Activin type IIa receptor mutant and pharmaceutical composition containing the mutant

Abstract

To provide extracellular activin receptor type IIa (ActRIIa) variants, and to provide pharmaceutical compositions containing the variants.SOLUTION: A polypeptide including an extracellular ActRIIa variant includes an extracellular ActRIIa variant fused to an Fc domain monomer or moiety. A pharmaceutical composition containing the variant is used to treat diseases and conditions involving muscle weakness and muscle atrophy (for example, Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia, or cancer cachexia), or metabolic diseases (for example, obesity, Type-1 diabetes, or Type-2 diabetes).SELECTED DRAWING: Figure 2A

Description

[Technical Field]

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

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

[0003] Excess weight is a growing problem in the United States, affecting approximately 25% of the population. Excessive weight leads to the dysfunction of various organs, including obesity, diabetes (e.g., type 1 and type 2 diabetes), cardiovascular disease, and some forms of cancer. Insulin resistance, also associated with obesity, occurs when pancreatic tissue requires high amounts of insulin. When pancreatic beta cells can no longer produce enough insulin to meet this demand, hyperglycemia occurs, leading to the development of type 2 diabetes. Adipocytes, which increase in number with obesity, are thought to play a role in this process. Despite the prevalence of obesity and metabolic diseases, such as diabetes (e.g., type 1 and type 2 diabetes) and insulin resistance, few treatment options are available. Summary of the Invention [Problem to be solved by the invention]

[0004] New treatments for these muscle and metabolic diseases are needed. [Means for solving the problem]

[0005] The present invention relates to polypeptides comprising extracellular activin receptor type IIa (ActRIIa) variants. In some embodiments, the polypeptides of the present invention comprise an extracellular ActRIIa variant fused to the N- or C-terminus of an Fc domain monomer or moiety. Such moieties may be attached by amino acids or other covalent bonds and increase the stability of the polypeptide. Polypeptides comprising an extracellular ActRIIa variant fused to an Fc domain monomer can also form dimers (e.g., homodimers or heterodimers) through interaction between two Fc domain monomers. The polypeptides of the present invention can be used to increase muscle mass and strength in subjects with diseases or conditions involving muscle weakness and atrophy, such as Duchenne muscular dystrophy (DMD), facioscapulohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia. The polypeptides of the present invention can also be used to reduce weight, reduce body fat, increase glucose clearance, improve insulin sensitivity, or lower fasting insulin levels in subjects who have or are at risk of developing a metabolic disease (e.g., obesity, type 1 diabetes, or type 2 diabetes). Additionally, the polypeptides of the present invention can also be used to affect myostatin, activin, and / or bone morphogenetic protein 9 (BMP9) signaling in subjects who have or are at risk of developing a disease or condition associated with muscle weakness and muscle wasting, or a metabolic disease.

[0006] In one aspect, the present invention provides a polypeptide comprising an extracellular activin type IIa receptor (ActRIIa) variant, said variant comprising: GAILGRSETQECLX1X2NANWX3X4X5X6TNQTGVEX7CX8GX9X 10 X 11 X 12 X 13 X 14 HCX 15 ATWX 16 NISGSIEIVX 17 X 18 GCX 19 X 20 X 21 DX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 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 is K or Q;X 11 is D or A;X 12 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 is W or A;X 20 is 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 X27 is E or Q, and the mutant has at least one amino acid substitution relative to a wild-type extracellular ActRIIa having the sequence of SEQ ID NO: 73 or an extracellular ActRIIa having any one of the sequences of SEQ ID NOs: 76 to 96. Concerning polypeptides.

[0007] In some embodiments, the variant is GAILGRSETQECLFX2NANWX3X4X5X6TNQTGVEX7CX8GX9KX 11 X 12 X 13 X 14 HCX 15 ATWX 16 NISGSIEIVX 17 X 18 GCX 19 X 20 X 21 DX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 2) 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 is defined in the same way as above.

[0008] In some embodiments, the variant is: GAILGRSETQECLFX2NANWEX4X5RTNQTGVEX7CX8GX9KDKRX14 HCX 15 ATWX 16 NISGSIEIVKX 18 GCWLDDX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 3) wherein 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 is defined as above.

[0009] In some embodiments, the variant is: GAILGRSETQECLFX2NANWEX4DRTNQTGVEX7CX8GX9KDKRX 14 HCX 15 ATWX 16 NISGSIEIVKX 18 GCWLDDX 22 NCYDRTDCVEX 23 KX 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 4) wherein X2, X4, X7, X8, X9, X 14 , X 15 , X 16 , X 18 , X 22 , X 23 , X 25 , X 26 , and X 27 is defined as above.

[0010] In some embodiments, the variant is: GAILGRSETQECLFX2NANWEX4DRTNQTGVEPCX8GX9KDKRX 14 HCFATWKNISGSIEIVKX 18 GCWLDDINCYDRTDCVEX 23 KX 25 X 26 PX 27 VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 5) wherein X2, X4, X8, X9, X 14 , X 18 , X 23 , X 25 , X 26 , and X 27 is defined as above.

[0011] In any of the above embodiments, X1 is F or Y. In any of the above embodiments, X2 is F or Y. In any of the above embodiments, X3 is E or A. In any of the above embodiments, X4 is K or L. In any of the above embodiments, X5 is D or E. In any of the above embodiments, X6 is R or A. In any of the above embodiments, X7 is P or R. In any of the above embodiments, X8 is Y or E. In any of the above embodiments, X9 is D or E. In any of the above embodiments, X 10 is K or Q. In any of the above embodiments, X 11 is D or A. In any of the above embodiments, X 12 is K or A. In any of the above embodiments, X 13 is R or A. In any of the above embodiments, X 14 is R or L. In any of the above embodiments, X 15 is F or Y. In any of the above embodiments, X 16 is K, R, or A. In any of the above embodiments, X 17 is K, A, Y, F, or I. In any of the above embodiments, X18 is Q or K. In any of the above embodiments, X 19 is W or A. In any of the above embodiments, X 20 is L or A. In any of the above embodiments, X 21 is D, K, R, A, F, G, M, N, or I. In any of the above embodiments, X 22 is I, F, or A. In any of the above embodiments, X 23 is K or T. In any of the above embodiments, X 24 is K or E. In any of the above embodiments, X 25 is D or E. In any of the above embodiments, X 26 is S or N. In any of the above embodiments, X 27 is E or Q. In any of the above embodiments, X 23 is T and X 24 is E and X 25 is E and X 26 is N. In any of the above embodiments, X 23 is T and X 24 is K and X 25 is E and X 26 is N. In any of the above embodiments, X 17 is K.

[0012] In any of the above embodiments, the mutant has the sequence of any one of SEQ ID NOs: 6-72. In any of the above embodiments, position X 24 The amino acid can be substituted with the amino acid K.

[0013] In any of the above embodiments, position X 24 can be substituted with the amino acid E. In any of the above embodiments, the polypeptides described herein may 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).

[0014] In any of the above-described embodiments, the polypeptides described herein may further comprise a moiety fused or covalently bound to the C-terminus of the polypeptide. In some embodiments, the moiety increases the stability 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.

[0015] In any of the above-described embodiments, the polypeptide described herein may further comprise an Fc domain monomer fused to the C-terminus of the polypeptide via 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 heterodimer) through interaction between two Fc domain monomers. In some embodiments, the Fc domain monomer has the sequence of SEQ ID NO:97.

[0016] In any of the above-described embodiments, the polypeptide described herein may further comprise an Fc domain fused to the C-terminus of the polypeptide via a linker. In some embodiments, the Fc domain is a wild-type Fc domain. In some embodiments, the wild-type Fc domain has the sequence of SEQ ID NO: 151. In some embodiments, the Fc domain comprises one or more amino acid substitutions. In some embodiments, the Fc domain comprising one or more amino acid substitutions does not form a dimer.

[0017] In any of the above embodiments, the polypeptides described herein may further comprise an albumin-binding peptide fused to the C-terminus of the polypeptide via a linker. In some embodiments, the albumin-binding peptide has the sequence of SEQ ID NO: 152.

[0018] In any of the above embodiments, the polypeptides described herein may further comprise a fibronectin domain fused to the C-terminus of the polypeptide via a linker. In some embodiments, the fibronectin domain peptide has the sequence of SEQ ID NO: 153.

[0019] In any of the above embodiments, the polypeptides described herein may further comprise human serum albumin fused to the C-terminus of the polypeptide via a linker. In some embodiments, the human serum albumin has the sequence of SEQ ID NO: 154.

[0020] In some embodiments, the linker is an amino acid spacer, hi 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).

[0021] In some embodiments, the amino acid spacer is selected from the group consisting of 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), G AGAGAGAGA (SEQ ID NO: 114), GSGSGSGSGS (SEQ ID NO: 115), GAGAGAGAGA (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), GGGAGGGGAGGGGA (SEQ ID NO: 128), GGGGGSGGGGSGGGGS (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 ID NO: 142), RPACKIPNDLKQKVMNH (SEQ ID NO: 143), GGSAGGSGSGSSGGSSGASGTGTAGTGSGSGTGSG (SEQ ID NO: 144), AAANSSIDLISVPVDSR (SEQ ID NO: 145), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 146), EAAAK (SEQ ID NO: 147), or PAPAP (SEQ ID NO: 148).

[0022] In any of the above embodiments, the polypeptides described herein have a serum half-life of at least 7 days. In any of the above embodiments, the polypeptides described herein have a K D In some embodiments, the polypeptide binds to human bone morphogenetic protein 9 (BMP9) at a specific binding site. In some embodiments, the polypeptide binds to activin and / or myostatin, and exhibits low (e.g., weak) binding to human BMP9. In some embodiments, the polypeptide does not substantially bind to human BMP9.

[0023] In any of the above embodiments, the polypeptides described herein have a K D It binds to human activin A at In any of the above embodiments, the polypeptides described herein have a K D It binds to human activin B at

[0024] In any of the above embodiments, the polypeptides described herein have a K D It binds to human GDF-11. In another aspect, the present invention relates to a nucleic acid molecule encoding a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa mutant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)). In another aspect, the present invention also relates to a vector comprising the nucleic acid molecule described herein.

[0025] In another aspect, the invention relates to a host cell expressing a polypeptide as described herein, which host cell comprises a nucleic acid molecule or vector as described in the previous two aspects, which nucleic acid molecule or vector is expressed in the host cell.

[0026] In another aspect, the invention relates to a method of making a polypeptide as described herein, the method comprising: a) providing a host cell comprising a nucleic acid molecule or vector as described herein; and b) expressing the nucleic acid molecule or vector in the host cell under conditions that allow the formation of the polypeptide.

[0027] In another aspect, the invention relates to a pharmaceutical composition comprising a polypeptide, nucleic acid molecule, or vector described herein and one or more pharmaceutically acceptable carriers or excipients, in some embodiments of the pharmaceutical composition, the polypeptide, nucleic acid molecule, or vector is in a therapeutically effective amount.

[0028] In another aspect, the present invention also relates to a construct (e.g., a homodimer) comprising two identical polypeptides, each comprising an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72) fused to the N-terminus or C-terminus of an Fc domain monomer (e.g., the sequence of SEQ ID NOS: 97). The two Fc domain monomers within these two polypeptides interact to form an Fc domain within the construct.

[0029] In another aspect, the present invention also relates to a construct comprising two different polypeptides (e.g., heterodimers) each comprising an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72), fused to the N-terminus or C-terminus of an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97). The two Fc domain monomers in the two polypeptides interact to form an Fc domain in the construct.

[0030] In another aspect, the present invention relates to a method for increasing muscle mass in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a polypeptide, nucleic acid molecule, or vector described herein, or a pharmaceutical composition described herein.

[0031] In some embodiments of the methods 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.

[0032] In another aspect, the invention relates to a method of affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting binding of myostatin, activin, and / or BMP9 to their receptors) in a subject having a disease or condition involving muscle weakness and muscle wasting, 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.

[0033] In another aspect, the invention relates to a method of treating a subject with 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.

[0034] In another aspect, the invention relates to 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.

[0035] In another aspect, the invention relates to 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.

[0036] In another aspect, the invention relates to a method of treating a subject with 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.

[0037] In another aspect, the present invention relates to a method for 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.

[0038] In another aspect, the present invention relates to a method for reducing 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.

[0039] In another aspect, the present invention relates to a method of lowering 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.

[0040] In another aspect, the present invention relates to 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.

[0041] In some embodiments of any of the above aspects, the subject has or is at risk of developing a metabolic disease. In some embodiments, the metabolic disease is selected from the group including obesity, type 1 diabetes, and type 2 diabetes.

[0042] In another aspect, the present invention relates to a method of affecting myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of myostatin, activin, and / or BMP9 to their receptors) in a subject having 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.

[0043] In another aspect, the present invention relates to a method for treating and / or preventing a metabolic disorder 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.

[0044] In some embodiments of any of the above aspects, the metabolic disease is selected from the group comprising 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.

[0045] In some embodiments of any of the above aspects, the method reduces the subject's body weight and / or rate of weight gain. In some embodiments of any of the above aspects, the method reduces the subject's amount of body fat and / or 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 obesity in the subject. In some embodiments of any of the above aspects, the method reduces the subject's epididymal and perirenal fat pad weight. In some embodiments of any of the above aspects, the method reduces the subject's amount of subcutaneous fat and / or visceral fat. 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 rate. 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 mass.

[0046] In some embodiments of any of the above aspects, the method increases muscle mass. In some embodiments of any of the above aspects, the method reduces or inhibits binding of activin and / or myostatin to their receptors.

[0047] In some embodiments of any of the above aspects, the polypeptide, nucleic acid, vector, or pharmaceutical composition is administered in an amount sufficient to increase muscle mass and / or strength, to affect myostatin, activin, and / or BMP9 signaling in a subject, or to reduce or inhibit binding of activin and / or myostatin to their receptors.

[0048] In some embodiments of any of the above aspects, the polypeptide, nucleic acid, vector, or pharmaceutical composition is administered in an amount sufficient to reduce body fat, reduce the amount of subcutaneous fat, reduce the amount of visceral fat, reduce obesity, reduce the weight of epididymal and perirenal fat pads, reduce percent body fat, reduce body weight, reduce the rate of weight gain, lower fasting insulin levels, lower blood glucose levels, increase insulin sensitivity, affect myostatin, activin, and / or BMP9 signaling in a subject, reduce adipocyte proliferation, reduce or inhibit the binding of activin and / or myostatin to their receptors, reduce LDL, reduce triglycerides, improve the serum lipid profile, modulate insulin biosynthesis and / or secretion from beta-cells, delay, postpone, or reduce the need for insulin, or increase glucose clearance.

[0049] In some embodiments of any of the methods described herein, the method does not cause a vascular complication (e.g., increased vascular permeability or leakage) in the subject. In some embodiments of any of the methods described herein, the method increases bone mineral density in the subject.

