQ ID NO: 44) fused to the N-terminus of hFc via a GGG linker; (10) pLEV113—ActRIIa / bmax1 fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 70); (11) pLEV113—ActRIIa / bmax2 (SEQ ID NO: 71) fused to the N-terminus of hFc via a GGG linker; and (12) pLEV113—ActRIIa / bmax1 (SEQ ID NO: 72) fused to the N-terminus of hFc via a GGG linker.

[0189] 100 μg of plasmid construct was delivered in a volume of 10% body weight over 5-8 seconds. The high volume and short duration injection provides the pressure required to introduce the plasmid into the hepatocytes where the plasmid will be expressed (especially the target protein is expressed under a strong and ubiquitous promoter). The target protein is secreted and circulates freely under the endogenous mechanism of the hepatocytes. The mice were weighed twice a week for 30 days, and the measurements were recorded as absolute body weight (BW) in grams and the percentage change in body weight from the baseline measurement (respectively). Figure 5A and 5B The muscles were also weighed at the end of the study and the measurements were recorded in grams ( Fig. 6A and 6B ).

[0190] Example 6-Dose effect of extracellular ActRIIa variants on body weight, muscle weight and muscle mass 8-week-old male C57BL / 6 mice were weight-matched into 9 groups (n=10 / group). Each group was administered 5 mL / kg of vehicle (Tris-buffered saline, pH 7.4) or one of 4 concentrations of ActRIIA / B-Fc (SEQ ID NO: 69 fused to the N-terminus of hFc via a GGG linker) or ActRllA / BΔ9-Fc (SEQ ID NO: 58 fused to the N-terminus of hFc via a GGG linker). The doses evaluated were 20 mg / kg, 8 mg / kg, 3 mg / kg and 1 mg / kg. Treatment was administered intraperitoneally (IP) twice a week for 4 weeks (8 doses), and the study was terminated on day 28 of the study. Body weight ( Fig. 7A and 7B ), and at study termination, each group underwent NMR imaging for lean and fat mass analysis ( Fig. 8A and 8B ), and the pectoralis and gastrocnemius muscle weights were collected and weighed ( Fig.9A and 9B ).

[0191] Example 7 - Effects of extracellular ActRIIa variants on obesity Adult male C57BL / 6 mice were assigned to weight-matched treatment groups (n=10 / group). All animals were maintained on a regular diet (Chow; Purina LabDiet 5001; St. Louis, MO) or a high-fat diet (HFD; Research DietsD12331; New Brunswick, NJ). The Chow- and HFD-fed groups were further divided into groups that were dosed twice a week with ActR11 variants or vehicles for a period of 60 days. Body weight was measured twice a week during treatment. Body composition was measured using MiniSpec LF50 at baseline (before administration of treatment and transfer to HFD), and then every other week until the end of the study. At the study termination date, target tissues (serum, plasma, muscle and fat depots) were surgically removed and weighed. Serum samples were subsequently evaluated for biomarkers of obesity, and plasma was evaluated for Hba1c levels.

[0192] Other Implementations 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.

[0193] 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.

[0194] Other implementations are within the following claims.

Claims

1. A polypeptide comprising an extracellular activin receptor type IIa (ActRIIa) variant having the following sequence: GAILGRSETQECLX1X2NANWX3X4X5X6TNQTGVEX7CX8GX9X 10 X 11 X 12 X 13 X 14 HCX 15 ATWX 16 NISGSIEIVX 17 X 18 GCX19X 20 X 21 DX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS(SEQ ID NO: 11, Where X1 is F or Y; X2 is F or Y; X3 is E or A; X4 is K or L; X5 is D or E; X6 is R or A; X7 is P or R; X8 is Y or E; X9 is D or E; X 10 It is K or Q; X 11 It is D or A; X 12 It is K or A; X 13 is R or A; X 14 is R or L; X 15 is F or Y; X 16 is K, R or A; X 17 is K, A, Y, F or I; X 18 is q or K; X 19 W or A; X 20 L or A; X 21 is D, K, R, A, F, G, M, N, or I; X 22 is I, F or A; X 23 is K or T; X 24 Is K or E; X 25 Is D or E; X 26 is S or N; and X 27 is E or Q, and wherein the variant has at least one amino acid substitution relative to wild-type extracellular ActRIIa having the sequence of SEQ ID NO: 73 or extracellular ActRIIa having any one of the sequences of SEQ ID NOs: 76-96.

2. A nucleic acid molecule encoding the polypeptide of claim 1.

3. A vector comprising the nucleic acid molecule of claim 2.

4. A host cell expressing the polypeptide of claim 1, wherein the host cell comprises the nucleic acid molecule of claim 2 or the vector of claim 3, wherein the nucleic acid molecule or the vector is expressed in the host cell.

5. A method for preparing the polypeptide of claim 1, wherein the method comprises: a) providing a host cell comprising the nucleic acid molecule of claim 2 or the vector of claim 3, and b) expressing said nucleic acid molecule or vector in said host cell under conditions which allow the formation of the polypeptide.

6. A pharmaceutical composition comprising the polypeptide of claim 1, the nucleic acid molecule of claim 2, or the vector of claim 3, and one or more pharmaceutically acceptable carriers or excipients.

7. A method of increasing muscle mass in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the polypeptide of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, or the pharmaceutical composition of claim 6.

8. A method of affecting myostatin, activin and / or BMP9 signaling in a subject having a disease or condition involving weakness and atrophy of muscle, comprising administering to the subject a therapeutically effective amount of the polypeptide of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, or the pharmaceutical composition of claim 6.

9. A method of treating a subject having Duchenne muscular dystrophy, comprising administering to the subject a therapeutically effective amount of the polypeptide of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, or the pharmaceutical composition of claim 6.

10. A method of treating a subject having facioscapulohumeral muscular dystrophy, comprising administering to the subject a therapeutically effective amount of the polypeptide of claim 1, the nucleic acid molecule of claim 2, the vector of claim 3, or the pharmaceutical composition of claim 6.

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