[0050] In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 69. In some embodiments, the variant having the sequence of SEQ ID NO: 69 has a nucleotide sequence at position X 17 Amino acid K, 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 variant having the sequence of SEQ ID NO: 69, optionally at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26 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), 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 a subject with or at risk of developing obesity, type 1 diabetes, or type 2 diabetes), by administering to the subject a variant having the amino acid sequence TEEN or TKEN, and / or a C-terminal extension thereof, or a pharmaceutical composition containing the variant.

[0051] In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 58. In some embodiments, the variant having the sequence of SEQ ID NO: 58 has a nucleotide sequence at position X 17 Amino acid K, position X 23 , X 24 , X25 , 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 variant having the sequence of SEQ ID NO: 58, optionally at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26 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), 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 a subject with or at risk of developing obesity, type 1 diabetes, or type 2 diabetes), by administering to the subject a variant having the amino acid sequence TEEN or TKEN, and / or a C-terminal extension thereof, or a pharmaceutical composition containing the variant.

[0052] In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 6. In some embodiments, the variant having the sequence of SEQ ID NO: 6 has a nucleotide sequence at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a variant having the sequence of SEQ ID NO: 6, optionally at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26 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), 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 a subject with or at risk of developing obesity, type 1 diabetes, or type 2 diabetes), by administering to the subject a variant having the amino acid sequence TEEN or TKEN, and / or a C-terminal extension thereof, or a pharmaceutical composition containing the variant.

[0053] In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 38. In some embodiments, the variant having the sequence of SEQ ID NO: 38 has a nucleotide sequence at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a variant having the sequence of SEQ ID NO: 38, optionally at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26 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), 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 a subject with or at risk of developing obesity, type 1 diabetes, or type 2 diabetes), by administering to the subject a variant having the amino acid sequence TEEN or TKEN, and / or a C-terminal extension thereof, or a pharmaceutical composition containing the variant.

[0054] In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 41. In some embodiments, the variant having the sequence of SEQ ID NO: 41 has a nucleotide sequence at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a variant having the sequence of SEQ ID NO: 41, optionally at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26 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), 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 a subject with or at risk of developing obesity, type 1 diabetes, or type 2 diabetes), by administering to the subject a variant having the amino acid sequence TEEN or TKEN, and / or a C-terminal extension thereof, or a pharmaceutical composition containing the variant.

[0055] In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 44. In some embodiments, the variant having the sequence of SEQ ID NO: 44 has a nucleotide sequence at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a variant having the sequence of SEQ ID NO: 44, optionally at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26 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), 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 a subject with or at risk of developing obesity, type 1 diabetes, or type 2 diabetes), by administering to the subject a variant having the amino acid sequence TEEN or TKEN, and / or a C-terminal extension thereof, or a pharmaceutical composition containing the variant.

[0056] In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 70. In some embodiments, the variant having the sequence of SEQ ID NO: 70 has a nucleotide sequence at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a variant having the sequence of SEQ ID NO: 70, optionally at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26 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), 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 a subject with or at risk of developing obesity, type 1 diabetes, or type 2 diabetes), by administering to the subject a variant having the amino acid sequence TEEN or TKEN, and / or a C-terminal extension thereof, or a pharmaceutical composition containing the variant.

[0057] In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 71. In some embodiments, the variant having the sequence of SEQ ID NO: 71 has a nucleotide sequence at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a variant having the sequence of SEQ ID NO: 71, optionally at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26 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), 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 a subject with or at risk of developing obesity, type 1 diabetes, or type 2 diabetes), by administering to the subject a variant having the amino acid sequence TEEN or TKEN, and / or a C-terminal extension thereof, or a pharmaceutical composition containing the variant.

[0058] In some embodiments of any of the above aspects, the variant has the sequence of SEQ ID NO: 72. In some embodiments, the variant having the sequence of SEQ ID NO: 72 has a nucleotide sequence at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26In some embodiments of any of the above aspects, the method comprises administering to a subject a therapeutically effective amount of a variant having the sequence of SEQ ID NO: 72, optionally at position X 17 Amino acid K, position X 23 , X 24 , X 25 , and X 26 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), 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 a subject with or at risk of developing obesity, type 1 diabetes, or type 2 diabetes), by administering to the subject a variant having the amino acid sequence TEEN or TKEN, and / or a C-terminal extension thereof, or a pharmaceutical composition containing the variant.

[0059] definition As used herein, the term "extracellular activin type IIa receptor (ActRIIa) mutant" refers to a soluble extracellular portion of the single-pass transmembrane receptor ActRIIa having at least one amino acid substitution relative to wild-type extracellular ActRIIa (e.g., the bolded portion of the sequence of SEQ ID NO: 75 shown below), or a peptide containing extracellular ActRIIa having any one of the sequences of SEQ ID NOs: 76 to 96. The sequence of the wild-type human ActRIIa precursor protein is shown below (SEQ ID NO: 75), with the signal peptide in italics and the extracellular portion in bold.

[0060] Wild-type human ActRIIa precursor protein (SEQ ID NO: 75):

[0061] [ka]

[0062] The extracellular ActRIIa variant may have the sequence of any one of SEQ ID NOs: 1-72. In certain embodiments, the extracellular ActRIIa variant has the 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).

[0063] As used herein, the term "extracellular ActRIIb mutant" refers to a peptide comprising a soluble extracellular portion of the single-pass transmembrane receptor ActRIIb, which has at least one amino acid substitution relative to wild-type extracellular ActRIIb (e.g., the sequence of SEQ ID NO: 74). The extracellular ActRIIb mutant may have the sequence of SEQ ID NO: 149 shown below.

[0064] Extracellular ActRIIb variant (SEQ ID NO: 149): GRGEAETRECIFYNANWEKDRTNQSGLEPCYGDQDKRRHCFASWKNSSGTIELVKQGCWLDDINCYDRQECVAKKDSPEVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT As used herein, the term "linker" refers to a bond between two elements, e.g., peptide or protein domains. The polypeptides described herein may include an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one of the sequences of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)) fused to a moiety. This moiety can increase the stability or improve the pharmacokinetic properties of the polypeptide. This moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin) may be fused to the polypeptide via a linker. The linker can be a covalent bond or a spacer. The term "bond" refers to any type of bond created by a chemical bond, e.g., an amide bond or a disulfide bond, or a chemical reaction, e.g., chemical conjugation. The term "spacer" refers to a moiety (e.g., a polyethylene glycol (PEG) polymer) or amino acid sequence (e.g., a sequence of 1 to 200 amino acids) that exists between two elements, e.g., peptide or protein domains, to provide space and / or flexibility between the two elements. An amino acid spacer is part of the primary sequence of a polypeptide (e.g., fused to a peptide spaced apart by the polypeptide backbone). For example, the formation of a disulfide bond between two hinge regions that form an Fc domain is not considered a linker.

[0065] As used herein, the term "Fc domain" refers to a dimer of two Fc domain monomers. An Fc domain comprises at least C H 2 domain and C H The Fc domain monomer has at least 80% sequence identity (e.g., at least 85%, 90%, 95%, 97%, or 100% sequence identity) with a human Fc domain comprising three domains. The Fc domain monomer comprises a second and a third antibody constant domain (C H 2 and C HIn some embodiments, the Fc domain monomer also includes a hinge domain. The Fc domain does not include any portion of an immunoglobulin that can serve as an antigen recognition region, such as a variable domain or a complementarity determining region (CDR). In a wild-type Fc domain, two Fc domain monomers are joined by two C H Dimers are formed through interactions between the three antibody constant domains and one or more disulfide bonds formed between the hinge domains of two dimerizing Fc domain monomers. In some embodiments, the Fc domain may be mutated to lack effector function, exemplified by a "dead Fc domain." In certain embodiments, each Fc domain monomer of the Fc domain is mutated to reduce interaction or binding between the Fc domain and an Fcγ receptor. H The Fc domain comprises an amino acid substitution within the antibody constant domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce or inhibit dimerization of the Fc domain. The Fc domain may be of any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain may be of an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain may also be a non-natural Fc domain, for example, a recombinant Fc domain.

[0066] As used herein, the term "albumin-binding peptide" refers to an amino acid sequence of 12 to 16 amino acids that has affinity for and functions to bind to serum albumin. Albumin-binding peptides may be of different origins, e.g., human, mouse, or rat. In some embodiments, the albumin-binding peptide has the sequence DICLPRWGCLW (SEQ ID NO: 152).

[0067] As used herein, the term "fibronectin domain" refers to a high molecular weight glycoprotein of the extracellular matrix, or a fragment thereof, that binds to transmembrane receptor proteins, such as integrins, and extracellular matrix components, such as collagen and fibrin. In some embodiments, the fibronectin domain is a fibronectin type III domain having amino acids 610-702 of the sequence of UniProt ID NO:P02751 (SEQ ID NO:153). In other embodiments, the fibronectin domain is an Adnectin protein.

[0068] As used herein, the term "human serum albumin" refers to the albumin protein present in human plasma. Human serum albumin is the most abundant protein in blood. Human serum albumin accounts for approximately half of serum proteins. In some embodiments, human serum albumin has the sequence of UniProt ID NO: P02768 (SEQ ID NO: 154).

[0069] As used herein, the term "fused" is used to refer to the combination or joining of two or more elements, components, or protein domains, e.g., peptides or polypeptides, by means including chemical conjugation, recombinant means, and chemical bonds, e.g., amide bonds. For example, two single peptides can be fused in tandem via chemical conjugation, chemical bonds, peptide linkers, or any other covalent bonding means to form one continuous protein structure, e.g., a polypeptide. In some embodiments of the polypeptides described herein, an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)) may be fused in tandem via a linker to the N-terminus or C-terminus of a moiety (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 97), a wild-type Fc domain (e.g., the sequence of SEQ ID NO: 151), an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide (e.g., the sequence of SEQ ID NO: 152), a fibronectin domain (e.g., the sequence of SEQ ID NO: 153), or human serum albumin (e.g., the sequence of SEQ ID NO: 154)). For example, an extracellular ActRIIa mutant is fused to a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin) via a peptide linker, in which the N-terminus of the peptide linker is fused to the C-terminus of the extracellular ActRIIa mutant via a chemical bond, e.g., a peptide bond, and the C-terminus of the peptide linker is fused to the N-terminus of the moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin) via a chemical bond, e.g., a peptide bond.

[0070] As used herein, the term "C-terminal extension" refers to the addition of one or more amino acids to the C-terminus of a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOS: 1-70 (e.g., SEQ ID NOS: 6-70)). The C-terminal extension can be 1 to 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 2-amino acid C-terminal extension) and the amino acid sequence NPVTPK (SEQ ID NO: 155) (a 6-amino acid C-terminal extension). Any amino acid sequence that does not disrupt the activity of the polypeptide can be used. SEQ ID NO: 71, i.e., the sequence of SEQ ID NO: 69 with the C-terminal extension of NP, and SEQ ID NO: 72, i.e., the sequence of SEQ ID NO: 69 with the C-terminal extension of NPVTPK, represent two of the possible ways in which the polypeptides of the present invention can be modified to include a C-terminal extension.

[0071] As used herein, the term "percent (%) identity" refers to the percentage of amino acid (or nucleic acid) residues in a candidate sequence, e.g., an extracellular ActRIIa variant, that are identical to the amino acid (or nucleic acid) residues in a reference sequence, e.g., wild-type extracellular ActRIIa (e.g., SEQ ID NO: 73), after aligning the sequences and introducing gaps as necessary to achieve the maximum percent identity (i.e., gaps can be introduced into one or both of the candidate and reference sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). Alignment for determining percent identity can be achieved in a variety of ways within the skill of the art, such as using publicly available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for assessing alignment, including the algorithms required to achieve maximum alignment across the entire length of the sequences being compared. In some embodiments, the percent amino acid (or nucleic acid) sequence identity of a given candidate sequence to, with, or against a given reference sequence (alternatively, this can be expressed as a given candidate sequence having or containing a particular percent amino acid (or nucleic acid) sequence identity to a given reference sequence) is calculated as follows: 100 x (fraction A / B) where A is the number of amino acid (or nucleic acid) residues assigned an identity score in the alignment of the candidate sequence with the reference sequence, and B is the total number of amino acid (or nucleic acid) residues in the reference sequence. In some embodiments, if the length of the candidate sequence is not equal to the length of the reference sequence, the percent amino acid (or nucleic acid) sequence identity of the candidate sequence to the reference sequence will not be equal to the percent amino acid (or nucleic acid) sequence identity of the reference sequence to the candidate sequence.

[0072] In certain embodiments, a reference sequence aligned for comparison to a candidate sequence may show 50% to 100% identity with the candidate sequence over the entire length of the candidate sequence or over a selected portion of consecutive amino acid (or nucleic acid) residues of the candidate sequence. The length of the candidate sequence aligned for comparison purposes is at least 30%, e.g., at least 40%, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. If a position in the candidate sequence is occupied by the same amino acid (or nucleic acid) residue as the corresponding position in the reference sequence, then the molecules are identical at that position.

[0073] As used herein, the term "serum half-life," in the context of administering a therapeutic protein to a subject, refers to the time required for the plasma concentration of the protein to decrease by half in the subject. Proteins may be redistributed or eliminated from the bloodstream, or may be degraded, for example, by proteolysis. As described herein, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)) exhibits a serum half-life of 7 days in humans.

[0074] As used herein, the term "metabolic disease" refers to a disease, disorder, or syndrome related to a subject's metabolism, such as breaking down carbohydrates, proteins, and fats in food to release energy, converting chemicals into other substances, and transporting them into cells for energy utilization and / or storage. Some symptoms of metabolic disease include high serum triglycerides, high low-density cholesterol (LDL), low high-density cholesterol (HDL), and / or elevated fasting insulin levels, elevated fasting plasma glucose, abdominal obesity (central obesity), and elevated blood pressure. Metabolic disease increases 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.

[0075] As used herein, the term "percent weight gain" refers to the percentage of weight gained compared to a subject's previous weight at a previous time point. Percent weight gain can be calculated as follows:

[0076] 100 × [(weight at later time point – weight at earlier time point) / (weight at earlier time point)] In the present invention, administering to a subject a polypeptide comprising an extracellular ActRIIa mutant (e.g., an extracellular ActRIIa mutant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa mutant (e.g., an extracellular ActRIIa mutant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)), or a vector containing such a nucleic acid molecule reduces the subject's weight gain rate (%).

[0077] As used herein, the term "appetite for food intake" refers to a subject's natural desire or need for food. A 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 the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)). In the present invention, administering to a subject a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)), or a vector containing such a nucleic acid molecule does not affect the subject's appetite for food intake.

[0078] As used herein, the term "adiposity" refers to fat stored in adipose tissue of a subject. In the present invention, administering to a subject a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one of the sequences of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one of the sequences of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)), or a vector containing such a nucleic acid molecule, alleviates obesity in the subject without affecting lean mass.

[0079] As used herein, the term "lean mass" refers to components of body composition, including, for example, lean mass, body fat, and body fluid. Lean mass is typically calculated by subtracting the weight of body fat and body fluid from total body weight. Typically, a subject's lean mass is between 60% and 90% of their total body weight. In the present invention, administering to a subject a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one of the sequences of SEQ ID NOS: 1 to 72 (e.g., SEQ ID NOS: 6 to 72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one of the sequences of SEQ ID NOS: 1 to 72 (e.g., SEQ ID NOS: 6 to 72)), or a vector containing such a nucleic acid molecule reduces obesity (i.e., fat) in a subject without affecting lean mass.

[0080] As used herein, the term "epididymal and perirenal fat pads" refers to densely packed adipocytes in the epididymis and around the kidney. In the present invention, administering to a subject a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)), or a vector containing such a nucleic acid molecule reduces the weight of the epididymal and perirenal fat pads of the subject.

[0081] As used herein, the term "fasting insulin" refers to a subject's insulin level during a period (i.e., 12 to 24 hours) when the subject has not consumed any food. 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 for developing metabolic diseases. Typically, in subjects suffering from type 1 diabetes, the subject's fasting insulin level is low compared to that of healthy subjects. In subjects suffering from insulin resistance (i.e., type 2 diabetes), the subject's fasting insulin level is high compared to that of healthy subjects. In the present invention, administering to a subject a polypeptide comprising an extracellular ActRIIa mutant (e.g., an extracellular ActRIIa mutant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa mutant (e.g., an extracellular ActRIIa mutant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)), or a vector containing such a nucleic acid molecule reduces the subject's fasting insulin level.

[0082] As used herein, the term "glucose clearance rate" refers to the rate at which glucose is removed from blood.Glucose clearance rate can be measured by glucose tolerance test (GTT).In GTT, a certain amount of glucose is administered to a subject, and then blood samples are taken to determine how quickly it is removed from blood.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.

[0083] As used herein, the term "serum lipid profile" refers to the measurement of the distribution of different types of lipids and lipoproteins in a subject's serum. Such measurement can be achieved by a series of blood tests. The types of lipids and lipoproteins in a subject's serum 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 in 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 indicators or risk factors for developing certain metabolic diseases, or in some serious medical cases, cardiovascular diseases. In the present invention, administering to a subject a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)), a nucleic acid molecule encoding a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)), or a vector containing such a nucleic acid molecule improves the subject's serum lipid profile so that cholesterol (particularly low-density lipoprotein) and triglyceride levels are reduced.

[0084] As used herein, the term "affinity" or "binding affinity" refers to the strength of the binding interaction between two molecules. Generally, binding affinity refers to the strength of the sum of non-covalent interactions between a molecule and its binding partner, for example, an extracellular ActRIIa mutant and BMP9 or activin A. Unless otherwise indicated, binding affinity refers to intrinsic binding affinity, which represents a 1:1 interaction between members of a binding pair. The binding affinity between two molecules is generally determined by the dissociation constant (K D ) or affinity constant (K A ) Two molecules that have low binding affinity for each other generally tend to bind slowly and dissociate easily, with a large K D Two molecules that have a high affinity for each other generally bind more easily and tend to remain bound longer, exhibiting a small K D The K of two interacting molecules is shown. D can be determined using methods and techniques well known in the art, such as, for example, surface plasmon resonance. D is k off / k on It is calculated as the ratio of

[0085] As used herein, the term "muscle mass" refers to a component of body composition. Typically, muscle mass is calculated by subtracting the weight of body fat and fluid from total body weight. The percentage of muscle mass can vary greatly between individuals depending on the subject's genetic makeup, age, race, and health status. Typically, a subject's muscle mass can be 20% to 50% of their total body weight.

[0086] As used herein, the phrase "affecting myostatin, activin, and / or BMP9 signaling" means altering the binding of myostatin, activin, and / or BMP9 to their receptors, 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 their receptors, e.g., ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa). As described herein, a polypeptide of the present 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)) has a weak binding affinity (e.g., a low K) for BMP9. D 200 pM or more).

[0087] As used herein, the term "vascular complications" refers to vascular disorders or any damage to blood vessels, for example, damage to the vascular wall. Damage to the vascular wall can lead to increased vascular permeability or leakage. The term "vascular permeability or leakage" refers to the ability of the vascular wall to allow small molecules, proteins, and cells to enter and exit the blood vessel. Increased vascular permeability or leakage can occur due to an increase in the gaps between endothelial cells lining the vascular wall (e.g., an increase in the size and / or number of gaps) and / or thinning of the vascular wall.

[0088] As used herein, the term "polypeptide" refers to a single polymer in which the monomers are amino acid residues covalently linked to one another through amide bonds. Polypeptide is intended to encompass any amino acid sequence that is naturally occurring, recombinant, or synthetically produced.

[0089] As used herein, the term "homodimer" refers to a molecular construct formed by two identical macromolecules, such as proteins or nucleic acids. These two identical monomers can form a homodimer through covalent or non-covalent bonds. For example, an Fc domain can be a homodimer of two Fc domain monomers if the two Fc domain monomers contain the same sequence. In another example, a polypeptide described herein comprising an extracellular ActRIIa variant fused to an Fc domain monomer can form a homodimer through the interaction of the two Fc domain monomers, which form the Fc domain in the homodimer.

[0090] As used herein, the term "heterodimer" refers to a molecular construct formed by two different macromolecules, such as proteins or nucleic acids. These two monomers can form a heterodimer through a covalent or non-covalent bond. For example, a polypeptide described herein comprising an extracellular ActRIIa variant fused to an Fc domain monomer can form a heterodimer through the interaction of two Fc domain monomers, each fused to a different ActRIIa variant, to form an Fc domain in the heterodimer.

[0091] As used herein, the term "host cell" refers to a vehicle containing necessary cellular components, e.g., organelles required for expressing a protein from a corresponding nucleic acid. The nucleic acid is generally contained within a nucleic acid vector, which can be introduced into the host cell by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, etc.). The host cell can be a prokaryotic cell, e.g., a bacterial cell, or a eukaryotic cell, e.g., a mammalian cell (e.g., a CHO cell or HEK293 cell).

[0092] As used herein, the term "therapeutically effective amount" refers to an amount of a polypeptide, nucleic acid, or vector of the present invention, or a pharmaceutical composition containing a polypeptide, nucleic acid, or vector of the present invention, that is effective to achieve a desired therapeutic effect in treating a patient with a disease, such as a muscle disease, or a disease or condition associated with muscle weakness and muscle atrophy, for example, Duchenne muscular dystrophy (DMD), faciocraniohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS), sarcopenia, or cancer cachexia. The term "therapeutically effective amount" also refers to an amount of a polypeptide, nucleic acid, or vector of the present invention, or a pharmaceutical composition containing a polypeptide, nucleic acid, or vector of the present invention, that is effective to achieve a desired therapeutic effect in treating a disease, such as a metabolic disease, or a condition associated with excess weight, excess body fat, hyperglycemia, elevated fasting insulin levels, or insulin resistance, for example, obesity, type 1 diabetes, or type 2 diabetes. In particular, a therapeutically effective amount of a polypeptide, nucleic acid, or vector avoids adverse side effects.

[0093] As used herein, the term "pharmaceutical composition" refers to a medicament or pharmaceutical formulation containing an active ingredient and excipients and diluents to make the active ingredient suitable for the method of administration. The pharmaceutical composition of the present invention contains pharmaceutically acceptable ingredients that are compatible with the polypeptide, nucleic acid, or vector. The pharmaceutical composition may be in tablet or capsule form for oral administration, or in aqueous form for intravenous or subcutaneous administration.

[0094] 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 other ingredients of the formulation and not deleterious to the recipient. In the present invention, a pharmaceutically acceptable carrier or excipient must provide sufficient pharmaceutical stability to 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 will vary depending on the mode of administration. For example, an aqueous carrier is generally used for intravenous administration, while a solid carrier is preferred for oral administration.

[0095] As used herein, the term "treating and / or preventing" refers to treating and / or preventing a disease, such as a metabolic disease (e.g., obesity, type 1 diabetes, and type 2 diabetes) or a muscle disease (e.g., DMD, FSHD, IBM, and ALS) using the methods and compositions of the present invention. Generally, treating a metabolic or muscle disease is performed after a subject has developed a metabolic or muscle disease and / or after the subject has already been diagnosed with a metabolic or muscle disease. Preventing 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 exhibit signs or mild symptoms that are determined by a physician to be indicative of or a risk factor for developing a metabolic or muscle disease, or may not have yet developed the metabolic or muscle disease but are determined by a physician to have a family history or a genetic predisposition to developing the metabolic or muscle disease.

[0096] As used herein, the term "subject" refers to a mammal, such as, preferably, a human. Mammals include, but are not limited to, humans and domestic and farm animals, such as monkeys (e.g., cynomolgus monkeys), mice, dogs, cats, horses, and cows. [Effects of the Invention]

[0097] According to the present invention, it is possible to provide an activin type IIa receptor mutant and a pharmaceutical composition containing the mutant. [Brief explanation of the drawings]

[0098] [Figure 1] FIG. 1 is a sequence alignment showing the wild-type sequences of extracellular ActRIIa and ActRIIb and the amino acid substitutions of ActRIIa mutants. [Figure 2A] 2A and 2B are scatter plots showing the effect of extracellular ActRIIa variants on body weight in mice receiving a single hydrodynamic injection of a plasmid construct encoding the indicated ActRIIa variant or a control plasmid. [Figure 2B] 2A and 2B are scatter plots showing the effect of extracellular ActRIIa variants on body weight in mice receiving a single hydrodynamic injection of a plasmid construct encoding the indicated ActRIIa variant or a control plasmid. [Figure 3A] 3A and 3B are bar graphs showing the effect of extracellular ActRIIa variants on muscle mass. [Figure 3B] 3A and 3B are bar graphs showing the effect of extracellular ActRIIa variants on muscle mass. [Figure 4A] 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 weekly for 4 weeks. [Figure 4B] FIG. 4B is a bar graph showing the effect of extracellular ActRIIa variants on individual muscle weight by histological analysis. [Figure 5A] Figure 5A is a scatter plot showing the effect of extracellular ActRIIa variants on body weight over the course of a study in which mice received a single hydrodynamic injection of a plasmid construct encoding the indicated ActRIIa variant or a control plasmid. [Figure 5B] FIG. 5B is a bar graph showing the effect of extracellular ActRIIa variants on body weight at the end of 28 days. [Figure 6A] 6A and 6B are bar graphs showing the effect of extracellular ActRIIa variants on body weight by tissue analysis. [Figure 6B] 6A and 6B are bar graphs showing the effect of extracellular ActRIIa variants on body weight by tissue analysis. [Figure 7A] 7A and 7B are scatter plots 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 4 weeks. [Figure 7B] 7A and 7B are scatter plots 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 4 weeks. [Figure 8A] 8A and 8B are bar graphs showing the effect of different doses of extracellular ActRIIa variants on muscle mass (FIG. 8A) and fat mass (FIG. 8B). [Figure 8B] 8A and 8B are bar graphs showing the effect of different doses of extracellular ActRIIa variants on muscle mass (FIG. 8A) and fat mass (FIG. 8B). [Figure 9A] 9A and 9B are bar graphs showing the effect of different doses of extracellular ActRIIa variants on muscle mass by histological analysis. [Figure 9B] 9A and 9B are bar graphs showing the effect of different doses of extracellular ActRIIa variants on muscle mass by histological analysis. DETAILED DESCRIPTION OF THE INVENTION

[0099] The present invention relates to polypeptides comprising extracellular activin type IIa receptor (ActRIIa) variants. In some embodiments, the polypeptides of the present invention comprise an extracellular ActRIIa variant fused to a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin). Polypeptides comprising an extracellular ActRIIa variant fused to an Fc domain monomer can also form dimers (e.g., homodimers or heterodimers) through interaction between two Fc domain monomers. The ActRIIa variants described herein have weaker or no binding affinity for bone morphogenetic protein 9 (BMP9) compared to activin and myostatin. The present invention also includes methods for treating diseases and conditions associated with muscle weakness and muscle atrophy in a subject by increasing muscle mass and strength, methods for treating or preventing metabolic diseases, or methods for affecting myostatin, activin and / or BMP9 signaling, by administering to the subject a polypeptide comprising an extracellular ActRIIa variant described herein.

[0100] I. Extracellular activin type IIa receptor (ActRIIa) mutant The activin type II receptor is a single-transmembrane domain receptor that regulates signals from ligands of the transforming growth factor β (TGF-β) superfamily. Ligands of the TGF-β superfamily are involved in many physiological processes in the host, such as muscle growth, vascular growth, cell differentiation, homeostasis, and bone formation. Examples of ligands of the TGF-β superfamily include activin, inhibin, growth differentiation factors (GDFs) (e.g., GDF8, also known as myostatin), and bone morphogenetic proteins (BMPs) (e.g., BMP9). Myostatin and activin are known to play a role in regulating skeletal muscle growth. For example, mice lacking myostatin exhibit a significant increase in skeletal muscle mass.

[0101] Activin is also highly expressed in adipose tissue, and elevated levels of myostatin and activin receptors have been observed in the subcutaneous and visceral fat of obese mice. Furthermore, myostatin has been shown to be elevated in skeletal muscle and plasma of obese and insulin-resistant women, and both type I and type II activin receptors have been linked to pancreatic function and diabetes. These data suggest that increased signaling through activin receptors, either through increased expression of activin ligands (e.g., activin, myostatin) or increased expression of the activin receptor itself, can lead to obesity and metabolic disorders such as type 1 and type 2 diabetes. Therefore, methods to reduce or inhibit this signaling could be used to treat obesity and metabolic disorders.

[0102] There are two types of activin type II receptors: ActRIIa and ActRIIb. Research has shown that BMP9 binds to ActRIIb with approximately 300-fold higher binding affinity than ActRIIa (see, for example, Townson et al., J. Biol. Chem. 287:27313, 2012). ActRIIa is known to have a longer half-life than ActRIIb. The present invention describes extracellular ActRIIa variants constructed by introducing amino acid residues of ActRIIb into ActRIIa with the aim of conferring the physiological properties conferred by ActRIIb while also maintaining the beneficial physiological and pharmacokinetic properties of ActRIIa. The optimal peptide will confer a significant increase in muscle mass while retaining a longer serum half-life and a low binding affinity to, for example, BMP9. Preferred ActRIIa variants also exhibit improved binding to activin and / or myostatin compared to wild-type ActRIIa, allowing them to compete with endogenous activin receptors for ligand binding and reduce or inhibit endogenous activin receptor signaling. These variants can be used to treat disorders in which activin receptor signaling is elevated, such as metabolic disorders, 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 variant to reduce or eliminate the variant's binding affinity to BMP9. The wild-type amino acid sequences of the extracellular portions of human ActRIIa and ActRIIb are shown below.

[0103] Human ActRIIa extracellular portion (SEQ ID NO: 73): GAILGRSETQECLFFNANWEKDRTNQTGVEPCYGDKDKRRHCFATWKNISGSIEIVKQGCWLDDINCYDRTDCVEKKDSPEVYFCCCEGNMCNEKFSYFPEMEVTQPTS Human ActRIIb extracellular portion (SEQ ID NO: 74): GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPT The polypeptides described herein include extracellular ActRIIa mutants having 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. Potential amino acid substitutions at 27 different positions can be introduced into the extracellular ActRIIa mutants (Table 1). In some embodiments, the extracellular ActRIIa mutants 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 may 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 the sequence of SEQ ID NO: 1) may contain amino acid substitutions at all 27 positions as listed in Table 1. In some embodiments, the extracellular ActRIIa variant may contain amino acid substitutions at multiple positions, for example, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, or 26, of the 27 positions listed in Table 1.

[0104] Amino acid substitutions can reduce or improve the activity and / or binding affinity of the ActRIIa variants of the present invention. To maintain polypeptide function, amino acid substitutions at positions X, X, and X of the sequences shown in Tables 1 and 2 (SEQ ID NOS: 1 to 72 (e.g., SEQ ID NOS: 6 to 72)) are preferred. 17It is important that the lysine (K) in GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 150) The ActRIIa mutant with the sequence 17 This indicates that substitution of lysine (K) with alanine (A) at position X is not tolerated. Thus, the variants in Tables 1 and 2 (e.g., ActRIIa variants of the present invention including SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)) have a lysine (K) at position X 17 It holds the amino acid K.

[0105] 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 ActRIIa variants containing the amino acid sequence TEEN at position X 24 Maintaining amino acid K at position X 23 , X 24 , X 25 , and X 26 Mutants having the amino acid sequence TKEN at 1000 bp have reduced BMP9 binding. The sequences TEEN and TKEN can be used interchangeably to provide reduced BMP9 binding in ActRIIa mutants of the invention (e.g., mutants in Tables 1 and 2, e.g., SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)).

[0106] ActRIIa variants of the present invention may further comprise a C-terminal extension (e.g., additional amino acids at the C-terminus). A C-terminal extension can include the addition of 1 to 6 additional amino acids (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) to the C-terminus of any of the variants shown in Tables 1 and 2 (e.g., SEQ ID NOs: 1 to 70 (e.g., SEQ ID NOs: 6 to 70)). One possible C-terminal extension that can be included in an ActRIIa variant of the present invention is the amino acid sequence NP. For example, a sequence including a C-terminal extension is SEQ ID NO: 71 (e.g., SEQ ID NO: 69 with the C-terminal extension NP). Another exemplary C-terminal extension that can be included in an ActRIIa variant of the present invention is the amino acid sequence NPVTPK (SEQ ID NO: 155). For example, a sequence including a C-terminal extension is SEQ ID NO: 72 (e.g., SEQ ID NO: 69 with the C-terminal extension NPVTPK).

[0107] [Table 1]

[0108] In some embodiments of the extracellular ActRIIa variant having the sequence of SEQ ID NO: 2, X3 is E, X6 is R, and X 11 is D and X 12 is K and X 13 is R and X 16 is K or R, and X 17 is K and X 19 is W and X 20 is L and X 21 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 sequences of SEQ ID NOs: 1-3, X 17 is K and X 23 is T and X 24 is E and X 25 is E and X 26is N. In some embodiments of the extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1-5, X 17 is K and X 23 is T and X 24 is K and X 25 is E and X 26 is N.

[0109] In some embodiments, the polypeptides described herein comprise an extracellular ActRIIa mutant having the sequence of any one of SEQ ID NOs: 6-72 (Table 2).

[0110] [Table 2-1]

[0111] [Table 2-2]

[0112] [Table 2-3]

[0113] [Table 2-4]

[0114] In some embodiments, a polypeptide of the present invention comprising an extracellular ActRIIa variant (e.g., any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) has a sequence at position X 17 It has amino acid K at position X. 17 Altering the amino acids in results in reduced activity. GAILGRSETQECLFYNANWELERTNQTGVERCEGEKDKRLHCYATWRNISGSIEIVAKGCWLDDFNCYDRTDCVETEENPQVYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 150) The ActRIIa mutant having the sequence: 17 This indicates that substitution of K with A in

[0115] In some embodiments, position X 23 , X 24 , X 25 , and X 26 The polypeptide of the present invention, comprising an extracellular ActRIIa variant (e.g., any one of SEQ ID NOS: 1 to 72 (e.g., SEQ ID NOS: 6 to 72)) having the sequence TEEN at position X 24 In some embodiments, at position X, the amino acid E may be substituted with the amino acid K. 23 , X 24 , X 25 , and X 26 The polypeptide of the present invention, which comprises an extracellular ActRIIa mutant (e.g., any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)) having the sequence TKEN at position X 24 At position X, the amino acid K may be substituted with the amino acid E. 23 , X 24 , X 25 , and X 26 Polypeptides having the sequence TEEN or TKEN at the amino acid sequence have reduced or weak binding to BMP9.

[0116] In some embodiments, a polypeptide of the present 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 the C-terminal extension 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 the C-terminal extension NPVTPK). The C-terminal extension can include the addition of 1 to 6 additional amino acids (e.g., 1, 2, 3, 4, 5, 6, or more additional amino acids) to the C-terminus.

[0117] In some embodiments, a polypeptide of the present invention comprising an extracellular ActRIIa variant may further comprise a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin), which can be fused to the N-terminus or C-terminus (e.g., the C-terminus) of the extracellular ActRIIa variant via a linker or other covalent bond. A polypeptide comprising an extracellular ActRIIa variant fused to an Fc domain monomer can form a dimer (e.g., a homodimer or heterodimer) through interaction between two Fc domain monomers, which combine to form an Fc domain in the dimer.

[0118] In some embodiments, the extracellular ActRIIa mutants described herein do not have any of the sequences of SEQ ID NOs: 76-96 shown in Table 3 below.

[0119] [Table 3-1]

[0120] [Table 3-2]

[0121] Furthermore, in some embodiments, the polypeptides described herein have a serum half-life in humans of at least 7 days. D The polypeptide can bind to bone morphogenetic protein 9 (BMP9) at a K of 10 pM or greater. D In some embodiments, the polypeptide can bind to activin A at position X. In some embodiments, the polypeptide does not bind to BMP9 or activin A. In some embodiments, the polypeptide binds to activin and / or myostatin and exhibits low (e.g., weak) binding to BMP9. In some embodiments, the polypeptide with low or weak binding to BMP9 can bind to activin A at position X. 23 , X 24 , X 25 , and X 26 has the sequence TEEN or TKEN.

[0122] Additionally, in some embodiments, the polypeptide has a K of about 200 pM or greater. D (e.g., K of about 200, 300, 400, 500, 600, 700, 800, or 900 pM or greater) D , e.g., a K of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nM or greater D , e.g., a K between about 200 pM and about 50 nM D In some embodiments, the polypeptide may bind to human BMP9 at a K of about 800 pM or less. In some embodiments, the polypeptide does not substantially bind to human BMP9. D (e.g., a K of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less) D , e.g., K between about 800 pM and about 200 pM DIn some embodiments, the polypeptide may bind to human activin A with a K of 800 pM or less. D (e.g., a K of about 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 pM or less) D , e.g., K between about 800 pM and about 200 pM D ) can bind to human activin B. The polypeptide has a K of approximately 5 pM or greater. D (e.g., a K of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 pM or more) D ) and can also bind to growth and differentiation factor 11 (GDF-11).

[0123] II. Fc Domain In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIa variant fused to an Fc domain monomer or a fragment of an Fc domain of an immunoglobulin to extend the serum half-life of the polypeptide. A polypeptide comprising an extracellular ActRIIa variant fused to an Fc domain monomer can form a dimer (e.g., a homodimer or a heterodimer) through interaction between two Fc domain monomers, which form an Fc domain in the dimer. As conventionally known in the art, an Fc domain is a protein structure found at the C-terminus of an immunoglobulin. An Fc domain is a C HThe Fc domain comprises two Fc domain monomers that form a dimer through interaction between three antibody constant domains. A wild-type Fc domain forms the minimal structure that binds to an Fc receptor, e.g., FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, FcγRIIIb, or FcγRIV. In some embodiments, the Fc domain can be mutated to lack effector function, typically resulting in a "dead" Fc domain. For example, the Fc domain may contain specific amino acid substitutions known to minimize interaction between the Fc domain and an Fcγ receptor. In some embodiments, the Fc domain is derived from an IgG1 antibody and contains amino acid substitutions L234A, L235A, and G237A. In some embodiments, the Fc domain is derived from an IgG1 antibody and contains amino acid substitutions D265A, K322A, and N434A. The above amino acid positions are defined according to Kabat (Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Kabat numbering of amino acid residues can be determined for a given antibody by aligning homologous regions of the antibody sequence with the "standard" Kabat numbering sequence. Furthermore, in some embodiments, the Fc domain does not induce any immune system-related response. For example, the Fc domain in a polypeptide dimer comprising an extracellular ActRIIa variant fused to an Fc domain monomer may be modified to reduce the interaction or binding between the Fc domain and the Fcγ receptor. The sequence of the Fc domain monomer that can be fused to the extracellular ActRIIa variant is shown below (SEQ ID NO: 97): THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEK TISKAKGQPREPQVYTLPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In some embodiments, the Fc domain is derived from an IgG1 antibody and includes amino acid substitutions L12A, L13A, and G15A relative to the sequence of SEQ ID NO: 97. In some embodiments, the Fc domain is derived from an IgG1 antibody and includes amino acid substitutions D43A, K100A, and N212A relative to the sequence of SEQ ID NO: 97. In some embodiments, an extracellular ActRIIa variant described herein (e.g., an extracellular ActRIIa variant having the 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 Fc domain monomer (e.g., SEQ ID NO: 97) by conventional genetic or chemical means, e.g., chemical conjugation. Optionally, 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.

[0124] In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIa variant fused to an Fc domain. In some embodiments, the Fc domain comprises one or more amino acid substitutions that reduce or inhibit dimerization of the Fc domain. In some embodiments, the Fc domain comprises a hinge domain. The Fc domain may be of the immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD. In addition, the Fc domain may be of an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain may also be a non-natural Fc domain, for example, a recombinant Fc domain.

[0125] Methods for creating Fc domains with reduced dimerization are known in the art. In some embodiments, C is modified to impair dimerization due to steric clashes. H 3-C H One or more amino acids with large side chains (e.g., tyrosine or tryptophan) may be introduced into the C3 dimer interface to remove favorable interactions. H 3-C H 3 One or more amino acids with small side chains (e.g., alanine, valine, or threonine) may be introduced into the dimer interface. H Methods for introducing amino acids with large or small side chains into the three domains are described, for example, in Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Application Publication No. 2006 / 0074225, U.S. Patent No. 8,216,805 and U.S. Patent No. 5,731,168, Ridgway et al. (Protein Eng. 9:617-612, 1996), Atwell et al. (J Mol Biol. 270:26-35, 1997), and Merchant et al. (Nat Biotechnol. 16:677-681, 1998), all of which are incorporated herein by reference in their entirety.

[0126] In yet another embodiment, a C between two Fc domains H 3-C H 3C that constitutes the interface H One or more amino acid residues in the three domains are substituted with positively charged amino acid residues (e.g., lysine, arginine, or histidine) or negatively charged amino acid residues (e.g., aspartic acid or glutamic acid) so that the interaction is electrostatically unfavorable due to the introduced amino acid of the particular charge. HMethods for introducing charged amino acids into the three domains are described, for example, in Ying et al. (J Biol Chem. 287:19399-19408, 2012), U.S. Patent Application Publication No. 2006 / 0074225, U.S. Patent Application Publication No. 2012 / 0244578, and U.S. Patent Application Publication No. 2014 / 0024111, all of which are incorporated herein by reference in their entirety.

[0127] In some embodiments of the invention, the Fc domain comprises 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, The Fc domain comprises one or more of the following: 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 set forth in SEQ ID NO: 151.

[0128] 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 the serum protein peptide can improve the pharmacokinetics of protein drugs.

[0129] By way of example, albumin-binding peptides that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the albumin-binding peptide comprises the sequence DICLPRWGCLW (SEQ ID NO: 152).

[0130] In the present invention, to extend the serum half-life of an extracellular ActRIIa mutant, an albumin-binding peptide can be linked to the N-terminus or C-terminus (e.g., the C-terminus) of an extracellular ActRIIa mutant described herein (e.g., an extracellular ActRIIa mutant having any one of the sequences of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)). In some embodiments, the albumin-binding peptide is linked to the N-terminus or C-terminus of the extracellular ActRIIa mutant directly or via a linker.

[0131] In some embodiments, an extracellular ActRIIa variant described herein (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N-terminus or C-terminus of an albumin-binding peptide (e.g., SEQ ID NO: 152) by conventional genetic or chemical means, for example, 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 believed that the inclusion of an albumin-binding peptide in the extracellular ActRIIa variant described herein may result in extended retention of the therapeutic protein through its binding to serum albumin.

[0132] IV. Fibronectin Domains In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIa variant fused to a fibronectin domain, which can improve the pharmacokinetics of protein drugs.

[0133] The fibronectin domain is, for example, a high-molecular-weight glycoprotein of the extracellular matrix, or a fragment thereof, that binds to transmembrane receptor proteins such as integrins and extracellular matrix components such as collagen and fibrin. In some embodiments of the present invention, to extend the serum half-life of an extracellular ActRIIa mutant, a fibronectin domain is linked to the N-terminus or C-terminus (e.g., the C-terminus) of an extracellular ActRIIa mutant described herein (e.g., an extracellular ActRIIa mutant having any one of the sequences of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)). The fibronectin domain can be linked to the N-terminus or C-terminus of the extracellular ActRIIa mutant directly or via a linker.

[0134] By way of example, fibronectin domains that can be used in the methods and compositions described herein are generally known in the art. In one embodiment, the fibronectin domain is a fibronectin type III domain having amino acids 610-702 of the sequence of UniProt ID NO:P02751 (SEQ ID NO:153). In another embodiment, the fibronectin domain is an Adnectin protein.

[0135] In some embodiments, an extracellular ActRIIa variant described herein (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N-terminus or C-terminus of a fibronectin domain (e.g., SEQ ID NO: 153) by conventional genetic or chemical means, e.g., 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 believed that the inclusion of a fibronectin domain in the extracellular ActRIIa variant described herein may result in extended retention of the therapeutic protein through its binding to integrins and extracellular matrix components such as collagen and fibrin.

[0136] V. Serum Albumin In some embodiments, the polypeptides described herein may comprise an extracellular ActRIIa variant fused to serum albumin. Binding to serum albumin can improve the pharmacokinetics of protein drugs.

[0137] Serum albumin is a globular protein that is the most abundant blood protein in mammals. It is produced in the liver and accounts for approximately half of serum proteins. Serum albumin is monomeric and soluble in blood. Some of the most important functions of serum albumin include transporting hormones, fatty acids, and other proteins in the body, buffering pH, and maintaining the osmotic pressure required for proper distribution of body fluids between blood vessels and body tissues. In a preferred embodiment, the serum albumin is human serum albumin. In some embodiments of the present invention, to extend the serum half-life of an extracellular ActRIIa variant described herein (e.g., an extracellular ActRIIa variant having any one of the sequences of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)), human serum albumin is linked to the N-terminus or C-terminus (e.g., C-terminus) of the extracellular ActRIIa variant. Human serum albumin can be linked to the N-terminus or C-terminus of the extracellular ActRIIa variant directly or via a linker.

[0138] By way of example, serum albumin that can be used in the methods and compositions described herein is generally known in the art. In one embodiment, the serum albumin comprises the sequence of UniProt ID NO: P02768 (SEQ ID NO: 154).

[0139] In some embodiments, an extracellular ActRIIa variant described herein (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) can be fused to the N-terminus or C-terminus of human serum albumin (e.g., SEQ ID NO: 154) by conventional genetic or chemical means, for example, chemical conjugation. If desired, a linker (e.g., a spacer) can be inserted between the extracellular ActRIIa variant and human serum albumin. Without being bound by theory, it is believed that the inclusion of human serum albumin in the extracellular ActRIIa variant described herein may result in extended retention of the therapeutic protein.

[0140] VI. Linker The polypeptides described herein may include an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) fused to a moiety via a linker. In some embodiments, the moiety increases the stability of the polypeptide. Exemplary moieties include an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin. In the present invention, the linker between a certain portion (e.g., an Fc domain monomer (e.g., the sequence of SEQ ID NO: 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 dimer formation), 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 the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)) can be an amino acid spacer containing 1 to 200 amino acids. Suitable peptide spacers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine, alanine, and serine. In some embodiments, the spacer may include a GA, GS, GG, GGA, GGS, GGG, GGGA (SEQ ID NO: 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) motif, e.g., multiple or repeated motifs.In some embodiments, the spacer can include 2 to 12 amino acids containing a GA or GS motif, e.g., GA, GS, GAGA (SEQ ID NO: 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 GSGSGSGSGSGSGS (SEQ ID NO: 117). In some embodiments, the spacer can include 3 to 12 amino acids containing a GGA or GGS motif, e.g., GGA, GGS, GGAGGA (SEQ ID NO: 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). Further, in some embodiments, the spacer can comprise 4 to 12 amino acids including the motifs 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 comprise the motifs GGGGA (SEQ ID NO: 101) or GGGGS (SEQ ID NO: 102), e.g., GGGGAGGGGAGGGGA (SEQ ID NO: 128) and GGGGSGGGGSGGGGGS (SEQ ID NO: 129).In some embodiments of the present invention, the amino acid spacer between a certain portion (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain having amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or serum albumin) and an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72)) may 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).

[0141] In some embodiments, the spacer may also include amino acids other than glycine, alanine, and serine, for example, 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), or GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 146). In some embodiments, the spacer can include a motif, e.g., a multiple or repeated motif, of EAAAK (SEQ ID NO: 147). ... n (wherein X can be any amino acid (e.g., A, K, or E) and n is 1 to 5), and can include motifs, e.g., multiple or repeated motifs, of proline-rich sequences such as PAPAP (SEQ ID NO: 148).

[0142] The length of the peptide spacer and amino acids used can be adjusted depending on the two proteins involved and the degree of flexibility desired in the final protein fusion polypeptide. The length of the spacer can be adjusted to ensure proper protein folding and avoid the formation of aggregates.

[0143] VII. Vectors, Host Cells, and Protein Production The polypeptides of the present invention can be produced from host cells. A host cell refers to a vehicle containing necessary cellular components, e.g., organelles required for expressing the polypeptides and fusion polypeptides described herein from the corresponding nucleic acids. These nucleic acids may be contained within a nucleic acid vector, which can be introduced into host cells by conventional techniques known in the art (e.g., transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, or infection). The choice of nucleic acid vector will depend in part on the host cell used. Generally, preferred host cells are of eukaryotic (e.g., mammalian) or prokaryotic (e.g., bacterial) origin.

[0144] Nucleic acid vector construction and host cells Nucleic acid sequences encoding the amino acid sequences of the polypeptides of the present invention can be prepared by various methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis and PCR mutagenesis. Nucleic acid molecules encoding the polypeptides of the present invention can be obtained using standard techniques, such as gene synthesis. Alternatively, nucleic acid molecules encoding wild-type extracellular ActRIIa can be mutated to contain specific amino acid substitutions using standard techniques in the art, such as QuikChange™ mutagenesis. Nucleic acid molecules can be synthesized using a nucleotide synthesizer or PCR technology.

[0145] The nucleic acid sequence encoding the polypeptide of the present invention can be inserted into a vector capable of replicating and expressing the nucleic acid molecule in a prokaryotic or eukaryotic host cell. Many vectors are available in the art and can be used for the purposes of the present invention. Each vector may contain various components, which can be adjusted and optimized to suit a specific host cell. For example, vector components include, but are not limited to, a replication origin, a selectable marker gene, a promoter, a ribosome binding site, a signal sequence, a nucleic acid sequence encoding a target protein, and a transcription termination sequence.

[0146] In some embodiments, mammalian cells can be used as host cells of the present invention. Examples of mammalian cell types include, but are not limited to, human embryonic kidney (HEK) (e.g., HEK293, HEK293F) cells, Chinese hamster ovary (CHO) cells, HeLa cells, COS cells, PC3 cells, Vero cells, MC3T3 cells, NS0 cells, Sp2 / 0 cells, VERY cells, BHK cells, MDCK cells, W138 cells, BT483 cells, Hs578T cells, HTB2 cells, BT20 cells, T47D cells, NS0 cells (a mouse myeloma cell line that does not endogenously produce immunoglobulin chains), CRL7O3O cells, and HsS78Bst cells. In some embodiments, Escherichia coli (E. coli) cells can also be used as host cells of the present invention. E. coli strains include, but are not limited to, E. coli 294 (ATCC® 31,446), E. coli λ1776 (ATCC® 31,537), E. coli BL21(DE3) (ATCC® BAA-1025), and E. coli RV308 (ATCC® 31,608). Different host cells have characteristic and specific mechanisms for post-translational processing and modification (e.g., glycosylation) of protein products. An appropriate cell line or host system can be selected to ensure proper modification and processing of the expressed polypeptide. The above-described expression vectors can be introduced into appropriate host cells using conventional techniques in the art, such as transformation, transfection, electroporation, calcium phosphate precipitation, and direct microinjection. Once the vector has been introduced into the host cells for protein production, the host cells are cultured in conventional nutrient media modified as necessary to induce promoters, select transformants, or amplify the gene encoding the desired sequence.Methods for the expression of 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).

[0147] Protein Production, Recovery and Purification Host cells used to produce the polypeptides of the present invention can be grown in media known in the art and suitable for culturing the selected host cells. Examples of suitable media for mammalian host cells include Minimal Essential Medium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Expi293™ Expression Medium, DMEM supplemented with fetal bovine serum (FBS), and RPMI-1640. Examples of suitable media for bacterial host cells include Luria broth (LB) with any necessary supplementation of a selection agent, e.g., ampicillin. Host cells are cultured at a suitable temperature, e.g., about 20°C to about 39°C, e.g., 25°C to about 37°C, preferably 37°C, and at a CO2 level, e.g., 5-10%. The pH of the medium is generally about 6.8 to 7.4, e.g., 7.0, depending primarily on the host organism. The expression vectors of the present invention use an inducible promoter, and protein expression is induced under conditions suitable for promoter activation.

[0148] In some embodiments, depending on the expression vector and host cell used, the expressed protein can be secreted from the host cell (e.g., mammalian host cell) into the cell culture medium. Protein recovery can include filtering the cell culture medium to remove cellular debris. These proteins may be further purified. The polypeptides of the present invention can be purified by any method known in the art of protein purification, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Proteins can be isolated and purified, for example, by appropriately selecting an affinity column, such as a Protein A column (e.g., POROS Protein A chromatography), in combination with a chromatography column (e.g., POROS HS-50 cation exchange chromatography), filtration, ultrafiltration, salting out, and dialysis.

[0149] In other embodiments, host cells can be disrupted, for example, by osmotic shock, sonication, or lysis, to recover the expressed protein. Once the cells are disrupted, the cellular debris can be removed by centrifugation or filtration. In some cases, the polypeptide can be linked to a marker sequence, such as a peptide, to facilitate purification. An example of a marker amino acid sequence is a hexahistidine peptide (His tag), which binds with micromolar affinity to nickel-functionalized agarose affinity columns. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767, 1984).

[0150] Alternatively, the polypeptides of the present invention can be produced by cells of a subject (e.g., a human) by administering a vector (e.g., a viral vector (e.g., a retroviral vector, an adenoviral vector, a poxvirus vector (e.g., a vaccinia virus vector, e.g., Modified Vaccinia Ankara (MVA)), an adeno-associated virus vector, and an alphavirus vector)) containing a nucleic acid molecule encoding a polypeptide of the present invention, e.g., in gene therapy. Once in the subject's cells (e.g., by transformation, transfection, electroporation, calcium phosphate precipitation, direct microinjection, infection, etc.), the vector promotes expression of the polypeptide, which is then secreted from the cells. If treatment of the disease or disorder is the desired outcome, no further action may be required. If collection of protein is desired, blood can be drawn from the subject and the protein can be purified from the blood by methods known in the art.

[0151] VIII. Pharmaceutical Compositions and Formulations The present invention relates to pharmaceutical compositions comprising a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72))). In some embodiments, the pharmaceutical compositions of the present invention comprise, as a therapeutic protein, a polypeptide comprising an extracellular ActRIIa variant having a C-terminal extension (e.g., 1, 2, 3, 4, 5, 6 or more additional amino acids) (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1 to 70 (e.g., SEQ ID NOs: 6 to 70)). In some embodiments, the pharmaceutical compositions of the present invention comprise a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)) fused to a moiety (e.g., an Fc domain monomer or dimer thereof, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or serum albumin) as a therapeutic protein. In some embodiments, the pharmaceutical compositions of the present invention comprising the polypeptides of the present invention can be used in combination with other agents (e.g., therapeutic biologics and / or small molecules) or compositions in therapy. In addition to a therapeutically effective amount of the polypeptide, the pharmaceutical composition may include one or more pharmaceutically acceptable carriers or excipients and can be formulated by methods known to those skilled in the art. In some embodiments, the pharmaceutical compositions of the present invention comprise a nucleic acid molecule (DNA or RNA, e.g., mRNA) encoding a polypeptide of the present invention or a vector containing such a nucleic acid molecule.

[0152] Acceptable carriers and excipients for pharmaceutical compositions are non-toxic to recipients at the dosage and concentration used.Acceptable carriers and excipients may include buffers such as phosphate, citrate, HEPES, and TAE; antioxidants such as ascorbic acid and methionine; preservatives such as hexamethonium chloride, octadecyldimethylbenzylammonium chloride, resorcinol, and benzalkonium chloride; proteins such as human serum albumin, gelatin, dextran, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, histidine, and lysine; and carbohydrates such as glucose, mannose, sucrose, and sorbitol.The pharmaceutical composition of the present invention can be administered parenterally in the form of an injection preparation.Injectable pharmaceutical compositions can be prepared using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles include, but are not limited to, sterile water, saline, and cell culture medium (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-Modified Eagle Medium (α-MEM), F-12 medium). Formulation methods are known in the art, see, for example, Banga (ed.), Therapeutic Peptides and Proteins: Formulation, Processing and Delivery Systems (3rd Edition), Taylor & Francis Group, CRC Press (2015).

[0153] The pharmaceutical compositions of the present invention may be formulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules. The pharmaceutical compositions of the present invention may also be formulated in other drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules. Such techniques are described in Remington: The Science and Practice of Pharmacy, 22nd Edition (2012). Pharmaceutical compositions used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0154] The pharmaceutical compositions of the present invention may also be prepared as sustained-release formulations. Suitable sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the polypeptides of the present invention. Examples of sustained-release matrices include polyesters, hydrogels, polyactides, copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., LUPRON DEPOT™), and poly-D-(-)-3-hydroxybutyric acid. Some sustained-release formulations enable release of molecules over several months, e.g., 1 to 6 months, while other formulations release the pharmaceutical compositions of the present invention over shorter periods, e.g., days to weeks.

[0155] The pharmaceutical composition can be prepared in a unit dosage form, if necessary. The amount of the active ingredient, e.g., the polypeptide of the present invention, contained in the pharmaceutical preparation is such that an appropriate dose is provided within the specified range (e.g., a dose within the range of 0.01 to 100 mg / kg body weight).

[0156] Pharmaceutical compositions for gene therapy may be in an acceptable diluent or may comprise a slow-release matrix in which the gene delivery vehicle is embedded. When hydrodynamic injection is used as the delivery method, pharmaceutical compositions containing nucleic acid molecules encoding the polypeptides described herein or vectors (e.g., viral vectors) containing the nucleic acid molecules are easily delivered intravenously in large liquid volumes. Vectors that can be used as in vivo gene delivery vehicles include, but are not limited to, retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors, e.g., mutant vaccinia Ankara), adeno-associated virus vectors, and alphavirus vectors.

[0157] IX. Route, Dosage, and Administration The pharmaceutical composition comprising the polypeptide of the present invention as therapeutic protein can be prepared for, for example, intravenous administration, parenteral administration, subcutaneous administration, intramuscular administration, intraarterial administration, intrathecal administration or intraperitoneal administration.The pharmaceutical composition can also be prepared for or administered via oral administration, nasal administration, spray administration, aerosol administration, rectal or vaginal administration.For injection preparations, various effective pharmaceutical carriers are known in the art.For example, see ASHP Handbook on Injectable Drugs, Toissel, 18th Edition (2014).

[0158] In some embodiments, pharmaceutical compositions comprising nucleic acid molecules encoding polypeptides of the present invention or vectors containing such nucleic acid molecules can be delivered by gene delivery. Methods of gene delivery are well known to those skilled in the art. Vectors that can be used for in vivo gene delivery and expression include, but are not limited to, retroviral vectors, adenoviral vectors, poxvirus vectors (e.g., vaccinia virus vectors, e.g., mutated vaccinia Ankara (MVA)), adeno-associated virus vectors, and alphavirus vectors. In some embodiments, mRNA molecules encoding polypeptides of the present invention can be directly administered to a subject.

[0159] In some embodiments of the present invention, nucleic acid molecules encoding the polypeptides described herein or vectors containing such nucleic acid molecules can be administered using a hydrodynamic injection platform. In hydrodynamic injection, nucleic acid molecules encoding the polypeptides described herein are placed under the control of a strong promoter within an engineered plasmid (e.g., a viral plasmid). Plasmids are often easily delivered intravenously in large fluid volumes. Hydrodynamic injection uses controlled hydrodynamic pressure within a vein to increase cell permeability, so that the high pressure resulting from the rapid injection of a large fluid volume results in extravasation of the fluid and plasmid from the vein. Expression of nucleic acid molecules is primarily driven by the liver. In mice, hydrodynamic injection is often performed by injecting the plasmid into the tail vein. In certain embodiments, mRNA molecules encoding the polypeptides described herein can be administered using hydrodynamic injection.

[0160] The dosage of the pharmaceutical composition of the present invention depends on factors including the route of administration, the disease being treated, and the subject's physical characteristics, such as age, weight, and health status. The pharmaceutical composition of the present invention may contain a polypeptide of the present invention at a dose ranging from 0.01 to 500 mg / kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg / kg), or in more specific embodiments, about 0.1 to about 30 mg / kg, and even more specific embodiments, about 0.3 to about 30 mg / kg. The dosage can be adjusted by a physician according to conventional factors, such as the extent of the subject's disease and various parameters.

[0161] Pharmaceutical compositions are administered in a manner appropriate for the dosage form and in a therapeutically effective amount to improve or remedy symptoms. Pharmaceutical compositions are administered in a variety of dosage forms, including intravenous, subcutaneous, and oral dosage forms (e.g., ingestible solutions, drug-release capsules). Generally, therapeutic proteins are administered at 0.1-100 mg / kg, e.g., 1-50 mg / kg. Pharmaceutical compositions comprising the polypeptides of the present invention can be administered to a subject in need thereof, for example, daily, weekly, biweekly, monthly, bimonthly, quarterly, semi-annually, yearly, or one or more times (e.g., 1-10 times or more) as medically necessary. In some embodiments, pharmaceutical compositions comprising the polypeptides of the present invention can be administered to a subject in need thereof weekly, biweekly, monthly, bimonthly, or quarterly. Doses can be provided in single or multiple dosing regimens. The timing between doses can be decreased as the medical condition improves or increased as the patient's health declines.

[0162] X. Treatment method The present invention is based on the discovery that amino acid substitutions from the extracellular portion of ActRIIb to the extracellular portion of ActRIIa result in ActRIIa variants with improved properties. ActRIIa variants created by introducing residues from ActRIIb into ActRIIa retain the beneficial properties of ActRIIa, such as longer serum half-life and lower binding affinity for BMP9, while acquiring some of the beneficial properties of ActRIIb, such as enhanced 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 create useful therapeutics that can compete with endogenous activin receptors for ligand binding. Because ActRIIa variants contain the extracellular portion of the receptor, they are soluble and can 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 associated with elevated activin signaling (e.g., diseases or conditions associated with increased expression of activin receptors or activin receptor ligands). For example, loss of myostatin has been shown to increase skeletal muscle mass, suggesting that myostatin inhibits skeletal muscle growth. Consequently, treatment with therapeutic agents that bind to myostatin and reduce its interaction with endogenous receptors may be a viable approach to increasing 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 demonstrate that the extracellular ActRIIa variants described herein increase muscle mass and can be used to treat subjects with diseases or conditions that result in muscle weakness or muscle atrophy.

[0163] Furthermore, these data provide a compelling reason why the extracellular ActRIIa variants of the present invention can be used 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 one way to reduce body fat and / or body weight, indicating that the extracellular ActRIIa variants described herein can be used to indirectly treat metabolic diseases (e.g., obesity, type 1 diabetes, and type 2 diabetes) by increasing muscle mass. However, because activin receptors and activin receptor ligands have been shown to be elevated in obese mice and humans, the extracellular ActRIIa variants described herein can be used to treat obesity by reducing elevated activin receptor signaling (e.g., by binding to and sequestering endogenous activin receptor ligands, such as activin and myostatin).

[0164] The present invention provides compositions and therapeutic methods that can be used to increase muscle mass and strength in subjects in need thereof. In some embodiments, the subject may have a disease that causes muscle weakness or muscle atrophy (e.g., skeletal muscle weakness or atrophy). In some embodiments, the methods described herein relate to affecting myostatin, activin, and / or BMP9 signaling in subjects with diseases or conditions involving muscle weakness and atrophy. In some embodiments, polypeptides comprising extracellular ActRIIa variants described herein reduce or inhibit the binding of myostatin, activin, and / or BMP9 to their 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 the binding of myostatin, activin, and / or BMP9 to their receptors (e.g., ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa))) increases muscle mass in a subject.

[0165] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the 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 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 accompanied by muscle weakness and muscle atrophy (e.g., Duchenne muscular dystrophy (DMD), faciocraniohumeral muscular dystrophy (FSHD), inclusion body myositis (IBM), amyotrophic lateral sclerosis (ALS) sarcopenia, or cancer cachexia).

[0166] The present invention also includes methods for 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 the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72))).

[0167] 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 diabetes and type 2 diabetes). In some embodiments, the subject may suffer from 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 diabetes and type 2 diabetes), or a disease or condition that results in obesity. In some embodiments, a polypeptide comprising an extracellular ActRIIa variant described herein reduces or inhibits binding of myostatin, activin, and / or BMP9 to their receptors (e.g., ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa)). In some embodiments, acting on myostatin, activin, and / or BMP9 signaling (e.g., reducing or inhibiting the binding of myostatin, activin, and / or BMP9 to their receptors (e.g., ActRIIa, ActRIIb, and BMPRII (e.g., ActRIIa))) results in a reduction in the subject's body fat (e.g., body fat mass or percentage), a reduction in the subject's weight or weight gain, a reduction in fasting insulin levels, an increase in glucose clearance, or an increase in insulin sensitivity (e.g., a reduction in insulin resistance).

[0168] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the 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 onset of obesity (e.g., in patients at risk of developing obesity (e.g., patients who are overweight, have a family history of obesity, or have other medical conditions or genetic risk factors associated with an increased risk of obesity)) and / or to treat patients already diagnosed with obesity. For example, administration of an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1-72 (e.g., SEQ ID NOs: 6-72)) to a subject can help the subject lose weight by reducing fat mass. In some embodiments, the extracellular ActRIIa variant reduces fat mass while maintaining or increasing lean mass.

[0169] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72))) can be used to prevent the onset of diabetes (e.g., type 1 diabetes and type 2 diabetes) and / or to treat patients already diagnosed with diabetes. Patients at high risk of developing diabetes (e.g., individuals with a genetic predisposition, a family history of diabetes, prediabetes, association with other autoimmune diseases, or other metabolic diseases) may be prophylactically administered a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72))), thereby maintaining the normal function and health of β-cells and preventing or delaying 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 the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72))) may be administered to an individual before they are diagnosed with diabetes (e.g., type 1 diabetes and type 2 diabetes) or before they are diagnosed with clinical symptoms of diabetes (e.g., high blood glucose levels, high fasting insulin levels, insulin resistance, polyuria, polydipsia, polyphagia). In some embodiments, the extracellular ActRIIa polypeptide can be administered to a patient before they require insulin. In yet other embodiments, administration of the extracellular ActRIIa polypeptide can delay or postpone the need for insulin treatment in a diabetic patient. For example, administration of the extracellular ActRIIa polypeptide of the present invention to a subject can help increase the rate of glucose clearance from the blood.

[0170] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72))) may be administered to a subject to prevent and / or treat the onset of obesity or diabetes (e.g., type 1 diabetes and type 2 diabetes) in a patient, or to affect myostatin, activin, and / or BMP9 signaling (e.g., to reduce or inhibit binding of activin, myostatin, and / or BMP9 to their receptors). In some embodiments, the methods described herein reduce body fat (e.g., reduce the amount of subcutaneous fat and / or visceral fat, reduce adiposity, reduce the weight of epididymal and perirenal fat pads, or reduce the percentage of body fat). In some embodiments, the methods described herein reduce body weight or reduce weight gain (e.g., reduce the rate of weight gain). In some embodiments, the methods described herein reduce adipocyte proliferation. In some embodiments, the methods described herein lower LDL. In some embodiments, the methods described herein reduce triglycerides. In some embodiments, the methods described herein improve the serum lipid profile of a subject. In some embodiments, the methods described herein reduce body fat without reducing lean mass (e.g., without affecting or increasing lean 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 blood glucose levels) and / or 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 modulate insulin biosynthesis and / or secretion from beta-cells. In some embodiments, the methods described herein do not affect appetite for food intake.In some embodiments, the methods described herein do not cause vascular complications in a subject, such as increased vascular permeability or leakage.

[0171] In some embodiments, a polypeptide described herein (e.g., a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOs: 1 to 72 (e.g., SEQ ID NOs: 6 to 72))) reduces body fat, reduces body weight, or increases insulin sensitivity and / or glucose clearance by increasing muscle mass.

[0172] In any of the methods described herein, a polypeptide comprising an extracellular ActRIIa variant (e.g., an extracellular ActRIIa variant having the sequence of any one of SEQ ID NOS: 1-71 (e.g., SEQ ID NOS: 6-71)) and further comprising a C-terminal extension of 1 to 6 amino acids (e.g., 1, 2, 3, 4, 5, 6, or more amino acids) may 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 the sequence of any one of SEQ ID NOS: 1-72 (e.g., SEQ ID NOS: 6-72)) fused to a moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimer formation), an albumin-binding peptide, a fibronectin domain, or human serum albumin) may be used as a therapeutic protein. Nucleic acids encoding the polypeptides described herein, or vectors containing the nucleic acids, may also be administered according to any of the methods described herein. In any of the methods described herein, the polypeptide, nucleic acid, or vector can be administered as part of a pharmaceutical composition. [Example]

[0173] Example 1: Effect of extracellular ActRIIa mutants on body weight C57B1 / 6 mice received a single hydrodynamic injection (n=10 / group) of a plasmid construct encoding one of the following six polypeptides:

[0174] (1) human Fc (hFc), (2) extracellular ActRIIa fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 73); (3) extracellular ActRIIb (SEQ ID NO: 74) fused to the N-terminus of hFc via a GGG linker; (4) an extracellular ActRIIa variant (SEQ ID NO: 69) fused to the N-terminus of hFc via a GGG linker; and (5) An extracellular ActRIIb variant fused to the N-terminus of hFc via a GGG linker (sequence number 149).

[0175] 100 μg of the plasmid construct was delivered over 5–8 seconds at a volume of 10% of body weight. This high-volume, short-duration injection provided the necessary pressure to introduce the plasmid into hepatocytes where it would be expressed, particularly the protein of interest, under a strong, ubiquitous promoter. The protein of interest was secreted by the hepatocyte's endogenous machinery and circulated freely. Mice were weighed twice weekly for 30 days, and measurements were recorded as absolute body weight (BW) in grams and as percent weight change from baseline (Figures 2A and 2B, respectively).

[0176] Example 2: Effect of extracellular ActRIIa mutants on muscle mass Mice received a single hydrodynamic injection (n=10 / group) of a plasmid construct encoding one of the following six polypeptides:

[0177] (1) human Fc (hFc), (2) extracellular ActRIIa fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 73); (3) extracellular ActRIIb (SEQ ID NO: 74) fused to the N-terminus of hFc via a GGG linker; (4) an extracellular ActRIIa variant (SEQ ID NO: 69) fused to the N-terminus of hFc via a GGG linker; and (5) An extracellular ActRIIb variant fused to the N-terminus of hFc via a GGG linker (sequence number 149).

[0178] 100 μg of the plasmid construct was delivered over 5–8 seconds at a volume of 10% of body weight. This high-volume, short-duration injection provided the necessary pressure to introduce the plasmid into hepatocytes where it would be expressed, particularly the protein of interest, under a strong and ubiquitous promoter. The protein of interest was secreted by endogenous hepatocyte machinery and circulated freely. On days 0 (baseline), 14, and 28 of the study, mice underwent NMR analysis for determination of lean mass using a MiniSpec LF90 NMR analyzer (Bruker, The Woodlands, TX). The percent change in lean mass from baseline was recorded on days 14 and 28 (Figures 3A and 3B).

[0179] Example 3: Effect of extracellular ActRIIa mutants on body weight when administered as purified recombinant proteins Female C57Bl / 6 mice (Taconic Biosciences, Hudson, NY) were injected intraperitoneally twice weekly for 4 weeks with either Tris-buffered saline vehicle or one of the following five purified recombinant polypeptides at a dose of 10 mg / kg (n=10 / group).

[0180] (1) Tris-buffered saline, (2) extracellular ActRIIa fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 73); (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 / b9Δ9 mutant (SEQ ID NO: 58) fused to the N-terminus of hFc via a GGG linker; and (6) Extracellular ActRIIa / bΔ9min mutant fused to the N-terminus of hFc via a GGG linker (SEQ ID NO: 6).

[0181] Purified recombinant protein was produced by transient expression in HEK293 cells and purified from conditioned medium using protein A sepharose chromatography. After 4 weeks of treatment, mice were humanely sacrificed and autopsies were performed. Autopsies included weight collection for the whole body, as well as the gastrocnemius, pectoralis, and quadriceps muscles. Statistical analysis of muscle and body weight data was performed using GraphPad Prism 7 (GraphPad Software, La Jolla, CA) (Figures 4A and 4B, respectively).

[0182] Example 4: Evaluation of ActRIIa variant binding affinity by surface plasmon resonance (SPR) Biacore 3000 was used to measure the kinetics of interactions between ActRIIa variants and the ligands activin A, activin B, growth differentiation factor 11 (GDF11), and BMP-9. ActRIIa variants were expressed and purified as described in Example 3. ActRIIa variants were immobilized on chips (CM4 or CM5) using a capture antibody (anti-mouse from GEGE) in flow cells 2-4 to ensure proper orientation. Flow cell 1 was used as a reference cell to subtract nonspecific binding and bulk effects. HBS-EP+ buffer (GE Healthcare™) was used as the running buffer. Each ligand was flowed at a constant concentration series at 40 μl / min to avoid mass transfer effects. The K of each interaction was calculated. D Data were analyzed using Scrubber2 with BioLogic™ software to calculate (Table 4).

[0183] [Table 4]

[0184] Example 5: Effects of extracellular ActRIIa mutants on body weight and muscle weight C57Bl / 6 mice received a single hydrodynamic injection (n=10 / group) of a plasmid construct encoding one of the following 12 polypeptides:

[0185] (1) vehicle, (2) pLEV113-ActRIIa(19-127) (SEQ ID NO: 73) fused to the N-terminus of hFc via a GGG linker; (3) pLEV113-ActRIIb(41-155) (SEQ ID NO: 74) fused to the N-terminus of hFc via a GGG linker; (4) pLEV113-ActRIIa / b (SEQ ID NO: 69) fused to the N-terminus of hFc via a GGG linker; (5) pLEV113-ActRIIb / a (SEQ ID NO: 149) fused to the N-terminus of hFc via a GGG linker; (6) pLEV113-ActRIIa / b+ (SEQ ID NO: 150) fused to the N-terminus of hFc via a GGG linker; (7) pLEV113-ActRIIa / b-delta9m2 (SEQ ID NO: 38) fused to the N-terminus of hFc via a GGG linker; (8) pLEV113-ActRIIa / b-delta9m3 (SEQ ID NO: 41) fused to the N-terminus of hFc via a GGG linker; (9) pLEV113-ActRIIa / b-delta9m4 (SEQ ID NO: 44) fused to the N-terminus of hFc via a GGG linker; (10) pLEV113-ActRIIa / bmax1 (SEQ ID NO: 70) fused to the N-terminus of hFc via a GGG linker; (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.

[0186] 100 µg of the plasmid construct was delivered over 5–8 seconds at a volume of 10% of body weight. The high-volume, short-duration injection provided the necessary pressure to introduce the plasmid into hepatocytes where it would be expressed, particularly the protein of interest, under a strong and ubiquitous promoter. The protein of interest was secreted by endogenous hepatocyte machinery and circulated freely. Mice were weighed twice weekly for 30 days, and measurements were recorded as absolute body weight (BW) in grams and as percent weight change from baseline measurements (Figure 5A and 5B, respectively). Muscle was also weighed at the end of the study, and measurements were recorded in grams (Figure 6A and 6B).

[0187] Example 6: Dose effect of extracellular ActRIIa mutants on body weight, muscle weight, and muscle mass Eight-week-old male C57BL / 6 mice were weight-matched into nine groups (n=10 / group). Groups were administered 5 mL / kg of vehicle (Tris-buffered saline, pH 7.4) or one of four concentrations of ActRIIA / B-Fc (SEQ ID NO: 69 fused to the N-terminus of hFc via a GGG linker) or ActRIIA / 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 weekly for four weeks (8 doses), and the study was terminated on study day 28. Body weights were recorded on the day of dosing (Figures 7A and 7B) throughout the study, and at the end of the study, groups were NMR imaged for lean and fat mass analysis (Figures 8A and 8B), and pectoral and gastrocnemius muscle weights were collected and weighed (Figures 9A and 9B).

[0188] Example 7: Effects of extracellular ActRIIa mutants on obesity Adult male C57BL / 6 mice were assigned to weight-matched treatment groups (n=10 / group). All animals were maintained on a regular chow diet (Chow; Purina LabDiet 5001, St. Louis, MO) or a high-fat diet (HFD; Research Diets® D12331, New Brunswick, NJ). The chow and HFD groups were further divided into groups receiving either the ActRII mutant or vehicle twice weekly for 60 days. Body weight was measured twice weekly during treatment. Body composition was measured using a MiniSpec LF50 at baseline (before treatment and transition to an HFD) and then every other week until the end of the study. At the end of the study, tissues of interest (serum, plasma, muscle, and fat depots) were surgically removed and weighed. Serum samples were then assessed for biomarkers of obesity and plasma for Hba1c levels.

[0189] Other embodiments While the invention has been described in terms of particular embodiments thereof, it will be understood that further modifications are possible, and that this application is intended to cover any variations, uses, or adaptations of the invention which generally follow from the principles of the invention, including such departures from the present disclosure as are within known or related practice within the art to which this invention pertains and which may fall within the essential characteristics set forth above.

[0190] The entire contents of all publications, patents, and patent applications are incorporated herein by reference to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference in its entirety.

[0191] Other embodiments are within the scope of the following claims. The technical concepts that can be understood from the above-described embodiment will be described below as supplementary notes. [Appendix 1] containing an extracellular activin type IIa receptor (ActRIIa) mutant, The mutant GAILGRSETQECLX1X2NANWX3X4X5X6TNQTGVEX7CX8GX9X 10 X 11 X 12 X 13 X 14 HCX 15 ATWX 16 NISGSIEIVX 17 X 18 GCX 19 X 20 X 21 DX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 X6 is R; X7 is R or P; X8 is E; X9 is E; X1 is F or Y; X2 is Y; X3 is E; X4 is L; X5 is E or D; X6 is R; X7 is R or P; X8 is E; X9 is E; 10 is K or Q;X 11 is D;X 12 is K;X 13 is R;X 14 is L;X 15 is Y or F;X 16 is R or K; X 17 is K;X 18 is K;X 19 is W;X 20 is L;X 21 is D;X 22 is F or I;X 23 is T;X 24 is E or K;X 25 is E;X 26 is N; and X 27 is Q, a polypeptide.

[0192] [Appendix 2] X1 is F and X 10 is K. [Appendix 3] The polypeptide according to Appendix 1, wherein the mutant has any one of the sequences set forth in SEQ ID NOs: 6 to 72.

[0193] [Appendix 4] 4. The polypeptide of any one of appendices 1 to 3, further comprising a C-terminal extension of one or more amino acids.

[0194] [Appendix 5] 5. The polypeptide of claim 4, wherein the C-terminal extension is NP or NPVTPK. [Appendix 6] 6. The polypeptide of any one of appendix 1 to 5, further comprising an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions, an albumin-binding peptide, a fibronectin domain, or human serum albumin fused to the C-terminus of the polypeptide via a linker.

[0195] [Appendix 7] 7. The polypeptide of claim 6, wherein the linker is an amino acid spacer. [Appendix 8] A nucleic acid molecule encoding the polypeptide according to any one of appendices 1 to 7.

[0196] [Appendix 9] A vector comprising the nucleic acid molecule of Appendix 8. [Appendix 10] A pharmaceutical composition comprising a polypeptide according to any one of Appendixes 1 to 7, a nucleic acid molecule according to Appendix 8, or a vector according to Appendix 9, and one or more pharmaceutically acceptable carriers or excipients.

[0197] [Appendix 11] 10. A pharmaceutical composition for treating a subject having a disease or condition involving muscle weakness or muscle atrophy, said pharmaceutical composition comprising a therapeutically effective amount of a polypeptide according to any one of Appendixes 1 to 7, a nucleic acid molecule according to Appendix 8, or a vector according to Appendix 9, wherein said pharmaceutical composition is administered to said subject.

[0198] [Appendix 12] 12. The pharmaceutical composition of claim 11, wherein the disease or condition is Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, amyotrophic lateral sclerosis, sarcopenia, cancer cachexia, or inclusion body myositis.

[0199] [Appendix 13] 10. A pharmaceutical composition for increasing muscle mass in a subject in need thereof, comprising a therapeutically effective amount of a polypeptide according to any one of Appendixes 1 to 7, a nucleic acid molecule according to Appendix 8, or a vector according to Appendix 9, wherein the pharmaceutical composition is administered to the subject.

[0200] [Appendix 14] 14. The pharmaceutical composition of claim 13, wherein the subject has Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia, or cancer cachexia.

[0201] [Appendix 15] 10. A pharmaceutical composition for treating a subject having Duchenne muscular dystrophy, cystic facial brachial muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia, or cancer cachexia, the pharmaceutical composition comprising a therapeutically effective amount of a polypeptide described in any one of Appendixes 1 to 7, a nucleic acid molecule described in Appendix 8, or a vector described in Appendix 9, the pharmaceutical composition being administered to the subject.

[0202] [Supplementary Note 16] The extracellular activin type IIa receptor (ActRIIa) mutant, wherein the mutant is GAILGRSETQECLX1X2NANWX3X4X5X6TNQTGVEX7CX8GX9X 10 X 11 X 12 X 13 X 14 HCX 15 ATWX 16 NISGSIEIVX 17 X 18 GCX 19 X 20 X 21 DX22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 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 is K or Q;X 11 is D or A;X 12 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 is W or A;X 20 is 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 the mutant 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 to 96.

[0203] [Appendix 17] The variant is GAILGRSETQECLFX2NANWX3X4X5X6TNQTGVEX7CX8GX9KX 11 X 12 X 13 X 14 HCX 15 ATWX 16NISGSIEIVX 17 X 18 GCX 19 X 20 X 21 DX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 17. The polypeptide of claim 16, having the sequence VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 2).

[0204] [Appendix 18] The mutant is GAILGRSETQECLFX2NANWEX4X5RTNQTGVEX7CX8GX9KDKRX 14 HCX 15 ATWX 16 NISGSIEIVKX 18 GCWLDDX 22 NCYDRTDCVEX 23 X 24 X 25 X 26 PX 27 18. The polypeptide of claim 16 or 17, having the sequence VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 3).

[0205] [Appendix 19] The variant is GAILGRSETQECLFX2NANWEX4DRTNQTGVEX7CX8GX9KDKRX 14 HCX 15 ATWX 16 NISGSIEIVKX 18 GCWLDDX 22 NCYDRTDCVEX 23 KX 25 X 26 PX 27 19. The polypeptide according to any one of appendices 16 to 18, having the sequence VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 4).

[0206] [Appendix 20] The variant is GAILGRSETQECLFX2NANWEX4DRTNQTGVEPCX8GX9KDKRX 14HCFATWKNISGSIEIVKX 18 GCWLDDINCYDRTDCVEX 23 KX 25 X 26 PX 27 20. The polypeptide according to any one of appendices 16 to 19, having the sequence VYFCCCEGNMCNEKFSYFPEMEVTQPTS (SEQ ID NO: 5).

[0207] [Appendix 21] The polypeptide described in Appendix 16, wherein X1 is F. [Appendix 22] The polypeptide described in Appendix 16, wherein X1 is Y. [Appendix 23]X 10 23. The polypeptide of claim 16, 21, or 22, wherein

[0208] [Appendix 24]X 10 23. The polypeptide of claim 16, 21, or 22, wherein [Appendix 25] The polypeptide according to any one of Appendices 16 to 24, wherein X2 is F. [Appendix 26] The polypeptide according to any one of Appendices 16 to 24, wherein X2 is or Y.

[0209] [Appendix 27] The polypeptide described in any one of Appendices 16, 17, and 21 to 26, wherein X3 is E. [Appendix 28] The polypeptide described in any one of Appendices 16, 17, and 21 to 26, wherein X3 is A.

[0210] [Appendix 29] The polypeptide described in any one of Appendices 16 to 28, wherein X4 is K. [Appendix 30] The polypeptide according to any one of Appendices 16 to 28, wherein X4 is L. [Appendix 31] The polypeptide described in any one of Appendices 16, 17, 18, and 21 to 30, wherein X5 is D.

[0211] [Appendix 32] The polypeptide described in any one of Appendices 16, 17, 18, and 21 to 30, wherein X5 is E. [Appendix 33] A polypeptide described in any one of Appendices 16, 17, and 21 to 32, wherein X6 is R.

[0212] [Appendix 34] A polypeptide described in any one of Appendices 16, 17, and 21 to 32, wherein X6 is A. [Appendix 35] The polypeptide according to any one of Appendices 16 to 19 and 21 to 34, wherein X7 is P.

[0213] [Appendix 36] The polypeptide described in any one of Appendices 16 to 19 and 21 to 34, wherein X7 is R. [Appendix 37] The polypeptide described in any one of Appendices 16 to 36, wherein X8 is Y.

[0214] [Appendix 38] The polypeptide described in any one of Appendices 16 to 36, wherein X8 is E. [Appendix 39] The polypeptide described in any one of Appendices 16 to 38, wherein X9 is D. [Appendix 40] The polypeptide described in any one of Appendices 16 to 38, wherein X9 is E.

[0215] [Appendix 41]X 11 The polypeptide of any one of Appendices 16, 17 and 21 to 40, wherein [Appendix 42]X 11 The polypeptide according to any one of Appendices 16, 17 and 21 to 40, wherein is A.

[0216] [Appendix 43]X 12 The polypeptide of any one of appendices 16, 17 and 21 to 42, wherein [Appendix 44]X 12 The polypeptide according to any one of appendices 16, 17 and 21 to 42, wherein

[0217] [Appendix 45]X 13 The polypeptide of any one of appendices 16, 17 and 21 to 44, wherein [Appendix 46]X 13The polypeptide of any one of appendices 16, 17 and 21 to 44, wherein is A.

[0218] [Appendix 47]X 14 The polypeptide according to any one of Appendices 16 to 46, wherein is R. [Appendix 48]X 14 The polypeptide according to any one of appendices 16 to 46, wherein

[0219] [Appendix 49]X 15 The polypeptide according to any one of Appendices 16 to 19 and 21 to 48, wherein [Appendix 50]X 15 is Y.

[0220] [Appendix 51]X 16 The polypeptide according to any one of Appendices 16 to 19 and 21 to 50, wherein [Appendix 52]X 16 The polypeptide according to any one of Appendices 16 to 19 and 21 to 50, wherein

[0221] [Appendix 53]X 16 The polypeptide according to any one of Appendices 16 to 19 and 21 to 50, wherein is A. [Appendix 54]X 17 The polypeptide of any one of appendices 16, 17 and 21 to 53, wherein

[0222] [Appendix 55]X 17 The polypeptide of any one of appendices 16, 17 and 21 to 53, wherein is A. [Appendix 56]X 17 is Y.

[0223] [Appendix 57]X 17 The polypeptide of any one of appendices 16, 17 and 21 to 53, wherein [Appendix 58]X17 The polypeptide of any one of appendices 16, 17 and 21 to 53, wherein

[0224] [Appendix 59]X 18 The polypeptide according to any one of appendices 16 to 58, wherein [Appendix 60]X 18 The polypeptide according to any one of Appendices 16 to 58, wherein

[0225] [Appendix 61]X 19 is W. [Appendix 62]X 19 The polypeptide of any one of appendices 16, 17 and 21 to 60, wherein is A.

[0226] [Appendix 63]X 20 The polypeptide of any one of appendices 16, 17 and 21 to 62, wherein [Appendix 64]X 20 The polypeptide of any one of appendices 16, 17 and 21 to 62, wherein is A.

[0227] [Appendix 65]X 21 The polypeptide of any one of appendices 16, 17 and 21 to 64, wherein [Appendix 66]X 21 The polypeptide of any one of appendices 16, 17 and 21 to 64, wherein

[0228] [Appendix 67]X 21 The polypeptide of any one of appendices 16, 17 and 21 to 64, wherein [Appendix 68]X 21 The polypeptide of any one of appendices 16, 17 and 21 to 64, wherein is A.

[0229] [Appendix 69]X 21 The polypeptide of any one of appendices 16, 17 and 21 to 64, wherein [Appendix 70]X 21 is G.

[0230] [Appendix 71]X 21 The polypeptide of any one of appendices 16, 17 and 21 to 64, wherein [Appendix 72]X 21 The polypeptide of any one of appendices 16, 17 and 21 to 64, wherein is N.

[0231] [Appendix 73]X 21 The polypeptide of any one of appendices 16, 17 and 21 to 64, wherein [Appendix 74]X 22 The polypeptide according to any one of Appendices 16 to 17 and 21 to 73, wherein

[0232] [Appendix 75]X 22 The polypeptide according to any one of Appendices 16 to 19 and 21 to 73, wherein [Appendix 76]X 22 The polypeptide according to any one of Appendices 16 to 19 and 21 to 73, wherein

[0233] [Appendix 77]X 23 The polypeptide according to any one of Appendices 16 to 76, wherein is K. [Appendix 78]X 23 The polypeptide according to any one of Appendices 16 to 76, wherein is T.

[0234] [Appendix 79]X 24 The polypeptide of any one of appendices 16, 17, 18, and 21-78, wherein [Appendix 80]X 24 The polypeptide of any one of appendices 16, 17, 18, and 21-78, wherein is E.

[0235] [Appendix 81]X 25 The polypeptide according to any one of Appendices 16 to 80, wherein is D. [Appendix 82]X 25 The polypeptide according to any one of Appendices 16 to 80, wherein is E.

[0236] [Appendix 83]X 26 The polypeptide according to any one of Appendices 16 to 82, wherein is S. [Appendix 84]X 26 The polypeptide according to any one of Appendices 16 to 82, wherein is N.

[0237] [Appendix 85]X 27 The polypeptide according to any one of Appendices 16 to 84, wherein is E. [Appendix 86]X 27 The polypeptide according to any one of Appendices 16 to 84, wherein

[0238] [Appendix 87]X 23 is T and X 24 is E and X 25 is E and X 26 The polypeptide according to any one of Appendices 16 to 86, wherein is N. [Appendix 88]X 23 is T and X 24 is K and X 25 is E and X 26 The polypeptide according to any one of Appendices 16 to 86, wherein is N.

[0239] [Appendix 89]X 17 The polypeptide according to any one of appendices 16 to 88, wherein [Appendix 90] The polypeptide according to Appendix 16, wherein the mutant has any one of the sequences of SEQ ID NOs: 21 to 87.

[0240] [Appendix 91] Position X 24 The polypeptide according to any one of Appendices 16 to 90, wherein the amino acid is substituted with the amino acid K. [Appendix 92] Position X 24 The polypeptide according to any one of Appendices 16 to 90, wherein the amino acid is substituted with the amino acid E.

[0241] [Appendix 93] The polypeptide of any one of Appendices 16 to 92, further comprising a C-terminal extension of one or more amino acids. [Appendix 94] The polypeptide of Appendices 93, wherein the C-terminal extension is NP.

[0242] [Appendix 95] The polypeptide of Appendices 93, wherein the C-terminal extension is NPVTPK. [Appendix 96] The polypeptide described in any one of Appendices 16 to 95, further comprising an Fc domain monomer fused to the C-terminus of the polypeptide via a linker.

[0243] [Appendix 97] The polypeptide described in Appendix 96, wherein the Fc domain monomer comprises the sequence of SEQ ID NO: 97. [Appendix 98] The polypeptide described in any one of Appendices 16 to 95, further comprising a wild-type Fc domain fused to the C-terminus of the polypeptide via a linker.

[0244] [Appendix 99] The polypeptide described in Appendix 98, wherein the wild-type Fc domain comprises the sequence of SEQ ID NO: 151. [Appendix 100] The polypeptide described in any one of Appendices 16 to 95, further comprising an Fc domain having an amino acid substitution fused to the C-terminus of the polypeptide via a linker.

[0245] [Appendix 101] The polypeptide described in Appendix 100, wherein the Fc domain does not form a dimer. [Appendix 102] The polypeptide according to any one of Appendices 16 to 95, further comprising an albumin-binding peptide fused to the C-terminus of the polypeptide via a linker.

[0246] [Appendix 103] The polypeptide of Appendices 102, wherein the albumin-binding peptide comprises the sequence of SEQ ID NO: 152. [Appendix 104] A polypeptide described in any one of Appendices 16 to 95, further comprising a fibronectin domain fused to the C-terminus of the polypeptide via a linker.

[0247] [Appendix 105] The polypeptide described in Appendix 104, wherein the fibronectin domain comprises the sequence of SEQ ID NO: 153. [Appendix 106] The polypeptide according to any one of Appendices 16 to 95, further comprising human serum albumin fused to the C-terminus of the polypeptide via a linker.

[0248] [Appendix 107] The polypeptide described in Appendix 106, wherein the human serum albumin comprises the sequence of SEQ ID NO: 154. [Appendix 108] A polypeptide according to appendix 96 or 97, which forms a dimer.

[0249] [Appendix 109] The polypeptide described in any one of Appendices 96 to 108, wherein the linker is an amino acid spacer. [Appendix 110] The polypeptide of Appendices 109, wherein 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).

[0250] [Appendix 111] A polypeptide described in any one of Appendices 16 to 110, having a serum half-life of at least 7 days. [Appendix 112] K of 200 pM or more D 112. The polypeptide according to any one of appendices 16 to 111, which binds to human bone morphogenetic protein 9 (BMP9) at

[0251] [Appendix 113] A polypeptide described in Appendix 112, which binds to at least one of activin and myostatin and has low or weak binding to human BMP9. [Appendix 114] A polypeptide according to appendix 112 or 113, which does not substantially bind to human BMP9.

[0252] [Appendix 115] K below 800 pM D The polypeptide according to any one of appendices 16 to 114, which binds to human activin A at [Appendix 116] K below 800 pM D The polypeptide according to any one of appendices 16 to 115, which binds to human activin B at

[0253] [Appendix 117] K above 5 pM D 117. The polypeptide according to any one of appendices 16 to 116, which binds to human GDF-11 at [Appendix 118] A nucleic acid molecule encoding the polypeptide according to any one of Appendices 16 to 117.

[0254] [Appendix 119] A vector comprising the nucleic acid molecule of Appendices 118. [Appendix 120] A host cell expressing a polypeptide according to any one of Appendices 16 to 117, comprising the nucleic acid molecule according to Appendices 118 or the vector according to Appendices 119, wherein the nucleic acid molecule or vector is expressed in the host cell.

[0255] [Appendix 121] A method for producing the polypeptide according to any one of Appendices 16 to 117, comprising: a) providing a host cell comprising the nucleic acid molecule of Appendix 118 or the vector of Appendix 119, and b) expressing said nucleic acid molecule or vector in a host cell under conditions that allow the formation of said polypeptide.

[0256] [Appendix 122] A pharmaceutical composition comprising a polypeptide described in any one of Appendices 16 to 117, a nucleic acid molecule described in Appendices 118, or a vector described in Appendices 119, and one or more pharmaceutically acceptable carriers or excipients.

[0257] [Appendix 123] The pharmaceutical composition described in Appendix 122, wherein the polypeptide is in a therapeutically effective amount. [Appendix 124] A method of increasing muscle mass in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 16 to 117, a nucleic acid molecule described in Appendices 118, a vector described in Appendices 119, or a pharmaceutical composition described in Appendices 122 or 123.

[0258] [Appendix 125] The method of Appendices 124, wherein the subject has Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia, or cancer cachexia.

[0259] [Appendix 126] A method for affecting the signal transduction of at least one of myostatin, activin, and BMP9 in a subject having a disease or condition accompanied by muscle weakness and muscle atrophy, the method comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 16 to 117, a nucleic acid molecule described in Appendices 118, a vector described in Appendices 119, or a pharmaceutical composition described in Appendices 122 or 123.

[0260] [Appendix 127] The method of Appendices 126, wherein the disease or condition is Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, inclusion body myositis, amyotrophic lateral sclerosis, sarcopenia, or cancer cachexia.

[0261] [Appendix 128] A method of treating a subject having Duchenne muscular dystrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 16 to 117, a nucleic acid molecule described in Appendices 118, a vector described in Appendices 119, or a pharmaceutical composition described in any of Appendices 122 or 123.

[0262] [Appendix 129] A method of treating a subject having cystic facial brachial muscular dystrophy, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 16 to 117, a nucleic acid molecule described in Appendices 118, a vector described in Appendices 119, or a pharmaceutical composition described in any of Appendices 122 or 123.

[0263] [Appendix 130] A method of treating a subject having inclusion body myositis, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 16 to 117, a nucleic acid molecule described in Appendices 118, a vector described in Appendices 119, or a pharmaceutical composition described in any of Appendices 122 or 123.

[0264] [Appendix 131] A method of treating a subject having amyotrophic lateral sclerosis, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 16 to 117, a nucleic acid molecule described in Appendices 118, a vector described in Appendices 119, or a pharmaceutical composition described in any of Appendices 122 or 123.

[0265] [Appendix 132] A method for reducing body fat in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 16 to 117, a nucleic acid molecule described in Appendices 118, a vector described in Appendices 119, or a pharmaceutical composition described in any of Appendices 122 or 123.

[0266] [Appendix 133] A method for reducing body weight in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 16 to 117, a nucleic acid molecule described in Appendices 118, a vector described in Appendices 119, or a pharmaceutical composition described in any of Appendices 122 or 123.

[0267] [Appendix 134] A method of lowering blood glucose in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 16 to 117, a nucleic acid molecule described in Appendices 118, a vector described in Appendices 119, or a pharmaceutical composition described in any of Appendices 122 or 123.

[0268] [Appendix 135] A method of increasing insulin sensitivity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 16 to 117, a nucleic acid molecule described in Appendices 118, a vector described in Appendices 119, or a pharmaceutical composition described in any of Appendices 122 or 123.

[0269] [Appendix 136] The method of any one of Appendices 1132 to 135, wherein the subject has or is at risk of developing a metabolic disorder. [Appendix 137] A method for affecting the signaling of at least one of myostatin, activin, and BMP9 in a subject having or at risk of developing a metabolic disease, the method comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 16-117, a nucleic acid molecule described in Appendices 118, a vector described in Appendices 119, or a pharmaceutical composition described in any of Appendices 122 or 123.

[0270] [Appendix 138] A method of at least one of treating and preventing a metabolic disease in a subject, the method comprising administering to the subject a therapeutically effective amount of a polypeptide described in any one of Appendices 16 to 117, a nucleic acid molecule described in Appendices 118, a vector described in Appendices 119, or a pharmaceutical composition described in any of Appendices 122 or 123.

[0271] [Appendix 139] The method according to any one of Appendices 136 to 138, wherein the metabolic disease is selected from the group consisting of obesity, type 1 diabetes, and type 2 diabetes. [Appendix 140] The method of Appendices 139, wherein the metabolic disease is obesity.

[0272] [Appendix 141] The method of Appendices 139, wherein the metabolic disease is type 1 diabetes. [Appendix 142] The method described in Appendices 139, wherein the metabolic disease is type 2 diabetes. [Appendix 143] The method of any one of Appendices 132 to 142, wherein the method reduces at least one of the subject's weight and rate of weight gain.

[0273] [Appendix 144] The method of any one of Appendices 132 to 143, wherein the method reduces at least one of the amount of body fat and the percentage of body fat in the subject. [Appendix 145] The method of any one of Appendices 132 to 144, wherein the method does not affect the subject's appetite for food intake.

[0274] [Appendix 146] The method of any one of Appendices 132 to 130, wherein the method reduces obesity in the subject. [Appendix 147] The method of any one of Appendices 132 to 146, wherein the method reduces the weight of the epididymal and perirenal fat pads of the subject.

[0275] [Appendix 148] The method of any one of Appendices 132 to 147, wherein the method reduces the amount of at least one of subcutaneous fat and visceral fat in the subject. [Appendix 149] The method of any one of Appendices 132 to 148, wherein the method reduces fasting insulin levels in the subject.

[0276] [Appendix 150] The method of any one of Appendices 132 to 149, wherein the method reduces blood glucose levels in the subject. [Appendix 151] The method of any one of Appendices 132 to 150, wherein the method increases insulin sensitivity in the subject.

[0277] [Appendix 152] The method of any one of Appendices 132 to 151, wherein the method increases the glucose clearance rate of the subject. [Appendix 153] The method of any one of Appendices 132 to 152, wherein the method improves the serum lipid profile of the subject.

[0278] [Appendix 154] The method according to any one of Appendices 132 to 153, wherein the method does not reduce lean mass. [Appendix 155] The method described in any one of Appendices 124 to 154, wherein the method increases muscle mass.

[0279] [Appendix 156] A method described in any one of Appendices 124 to 155, wherein the method reduces or inhibits binding of at least one of activin and myostatin to its corresponding receptor.

[0280] [Appendix 157] The method of any one of Appendices 124-131 and 155-156, wherein the polypeptide, nucleic acid, vector, or pharmaceutical composition is administered in an amount sufficient to increase at least one of muscle mass and strength, or to affect at least one of myostatin, activin, and BMP9 in the subject, or to reduce or inhibit binding of at least one of activin and myostatin to their corresponding receptors.

[0281] [Appendix 158] The method of any one of Appendices 132-156, wherein 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 epididymal and perirenal fat pad weight, reduce body fat percentage, reduce body weight, reduce weight gain rate, lower fasting insulin levels, lower blood glucose levels, increase insulin sensitivity, affect signal transduction of at least one of myostatin, activin, and BMP9 in the subject, reduce adipocyte proliferation, reduce or inhibit binding of at least one of activin and myostatin to their corresponding receptors, reduce LDL, reduce triglycerides, improve serum lipid profile, modulate at least one of insulin biosynthesis and secretion from beta-cells, delay, postpone, or reduce the need for insulin, or increase glucose clearance.

[0282] [Appendix 159] The method of any one of Appendices 124 to 158, wherein the method does not cause vascular complications in the subject. [Appendix 160] The method of Appendices 159, wherein the method does not increase vascular permeability or leakage.

[0283] [Appendix 161] The method of any one of Appendices 124 to 160, wherein the method increases bone mineral density in the subject.

Claims

1. A composition comprising a homodimer of a polypeptide comprising an extracellular activin receptor type IIa (ActRIIa) variant, wherein the ActRIIa variant has the sequence of SEQ ID NO: 69 and is fused at its C-terminus to a human IgG1 Fc domain monomer.

2. The composition described in claim 1, wherein the Fc domain monomer is fused to the C-terminus of the ActRIIa mutant via a linker.

3. The composition described in claim 2, wherein the linker is an amino acid linker.

4. The amino acid linker is selected from the group consisting 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), 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), GAGAGAGGA (SEQ ID NO: 112), No. 112), GSGSGSGGS (SEQ ID NO: 113), GAGAGAGAG (SEQ ID NO: 114), GSGSGSGSG (SEQ ID NO: 115), GAGAGAGAGAG (SEQ ID NO: 116), GSGSGSGSGGS (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), GGSGGSGGSGGGS (SEQ ID NO: 123), GGAGGGAG (SEQ ID NO: 124), GGSGG GSG (SEQ ID NO: 125), GGAGGGAGGGAG (SEQ ID NO: 126), GGSGGGSGGGGSG (SEQ ID NO: 127), GGGGAGGGGAGGGGA (SEQ ID NO: 128), GGGGSGGGGSGGGGS (SEQ ID NO: 129), GGGAG (SEQ ID NO: 130), GGGAGG (SEQ ID NO: 131), GGGAGGG (SEQ ID NO: 132), 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), AEA AAKEAAAKA (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), GGSGGGSEGGGGSEGGGGSEGGGGSEGGGGSEGGGGSGGGS (SEQ ID NO: 146),The composition of claim 3, having the amino acid sequence of EAAAK (SEQ ID NO: 147) or PAPAP (SEQ ID NO: 148).

5. The composition described in claim 4, wherein the amino acid linker has an amino acid sequence of GGG.

6. A pharmaceutical composition comprising the composition according to any one of claims 1 to 5 and one or more pharmaceutically acceptable carriers or excipients.

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